PH dependent antibody with prolonged half-life period

Abstract

The present invention provides a pH dependent antibody having an extended half-life. In particular, the invention relates to an anti-ANGPTL3 antibody which is engineered and has pH-dependent antigen binding. The compound can be used for treating lipid metabolism disorder diseases such as homozygous family hypercholesterolemia patients (HoFH), refractory hypercholesterolemia and serious hypertriglyceridemia.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, it relates to a pH-dependent antibody with an extended half-life. Background Technology

[0002] Human lipid metabolism is a complex process involving numerous enzymes, receptors, and transport proteins. Clinically available lipid-lowering drugs can be broadly classified into two categories: those that primarily lower cholesterol and those that lower triglycerides. Some lipid-lowering drugs can lower both cholesterol and triglycerides. For severe hyperlipidemia, multiple lipid-lowering drugs often need to be used in combination to achieve good therapeutic effects. Furthermore, different symptoms require drugs with different mechanisms of action. In conclusion, in an era where statins remain the cornerstone and the lipid-lowering drug market is currently booming, there is still a need to discover safe and effective new lipid-lowering drugs.

[0003] The binding of the Fc region of IgG antibodies to FcRn prolongs their half-life. The binding of the Fc region of IgG to FcRn is pH-dependent; it binds to FcRn with high affinity under acidic intracellular conditions and rapidly dissociates from FcRn under slightly alkaline conditions in the blood. This receptor-mediated recycling mechanism prolongs the half-life of IgG. Henne et al. modified the Fc region of their modified anti-PCSK9 recirculating antibody. In tests of FcRn affinity, the mutant showed a 7-fold increase in affinity compared to the parent antibody, and a half-life twice that of the parent antibody in cynomolgus monkeys, indicating that modifications to the Fc region to enhance affinity for FcRn under acidic conditions can improve antibody half-life. The mutation sites M252Y / S254T / T256E (called YTE) identified by MedImmune can extend the half-life of IgG in cynomolgus monkeys to nearly four times. IgG type antibodies can be captured by FcRn and circulate to the extracellular space in the acidic environment of the endosome, which is the main reason why this type of antibody is not subject to nonspecific clearance.

[0004] There remains a need in this field for pH-dependent antibodies with longer half-lives and better efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a pH-dependent antibody with an extended half-life.

[0006] In a first aspect of the invention, a human antibody or antigen-binding fragment thereof having pH-dependent antigen binding is provided, comprising a light chain variable region and a heavy chain variable region of everxumab or a variant having the same or similar biological activity therewith, wherein at least one CDR domain of the light chain variable region and / or the heavy chain variable region is mutated by replacing one or more amino acids within the CDR domain with histidine residues, thereby producing a mutated everxumab or an everxumab variant, the variant inducing pH-dependent antigen binding, wherein the mutated everxumab exhibits an affinity constant (K0) for binding to the antigen at pH 6 as measured by Biolayer Interferometry. D The ratio of the affinity constant of the antigen bound at pH 7.2 to that bound at pH 6 is K1, and the ratio of the affinity constant of the non-mutated everzumab bound to the antigen at pH 6 to that bound at pH 7.2 is K0, where K1 / K0 is r, and r≥1.2.

[0007] In another preferred embodiment, r ≥ 10, more preferably ≥ 20, more preferably ≥ 50, and even more preferably ≥ 100.

[0008] In another preferred embodiment, r ≤ 5000, more preferably ≤ 3000.

[0009] In another preferred embodiment, the human antibody or antigen-binding fragment thereof having pH-dependent antigen binding comprises a variant having the same or similar biological activity as the light chain variable region and heavy chain variable region of the human antibody evinacumab, the variant causing pH-dependent antigen binding, wherein,

[0010] The ratio of antigen affinity constants of the mutated everxumab at pH 6 and pH 7, measured by Biolayer Interferometry (Octet Red), is K1, and the ratio of antigen affinity constants of the non-mutated everxumab is K0, where K1 / K0 is r, and r≥1.2.

[0011] In another preferred embodiment, r is 1.2-10, more preferably, r is 1.2-5.5, and even more preferably, r is 1.5-5.5.

[0012] In another preferred embodiment, r ≥ 5.

[0013] In another preferred embodiment, the human antibody refers to a humanized anti-ANGPTL3 antibody.

[0014] In another preferred embodiment, the antigen or the pH-dependent antigen is ANGPTL3.

[0015] In another preferred embodiment, the mutation of the at least one, two, or three CDR domains by replacing one or more amino acids within the CDR domain with histidine residues includes mutations of one, two, three, four, or five amino acids.

[0016] In another preferred embodiment, the mutated everoumab retains 4-5 identical CDR domains compared to the original everoumab, and 1-2 CDR domains have amino acid mutations.

[0017] In another preferred embodiment, the amino acid mutation includes a derived sequence that has optionally been added, deleted, modified, and / or substituted with at least one amino acid and is capable of retaining binding affinity.

[0018] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an HCDR1 sequence selected from the following: SEQ ID NO.7-11.

[0019] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an HCDR2 sequence selected from the following: SEQ ID NO.12-28, 78, 79.

