TW201904996A - PCSK9 antibody, antigen-binding fragment thereof and medical use thereof - Google Patents

Abstract

The present invention relates to a PCSK9 antibody, antigen-binding fragments, and pharmaceutical use thereof. Furthermore, the present invention relates to chimeric and humanized antibodies comprising the CDR regions of the present PCSK9 antibody, as well as pharmaceutical compositions comprising the present PCSK9 antibody and antigen-binding fragments thereof, and their use as hypolipidemic drugs. In particular, the present invention relates to a humanized PCSK9 antibody, and its use in preparation of a medicament for the treatment of PCSK9 mediated disease.

Description

   PCSK9 antibody, its antigen-binding fragment and its medical use   

The invention relates to a PCSK9 antibody, an antigen-binding fragment of a PCSK9 antibody, a chimeric antibody comprising a CDR region of the PCSK9 antibody, a humanized antibody, a pharmaceutical composition comprising the PCSK9 antibody and an antigen-binding fragment thereof, and a lipid-lowering drug thereof. the use of.

Hypercholesterolemia is a disease of abnormal lipid metabolism characterized by elevated serum cholesterol levels. It is mainly manifested by elevated serum cholesterol levels, which causes cholesterol to accumulate in blood vessels and form atherosclerosis. A large number of clinical and experimental studies have confirmed that abnormal lipid metabolism is closely related to the occurrence and development of coronary heart disease. Therefore, reducing the concentration of cholesterol in the blood has become a major means of treating and preventing atherosclerosis.

At present, statins are the main drugs that regulate lipid metabolism in clinical practice. Liptor, as the most widely used cholesterol-lowering drug in the world, is also the best-selling drug in the history of medicine. By blocking the liver's enzymes that produce cholesterol, it reduces the production of cholesterol and increases the liver's intake of more cholesterol from the blood. , Thereby reducing the cholesterol concentration in the blood. However, Lipitor has its shortcomings. First, Lipitor can reduce LDL by 30% -40%, but there are still many patients whose blood lipid cannot be reduced to an effective concentration (LDL concentration <50mg / dL); Secondly, the response rate of patients to Lipitor is also different. Therefore, many patients need a more effective lipid-lowering drug.

Familial hypercholesterolemia (FM) is an autosomal dominant single-dominant hereditary disease with clinical features characterized by a significant increase in total blood cholesterol and low density lipoprotein-cholesterol (LDL-c). High, xanthomas, corneal arches, and early-onset cardiovascular disease. The low-density lipoprotein receptor (LDLR) gene mutation causes its deficiency or deficiency, and LDL-c cannot be successfully transported to the liver to clear, which leads to an increase in the level of LDL-c in the blood. Three genes have been identified to be related to the occurrence of FM. They are LDLR gene, apolipoprotein B100 gene and PCSK9 (proprotein convertase subtilisin / kexin type 9) gene.

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a proprotein convertase, which belongs to the protease K subfamily of the subtilase family that is secreted. The encoded protein is synthesized as a soluble zymogen and processed into an active PCSK9 in the endoplasmic reticulum through autocatalytic intramolecular processing. Studies have shown that PCSK9 can promote the degradation of LDL receptors and increase the level of LDL cholesterol in plasma, while LDL receptors mediate the LDL endocytosis in the liver, which is the main way to clear LDL from the circulatory system. Studies have found that 12.5% of patients with hypercholesterolemia (ADH) have detected mutations in the PCSK9 gene. There are various forms of PCSK9 mutations. According to the different effects of mutations on PCSK9 regulating LDL-C levels, they can be divided into two categories: loss-of-function and gain-of-function. Among them, loss-of-function mutations are related to low blood cholesterol levels, which can prevent the occurrence of coronary atherosclerotic heart disease. The PCSK9 mutation rate of low cholesterol in African population is higher than that of other races. PCSK9 mutants increase plasma cholesterol levels by increasing PCSK9 function and decreasing LDLR expression, which can lead to severe hypercholesterolemia and early-onset coronary atherosclerotic heart disease. The currently-obtained PCSK9 mutations include: D374Y, S127R, F216L, N157K, R306S and so on. Among them, compared with the PCSK9 wild type, the cell surface LDLR of the D374Y mutant was reduced by 36%, and the S127R mutation was correspondingly reduced by 10%.

The stability of antibody drugs is one of the key factors affecting the drugability of antibodies. As a product of recombinant gene expression, antibody drugs undergo a variety of physical and chemical degradations during their production, transportation, storage, and in vivo use, such as disulfide. Bond mismatch, oxidation, deamination, isomerization, etc. As a result, changes in the charge groups on the surface of the antibody, which indirectly lead to structural changes in the antibody, will ultimately affect the physicochemical properties of the antibody drug and biological functions in vivo and in vitro. Among them, isomerization and deamidation are two common chemical degradation pathways of antibody molecules, which seriously affect the stability, biological function and bioavailability of antibodies (Electrophoresis. 2010 Jun; 31 (11): 1764-72 .).

The aspartic acid (Asp) site in the antibody is prone to non-enzymatic post-translational modification. This modification causes Asp to undergo a process of cyclization of sulfenimine and eventually forms isomerized Asp. At present, Asp isomerization has been found in many antibodies as a common protein degradation pathway. It has been reported in the literature that Asp isomerization of the CDR region of an antibody can significantly reduce the affinity and chemical stability of the antibody, and ultimately affect the application potential of the antibody in disease treatment. (Biotechnol Bioeng. 2010 Feb 15; 105 (3): 515-523; MAbs. 2014 Mar-Apr; 6 (2): 327-39.). Therefore, the purpose of reducing Asp isomerization or mutating Asp sites at specific sites in the CDR region of antibodies is expected to be one of the means to improve the stability and function of antibodies.

At present, PCSK9, as a new target with great potential, has become a focus of research on hypercholesterolemia, which is of great significance for understanding the mechanism of cholesterol metabolism and seeking new treatments. Several multinational pharmaceutical companies are developing monoclonal antibodies against PCSK9. Related patents include WO2011111007, WO2011072263, WO2012101251, WO2012088313, WO2013039958, WO2013016648, WO2013008185, and so on.

The invention provides a PCSK9 antibody with higher affinity, higher selectivity, higher biological activity and chemical stability.

The present invention provides a PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9. The PCSK9 antibody or antigen-binding fragment thereof comprises the following CDR regions: i) the sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs: 12, 13, and 31; ii) The sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 15, 16 and 17, respectively.

In a preferred embodiment of the present invention, the HCDR3 sequence shown in SEQ ID NO: 31 is QYDYX ¹ EX ² WYFDV, wherein: X ¹ may be selected from D, E, H, M, N, or Q; X ² may It is selected from D, E, H, M, N or Q; but X ¹ and X ² cannot be D at the same time.

In another preferred embodiment of the present invention, the sequence of HCDR3 is selected from the sequence shown in any one of SEQ ID NOs: 38-47.

In another preferred embodiment of the present invention, wherein the PCSK9 antibody or antigen-binding fragment thereof comprises the following CDR regions: a) the sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 38, respectively; The sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively; b) the sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 39, respectively; The sequences are shown in SEQ ID NOs: 15, 16, and 17, respectively; c) The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 40, respectively; the sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO, respectively. : 15, 16, and 17; d) The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 41, and the sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively. E) The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 42, respectively, and the sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively; f) HCDR1 The sequences of HCDR2, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 43, respectively. The sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively; g) HCD The sequences of R1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 44, respectively. The sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively; h) the sequences of HCDR1, HCDR2, and HCDR3 The sequences are shown in SEQ ID NOs: 12, 13, and 45, and the sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively. I) The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO, respectively. : 12, 13, and 46, the sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 15, 16, and 17, respectively; or g) the sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and The sequences of LCDR1, LCDR2, and LCDR3 are shown in FIG. 47 as shown in SEQ ID NOs: 15, 16, and 17, respectively.

In another preferred embodiment of the present invention, the PCSK9 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody or a murine antibody, a chimeric antibody or an humanized antibody. .

In another preferred embodiment of the present invention, the light chain variable region of the PCSK9 antibody further comprises a light chain FR region of a murine κ chain or a murine κ chain variant; wherein the heavy chain variable region of the PCSK9 antibody It further comprises a heavy chain FR region of a murine IgG1 or a murine IgG1 variant.

In another preferred embodiment of the present invention, the light chain of the PCSK9 antibody further comprises a mouse-derived κ chain or a light chain constant region containing a mouse-derived κ chain variant; wherein the heavy chain of the PCSK9 antibody further comprises a mouse-derived IgG1 Or the heavy chain constant region of a murine IgG1 variant.

In another preferred embodiment of the present invention, the sequence of the heavy chain FR region on the heavy chain variable region of the humanized antibody is derived from the combined sequence of human germline heavy chain IGHV1-2 * 02 and hjh2 or a mutation thereof Sequence; the heavy chain FR region sequence includes the human germline heavy chain IGHV1-2 * 02 FR1, FR2, FR3 region sequence and hjh2 FR4 region sequence or the human germline heavy chain IGHV1-2 * 02 FR1, FR2, FR3 Mutant sequence of the region sequence and the FR4 region sequence of hjh2.

