Anti-ASGR1 monoclonal antibody and application thereof

Abstract

The invention discloses an anti-ASGR1 monoclonal antibody and an application thereof. The present invention further discloses the use of a combination of an ASGR1 inhibitor comprising the anti-ASGR1 monoclonal antibody and a second lipid-lowering drug in reducing the level of total cholesterol and / or triglyceride in blood and / or liver or in treating and / or preventing cardiovascular diseases. The invention proves that the combination of the ASGR1 inhibitor and the second lipid-lowering drug has the effect of synergistically reducing total cholesterol and triglyceride in serum and liver.

Description

[0001] This application is a divisional application of the invention patent application with application number 202210440150.9, application date April 25, 2022, and invention name “Anti-ASGR1 monoclonal antibodies and their applications”. Technical Field

[0002] The present invention relates to anti-ASGR1 monoclonal antibodies and also to the combined use of an ASGR1 inhibitor (eg, anti-ASGR1 monoclonal antibodies) and another lipid-lowering drug. Background Art

[0003] Statins, which are usually used to reduce low-density lipoprotein cholesterol (LDL-c), are the first-line drugs used to lower cholesterol. The statins currently approved by the FDA mainly include lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pitavastatin, among which atorvastatin and rosuvastatin are the most widely used. However, related studies have also found that long-term use of statins can cause patients to develop statin resistance and produce liver toxicity. Some patients are intolerant to statins and have side effects such as muscle weakness, rhabdomyolysis, and diabetes. Moderate-intensity statins cannot meet the clinical needs of intensive lipid-lowering; and the efficacy of high-dose statins only increases by 6% after doubling the dose, but the adverse reactions double, which has great limitations in clinical application.

[0004] Ezetimibe (EZ) is the first cholesterol absorption inhibitor. Its main function is to inhibit the absorption of cholesterol from diet and bile by the brush border of the small intestine mediated by NPC1L1, but it does not affect the absorption of other fat-soluble nutrients. It is mainly used for hypercholesterolemia. EZ treatment reduces the accumulation of lipids in the liver, reduces cholesterol, triglycerides and LDL-c in the blood, and increases high-density lipoprotein cholesterol (HDL-c). It has no significant effect on fat-soluble vitamins such as vitamin A, vitamin D and vitamin E. However, when ezetimibe is used alone, the degree of reduction in LDL-c plasma concentration is weak (about 20%), so it is often used in combination with statins in clinical practice. In four multicenter, double-blind, placebo-controlled, 12-week studies, 1,187 patients with primary hypercholesterolemia were treated with ezetimibe 10 mg per day alone or in combination with atorvastatin, simvastatin, pravastatin, and lovastatin. The degree of LDL-c reduction in patients taking the combination drug was unrelated to the type and dose of statins. The effect of combining ezetimibe with the minimum dose of statin in lowering LDL-c is better than that of high-dose statin alone. It is the only lipid-lowering drug compound preparation in China. The tablet contains two widely used lipid-lowering drugs in clinical practice, simvastatin and ezetimibe, and is suitable for patients with hypercholesterolemia. Ezetimibe simvastatin tablets have a dual mechanism of action. While inhibiting the synthesis of cholesterol in the liver, it also inhibits the absorption of cholesterol in the small intestine, and can effectively reduce LDL-c by more than 50%.

[0005] Although the combined administration of ezetimibe and statin lipid-lowering drugs reduces the dosage of statins to a certain extent, thereby reducing their toxic side effects, the combined administration still has common side effects, such as abnormal liver function tests, such as increased alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST); increased blood creatine kinase (CK), and uncommonly increased blood bilirubin; increased blood uric acid; increased γ-glutamyl transpeptidase; increased international normalized ratio; the occurrence of proteinuria; weight loss, etc.