[0020] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an HCDR3 sequence selected from the following: SEQ ID NO.29-45, 80, 81.

[0021] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an LCDR1 sequence selected from the following: SEQ ID NO.46-56.

[0022] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an LCDR2 sequence selected from the following: SEQ ID NO. 57-63, 73-77.

[0023] In another preferred embodiment, the mutated ivexumab or ivexumab variant comprises an LCDR3 sequence selected from the following: SEQ ID NO.64-72.

[0024] In another preferred embodiment, the binding fragment comprises a sequence selected from the group consisting of the remaining sequences identical to the CDR region sequence of non-mutated ivexumab:

[0025] As shown in SEQ ID NO: 7, HCDR1,

[0026] HCDR2, as shown in SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 78, or SEQ ID NO: 79.

[0027] HCDR3, as shown in SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45, or SEQ ID NO: 80.

[0028] LCDR2 as shown in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 76

[0029] LCDR3 as shown in SEQ ID NO: 70 or SEQ ID NO: 71, or a combination thereof.

[0030] In another preferred embodiment, the binding fragment comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein,

[0031] The HCDR1 sequence is SEQ ID NO: 7;

[0032] The HCDR2 sequence is SEQ ID NO: 2;

[0033] The HCDR3 sequence is SEQ ID NO: 3;

[0034] The LCDR1 sequence is SEQ ID NO: 4;

[0035] The LCDR2 sequence is SEQ ID NO: 5; and

[0036] The LCDR3 sequence is SEQ ID NO: 6; or

[0037] The HCDR1 sequence is SEQ ID NO: 1;

[0038] The HCDR2 sequence is SEQ ID NO: 15;

[0039] The HCDR3 sequence is SEQ ID NO: 3;

[0040] The LCDR1 sequence is SEQ ID NO: 4;

[0041] The LCDR2 sequence is SEQ ID NO: 5; and

[0042] The LCDR3 sequence is SEQ ID NO: 6; or

[0043] The HCDR1 sequence is SEQ ID NO: 1;

[0044] The HCDR2 sequence is SEQ ID NO: 16;

[0045] The HCDR3 sequence is SEQ ID NO: 3;

[0046] The LCDR1 sequence is SEQ ID NO: 4;

[0047] The LCDR2 sequence is SEQ ID NO: 5; and

[0048] The LCDR3 sequence is SEQ ID NO: 6; or

[0049] The HCDR1 sequence is SEQ ID NO: 1;

[0050] The HCDR2 sequence is SEQ ID NO: 34;

[0051] The HCDR3 sequence is SEQ ID NO: 3;

[0052] The LCDR1 sequence is SEQ ID NO: 4;

[0053] The LCDR2 sequence is SEQ ID NO: 5; and

[0054] The LCDR3 sequence is SEQ ID NO: 6; or

[0055] The HCDR1 sequence is SEQ ID NO: 1;

[0056] The HCDR2 sequence is SEQ ID NO: 2;

[0057] The HCDR3 sequence is SEQ ID NO: 37;

[0058] The LCDR1 sequence is SEQ ID NO: 4;

[0059] The LCDR2 sequence is SEQ ID NO: 5; and

[0060] The LCDR3 sequence is SEQ ID NO: 6; or

[0061] The HCDR1 sequence is SEQ ID NO: 1;

[0062] The HCDR2 sequence is SEQ ID NO: 2;

[0063] The HCDR3 sequence is SEQ ID NO: 38;

[0064] The LCDR1 sequence is SEQ ID NO: 4;

[0065] The LCDR2 sequence is SEQ ID NO: 5; and

[0066] The LCDR3 sequence is SEQ ID NO: 6; or

[0067] The HCDR1 sequence is SEQ ID NO: 1;

[0068] The HCDR2 sequence is SEQ ID NO: 2;

[0069] The HCDR3 sequence is SEQ ID NO: 3;

[0070] The LCDR1 sequence is SEQ ID NO: 4;

[0071] The LCDR2 sequence is SEQ ID NO: 5; and

[0072] The LCDR3 sequence is SEQ ID NO: 70 or

[0073] The HCDR1 sequence is SEQ ID NO: 1;

[0074] The HCDR2 sequence is SEQ ID NO: 2;

[0075] The HCDR3 sequence is SEQ ID NO: 3;

[0076] The LCDR1 sequence is SEQ ID NO: 4;

[0077] The LCDR2 sequence is SEQ ID NO: 5; and

[0078] The LCDR3 sequence is SEQ ID NO: 71 or

[0079] The HCDR1 sequence is SEQ ID NO: 1;

[0080] The HCDR2 sequence is SEQ ID NO: 2;

[0081] The HCDR3 sequence is SEQ ID NO: 80;

[0082] The LCDR1 sequence is SEQ ID NO: 4;

[0083] The LCDR2 sequence is SEQ ID NO: 5; and

[0084] The LCDR3 sequence is SEQ ID NO: 6 or

[0085] The HCDR1 sequence is SEQ ID NO: 1;

[0086] The HCDR2 sequence is SEQ ID NO: 2;

[0087] The HCDR3 sequence is SEQ ID NO: 80;

[0088] The LCDR1 sequence is SEQ ID NO: 4;