In another preferred embodiment of the present invention, the humanized antibody contains the heavy chain variable region shown in SEQ ID NO: 32; or a variant of the heavy chain variable region shown in SEQ ID NO: 32; Wherein, the variant of the heavy chain variable region shown in SEQ ID NO: 32 is a change having 1-10 amino acid insertions, deletions or substitutions on the heavy chain variable region shown in SEQ ID NO: 32. body. The amino acid insertion, deletion or substitution can be improved by using the existing technology to improve the performance of the antibody such as affinity and half-life, such as modifying the amino acid of the CDR region with affinity maturity, or modifying the FR region with back mutation. Amino acids.

In another preferred embodiment of the present invention, the heavy chain FR region sequence of the humanized antibody has 0 to 10 amino acid back mutations, preferably one or more selected from T30N, R87T, R72A, T74K, M48I, V68A, M70L, R38K and R67K amino acid back mutations.

In another preferred embodiment of the present invention, the humanized antibody comprises a heavy chain variable region selected from a sequence selected from any one of SEQ ID NOs: 32-37.

In another preferred embodiment of the present invention, the light chain FR region sequence on the light chain variable region of the humanized antibody is derived from the combined sequence of the human germline light chain IGKV1-39 * 01 and hjk2.1 and Its mutated sequence; the light chain FR region sequence includes the human germline light chain IGKV1-39 * 01 FR1, FR2, FR3 region sequence and hjk2.1 FR4 region sequence or the human germline light chain IGKV1-39 * 01 FR1 Sequences of FR2, FR2, FR3 and hjk2.1.

In another preferred embodiment of the present invention, the humanized antibody further comprises a light chain variable region as shown in any one of SEQ ID NOs: 24-27.

In another preferred embodiment of the present invention, the humanized antibody comprises a heavy chain variable region sequence and / or a light chain variable region sequence, and the heavy chain variable region sequence is selected from the group consisting of SEQ ID NOs: 32-37 Any of the sequences shown; the light chain variable region sequence is selected from any of the sequences shown in SEQ ID NOs: 24-27.

In another preferred embodiment of the present invention, the PCSK9 antibody comprises a heavy chain variable region selected from the sequence shown in any one of SEQ ID NOs: 32-37 and any sequence shown in SEQ ID NOs: 24-27 Light chain variable region shown.

In another preferred embodiment of the present invention, the PCSK9 antibody comprises a heavy chain variable region selected from the sequence shown in any one of SEQ ID NOs: 48-57 and any one of SEQ ID NOs: 24-27 Light chain variable region as shown in the sequence.

In another preferred embodiment of the present invention, the PCSK9 antibody comprises: 1) a heavy chain variable region shown in SEQ ID: 48 and a light chain variable region shown in SEQ ID: 24; 2) SEQ ID: The heavy chain variable region shown at 48 and the light chain variable region shown at SEQ ID: 25; 3) The heavy chain variable region shown at SEQ ID: 48 and the light chain variable region shown at SEQ ID: 26 4) The heavy chain variable region shown in SEQ ID: 48 and the light chain variable region shown in SEQ ID: 27; 5) The heavy chain variable region shown in SEQ ID: 49 and SEQ ID: 24 6) the heavy chain variable region shown in SEQ ID: 49 and the light chain variable region shown in SEQ ID: 25; 7) the heavy chain variable region shown in SEQ ID: 49 and The light chain variable region shown in SEQ ID: 26; 8) the heavy chain variable region shown in SEQ ID: 49 and the light chain variable region shown in SEQ ID: 27; 9) the light chain variable region shown in SEQ ID: 50 Heavy chain variable region and light chain variable region shown in SEQ ID: 24; 10) Heavy chain variable region shown in SEQ ID: 50 and light chain variable region shown in SEQ ID: 25; 11) SEQ The heavy chain variable region shown in ID: 50 and the light chain variable region shown in SEQ ID: 26; 12) The heavy chain variable region shown in SEQ ID: 50 and the light chain shown in SEQ ID: 27 13) the heavy chain variable region shown in SEQ ID: 51 and the light chain variable region shown in SEQ ID: 24; 14) the heavy chain variable region shown in SEQ ID: 51 and SEQ ID: 25 The light chain variable region shown; 15) the heavy chain variable region shown by SEQ ID: 51 and the light chain variable region shown by SEQ ID: 26; 16) the heavy chain variable region shown by SEQ ID: 51 Region and the light chain variable region shown in SEQ ID: 27; 17) the heavy chain variable region shown in SEQ ID: 52 and the light chain variable region shown in SEQ ID: 24; 18) SEQ ID: 52 The heavy chain variable region shown in the figure and the light chain variable region shown in SEQ ID: 25; 19) the heavy chain variable region shown in SEQ ID: 52 and the light chain variable region shown in SEQ ID: 26; 20 ) The heavy chain variable region shown in SEQ ID: 52 and the light chain variable region shown in SEQ ID: 27; 21) The heavy chain variable region shown in SEQ ID: 53 and the light chain shown in SEQ ID: 24 Chain variable region; 22) the heavy chain variable region shown in SEQ ID: 53 and the light chain variable region shown in SEQ ID: 25; 23) the heavy chain variable region shown in SEQ ID: 53 and SEQ ID : 26 light chain variable region; 24) heavy chain variable region shown in SEQ ID: 53 and light chain variable region shown in SEQ ID: 27; 25) heavy chain shown in SEQ ID: 54 Variable region The light chain variable region shown in SEQ ID: 24; 26) the heavy chain variable region shown in SEQ ID: 54 and the light chain variable region shown in SEQ ID: 25; 27) the light chain variable region shown in SEQ ID: 54 Heavy chain variable region and light chain variable region shown in SEQ ID: 26; 28) Heavy chain variable region shown in SEQ ID: 54 and light chain variable region shown in SEQ ID: 27; 29) SEQ The heavy chain variable region shown in ID: 55 and the light chain variable region shown in SEQ ID: 24; 30) The heavy chain variable region shown in SEQ ID: 55 and the light chain shown in SEQ ID: 25 may Variable region; 31) the heavy chain variable region shown in SEQ ID: 55 and the light chain variable region shown in SEQ ID: 26; 32) the heavy chain variable region shown in SEQ ID: 55 and SEQ ID: 27 The light chain variable region shown; 33) the heavy chain variable region shown in SEQ ID: 56 and the light chain variable region shown in SEQ ID: 24; 34) the heavy chain variable shown in SEQ ID: 56 Region and the light chain variable region shown in SEQ ID: 25; 35) the heavy chain variable region shown in SEQ ID: 56 and the light chain variable region shown in SEQ ID: 26; 36) SEQ ID: 56 The heavy chain variable region shown in the figure and the light chain variable region shown in SEQ ID: 27; 37) the heavy chain variable region shown in SEQ ID: 57 and the light chain variable region shown in SEQ ID: 24; 38 ) SEQ ID: The heavy chain variable region shown in 57 and the light chain variable region shown in SEQ ID: 25; 39) The heavy chain variable region shown in SEQ ID: 57 and the light chain variable region shown in SEQ ID: 26 And 40) the heavy chain variable region shown in SEQ ID: 57 and the light chain variable region shown in SEQ ID: 27.

In another preferred embodiment of the present invention, the heavy chain of the PCSK9 antibody further comprises a heavy chain constant region of human IgG1 or a variant thereof; preferably an amino acid mutation prolongs the half-life of the antibody in the serum of human IgG1 The heavy chain constant region of the variant, more preferably the heavy chain constant region of a human IgG1 variant that introduces a YTE mutation. The light chain of the PCSK9 antibody further comprises a light chain constant region of a human-derived kappa or a variant thereof.

In another preferred embodiment of the present invention, the humanized antibody comprises a heavy chain constant region shown in SEQ ID NO: 28 or SEQ ID NO: 29 and a light chain constant region shown in SEQ ID NO: 30.

The present invention further provides a pharmaceutical composition containing a therapeutically effective amount of the PCSK9 antibody or antigen-binding fragment thereof specifically binding to PCSK9 as described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The present invention further provides a DNA molecule encoding a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9 as described above.

The invention further provides an expression vector for the DNA molecule as described above.

The present invention further provides a host cell transformed by the expression vector as described above, the host cell is selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian cells.

The present invention further provides a method for producing a PCSK9 antibody or an antigen-binding fragment thereof as described above, the method comprising culturing a host cell as described in the aforementioned item in a culture to form and accumulate the PCSK9 antibody as described above Or its antigen-binding fragment, and the accumulated PCSK9 antibody or its antigen-binding fragment is recovered from the culture.

The invention further provides a method for immunodetection or determination of human PCSK9, which method comprises the step of specifically binding human PCSK9 using a PCSK9 antibody or an antigen-binding fragment thereof as described above under conditions suitable for specific binding to human PCSK9. .

The present invention further provides a reagent for detecting or measuring human PCSK9, which reagent comprises the PCSK9 antibody or an antigen-binding fragment thereof as described above.

The invention further provides a use of a PCSK9 antibody or an antigen-binding fragment thereof or a pharmaceutical composition as described above that specifically binds PCSK9 as described above in the manufacture of a medicament for treating a PCSK9-mediated disease or disorder, wherein the disease Or conditions are preferably cholesterol-related diseases (which include "serum cholesterol-related diseases"); more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's Symptoms and general dyslipidemia; preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention further provides a method for treating a PCSK9-mediated disease or condition, as described above, with a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9, the method comprising administering an effective amount of a specific binding to an individual. PCSK9 antibody or antigen-binding fragment thereof of PCSK9. The disease or condition is preferably a cholesterol-related disease; more preferably, hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general abnormalities Lipidemia; preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention further provides a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9 as described above or a pharmaceutical composition as described above for use in the preparation of a reagent for diagnosing a PCSK9-mediated disease or disorder.