[0006] WO 2017058944 A1, WO 2022006327 A1 and US20210163601 A1 disclose methods for treating and preventing cardiovascular diseases by administering ASGR-1 antigen binding proteins, wherein anti-ASGR1 antibodies are disclosed that can reduce the level of low-density lipoprotein cholesterol (LDL-c) and / or non-high-density lipoprotein cholesterol (Non-HDL-C). However, no ASGR1 antibodies are currently approved for marketing for the treatment of simple hypercholesterolemia or other cardiovascular diseases. Summary of the invention

[0007] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that binds to ASGR1, wherein the monoclonal antibody inhibits or blocks the binding of ASGR1 to its natural ligand and / or the endocytosis of ASGR1, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NO: 4-6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NO: 7-9 or an equivalent thereof; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 10-12 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 13-15 or an equivalent thereof; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 16-18 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 19-21 or an equivalent thereof; (d) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 22-24 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 25-27 or an equivalent thereof; (e) the light chain variable region comprises the sequence shown in SEQ ID NO: 28 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29 or its equivalent; (f) the light chain variable region comprises the sequence shown in SEQ ID NO: 30 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31 or its equivalent; (g) the light chain variable region comprises the sequence shown in SEQ ID NO: 32 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33 or its equivalent; and (h) the light chain variable region comprises the sequence shown in SEQ ID NO: 34 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 35 or its equivalent.

[0008] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.

[0009] In a third aspect, the present invention provides a method for lowering the level of cholesterol and / or triglycerides in the blood or treating and / or preventing cardiovascular disease, the method comprising administering to a subject a therapeutically effective amount of any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof, or a pharmaceutical composition comprising any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof.

[0010] In a fourth aspect, the present invention provides the use of the above monoclonal antibody or antigen-binding fragment thereof or the above pharmaceutical composition in the preparation of a drug for lowering the level of cholesterol and / or triglycerides in the blood or for treating and / or preventing cardiovascular diseases.

[0011] In a fifth aspect, the present invention provides the above monoclonal antibody or antigen-binding fragment thereof or the above pharmaceutical composition for use in lowering the level of cholesterol and / or triglycerides in the blood or for treating and / or preventing cardiovascular diseases.

[0012] In a sixth aspect, the present invention provides a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. In another aspect, the present invention also provides a vector comprising the aforementioned nucleotide sequence. In another aspect, the present invention also provides a host cell comprising the aforementioned vector. In addition, the present invention also provides a cell line for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, a recombinant expression vector comprising the nucleotide of the present invention, and a method for preparing an antibody by culturing an antibody-producing cell line.

[0013] In any of the above aspects, the monoclonal antibody is preferably a humanized antibody or a chimeric antibody.

[0014] The inventors also found that the combined administration of an ASGR1 inhibitor and another lipid-lowering drug produced a significant synergistic lipid-lowering effect, with both total cholesterol levels and triglyceride levels in the blood and / or liver being significantly reduced.

[0015] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor, a therapeutically effective amount of a second lipid-lowering drug, and a pharmaceutically acceptable carrier.

[0016] In an eighth aspect, the present invention provides a medicine kit comprising a first pharmaceutical composition and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a therapeutically effective amount of an ASGR1 inhibitor and a pharmaceutically acceptable carrier, and the second pharmaceutical composition comprises a therapeutically effective amount of a second lipid-lowering drug and a pharmaceutically acceptable carrier.

[0017] In a ninth aspect, the present invention provides use of a combination of an ASGR1 inhibitor and a second lipid-lowering drug in the preparation of a medicament for lowering the level of total cholesterol and / or triglycerides in blood and / or liver or for treating and / or preventing cardiovascular disease.

[0018] In a tenth aspect, the present invention provides a combination of an ASGR1 inhibitor and a second lipid-lowering drug for use in lowering the level of total cholesterol and / or triglycerides in the blood and / or liver, or for treating and / or preventing cardiovascular disease.

[0019] In an eleventh aspect, the present invention provides a method for lowering the level of total cholesterol and / or triglycerides in blood and / or liver or treating and / or preventing cardiovascular disease, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor and a therapeutically effective amount of a second lipid-lowering drug.

[0020] In any of the above aspects, the ASGR1 inhibitor is preferably selected from anti-ASGR1 monoclonal antibodies or antigen-binding fragments thereof, nucleic acids encoding nucleic acids targeting ASGR1, nucleic acid aptamers targeting ASGR1, and combinations thereof.