[0089] The LCDR2 sequence is SEQ ID NO: 61; and

[0090] The LCDR3 sequence is SEQ ID NO: 6 or

[0091] The HCDR1 sequence is SEQ ID NO: 1;

[0092] The HCDR2 sequence is SEQ ID NO: 2;

[0093] The HCDR3 sequence is SEQ ID NO: 80;

[0094] The LCDR1 sequence is SEQ ID NO: 4;

[0095] The LCDR2 sequence is SEQ ID NO: 62; and

[0096] The LCDR3 sequence is SEQ ID NO: 6 or

[0097] The HCDR1 sequence is SEQ ID NO: 1;

[0098] The HCDR2 sequence is SEQ ID NO: 21;

[0099] The HCDR3 sequence is SEQ ID NO: 3;

[0100] The LCDR1 sequence is SEQ ID NO: 4;

[0101] The LCDR2 sequence is SEQ ID NO: 74; and

[0102] The LCDR3 sequence is SEQ ID NO: 6 or

[0103] The HCDR1 sequence is SEQ ID NO: 1;

[0104] The HCDR2 sequence is SEQ ID NO: 2;

[0105] The HCDR3 sequence is SEQ ID NO: 45;

[0106] The LCDR1 sequence is SEQ ID NO: 4;

[0107] The LCDR2 sequence is SEQ ID NO: 74; and

[0108] The LCDR3 sequence is SEQ ID NO: 6 or

[0109] The HCDR1 sequence is SEQ ID NO: 1;

[0110] The HCDR2 sequence is SEQ ID NO: 2;

[0111] The HCDR3 sequence is SEQ ID NO: 45;

[0112] The LCDR1 sequence is SEQ ID NO: 4;

[0113] The LCDR2 sequence is SEQ ID NO: 76; and

[0114] The LCDR3 sequence is SEQ ID NO: 6 or

[0115] The HCDR1 sequence is SEQ ID NO: 1;

[0116] The HCDR2 sequence is SEQ ID NO: 79;

[0117] The HCDR3 sequence is SEQ ID NO: 3;

[0118] The LCDR1 sequence is SEQ ID NO: 4;

[0119] The LCDR2 sequence is SEQ ID NO: 76; and

[0120] The LCDR3 sequence is SEQ ID NO: 6 or

[0121] The HCDR1 sequence is SEQ ID NO: 1;

[0122] The HCDR2 sequence is SEQ ID NO: 78;

[0123] The HCDR3 sequence is SEQ ID NO: 3;

[0124] The LCDR1 sequence is SEQ ID NO: 4;

[0125] The LCDR2 sequence is SEQ ID NO: 73; and

[0126] The LCDR3 sequence is SEQ ID NO: 6.

[0127] In another preferred embodiment, the mutant ivexumab comprises a variable heavy chain sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 88 or the amino acid sequence shown in SEQ ID NO. 90.

[0128] In another preferred embodiment, the mutant ivexumab comprises a variable light chain sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO. 89 or the amino acid sequence shown in SEQ ID NO. 91.

[0129] In another preferred embodiment, the mutant everxumab comprises the following variable regions: a heavy chain variable region as shown in SEQ ID NO. 88 and a light chain variable region as shown in SEQ ID NO. 89.

[0130] In another preferred embodiment, the mutant everxumab comprises the following variable regions: a heavy chain variable region as shown in SEQ ID NO. 90 and a light chain variable region as shown in SEQ ID NO. 91.

[0131] In another preferred embodiment, the human antibody or its antigen-binding fragment comprises a constant region of human heavy chain IgG1.

[0132] In another preferred embodiment, the constant region of human heavy chain IgG1 includes wild-type and mutant types; more preferably, the sequence is as shown in SEQ ID NO:95.

[0133] In another preferred embodiment, the Fc portion of the constant region of IgG1 is mutated at one or more amino acid positions to produce an antibody with significantly enhanced affinity for FCRN under acidic conditions.

[0134] In another preferred embodiment, the human antibody or its antigen-binding fragment contains a mutant sequence in the IgG Fc region.

[0135] In another preferred embodiment, the IgG Fc region mutation sequence has an amino acid sequence selected from those shown in SEQ ID NO. 82-87.

[0136] In a second aspect of the invention, an isolated nucleic acid molecule is provided that encodes the human antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0137] In a third aspect of the invention, an expression vector comprising a nucleic acid molecule as described in the second aspect of the invention is provided.

[0138] In a fourth aspect of the invention, an isolated host cell comprising an expression vector as described in the third aspect of the invention is provided.

[0139] In a fifth aspect of the invention, a method for producing a human antibody or an antigen-binding fragment thereof is provided, comprising culturing a host cell as described in the fourth aspect of the invention under conditions permissible for producing the antibody or a fragment thereof, and recovering the antibody or a fragment thereof thus produced.

[0140] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0141] (i) the antibody or a fragment thereof as described in the first aspect of the present invention; and

[0142] (ii) Pharmaceutically acceptable carriers.

[0143] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0144] In another preferred embodiment, the pharmaceutical composition is an injection.

[0145] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an antibody or fragment thereof, or a combination thereof, of the first aspect of the present invention and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentages are percentages by mass of the pharmaceutical composition.