Exemplary diseases that can be diagnosed using the PCSK9 antibody or antigen-binding fragment thereof of the present invention include cholesterol-related diseases (which include "serum cholesterol-related diseases"), which include any one or more of the following: hypercholesterolemia, heart disease, metabolism Syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general dyslipidemia (which is shown, for example, as elevated total serum cholesterol, elevated LDL, elevated triglycerides, Increased very low density lipoprotein (VLDL) and / or low HDL).

In one aspect, the invention provides a method of treating or preventing hypercholesterolemia and / or at least one of the following symptoms in a subject: dyslipidemia, atherosclerosis, cardiovascular disease (CVD) or coronary heart disease, The method includes administering to the individual an effective amount of a PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9. The present invention also provides the use of an effective amount of an anti-PCSK9 antibody and its antigen-binding fragment to antagonize extracellular or circulating PCSK9 in the preparation of a medicament for the treatment or prevention of hypercholesterolemia and / or at least one of the following symptoms in an individual: abnormal Lipidemia, atherosclerosis, CVD or coronary heart disease.

The PCSK9 antibody or antigen-binding fragment thereof specifically binding to PCSK9 provided by the present invention further has the characteristics of eliminating isomerization of CDR regions and has better stability.

FIG. 1 is a schematic diagram of primer design in the construction of an antibody vector of the present invention.

Figure 2 is a schematic diagram of the construction of an antibody vector of the present invention.

Figure 3 shows the binding ability curves of mutants at the D103 site in the HCDR3 region of different h001-4-YTE antibodies with wild-type PCSK9 protein. The results show that amino acid substitution at the D103 site does not affect the binding activity of the antibody to wild-type PCSK9.

Figure 4 shows the binding ability curves of mutants at the D105 site in the HCDR3 region of different h001-4-YTE antibodies and wild-type PCSK9 protein. ability.

Figure 5 shows the changes in LDL uptake by HepG2 cells at different h001-4-YTE anti-PCSK9 antibody concentrations. The data show that PCSK9 antibodies can promote LDL uptake by HepG2 cells.

Figure 6 shows the changes in LDL uptake of HepG2 cells at different h001-4-WT anti-PCSK9 antibody concentrations. The data show that PCSK9 antibodies can promote LDL uptake by HepG2 cells.

FIG. 7 is a graph showing changes in the concentration of LDL-c in the serum of mice injected with h001-4-WT anti-PCSK9 antibody over time (*: p <0.05, vs IgG, **: p <0.01, vs IgG). The data show that PCSK9 antibody can reduce the LDL-c concentration in the serum of mice overexpressing human PCSK9.

Figure 8 is the change of the concentration of LDL-c in the serum of mice injected with h001-4-WT anti-PCSK9 antibody relative to the IgG group. The data showed that compared with the IgG group, the PCSK9 antibody could reduce the LDL-c concentration in the serum of mice overexpressing human PCSK9.

FIG. 9 is a diagram showing the drug efficacy and drug metabolism detection of the antibody of the present invention in cynomolgus monkeys. Figure 9 shows that both h001-4-WT and h001-4-YTE can significantly reduce the LDL content in cynomolgus monkeys, and the reduction duration of h001-4-YTE is better than h001-4-WT.

Definition of Terms

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are described in J. biol. Chem, 243, p3558 (1968).

The "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa chains or lambda chains by different constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain according to the present invention may further comprise a light chain constant region, which comprises a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain according to the present invention may further comprise a heavy chain constant region, the heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (Fc region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions. The sequence from amine end to carboxy end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) or the numbering rules of Kabat and chothia. (HCDR1).

The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, and preferably humanized antibodies.

The term "murine antibody" in the present invention is a monoclonal antibody to human PCSK9 prepared according to the knowledge and skill in the art. The test article is injected with the PCSK9 antigen during preparation, and a fusion tumor expressing an antibody having a desired sequence or functional property is isolated. In a preferred embodiment of the present invention, the mouse-derived PCSK9 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived κ, lambda chain or a variant thereof, or further include a mouse-derived IgG1, IgG2, The heavy chain constant region of IgG3 or a variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody. To establish a chimeric antibody, select a fusion tumor that secretes a mouse-specific monoclonal antibody, then select the variable region gene from the mouse fusion tumor cell, and then select the constant region gene of the human antibody according to the needs. The variable region gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain of the PCSK9 chimeric antibody further comprises a light chain Fc region of a human-derived kappa, lambda chain, or a variant thereof. The antibody heavy chain of the PCSK9 chimeric antibody further comprises a heavy chain Fc region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or an amino acid is used Mutations (such as YTE mutations) of IgG1, IgG2 or IgG4 variants that extend the half-life of the antibody in serum.

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline Antibodies produced in antibody framework sequences. It can overcome the strong antibody variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components. Such framework sequences can be obtained from a public DNA library including germline antibody gene sequences or published references. Germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase ), and in Kabat , EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain the activity. The humanized antibody of the present invention also includes a humanized antibody that further undergoes affinity maturation of CDRs by phage display. In a preferred embodiment of the present invention, the CDR sequence of the PCSK9 humanized antibody mouse is selected from the sequence shown in SEQ ID NO: 12, 13, 31, 15, 16, or 17; the human antibody variable region The framework was designed and selected, in which the light chain FR region sequence on the antibody light chain variable region is derived from the human germline light chain IGKV1-39 * 01 and hjk2.1 combination sequence; wherein the antibody heavy chain variable region The heavy chain FR region sequence is derived from the combined sequence of the human germline heavy chain IGHV1-2 * 02 and hjh2. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region can be subjected to a minimum of reverse mutations to maintain the activity.

The "antigen-binding fragment" in the present invention refers to a Fab fragment, Fab 'fragment, F (ab') ₂ fragment, and Fv fragment ScFv fragment that binds to human PCSK9; the Fv fragment contains an antibody heavy chain Variable and light chain variable regions, but without constant regions, and the smallest antibody fragment with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The variable regions of two antibodies can also be linked into a polypeptide chain with different linkers, which is called a single chain antibody (single chain antibody) or a single chain Fv (sFv). The term "combining with PCSK9" in the present invention refers to the ability to interact with human PCSK9. The term "antigen-binding site" of the present invention refers to a three-dimensional spatial site on the antigen that is discontinuous and is recognized by the antibody or antigen-binding fragment of the present invention.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain, which region contains at least a portion of a constant region. The term includes natural sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest Sequence), 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc region is required for the effector function of the antibody. Effector functions include initiation of complement-dependent cytotoxicity (CDC), initiation of phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), and transport of antibodies through cell transduction via the cell barrier. In addition, the Fc region is essential for maintaining the serum half-life of IgG-like antibodies (Ward and Ghetie, Ther. Immunol. 2: 77-94 (1995)). Studies have found that the serum half-life of IgG antibodies is mediated by the binding of Fc and nascent Fc receptors (FcRn). FcRn is a heterodimer consisting of a transmembrane alpha chain and a soluble beta chain (β2-microglobulin). U.S. Patent No. 6,165,745 discloses a method for producing antibodies with reduced biological half-life by introducing mutations into DNA fragments encoding the antibodies. The mutation includes an amino acid substitution at position 253, 310, 311, 433, or 434 of the Fc-hinge domain. U.S. Patent No. 6,277,375 B1 discloses a composition containing a mutant IgG molecule that has an increased serum half-life relative to wild-type IgG, wherein the mutant IgG molecule contains the following amino acid substitutions: threonine replaces leucine at position 252, Substitution of serine at position 254 or phenylalanine at position 256 (M252Y, S254T and T256E). Mutant IgGs with amino acid substitutions at positions 433, 435, or 436 are also disclosed. U.S. Patent No. 6,528,624 discloses a variant of an antibody containing an IgG Fc region at one or more amino acid positions (positions 270, 322, 326, 327, 329, 331, 333, and 334) of the human IgG Fc region With amino acid substitution. PCT Publication No. WO 02 / 060919A2 discloses a modified IgG containing an IgG constant region containing one or more amino acid modifications relative to a wild-type IgG constant region, wherein the modified IgG is constant with a wild-type IgG Regions have an increased half-life compared to IgG, and one or more amino acid modifications are located at one or more of the following positions: 251, 253, 255, 285-290, 308-314, 385-389, and 428-435. Specifically, the "YTE" or "YET mutation" described herein refers to a mutation combination of the Fc region of IgG1, which is used to promote the binding of the Fc region to human FcRn and extend the half-life of the antibody in human serum. The YTE mutant contains a combination of three "YTE mutants": M252Y, S254T, and T256E. The residue numbering is according to the EU numbering system, which is also known as the EU index, as described in Kabat et al. (Refer to US Patent No. 7,658,921 ) Numbering of IgG heavy chains. Compared with wild-type antibodies, YTE mutant antibodies greatly extend the half-life of antibodies in serum, such as Dall'Acqua et al., J. Biol. Chem. 281: 23514-24 (2006) and US Patent No. 7,083,784.