[0146] In a seventh aspect of the invention, there is provided the use of a human antibody or antigen-binding fragment thereof from any of the first aspects of the invention, or a pharmaceutical composition from the sixth aspect of the invention, for the preparation of a medicament for treating lipid metabolism disorders.

[0147] In another preferred embodiment, the lipid metabolism disorder includes: hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherosclerotic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia, including severe hypertriglyceridemia with TG>1000mg / dL, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipid metabolism disorders, lipoatrophy, etc., preferably patients with hypercholesterolemia (HoFH), refractory hypercholesterolemia, and severe hypertriglyceridemia.

[0148] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0149] none Detailed Implementation

[0150] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that this invention preferably relates to the anti-ANGPTL3 antibody evinacumab monoclonal antibody or its biologically active variants and fragments, wherein the original evinacumab monoclonal antibody or its variants or fragments are engineered by mutations in the amino acid sequence of the variable region and the Fc fragment sequence. Specifically, this invention relates to evinacumab monoclonal antibody or its biologically active variants or fragments, wherein the CDR domain is mutated by substituting one or more amino acid residues with histidine residues, and the Fc region is mutated by substituting multiple amino acid residues. This invention was completed based on this discovery.

[0151] the term

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0153] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0154] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0155] Angiopoietin-like protein 3 (ANGPTL3)

[0156] ANGPTL3, belonging to the vascular endothelial growth factor family, is synthesized and secreted by the liver. It is a recognized regulator of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and is also considered a significant factor in cardiovascular disease (CVD). Studies have shown that individuals with loss-of-function (LOF) mutations in ANGPTL3 and / or reduced plasma ANGPTL3 levels have a reduced risk of CVD. Furthermore, circulating ANGPTL3 concentrations were found in healthy controls with lower levels than in subjects experiencing heart attacks. Additionally, individuals completely lacking ANGPTL3 showed no signs of atherosclerosis compared to control subjects with a mean total atherosclerotic plaque burden of approximately 39%. Therefore, treatment that lowers plasma ANGPTL3 levels could be a significant advance in cardiovascular therapy for individuals at risk of ischemic heart disease, who have elevated LDL-C and plasma triglycerides, typically reflecting elevated residual cholesterol in the plasma. The amino acid sequence of human ANGPTL3 is shown in SEQ ID NO. 94.

[0157] Evinacumab

[0158] Evinacumab (trade name EVKEEZATM) is a fully human monoclonal antibody targeting the IgG4 subtype of angiopoietin-like protein 3 (ANGPTL3). Its mechanism of action differs from existing drugs. When used in combination with maximally tolerated lipid-lowering therapy, it can reduce dangerously high cholesterol levels in patients with familial hypercholesterolemia (a common, difficult-to-treat hereditary disease) to normal levels. It is FDA-approved for the treatment of homozygous familial hypercholesterolemia (HoFH) in children aged 12 years and older or adults. The recommended dose of evinacumab is 15 mg / kg intravenously every 4 weeks. The clinical dose of this antibody is very high, and the intravenous administration leads to poor patient adherence. The amino acid sequence of the CDR region of evinacumab is shown in Table 1.

[0159] pH-dependent antibody of the present invention

[0160] As used herein, the terms "pH-dependent antibody of the present invention," "pH-dependent antigen-binding human antibody of the present invention," "antibody of the present invention," "mutated antibody against ANGPTL3 of the present invention," "evinacumab variant of the present invention," "mutated evixumab of the present invention," and "evixumab variant of the present invention" are used interchangeably to refer to the mutated evixumab in the first aspect of the present invention. It should be understood that the term also includes the ANGPTL3 antigen-binding fragment.

[0161] In vivo, antibody clearance of antigens primarily involves the antibody binding to the antigen, followed by endocytosis of the antibody-antigen complex within the cell. Inside the cell, the antibody-antigen complex is either degraded by lysosomes or, after binding to FcRn, carried to the cell surface and released extracellularly. Therefore, antibody binding to antigens is a one-time event. pH-dependent antibodies, on the other hand, are created by modifying the variable region of the antibody (mainly by replacing some amino acids in the antibody's CDR region with histidine), allowing the antibody to bind antigens at pH 7.4 (neutral environment) and release antigens at pH 6.0 (acidic environment of the lysosome). When the pH-dependent antibody-antigen complex enters the lysosome, it is either degraded by the lysosome or, after release, degraded by the lysosome. The antibody then recycles to the extracellular space via FcRn. Extracellularly, the antibody can bind to the antigen again, allowing for multiple reuses and reducing the dosage of antibody drugs.

[0162] The recycling of pH-dependent antibodies requires the participation of FcRn. In this invention, the Fc region of the pH-dependent antibody is modified to enhance its affinity for FcRn, thereby enabling the pH-dependent antibody to have a dual recycling function and further prolonging the half-life of the pH-dependent antibody.

[0163] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies. Preferably, it may contain a mutant sequence in the IgG Fc region. Preferably, the mutation improves the antibody half-life, thereby protecting such antibodies from nonspecific clearance. The IgG Fc region contains at least one mutation.

[0164] Enhancing the binding of IgG antibody Fc to FcRn can prolong its half-life. The binding of the IgG Fc region to FcRn is pH-dependent; it binds to FcRn with high affinity under acidic intracellular conditions and rapidly dissociates from FcRn under slightly alkaline conditions in the blood. This receptor-mediated recycling mechanism prolongs the half-life of IgG. The mutation site M252Y / S254T / T256E (called YTE) identified by MedImmune can extend the half-life of IgG in cynomolgus monkeys by nearly four times. In the acidic endosome environment, IgG antibodies can be captured by FcRn and circulate extracellularly, protecting this type of antibody from nonspecific clearance.

[0165] In some embodiments, IgG is a subclass of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of IgG; in some embodiments, the Fc region has one or more amino acid substitutions that enhance the binding of the Fc region to the Fc receptor. In some embodiments, the Fc region has YTE mutations (M252Y, S254T, and T256E), L234A, L235A mutations, and / or S228P mutations, the mutations being numbered according to the EU index.

[0166] The pH-dependent antibody of the present invention, based on mutation and screening of evinacumab, and with modification of its Fc region, further prolongs its half-life, providing a more effective and better-adhered treatment for lipid metabolism disorders such as refractory hypercholesterolemia and severe hypertriglyceridemia.

[0167] The pH dependence of the evinacumab variant of this invention corresponds to the affinity constant (K) for binding at pH 6. D The ratio of the affinity constant to the binding affinity constant at pH 7.2.

[0168] The histidine-mutated everostumab of the present invention is suitable for treating the same conditions and diseases as approved and commercially available non-histidine-mutated everostumab, such as hyperlipidemia, hyperlipoproteinemia, and dyslipidemia, including atherosclerotic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia including severe hypertriglyceridemia with TG > 1000 mg / dL, hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipid metabolism disorders, lipoatrophy, etc., caused by factors such as: decreased LPL activity and / or LPL deficiency, decreased LDL receptor (LDLR) activity and / or LDL receptor deficiency (e.g., homozygous familial hypercholesterolemia characterized by LDLR- / -), ApoC2 alterations, ApoE deficiency, increased ApoB, increased production and / or decreased elimination of very low-density lipoprotein (VLDL), certain drug treatments (e.g., dyslipidemia caused by glucocorticoid therapy), any genetic predisposition, diet, lifestyle, etc.

[0169] The histidine-mutated everostumab of this invention can also prevent or treat diseases associated with or caused by hyperlipidemia, superlipoproteinemia, and / or dyslipidemia, including but not limited to cardiovascular diseases or disorders such as atherosclerosis, aneurysm, hypertension, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, etc.; acute pancreatitis; non-alcoholic steatohepatitis (NASH); glycemic disorders such as diabetes; obesity, etc. The drug is preferably administered via subcutaneous injection.

[0170] Like commercially available drugs, the histidine-mutated everostumab according to the present invention can be used alone or in combination with a second therapeutic agent supporting the treatment, such as (1) 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.; (2) inhibitors of cholesterol absorption and / or bile acid reabsorption; (3) niacin that increases lipoprotein catabolism; (4) fibrates or amphoteric acids that lower low-density lipoprotein (LDL) levels, improve high-density lipoprotein (HDL) and TG levels, and reduce the number of non-fatal heart attacks; and (5) in cholesterol... The second therapeutic agent can be an activator of LXR transcription factors that play a role in the clearance of 22-hydroxycholesterol, or certain fixed combinations, such as ezetimibe plus simvastatin; a statin plus bile resins (such as cholestyramine, colestipol, or colesvelam); a fixed combination of niacin and a statin (such as niacin plus lovastatin); or a fixed combination with other lipid-lowering agents such as omega-3 fatty acid ethyl esters (such as omacor). In addition, the second therapeutic agent can be one or more other inhibitors of ANGPTL3, as well as inhibitors of other molecules such as ANGPTL4, ANGPTL5, ANGPTL6, and proprotein convertase subtilisin 9 (PCSK9), which are involved in lipid metabolism, particularly the homeostasis of cholesterol and / or triglycerides. Inhibitors of these molecules include small molecules and antibodies that specifically bind to and block the activity of these molecules.

[0171] Antibody

[0172] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0173] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0174] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0175] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0176] In this invention, the term "antigen-binding fragment" refers to a fragment with antigen-binding capability, and includes Fab, F(ab′), F(ab′)2, Fv, etc. In antibody fragments, Fab (antigen-binding fragment) has a structure containing a light chain variable region and a heavy chain variable region, a light chain constant region and a heavy chain first constant region (CH1), and has an antigen-binding site. Fab′ differs from Fab in that it has a hinge region including at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. In F(ab′)2 antibody, the cysteine ​​residues in the hinge region of Fab′ form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragment size are known in the prior art, where Fv only has heavy chain and light chain variable regions. Double-chain variable fragments (dcFv) are non-covalently linked to the heavy chain and light chain variable regions, while single-chain variable fragments (scFv) are typically covalently linked to the heavy chain variable region or C-terminus via peptide linkers to form dimers, such as double-chain Fv. These antibody fragments can be obtained using proteases (e.g., Fab can be obtained by cleaving an intact antibody with papain, while the F(ab′)2 fragment can be obtained by cleaving with pepsin), or they can be prepared using genetic recombination techniques.