Methods of producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human PCSK9 or fragments thereof, and the resulting antibodies can be coated, purified, and can be subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human-derived FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained by comparing the IMGT human antibody variable region germline gene database and MOE software from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351 Get on.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminal site in the Fc region. Stable pure strains were obtained by expressing antibodies specifically binding to human PCSK9. Positive pure strains were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium in which the antibody is secreted can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was further extracted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods. Treatment of cells includes contact of the reagent with the cells, and contact of the reagent with the fluid, wherein the fluid is in contact with the cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, that the patient has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or inhibit the development of such symptoms to any clinically measured extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a number of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired effect in the patient. It can be assessed whether the symptoms of the disease have been alleviated by any clinical testing method commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the invention (e.g., treatment methods or articles of manufacture) may not be effective in relieving symptoms of each target disease, any statistical test method known in the art such as the Studentt test, Chi-square test, Un according to Mann and Whitney The test, Kruskal-Wallis test (H test), Jonckheere-Terpstra check sum Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of patients.

"Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in other amino acids in proteins with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), This allows frequent changes to be made without altering the biological activity of the protein. Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. ., P. 224, (4th edition)). In addition, structural or functional similar amino acid substitutions are unlikely to disrupt biological activity.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of the method of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside the organism, cell, or human as appropriate. "Endogenous" refers to a substance that is produced in a cell, organism, or human body as appropriate.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same fluorenyl or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position of. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100. For example, when the sequences are optimally aligned, if 10 positions in the two sequences have 6 matches or are homologous, then the two sequences are 60% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include descendants. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of metastases. It should also be understood that, due to intentional or unintentional mutations, all descendants cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells. Where different names are meant, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a specific amount of nucleic acid, RNA, and / or DNA is amplified in a trace amount as described, for example, in US Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end of the destination region or beyond, so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5 'terminal nucleotides of the two primers may coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plastid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample. The method includes using known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acids.

"As needed" or "as needed" means that the event or environment described later can, but does not have to occur, and the description includes situations where the event or environment occurs or does not occur. For example, "including 1-3 antibody heavy chain variable regions as needed" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as a physiological / pharmaceutically acceptable carrier And excipients. The purpose of the pharmaceutical composition is to promote the administration to the living body, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

Examples and test examples

The present invention is further described below with reference to examples, but these examples do not limit the scope of the present invention. The experimental methods without specific conditions in the examples of the present invention usually follow conventional conditions, such as the manual of antibody technology experiments and molecular cloning manuals of Cold Spring Harbor; or according to the conditions recommended by the raw material or commercial product manufacturers. The reagents without specific sources are conventional reagents purchased on the market.

Example 1. Preparation of PCSK9 antigen and detection protein

Protein design and expression

Using human preprotein converting enzyme subtilisin 9 (human PCSK9, Uniprot number: Q8MBP7) as the template of the PCSK9 of the present invention, the amino acid sequence of the antigen and detection protein of the present invention is designed, and optionally, the fusion is based on the PCSK9 protein Different tags, such as his tags or immune-promoting peptides, such as PADRE peptides, were cloned into pTT5 vectors (Biovector, Cat #: 102762) or pTargeT vectors (promega, A1410), respectively, and transiently expressed in 293 cells or CHO- S was stably expressed and purified to obtain an antigen encoding the present invention and a protein for detection.

PCSK9 with His tag: PCSK9-His6, for immunogenic mice or detection reagents; SEQ ID NO: 1

Note: The bottom line is the signal peptide, and the italics is His6-tag (6-histidine tag).

PCSK9 with PADRE peptide and His tag: PCSK9-PADRE-His6, as immunogen, the PADRE peptide contained can promote immunity; SEQ ID NO: 2

Note: The underlined part is the signal peptide, the double underlined part is the linker, the dotted line part is the PADRE peptide, and the italic part is His6-tag.

PCSK9 and his tag fusion protein with TEV digestion site: PCSK9-TEV-His6. N-PCSK9 (N-terminal PCSK9 domain) can be obtained by TEV digestion as an immunogen; SEQ ID NO: 3

Note: The underlined part is the signal peptide, the double underlined part is the TEV digestion site, and the italicized part is His6-tag.

PCSK9-D374Y mutant protein with his tag: PCSK9-D374Y-His6, as detection reagent; SEQ ID NO: 4

Note: The bottom line is the signal peptide and the italics are His6-tag.

PCSK9: PCSK9 protein inserted with biotin-receiving peptide BP15 and his tag: PCSK9-BP15-His6. As a detection reagent, the BP15 peptide position can be biotin-labeled during the expression process, eliminating in vitro biotin labeling and possible conformational changes; SEQ ID NO: 5

Note: The underlined part is the signal peptide, the double underlined part is the biotin accepting peptide, and the italicized part is His6-tag.

PCSK9-Y: PCSK9 D374Y mutant protein inserted with biotin acceptor peptide and his tag: PCSK9-D374Y-BP15-His6, detection protein; SEQ ID NO: 6

Note: The underlined part is the signal peptide, the double underlined part is the biotin accepting peptide, and the italicized part is His6-tag.

Fragment of the extracellular domain of PCSK9 receptor protein LDLR with Flag tag and His tag: LDLR-ECD-Flag-His6, detection reagent; SEQ ID NO: 7

Note: The underlined part is the signal peptide, the double underlined part is the Flag tag, and the italicized part is His6-tag.

LDLR-Fc: shortened form of LDLR extracellular domain fragment and hIgG1-Fc fusion protein (with PCSK9 binding activity): LDLR-sECD-Fc (hIgG1) as a detection reagent; SEQ ID NO: 8

Note: The underlined part is the signal peptide, the double underlined part is the shortened form of LDLR extracellular domain fragment (LDLR-sECD) with binding activity to PCSK9, and the italicized part is the hIgG1-Fc part.

A more shortened form of the LDLR extracellular domain fragment and hIgG1-Fc fusion protein (with binding activity to pCSK9): LDLR-ssECD-Fc (hIgG1) as a detection reagent; SEQ ID NO: 9

Note: The underlined part is the signal peptide, the double underlined part is a more shortened form of LDLR extracellular domain fragment (LDLR-ssECD) with binding activity to PCSK9, and the italicized part is the hIgG1-Fc part.

Example 2. Purification of PCSK9 and LDLR-related recombinant proteins. Purification of recombinant proteins and purification of fusion tumor antibodies and recombinant antibodies.

1. Purification steps of His-tagged recombinant protein:

The cell expression supernatant sample was centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse the column volume 2-5 times. The replaced supernatant sample was applied to an IMAC column. The column was rinsed with 5 mM imidazole in PBS until the A ₂₈₀ reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound foreign proteins, and the effluent was collected. The target protein was further extracted with 300 mM imidazole in PBS, and the eluted peaks were collected. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE). The mobile phase was PBS. Depolymerize peaks and collect spikes. The obtained protein was identified as correct by electrophoresis, peptide mapping, and LC-MS. PCSK9-His6 with His tag (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), PCSK9-TEV-His6 (SEQ ID NO: 3) PCSK9-D374Y-His6 (SEQ ID NO) : 4), PCSK9-BP15-His6 (SEQ ID NO: 5), PCSK9-D374Y-BP15-His6 (SEQ ID NO: 6) are used for the immunogen or detection reagent of the antibody of the present invention. Among them, PCSK9-TEV-His6 is purified by TEV enzyme, and the digested product is then combined with IMAC column to remove TEV enzyme. PCSK9-TEV-His6, which has not been digested completely, or a C-terminal domain with a His tag that has been excised The fragment was concentrated in the IMAC effluent to obtain a PCSK9 fragment (abbreviated as N-pCSK9) that retained only the N-terminal domain, and was used as an immunogen for mouse immunization.

2. Purification steps of LDLR-ECD-Flag-His6 (SEQ ID NO: 7) recombinant protein with His tag and Flag tag:

Centrifuge the sample at high speed to remove impurities and concentrate to the appropriate volume. The flag affinity column was equilibrated with 0.5 × PBS, and the column volume was washed 2-5 times. The dedifferentiated cell expression supernatant sample was applied to a column. The column was rinsed with 0.5 × PBS until the A ₂₈₀ reading dropped to baseline. The column was rinsed with 0.3M NaCl in PBS, and the foreign proteins were washed and collected. The target protein was extracted with 0.1M acetic acid (pH 3.5-4.0), and collected, and the pH was adjusted to neutral. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE). The mobile phase was PBS. Depolymerize the peaks, collect the extracted peaks and collect the samples for electrophoresis, peptide mapping, and LC-MS identification. LDLR-ECD-Flag-His6 (SEQ ID NO: 7) with a FLAG / His6 tag was obtained and used for performance testing of the antibody of the present invention.

3. Purification steps of LDLR Fc fusion protein:

The cell expression supernatant sample was centrifuged at high speed to remove impurities, concentrated to an appropriate volume, and applied to a Protein A column. Flush the column with PBS until the A ₂₈₀ reading drops to baseline. The target protein was extracted with 100 mM sodium acetate pH 3.0, and neutralized with 1 M Tris-HCl. The extracted sample was appropriately concentrated and further purified by Superdex200 (GE) gel chromatography equilibrated with PBS. The depolymerized peaks were collected and used for equipment. This method was used to purify LDLR-sECD-Fc (hIgG1) (SEQ ID NO: 8) and LDLR-ssECD-Fc (hIgG1) (SEQ ID NO: 9). Both can be used as a PCSK9 antibody functional test.

Example 3 Preparation of anti-human PCSK9 fusion tumor monoclonal antibody

1.Immunity

Anti-human PCSK9 monoclonal antibodies were produced by immunizing mice. SJL white mice for experiments, female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Jing) 2012-0001). Rearing environment: SPF level. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12 / 12-hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. The adapted mice were immunized according to two protocols (A / B), 6-10 mice per group. Immunization antigens were human PCSK9-His6 (SEQ ID NO: 1), pCSK9-PADRE-His6 (SEQ ID NO: 2) and N-PCSK9 (SEQ ID NO: 3) with His tags.

Scheme A was emulsified with Freund's adjuvant (sigma Lot Num: F5881 / F5506): Freund's complete adjuvant (CFA) was used for the first immunization, and Freund's incomplete adjuvant (IFA) was used for the rest of the immunization. The ratio of antigen to adjuvant was 1: 1, 100 μg / head (first immunization), and 50 μg / head (boosting). On day 0, 100 μg / post-emulsified antigen was injected intraperitoneally (IP), once every two weeks after the first immunization, for a total of 6-8 weeks.

Protocol B was cross immunized with Tiermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161). The ratio of antigen to adjuvant (titermax) is 1: 1, the ratio of antigen to adjuvant (Alum) is 3: 1, 10-20 μg / head (first immunization), and 5 μg / head (boosting). On the 0th day, intraperitoneal (IP) injection of 20/10 μg / post-emulsified antigen, once a week after the first immunization, Titermax and Alum were used alternately for a total of 6-11 weeks. Four weeks after immunization, antigens were selected for injection on the back or intraperitoneally, depending on back clumps and abdominal swelling.

Cell fusion

Sera with high antibody titers (see Test Examples 1 and 2 below, combined with the ELISA method of PCSK9) and titers tending to platelet were spleen-cell fused, and the selected mice were sprinted 72 hours before fusion to PCSK9- His6 10 μg / head, injected intraperitoneally. Spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC® CRL-8287 ^™ ) using an optimized PEG-mediated fusion step to obtain fusion tumor cells. Good fusion hybridoma cells HAT complete medium (containing 20% FBS, 1 × HAT and 1 × OPI of RPMI-1640 medium) were resuspended, aliquoted into 96-well cell culture plates (1 × 10 ⁵ / 150μl / hole ), 37 ° C, 5% CO ₂ incubation. On the fifth day after fusion, HAT complete medium was added, 50 μl / well, and incubated at 37 ° C. and 5% CO ₂ . From day 7 to day 8 after fusion, according to the cell growth density, the medium is completely changed. The medium is HT complete medium (RPMI-1640 medium containing 20% FBS, 1 × HT and 1 × OPI), 200 μl / well, 37 ° C, 5% CO ₂ incubation.

3.Screening of fusion tumor cells

On days 10-11 after fusion, an ELISA method was performed to detect PCSK9 or PCSK9-Y in accordance with cell growth density (see Test Examples 1 and 2). The positive well cells combined with ELISA were tested by PCSK9 or PCSK9-Y combined with LDLR to block ELISA (see Test Examples 3 and 4), the positive wells were exchanged, and the cells were expanded into the 24-well plate in time according to the cell density. The cell lines transferred to the 24-well plate were re-tested for seed conservation and first colonization. The first secondary selection screen (see Test Examples 1 and 2) is positive for seed conservation and the second secondary selection. The second selection was positive (see Test Examples 1 and 2) for seed conservation and protein expression. Fusion tumor cells that have the effect of blocking the binding of PCSK9 or PCSK9-Y to LDLR (see Test Examples 3 and 4) were obtained by multiple fusions.

The fusion tumor clone mAb-001 was obtained through screening experiments and binding experiments. Antibodies were further prepared by serum-free cell culture. The antibodies were purified according to the purification examples for use in the detection examples.

4. Sequence determination of fusion tumor positive pure strains

The process of selecting a sequence from a positive fusion tumor is as follows. Fusion tumor cells in the logarithmic growth phase were collected, RNA was extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the instructions of the kit, and reverse transcription was performed using a PrimeScript ^™ Reverse Transcriptase kit (Takara, Cat No. 2680A). The reverse-transcribed cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing. The amino acid sequences corresponding to the heavy and light chain variable region DNA sequences of mAb-001 were obtained, and the sequence of the mouse anti-variable region of the fusion tumor pure strain mAb-001 was determined as follows:> mAb-001 VH SEQ ID NO: 10

> mAb-001 VL SEQ ID NO: 11

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics in the sequence, and CDR sequences in the bottom line.

Example 4 Humanization of anti-human PCSK9 fusion tumor monoclonal antibodies

1. Selection of the humanized framework of the fusion tumor pure strain mAb-001. By comparing the germline gene database and the MOE software of the heavy and light chain variable regions of IMGT human antibodies, the heavy and light chains with high homology to mAb-001 can be selected. The germline gene of the variable region was used as a template, and the CDRs of the two mouse-derived antibodies were transplanted into the corresponding human-derived templates to form a variable region sequence of the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Wherein amino acid residues are identified and annotated by the Kabat numbering system.

The humanized light chain template of the mouse antibody mAb-001 is IGKV1-39 * 01 and hjk2.1, and the humanized heavy chain template is IGHV1-2 * 02 and hjh2. After humanization, the humanized antibody h001- The variable region sequence of 1 is as follows:> h001-1 VH SEQ ID NO: 18

> h001-1 VL SEQ ID NO: 24

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequence in italics in the sequence, and the CDR sequence in the bottom line.

2. The template selection and reverse mutation design of the fusion tumor pure strain mAb-001 are shown in Table 2 below; the humanized sequence combination of the fusion tumor pure strain after reverse mutation is shown in Table 4.

The specific sequence of the variable region of each mutant is shown in Table 3:

3. The above humanized variable region sequence is combined with the heavy chain constant region from human IgG and the light chain constant region of human kappa chain for antibodyization. The heavy chain constant region is derived from human IgG1 (such as SEQ ID NO: 28), the light chain constant region is from a human kappa chain (e.g., SEQ ID NO: 30). The corresponding humanized antibodies were obtained and tested by ELISA method (see Test Example 1) combined with PCSK9 and ELISA method (see Test Example 2) by PCSK9-Y; and positive well cells combined with ELISA detected by PCSK9 / LDLR-bound blocking ELISA test (see Test Example 4), and PCSK9-Y / LDLR-bound blocking ELISA test (see Test Example 3); the results are shown in Table 5-8.

The results show that the PCSK9 antibody obtained by the present invention has high binding activity with PCSK9 and PCSK9-Y; and can effectively block the binding between PCSK9 / PCSK9-Y and LDLR.

Example 5.Construction and expression of anti-human PCSK9 humanized antibody IgG1-YTE format

Example 4 The antibody was selected from the constant region of human heavy chain IgG1 / light chain kappa and each variable region to form a full-length antibody. The IgG1 heavy chain constant region mutation with YTE in the Fc segment can also be selected to increase the corresponding IgG1 antibody in the serum. (For example, replacing the heavy chain constant region of h001-4 with a YTE-mutated heavy chain constant region variant, the sequence is shown in SEQ ID NO: 29, and the antibody h001-4-YTE is obtained, correspondingly , H001-4 is also called h001-4 WT). Likewise, other known mutations in the art can be used to increase antibody performance.

Heavy chain constant region sequence (human IgG1): SEQ ID NO: 28

Heavy chain constant region sequence (IgG1-YTE): SEQ ID NO: 29

Light chain constant region sequence: SEQ ID NO: 30

1. Molecular selection of recombinant chimeric antibodies

After sequencing the positive antibody molecule obtained from the fusion tumor screening, the variable region coding gene sequence is obtained. Primer and tail primers were designed based on the sequence obtained. Using the sequenced gene as a template, the VH / VK gene fragments of each antibody were constructed by PCR, and then combined with the expression vector pHr (with signal peptide and hIgG1 / hkappa constant region gene (CH1-FC / CL) fragment). Homologous recombination was performed to construct a full-length recombinant plastid VH-CH1-FC-pHr / VL-CL-pHr to form a h001 chimeric antibody.

2. Molecular selection of humanized antibodies

The human-designed antibody sequence was codon-optimized to generate the human codon-preferred coding gene sequence. Primer PCR was designed to construct the antibody VH / VK gene fragments, and then combined with the expression vector pHr (with signal peptide and hIgG1 / hkappa constant region). Gene (CH1-FC / CL) fragment) was subjected to homologous recombination to construct a full-length humanized antibody expressing plastid VH-CH1-FC-pHr / VL-CL-pHr.

3. Expression and purification of recombinant chimeric antibodies and humanized antibodies

The plastids expressing the light and heavy chains of the antibodies were transfected into HEK293E cells at a ratio of 1: 1.2. After 6 days, the expression supernatants were collected, the impurities were removed by high-speed centrifugation, and purification was performed using a Protein A column. Flush the column with PBS until the A ₂₈₀ reading drops to baseline. The target protein was extracted with an acidic extraction solution of pH 3.0-pH 3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. The extracted sample was appropriately concentrated, and further purified using PBS balanced gel chromatography Superdex200 (GE) to remove the polymer and collect the monomer peaks for use in equipment.