[0177] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0178] Unless otherwise specified, the amino acid positions within the antibody molecules according to the invention are based on Kabat numbering.

[0179] As used herein, an "antibody variant" or "monoclonal antibody variant" includes an antibody with a modified amino acid sequence compared to the parent antibody but with the same or altered binding affinity to the target antigen. Antibody variants differ from parent antibodies in that one or more amino acid residues are replaced, deleted, or added at specific positions within the variable domains (including the CDR domain) and / or constant regions of the antibody to modify certain properties of the antibody, such as binding affinity and / or receptor function, such as ADCC, FcRn binding, etc. Histidine-mutated antibodies of the present invention without further modification are not referred to as "antibody variants" according to the present invention. Antibody variants according to the present invention exhibit 80-99% sequence homology compared to the parent antibody, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence homology, depending on the specific position of the amino acid residues to be replaced, deleted, or added.

[0180] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0181] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0182] Table A

[0183] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0184] Pharmaceutical Composition

[0185] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0186] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0187] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0188] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0189] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0190] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0191] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0192] The main advantages of this invention include

[0193] The antibody of this invention is a pH-dependent antibody. It is pH-dependent, and the binding force between the antibody and the antigen is weaker at pH 6.0. At the same time, the antibody has a higher affinity for FcRn, a longer half-life in animals, and better efficacy.

[0194] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0195] Example 1: Design of CDR region mutant sequence and protein preparation of evinacumab

[0196] To obtain evinacumab variants with pH-dependent binding properties, the heavy and light chain protein sequences of evinacumab antibodies are shown in SEQ ID NO.92-93, respectively. A series of mutant antibodies were constructed, with the amino acid sequence of the CDR region of evinacumab shown in Table 1 and the designed CDR region mutant sequences shown in Table 2.

[0197] Table 1. CDR region sequence of evinacumab

[0198] CDR amino acid sequence SEQ ID NO: HCDR1 DYAMN 1 HCDR2 AISGDGGSTYYADSVKG 2 HCDR3 DLRNTIFGVVIPDAFDI 3 LCDR1 RASQSIRSWLA 4 LCDR2 KASSLES 5 LCDR3 QQYNSYSYT 6

[0199] Table 2. CDR region mutation sequence of evinacumab

[0200]

[0201]

[0202] The complete antibody sequences corresponding to the mutation sequences in Table 2 (for example, antibody sequence number L3-007 means that, relative to the parent evinacumab antibody, the mutant antibody has HCDR2 as shown in SEQ ID NO.12, and the rest of the sequence is consistent with the parent antibody) were constructed into the expression vector, and antibody proteins were prepared by transient expression for subsequent affinity assays to analyze the pH-dependent binding properties of the mutant antibody.

[0203] Example 2: Affinity determination of mutant antibodies at pH 7.2 and pH 6.0.

[0204] The difference in dissociation behavior at pH 6.0 or pH 7.2 after binding to ANGPTL3 at pH 7.2 was evaluated using an initial Octet Red assay with immobilized antibodies. The KD values ​​of each histidine substitution variant anti-ANGPTL3 antibody bound to human ANGPTL3 at pH 7.2 (neutral) and pH 6.0 (acidic), and the corresponding pH 6.0 / pH 7.2 ratios, are shown in Table 3, with significant comparisons to the parental evinacumab antibody and the ratios.

[0205] Table 3. Affinity determination and ratio calculation of mutant antibodies

[0206]

[0207]

[0208] Example 3: Design of CDR region multi-point mutation and combinatorial mutation sequences and preparation of proteins

[0209] Based on the above CDR region mutation results, the following multi-point mutation and combined mutation sequences were designed (Table 4). The complete antibody sequences corresponding to the mutation sequences in Table 4 (for example, antibody sequence number L3-068 refers to the mutant antibody having LCDR2 as shown in SEQ ID NO.58 and HCDR2 as shown in SEQ ID NO.21, relative to the parent evinacumab antibody, while the remaining sequences are consistent with the parent antibody) were constructed into the expression vector. The antibody protein was prepared by transient expression and used for subsequent affinity assays to analyze the pH-dependent binding properties of the mutated antibody.

[0210] Table 4. CDR region mutation sequences

[0211]

[0212]

[0213]

[0214] Example 4: Affinity determination of mutant antibodies at pH 7.2 and pH 6.0.

[0215] The difference in dissociation behavior at pH 6.0 or pH 7.2 after binding to ANGPTL3 at pH 7.2 was evaluated using an initial Octet Red assay with immobilized antibodies. The KD values ​​of each histidine substitution variant anti-ANGPTL3 antibody bound to human ANGPTL3 at pH 7.2 (neutral) and pH 6.0 (acidic), and the corresponding pH 6.0 / pH 7.2 ratios, are shown in Table 5, with significant differences between the parental evinacumab antibody and the ratio.