Example 6: Heavy chain CDR3 mutants of h001 series antibodies

Isomerization of aspartic acid in antibodies is one of the main factors affecting the chemical stability of antibodies. In particular, aspartic acid isomerization modification of some CDR regions of antibodies is generally chosen to avoid or reduce mutations as much as possible. According to the accelerated stability test and computer analog antibody structure and hot spot prediction, it was found that aspartic acid in the DED site (ie, D103 / E104 / D105) of the HCDR3 heavy chain of h001 series antibodies is prone to isomerization. According to the amino acid properties With computer antibody structure analog technology, the amino acid at the above position can be substituted with any amino acid to effectively eliminate or reduce the isomerization of the site. Preferably, the heavy chain variable region of the h001 series antibody The CDR3 mutant is: QYDY X ¹ EX ² WYFDV (SEQ ID NO: 31), where X ¹ is the amino acid residue at position 103 of the heavy chain variable region of the h001 series antibody, and X ¹ may be selected from Asp, Glu, His, Met, Asn or Gln; X ² is the amino acid residue at position 105 of the heavy chain variable region of the h001 series antibody; X ² may be selected from Asp, Glu, His, Met, Asn or Gln. However, X ¹ and X ² cannot be Asp at the same time.

Further, the CDR3 containing the mutations at positions 103 and 105 and the FR region containing different back mutations can form the following heavy chain variable region:> h001_VH.1-CDR3 mutant (SEQ ID NO: 32)

> h001_VH.1A-CDR3 mutant (SEQ ID NO: 33)

> h001_VH.1B-CDR3 mutant (SEQ ID NO: 34)

> h001_VH.1C-CDR3 mutant (SEQ ID NO: 35)

> h001_VH.1D-CDR3 mutant (SEQ ID NO: 36)

> h001_VH.1E-CDR3 mutant (SEQ ID NO: 37)

Exemplarily, the specific mutant form of the CDR3 mutant (SEQ ID NO: 31) of the heavy chain of the h001 series antibody and the specific mutant form of h001 VH.1 are shown in the following mutants and Tables 5 and 6.

The following test method is used to verify the performance and beneficial effects of the antibodies of the present invention.

Test example 1: ELISA experiment of PCSK9 antibody binding to wild-type PCSK9 protein

The binding test of the PCSK9 antibody and PCSK9 of the present invention is detected by the amount of binding of the antibody to wild-type PCSK9 (WT-PCSK9, SEQ ID NO: 5) fixed on an ELISA plate.

Streptavidin (sigma, CAT # S4762) was diluted to 2 μg / ml with PBS, coated on a 96-well ELISA plate, and left at 4 ° C. overnight. After washing the plates, they were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skimmed milk powder) at 37 ° C for 2 hours. Wash the plate and add in-house produced biotin-labeled PCSK9 (bio-WT-PCSK9, diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skimmed milk powder) 100 μl / well, incubate at 37 ° C for 1 hour. Wash the plates, add anti-PCSK9 antibody samples diluted at different concentrations, and incubate at 37 ° C for 1 hour. The plate was washed again, and horseradish peroxidase-sheep anti-human (H + L) antibody (jackson, CAT # 109-035-088) was added, and incubated at 37 ° C for 1 hour. The plate was washed again, and tetramethylbenzidine solution was added to develop a color. Finally, add stop solution, measure OD450 on the microplate reader, and calculate its EC50 value.

The binding force ELISA test of the chimeric antibody and back-mutated antibody of the present invention with PCSK9 is shown in Table 7.

The results show that the PCSK9 antibody of the present invention has high binding activity to PCSK9.

In order to eliminate the isomerization problem caused by aspartic acid in the CDR region of the antibody, for example, in a specific embodiment, based on the antibody h001-4-YTE, the anti-weight chain CDR3 is mutated (h001-4D105H That is, on the basis of h001-4-YTE, the mutant of the 105th position in the variable region of the weight chain CDR3 was mutated to H, and other mutants were named by analogy), and the binding activity of different CDR3 mutants to PCSK9 was tested. Exemplary mutants are: h001-4-YTE D103E, h001-4-YTE D103H, h001-4-YTE D103M, h001-4-YTE D103N, h001-4-YTE D103Q, h001-4-YTE D105E, h001 -4-YTE D105H, h001-4-YTE D105M, h001-4-YTE D105N, h001-4-YTE D105Q, etc. The antibodies of each mutant were expressed and purified in small quantities, and the binding ability of each mutant to the wild-type PCSK9 protein was tested by referring to the experimental method of Test Example 1. The results are shown in Table 8, Figures 3 and 4.

The results showed that the amino acid substitution of D103 or D105 in the antibody heavy chain CDR3 still maintained the binding activity of the new antibody to wild-type PCSK9.

Test Example 2: ELISA experiment of PCSK9 antibody binding to PCSK9-Y

The binding test of the PCSK9 antibody and PCSK9-Y of the present invention is detected by the amount of binding of the antibody to PCSK9-Y (mutated PCSK9, SEQ ID NO: 6) fixed on an ELISA plate.

Streptavidin (sigma, CAT # S4762) was diluted to 2 μg / ml with PBS, coated on a 96-well ELISA plate, and left at 4 ° C. overnight. After washing the plates, they were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skimmed milk powder) at 37 ° C for 2 hours. Wash the plate and add in-house produced biotin-labeled PCSK9-Y (bio-PCSK9-Y, diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skimmed milk powder) 100 μl / well, 37 ° C Incubate for 1 hour. Wash the plates, add anti-PCSK9 antibody samples diluted at different concentrations, and incubate at 37 ° C for 1 hour. The plate was washed again, and horseradish peroxidase-sheep anti-human (H + L) antibody (jackson, CAT # 109-035-088) was added, and incubated at 37 ° C for 1 hour. The plate was washed again, and tetramethylbenzidine solution was added to develop a color. Finally, add stop solution, measure OD450 on the microplate reader, and calculate its EC50 value.

The binding force ELISA test of the chimeric antibody, back-mutated antibody of the present invention and mutant PCSK9 is shown in Table 9.

The results show that the PCSK9 antibody of the present invention has high binding activity to PCSK9-Y.

Test example 3: PCSK9 antibody blocked LDLR-FC / PCSK9-Y binding

The anti-PCSK9 antibody tested the ability to block the binding of LDLR-FC (SEQ ID NO: 8) and PCSK9-Y (mutant PCSK9, SEQ ID NO: 6). By detecting the presence of the antibody, PCSK9-Y and The amount of LDLR binding is determined.

LDLR-FC was diluted with phosphate buffer to 2 μg / ml, coated on a 96-well ELISA plate (Costar, CAT # 3590), and left at 4 ° C. overnight. After washing the plates, they were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skimmed milk powder) at 37 ° C for 2 hours. Wash the plate and add biotin-labeled PCSK9-Y (bio-PCSK9-Y, diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20, and 1% skim milk powder to a final concentration of 1 μg / ml), and antibody A 100 μl / well mixture of a sample (diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20, and 1% skimmed milk powder) was incubated at 37 ° C. for 1 hour. Wash the plate, add horseradish peroxidase-streptavidin (sigma, CAT # S2438), and incubate at 37 ° C for 1 hour. The plate was washed again, and tetramethylbenzidine solution was added to develop a color. Finally, stop solution was added, OD450 was measured on a microplate reader, and its IC50 value was calculated.

The blocking effect test of the chimeric antibody and back-mutated antibody of the present invention on LDLR-FC / PCSK9-Y binding is shown in Table 10:

The results show that the PCSK9 antibody of the present invention can effectively block the binding between PCSK9-Y and LDLR.

Using the method described above, the PCSK9 antibody of the present invention was tested for binding to other forms of LDLR-FC (internally produced, see SEQ ID NO: 7 or SEQ ID NO: 9) and PCSK9-Y (SEQ ID NO: 5). Blocking ability. Experiments show that the PCSK9 antibody of the present invention can effectively block the binding between PCSK9 and the shortened form of LDLR.

Test Example 4. PCSK9 antibody blocked LDLR-FC / PCSK9 binding

The PCSK9 antibody of the present invention tests the ability to block the binding of LDLR-FC (internally produced with the sequence of SEQ ID NO: 8) and PCSK9 (SEQ ID NO: 5). By detecting the presence of the antibody, PCSK9 and LDLR The amount of combination is determined.

LDLR-FC was diluted to 5 μg / ml with phosphate buffer, coated on a 96-well ELISA plate, and left at 4 ° C. overnight. After washing the plates, they were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skimmed milk powder) at 37 ° C for 2 hours. Wash the plate and add biotin-labeled PCSK9 (bio-WT-PCSK9, diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20, and 1% skimmed milk powder to a final concentration of 2 μg / ml) and antibody samples (using 100 μl / well of a mixture containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder in Tris buffer) was incubated at 37 ° C. for 1 hour. Wash the plate, add horseradish peroxidase-streptavidin (sigma, CAT # S2438), and incubate at 37 ° C for 1 hour. The plate was washed again, and tetramethylbenzidine solution was added to develop a color. Finally, stop solution was added, OD450 was measured on a microplate reader, and its IC50 value was calculated.

The blocking effect test of the chimeric antibody and back-mutated antibody of the present invention on the binding of LDLR-FC / PCSK9 is shown in Table 11.

The results show that the PCSK9 antibody of the present invention can effectively block the binding between PCSK9 and LDLR.

Using the method described above, the PCSK9 antibody of the present invention was tested for blocking the binding of other forms of LDLR-FC (internally produced, see SEQ ID NO: 7 or SEQ ID NO: 9) and PCSK9 (SEQ ID NO: 5). Ability, experiments show that the PCSK9 antibody of the present invention can effectively block the binding between PCSK9 and the shortened form of LDLR.