[0216] Table 5. Affinity determination and ratio calculation of mutant antibodies

[0217]

[0218]

[0219] Example 5: Design and Expression of Mutant Sequences in the Fc Region of IgG

[0220] Based on the mutation results of the CDR region, the Fc region mutation sequences (Table 6) were designed as shown in the table below. The sequences (Fc1-Fc6) in the table below were combined with the variable region and hinge region sequences of the L3-007 or L3-065 antibody mentioned above to form complete antibody sequences. Antibody proteins were prepared by transient expression and used for subsequent affinity assays to analyze the pH-dependent binding properties of the mutated antibody.

[0221] Table 6. Mutant sequences in the Fc region of IgG

[0222]

[0223]

[0224] Example 6: Affinity determination of IgG Fc region mutant antibody at pH 7.2 and pH 6.0.

[0225] The difference in dissociation behavior at pH 6.0 or pH 7.2 after binding to ANGPTL3 at pH 7.2 was evaluated using an initial Octet Red assay with immobilized antibodies. The KD values ​​of each IgG Fc region mutant anti-ANGPTL3 antibody bound to human ANGPTL3 at pH 7.2 (neutral) and pH 6.0 (acidic), and the corresponding pH 6.0 / pH 7.2 ratios, are shown in Table 7.

[0226] Table 7. Affinity determination and ratio calculation of IgG Fc region mutant antibodies

[0227]

[0228]

[0229] The results showed that L3-007-Fc4 and L3-124-Fc4 had a synergistic effect in improving the KD ratio at pH 6.0 and pH 7.4.

[0230] Example 7: Affinity of IgG Fc region mutant antibody with recombinant human and monkey FCRN was determined at pH 7.2 and pH 6.0.

[0231] The purpose of the IgG Fc region mutant antibody is to enhance the antibody's affinity for FCRN under acidic conditions (pH 6.0) without altering the binding and dissociation of the antibody with FCRN under neutral conditions (pH 7.2) and without affecting the antibody's ability to bind to the antigen. As evaluated in the previous embodiment, antibody-antigen binding was unaffected. This application will evaluate the affinity of the mutant antibody for recombinant human and monkey FCRN. The results are shown in Table 8. The L3-007-Fc4 and L3-124-Fc4 antibodies showed significantly stronger affinity for FCRN than the parent antibody evinacumab under acidic conditions (pH 6.0).

[0232] Table 8. Affinity determination of IgG Fc region mutant antibodies with FCRN from different species

[0233]

[0234] Example 8: Affinity determination of IgG Fc region mutant antibody with recombinant human FcγRIIIa

[0235] FcγRIIIa is a key receptor that causes ADCC. This invention will evaluate the affinity of IgG Fc region mutant antibodies for recombinant human FcγRIIIa.

[0236] The results are shown in Table 9. Under neutral conditions, the parental antibody evinacumab has a strong affinity for recombinant human FcγRIIIa, which may have an ADCC effect; while L3-007-Fc4 and L3-124-Fc4 do not bind to recombinant human FcγRIIIa, which may not have an ADCC effect.

[0237] Table 9. Affinity determination of IgG Fc region mutant antibody with recombinant human FcγRIIIa

[0238]

[0239] Example 9 Animal Pharmacodynamics and Pharmacokinetics Experiment

[0240] The efficacy and in vivo half-life of L3-007-Fc4, L3-124-Fc4, and the positive control drug Evinacumab in reducing LDL-c were evaluated in male dyslipidemia-producing cynomolgus monkeys. Animals were randomly divided into five experimental groups (n=3 per group) based on their day-1 LDL-c data: one group served as the positive control (Evinacumab 10 mg / kg); the other two groups received different doses of the compound: a low-dose group (1.0 mg / kg) and a high-dose group (3.0 mg / kg). The day of administration was designated as day 0. Blood lipids were measured in the cynomolgus monkeys on days 14, 28, and 35 following a single intravenous injection. Blood samples were collected from animals before administration, and at 3, 6, 12, and 24 hours after administration, as well as on days 2, 3, 5, 7, 10, 14, 21, and 28. Pharmacokinetic parameters were measured using ELISA and calculated using non-compartment analysis (NCA) with PhoenixWinNonlin 8.3.3.33 software.

[0241] Table 10 shows the pharmacodynamic results. L3-007-Fc4 and L3-124-Fc4 are superior to Evinacumab in terms of the strength and duration of LDL-C and TC reduction, and are used at lower doses. Table 11 shows the pharmacokinetic results. L3-007-Fc4 and L3-124-Fc4 have longer half-lives than Evinacumab.

[0242] Table 10 Results of the efficacy experiment in cynomolgus monkeys with dyslipidemia.

[0243]

[0244] Table 11. Pharmacokinetic Results in Cynomolgus Monkeys with Dyslipidemia

[0245]

[0246] The amino acid sequence of the mutant antibody (L3-007-Fc4 antibody) of the present invention from the N-terminus to the C-terminus is as follows:

[0247] L3-007-Fc4 antibody heavy chain variable region protein sequence: SEQ ID NO.88

[0248] EVQLVESGGGVIQPGGSLRLSCAASGFTFDDYAMNWVRQGPGKGLEWVSHISGDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAFFYCAKDLRNTIFGVVIPDAF DIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG

[0249] L3-007-Fc4 antibody light chain variable region protein sequence: SEQ ID NO.89

[0250] DIQMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0251] L3-124-Fc4 antibody heavy chain variable region protein sequence: SEQ ID NO.90

[0252] EVQLVESGGGVIQPGGSLRLSCAASGFTFDDYAMNWVRQGPGKGLEWVSHHSGDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAFFYCAKDLRNTIFGVVIPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDGEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG[[ID=**3**]] [[ID=**4**]]

[0253] [[ID=**5**]]The protein sequence of the light chain variable region of the L3-124-Fc4 antibody: SEQ ID NO.91[[ID=**6**]] [[ID=**7**]]

[0254] [[ID=**8**]]DIQMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYHHSSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[[ID=**9**]] [[ID=**10**]]

[0255] [[ID=**11**]]The protein sequence of the heavy chain of the Evinacumab antibody: SEQ ID NO.92[[ID=**12**]] [[ID=**13**]]

[0256] EVQLVESGGGVIQPGGSLRLSCAASGFTFDDYAMNWVRQGPGKGLEWVSAISGDGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAFFYCAKDLRNTIFGVVIPDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0257] Amino acid sequence of the light chain protein of evinacumab antibody: SEQ ID NO.93

[0258] DIQMTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0259] Amino acid sequence of human ANGPTL3: SEQ ID NO.94

[0260] SSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLES LLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNV KHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYS FYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

[0261] Human heavy chain IgG1 constant region sequence: SEQ ID NO.95

[0262] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pH-dependent antigen-binding human antibody or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region of everzumab or a variant having the same or similar biological activity, wherein at least one CDR domain of the light chain variable region and / or the heavy chain variable region is mutated by substituting one or more amino acids within the CDR domain with histidine residues, thereby producing a mutated everzumab or an everzumab variant. in, The mutated ivexumab's affinity constant (K₂O₅) for binding with the antigen at pH 6 was measured using Biolayer Interferometry. D The ratio of the affinity constant of the antigen bound at pH 7.2 to that bound at pH 6 is K1, and the ratio of the affinity constant of the non-mutated everzumab bound to the antigen at pH 6 to that bound at pH 7.2 is K0, where K1 / K0 is r, and r≥1.

2.

2. The human antibody or its antigen-binding fragment as described in claim 1, characterized in that, The human antibody or its antigen-binding fragment contains a mutant sequence in the IgG Fc region.

3. The human antibody or its antigen-binding fragment as described in claim 1, characterized in that, The binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), wherein, The HCDR1 sequence is SEQ ID NO: 7; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 5; and The LCDR3 sequence is SEQ ID NO: 6; or The HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 15; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 5; and The LCDR3 sequence is SEQ ID NO: 6; or The HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 16; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 5; and The LCDR3 sequence is SEQ ID NO: 6; or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 34; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 6; or the HCDR1 sequence is SEQ ID NO: 1; the HCDR2 sequence is SEQ ID NO: 2; and the HCDR3 sequence is SEQ ID NO: 37; the LCDR1 sequence is SEQ ID NO: 4; the LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 6; or the HCDR1 sequence is SEQ ID NO: 1; the HCDR2 sequence is SEQ ID NO: 2; and the HCDR3 sequence is SEQ ID NO: 38; the LCDR1 sequence is SEQ ID NO: 4; the LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 6; or the HCDR1 sequence is SEQ ID NO: 1; the HCDR2 sequence is SEQ ID NO: 2; and the HCDR3 sequence is SEQ ID NO: 3; the LCDR1 sequence is SEQ ID NO: 4; the LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 70; or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 71 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 80; The LCDR1 sequence is SEQ ID NO: 4; the LCDR2 sequence is SEQ ID NO: 5; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 80; the LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 61; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 80; the LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 62; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 21; the HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 74; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 45; the LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 74; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 2; The HCDR3 sequence is SEQ ID NO: 45; the LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 76; and the LCDR3 sequence is SEQ ID NO: 6 or the HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 79; the HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 76; and The LCDR3 sequence is SEQ ID NO: 6 or The HCDR1 sequence is SEQ ID NO: 1; The HCDR2 sequence is SEQ ID NO: 78; The HCDR3 sequence is SEQ ID NO: 3; The LCDR1 sequence is SEQ ID NO: 4; The LCDR2 sequence is SEQ ID NO: 73; and The LCDR3 sequence is SEQ ID NO:

6.

4. The human antibody or its antigen-binding fragment as described in claim 1, characterized in that, The mutant everxumab contains the following variable regions: a heavy chain variable region as shown in SEQ ID NO. 88 and a light chain variable region as shown in SEQ ID NO.

89.

5. The human antibody or its antigen-binding fragment as described in any one of claims 1-4, characterized in that, The IgG Fc region mutation sequence has an amino acid sequence selected from SEQ ID NO.82-87.

6. An isolated nucleic acid molecule encoding a human antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

7. An expression vector comprising the nucleic acid molecule as described in claim 6.

8. An isolated host cell comprising the expression vector as described in claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; and (ii) Pharmaceutically acceptable carriers.

10. Use of a human antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or the pharmaceutical composition of claim 9, characterized in that, This is used to prepare a drug for treating lipid metabolism disorders.

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