Test Example 5. LSK uptake experiment with PCSK9 antibody

HepG2 cells (Chinese Academy of Sciences Cell Bank, #CAT, TCHu72) were cultured in DMEM medium (Hyclone, #CAT SH30243.01B) (containing 10% fetal bovine serum, Gibco, #CAT 10099-141). When the cells covered 80-90%, 1.5 * 10 ⁴ cells / well were counted after digestion and blown up and spread on 96-well plates. After 24 hours, the medium was changed to DMEM, 10% lipoprotein-free serum (Millipore, CAT # LP4). After 48 hours, wash twice with phosphate buffer, and add a mixture containing PCSK9 (SEQ ID NO: 1, final concentration 10 μg / ml) and antibody samples (diluted to different concentrations in culture medium) pre-incubated at 4 ° C for 1 hour. And BODIPY-®LDL (Invitrogen, CAT # L3483) at a final concentration of 10 μg / ml, and incubated at 37 ° C. After 6 hours, the plate was washed twice with phosphate buffer, and the fluorescence value (EX485nm / EM535nm) was read with a microplate reader. Then add 50 μl / well CellTiter-Glo® Cell Activity Luminescence Detection Reagent (Promega, G7571) and read the chemiluminescence value. The results of LDL uptake are shown in Fig. 5 and Fig. 6. The data results show that the PCSK9 antibody of the present invention can promote the uptake of LDL by HepG2 cells.

Test example 6, BIAcore detection of PCSK9 antibody affinity test

Human Fab capture molecules were covalently coupled to a CM5 biosensor chip (Cat. # BR-1000-12, GE) according to this method in the human Fab capture kit (Cat. # 28-9583-25, GE) instructions. The antibody to be tested is affinity captured, and then the human PCSK9 antigen (His-tagged human PCSK9: PCSK9-His6, SEQ ID NO: 1) is passed on the surface of the wafer, and the reaction signal is detected by the Biacore instrument to obtain the binding and dissociation curve. The affinity values are obtained by fitting, see Table 12. After the dissociation of each loop was completed in the experiment, the biochip was washed and regenerated with the regeneration solution provided in the human Fab capture kit (GE).

The PCSK9 antibody of the present invention has strong affinity for human PCSK9 antigen.

The affinity of the PCSK9 antibody of the present invention and PCSK9-Y (SEQ ID NO: 4) was detected by a similar method as above, showing that the PCSK9 antibody of the present invention has a strong affinity for the PCSK9-Y antigen.

Test example 7, PCSK9 antibody in vivo efficacy test

In this experiment, a mouse model of overexpressing human PCSK9 was constructed, and PCSK9 antibody was injected into the tail vein to evaluate the effect of the PCSK9 antibody of the present invention on reducing LDL-c in mice overexpressing human PCSK9. Human IgG (human immunoglobulin purified from conventional normal human serum using conventional affinity chromatography methods such as ProteinA) was used as a blank control.

C57Bl / 6 mice (purchased from Shanghai Sybil-Bichem Laboratory Animals Co., Ltd.) were adapted to the laboratory environment for 5 days, and injected AAV-PCSK9 virus (Beijing Benyuan Zhengyang Gene Technology Co., Ltd.) into the tail vein, 4 × 10 ¹¹ vg. After injection of the virus, fasting overnight the day before the experiment, taking blood from the orbit, detecting LDL-c with HDL and LDL / VLDL cholesterol quantification kit (purchased from BioVision, article number # K613-100), randomly grouping according to the concentration of LDL-c, Six mice (n = 6) in each group were administered by tail vein injection, and the internally produced human IgG and h001-4-WT antibodies were administered at a dose of 10 mg / kg (PBS for human IgG and h001-4-WT antibodies) (Concentration, 1 mg / ml). Fasting was performed for 6 hours before blood collection. Orbital blood was collected at 24, 48, 72, and 96 hours after administration, and the samples were placed at 37 ° C for 1 hour, centrifuged at 3500 rpm for 10 minutes, and the serum was stored at -80 ° C.

After the last serum collection, the frozen serum was tested on the same day. HDL and LDL / VLDL cholesterol quantification kits were used to detect the concentration of LDL-c in the serum, and the kit instructions were followed.

The experimental results are shown in Figure 7. The serum LDL-c concentration in normal mice was about 12 mg / dl. After injection of AAV8-PCSK9 virus, the serum LDL-c concentration reached an average of 40 mg / dl. After the administration, the concentration of LDL-c in the h001-4-WT group decreased by 50% compared with the human IgG group after 24 hours of administration; the concentration of LDL-c in the h001-4-WT group decreased by 49% after 48 hours of administration After 72 hours of administration, the LDL-c concentration in the h001-4-WT group decreased by 32%; after 96 hours of administration, the LDL-c concentration in the h001-4-WT group decreased by 20%, as shown in Table 13 and Figure 8. Show.

In summary, h001-4-WT can reduce the concentration of LDL-c in the serum of mice overexpressing human PCSK9, and the effect lasts for 72 hours.

Test example 8, competitive experiment

In a competitive ELISA experiment, we plated an antibody overnight, and then added biotinylated pCSK9-his and 50 times the concentration of the competitive antibody. The coated antibody and the antibody in solution will competitively bind the antigen. The signal of the antigen on the plate is then detected. The results show that h001-4 and 21B12 (US8030457B2) can compete with antigens themselves, and there is no obvious competitive binding between h001-4 and 21B12, suggesting that the epitopes of the two are different. See Table 14 for specific results.

Test Example 9. In vivo efficacy and drug metabolism test of cynomolgus monkeys

In order to examine the effect and metabolism of the antibody of the present invention in vivo, an experiment in which a cynomolgus monkey was administered in vivo was tried, and h001-4-WT and h001-4-YTE were administered respectively. It was administered by intravenous injection at a dose of 3 mg / kg, with 3 male cynomolgus monkeys in each group. About 2 ~ 4ml / minute, slow bolus. Blood samples were taken at different time points to detect lipoproteins, especially low-density lipoprotein (LDL) and antibody concentrations in serum. The detection points for lipoproteins were before and after administration 1, 4, 8, 12, 16, 20, On 24 and 28 days, the PK blood collection points were before administration, 15 minutes, 30 minutes, 1 hour, 3 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72, 96, 120 hours, 144 hours, 168 hours, 336 hours, 504 hours, 672 hours.

The test results show (Figure 9) that h001-4-WT and h001-4-YTE can significantly reduce the LDL content in cynomolgus monkeys, and the reduction duration of h001-4-YTE is better than h001-4-WT.

Serum samples from blood spots of drug metabolism were tested for the contents of h001-4-WT and h001-4-YTE by ELISA. The method is described in Test Example 1. The results show that the half-life of h001-4-WT in cynomolgus monkey is 4 days The half-life of h001-4-YTE in cynomolgus monkeys is 7.3 days, and YTE has a significantly longer half-life in vivo than WT.

Test example 10, PCSK9 antibody isomerization level detection

After the PCSK9 antibody was placed in the buffer at 40 ° C to accelerate for a certain time, about 30 μg was taken out, denatured by adding high concentration guanidine hydrochloride, and then disulfide was reduced by adding DTT, and then replaced with 20 mM His buffer (pH 6.0), and trypsin was added. Reaction at 37 ° C overnight. The Q-Exactive mass spectrometer data-dependent acquisition mode was used to collect the primary and secondary MSs of the peptides, and the theoretical sequence was compared using the Pepfinder software. The aspartame demethylamine and methionine were set. Oxidation and isomerization of aspartic acid are variable modifications, and the specific digestion sites are K and R. After the analysis of the spectrum is completed, a list of modified and unmodified peptides is exported, and the proportion of modified sites is calculated according to their respective intensities.

Exemplarily, by comparison and analysis of LC-MS, it is found that the mutations of h001-4-YTE D103N, h001-4-YTE D105E, or h001-4-YTE D105N can effectively reduce or eliminate the occurrence of isomerization modification. The results are shown in Table 15. Show.

<110> Jiangsu Hengrui Pharmaceutical Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.

<120> PCSK9 antibody, its antigen-binding fragment, and its medical use

<130> 780045CPCT

<160> 57

<170> PatentIn version 3.5

<210> 1

<211> 698

<212> PRT

<213> Homo sapiens

<220>

<223> PCSK9 with His tag: PCSK9-His6

<400> 1

<210> 2

<211> 714

<212> PRT

<213> Homo sapiens

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<223> PCSK9 with PADRE peptide and His tag: PCSK9-PADRE-His6

<400> 2

<210> 3

<211> 704

<212> PRT

<213> Homo sapiens

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<223> PCSK9 and his-tag fusion protein with TEV restriction site: PCSK9-TEV-His6

<400> 3

<210> 4

<211> 698

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<213> Homo sapiens

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<223> PCSK9-D374Y mutant protein with his tag: PCSK9-D374Y-His6

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<223> PCSK9 D374Y mutant protein inserted with biotin acceptor peptide and his tag: PCSK9-D374Y-BP15-His6

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<211> 802

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<213> Homo sapiens

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<223> LDLR Extracellular Domain Fragment of PCSK9 Receptor Protein with Flag Tag and His Tag: LDLR-ECD-Flag-His6

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<223> LDLR extracellular domain fragment and hIgG1-Fc fusion protein in shortened form: LDLR-sECD-Fc (hIgG1)

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<223> A more shortened form of the LDLR extracellular domain fragment and hIgG1-Fc fusion protein: LDLR-ssECD-Fc (hIgG1)

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<213> Rat Source

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<223> mAb-001 VH

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<223> h001_1 VH.1

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<223> h001_VH.1A

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<223> Heavy chain constant region sequence (IgG1-YTE)

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<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

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<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

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<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

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<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

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<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

<221> MISC_FEATURE

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<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

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<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

<221> MISC_FEATURE

<222> (105) .. (105)

<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<400> 34

<210> 35

<211> 121

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<213> Homo sapiens

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<223> h001_VH.1C-CDR3 mutant heavy chain variable region

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<222> (103) .. (103)

<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

<221> MISC_FEATURE

<222> (105) .. (105)

<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<400> 35

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<223> h001_VH.1D-CDR3 mutant heavy chain variable region

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<222> (103) .. (103)

<223> "Xl" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1," "X2" cannot be Asp at the same time

<220>

<221> MISC_FEATURE

<222> (105) .. (105)

<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<400> 36

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<223> h001_VH.1E-CDR3 mutant

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<222> (103) .. (103)

<223> "X1" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<220>

<221> MISC_FEATURE

<222> (105) .. (105)

<223> "X2" can be selected from Asp, Glu, His, Met, Asn or Gln, but "X1" and "X2" cannot be Asp at the same time

<400> 37

<210> 38

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D103E mutant heavy chain CDR3 sequence

<400> 38

<210> 39

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D103H mutant heavy chain CDR3 sequence

<400> 39

<210> 40

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D103M mutant heavy chain CDR3 sequence

<400> 40

<210> 41

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D103N mutant heavy chain CDR3 sequence

<400> 41

<210> 42

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D103Q mutant heavy chain CDR3 sequence

<400> 42

<210> 43

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D105E mutant heavy chain CDR3 sequence

<400> 43

<210> 44

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D105H mutant heavy chain CDR3 sequence

<400> 44

<210> 45

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D105M mutant heavy chain CDR3 sequence

<400> 45

<210> 46

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D105N mutant heavy chain CDR3 sequence

<400> 46

<210> 47

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> D105Q mutant heavy chain CDR3 sequence

<400> 47

<210> 48

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D103E

<400> 48

<210> 49

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D103H

<400> 49

<210> 50

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D103M

<400> 50

<210> 51

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D103N

<400> 51

<210> 52

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D103Q

<400> 52

<210> 53

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D105E

<400> 53

<210> 54

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D105H

<400> 54

<210> 55

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D105M

<400> 55

<210> 56

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D105N

<400> 56

<210> 57

<211> 121

<212> PRT

<213> Homo sapiens

<220>

<223> h001_VH.1D105Q

<400> 57

Claims (31)

A PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9. The PCSK9 antibody or antigen-binding fragment thereof comprises the following CDR regions: i) The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 12, 13, and 31, respectively. Ii) The sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 15, 16 and 17, respectively; wherein the HCDR3 sequence shown in SEQ ID NO: 31 is QYDYX ¹ EX ² WYFDV, where: X ^{1 is} selected from D, E, H, M, N or Q; X ^{2 is} selected from D, E, H, M, N or Q; but X ¹ and X ² cannot be D at the same time.     The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 1 of the scope of the patent application, wherein the HCDR3 is selected from the sequence shown in any one of SEQ ID NOs: 38-47.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 1 or 2 of the scope of the patent application, wherein the PCSK9 antibody or antigen-binding fragment thereof is a mouse-derived antibody, a chimeric antibody or a humanized antibody or a mouse-derived antibody. Antigen-binding fragments of antibodies, chimeric antibodies or humanized antibodies.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the scope of the patent application, wherein the light chain variable region of the PCSK9 antibody further comprises a light chain FR of a mouse-derived κ chain or a mouse-derived κ chain variant Region; wherein the heavy chain variable region of the PCSK9 antibody further comprises a heavy chain FR region of a mouse IgG1 or a mouse IgG1 variant.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the scope of the patent application, wherein the light chain of the PCSK9 antibody further comprises a light chain constant region of a mouse-derived κ chain or a mouse-derived κ chain variant; The heavy chain of the PCSK9 antibody further comprises the heavy chain constant region of a mouse IgG1 or a mouse IgG1 variant.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the scope of patent application, wherein the sequence of the heavy chain FR region on the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain IGHV1 -2 * 02 and hjh2 combined sequence or its mutant sequence; the humanized antibody contains the human germline heavy chain IGHV1-2 * 02 FR1, FR2, FR3 region sequence and hjh2 FR4 region sequence or human germline heavy chain Mutated sequence of FR1, FR2, FR3 region sequence of IGHV1-2 * 02 and FR4 region sequence of hjh2.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 6 of the scope of the patent application, wherein the humanized antibody contains a heavy chain variable region or a variant thereof as shown in SEQ ID NO: 32, wherein This variant is an insertion, deletion or substitution of 1-10 amino acids on the variable region of the heavy chain shown in SEQ ID NO: 32.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 7 of the scope of patent application, wherein the variant has 1-10 on the FR region of the heavy chain variable region shown in SEQ ID NO: 32 Back amino acid mutations.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in claim range 8, wherein the back mutation is selected from the amino acid responses of T30N, R87T, R72A, T74K, M48I, V68A, M70L, R38K, and R67K One or more of the mutations.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 8 of the scope of the patent application, wherein the humanized antibody comprises a heavy chain shown in any one of SEQ ID NOs: 32-37. Variable zone.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the scope of patent application, wherein the sequence of the light chain FR region on the light chain variable region of the humanized antibody is derived from the human germline light chain IGKV1 -39 * 01 and hjk2.1 combination sequence or a mutant sequence thereof; the humanized antibody comprises a human germline light chain IGKV1-39 * 01 FR1, FR2, FR3 region sequence and hjk2.1 FR4 region sequence or human Mutant sequence of FR1, FR2, FR3 region sequence of germline light chain IGKV1-39 * 01 and FR4 region sequence of hjk2.1.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 11 of the scope of the patent application, wherein the humanized antibody comprises a light chain shown in any one of SEQ ID NOs: 24-27. Variable zone.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the patent application scope, wherein the humanized antibody comprises a heavy chain variable region sequence and a light chain variable region sequence, and the heavy chain variable region The sequence is selected from the sequence shown in any one of SEQ ID NOs: 32-37; the light chain variable region sequence is selected from the sequence shown in any one of SEQ ID NOs: 24-27.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 3 of the scope of patent application, wherein the PCSK9 antibody comprises a heavy chain variable region selected from the sequence shown in any one of SEQ ID NOs: 48-57 and The light chain variable region shown in any of SEQ ID NOs: 24-27.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in any of claims 3 to 14, wherein the heavy chain of the PCSK9 antibody further comprises a heavy chain constant region of human IgG1 or a variant thereof ; Wherein the light chain of the PCSK9 antibody further comprises a light chain constant region of human κ or a variant thereof.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 15 of the patent application scope, wherein the heavy chain of the PCSK9 antibody further comprises a human IgG1 variant that uses an amino acid mutation to extend the half-life of the antibody in the serum Heavy chain constant region.         The PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in item 16 of the scope of the patent application, wherein the heavy chain of the PCSK9 antibody further comprises a heavy chain constant region of a human IgG1 variant that introduces a YTE mutation.         The PCSK9 antibody or antigen-binding fragment thereof according to item 15 of the scope of patent application, wherein the humanized antibody comprises the heavy chain constant region shown in SEQ ID NO: 28 or 29 and the light chain shown in SEQ ID NO: 30. Chain constant region.         A pharmaceutical composition containing a therapeutically effective amount of a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 of the scope of patent application, and one or more pharmaceutically acceptable carriers , Diluent or excipient.         A DNA molecule encoding the PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 of the scope of the patent application.         An expression vector containing the DNA molecule according to item 20 of the patent application.         A host cell transformed with an expression vector according to item 21 of the scope of the patent application, the host cell being selected from a prokaryotic cell and a eukaryotic cell.         The host cell according to item 22 of the application, wherein the host cell is a eukaryotic cell.         The host cell of claim 22, wherein the host cell is a mammalian cell.         A PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 in the scope of patent application, or a pharmaceutical composition as described in claim 19 in the scope of patent application for treating PCSK9-mediated diseases Or method of the condition, the method comprising administering to an individual an effective amount of a PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9.         The method of claim 25, wherein the PCSK9-mediated disease or disorder is a cholesterol-related disease.         The method of claim 26, wherein the PCSK9-mediated disease or condition is hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alz Hemer disease and general dyslipidemia.         The method of claim 27, wherein the PCSK9-mediated disease or condition is hypercholesterolemia, dyslipidemia, atherosclerosis, CVD, or coronary heart disease.         A method for producing a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 of the scope of the patent application, the method comprising: Culturing in the culture to form and accumulate the PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 of the patent application scope, and recover the accumulated PCSK9 antibody from the culture Or an antigen-binding fragment thereof.         A method for immunodetection or determination of PCSK9, the method comprising the step of detecting PCSK9 using a PCSK9 antibody or an antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18.         A reagent for detecting or measuring human PCSK9, the reagent comprising the PCSK9 antibody or antigen-binding fragment thereof that specifically binds PCSK9 as described in any one of claims 1 to 18 of the scope of patent application.