[0021] In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence shown in SEQ ID NO: 1, and inhibits or blocks the binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or the endocytosis of human ASGR1. In some embodiments, the ASGR1 monoclonal antibody binds to the carbohydrate binding region of ASGR1 and inhibits or blocks the binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or the endocytosis of human ASGR1. In some embodiments, the carbohydrate binding region of ASGR1 comprises the sequence of SEQ ID NO: 2, or consists essentially of it, or consists of it. In some embodiments, the carbohydrate binding region of ASGR1 comprises the sequence of SEQ ID NO: 3, or consists essentially of it, or consists of it. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, D266, D267, R237, N209, H257, T259, and Y273 in SEQ ID NO: 1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1.

[0022] In some embodiments, the anti-ASGR1 monoclonal antibody or antigen-binding fragment thereof is the monoclonal antibody or antigen-binding fragment thereof described in the first aspect.

[0023] In any of the above aspects, the second lipid-lowering drug is preferably an HMGCR inhibitor, an ATP-citrate lyase (ACL) inhibitor, an NPC1L1 inhibitor and / or a PCSK9 inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pitavastatin. In some embodiments, the statin is atorvastatin. In some embodiments, the ACL inhibitor is bepedoic acid. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody or an interfering nucleic acid (such as siRNA or shRNA) targeting a PCSK9 encoding gene. In some embodiments, the PCSK9 inhibitor is Evolocumab, Alirocumab or Inclisiran.

[0024] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. Preparation of ASGR1 neutralizing antibodies. (a) Steps for preparing ASGR1 monoclonal neutralizing antibodies. Purified ASGR1 protein was used as an antigen to immunize rabbits. After the first round of screening, four B cell monoclonal clones were obtained as candidates. Then the coding region of the variable region of the antibody was sequenced, constructed into an antibody expression vector, and transfected into mammalian cells. Its effect was confirmed by protein immunoblotting and real-time quantitative PCR. Finally, the rabbit Fc fragment was replaced with the mouse Fc fragment. Finally, the neutralizing antibody 4B9 was selected for large-scale production and used in subsequent experiments. (b) Coomassie brilliant blue staining of purified ASGR1 protein in HEK293T cells; (c) Huh7 cells were incubated with purified neutralizing antibodies for 72 hours, and protein expression of LXRα was analyzed by protein immunoblotting; (d) Primary liver cells were isolated from 8-week-old wild-type C57B / L6 mice. Primary hepatocytes were incubated with different monoclonal neutralizing antibodies at different concentrations for 72 hours, and the cells were collected, lysed and subjected to protein immunoblotting analysis; (e) Primary hepatocytes were isolated from 8-week-old wild-type C57B / L6 mice. Primary hepatocytes were incubated with ASGR1 neutralizing antibody 4B9 for 72 hours, and the cells were collected, total RNA in the cells was extracted, and the target genes of LXR were detected by real-time quantitative PCR analysis; (f) Huh7 cells were transfected with the indicated plasmids. After 48 hours of transfection, the cells were collected, lysed, and the expression of each protein was analyzed by protein immunoblotting.

[0026] Figure 2. ASGR1 neutralizing antibodies increase cholesterol efflux and reduce blood lipids and liver fat. 8-week-old Asgr1 knockout mice and wild-type mice from the same litter were randomly divided into 4 groups as shown in the figure, with 6 mice in each group. Free drinking water and high-fat, high-cholesterol and bile salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol and 0.5% bile salt) were fed. At the same time, mice were intraperitoneally injected with control antibody or ASGR1 neutralizing antibody 4B9 every other day at a dose of 10 mg / kg / day. After 14 days, mice were uniformly starved for 4 hours before being killed. All data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, and bile acid metabolism in mouse liver, with cyclophilin as an internal reference; (c) total cholesterol in serum; (d) triglycerides in serum; (e) total cholesterol in liver; (f) triglycerides in liver; (g) volume of gallbladder; (h) cholesterol concentration in bile; (i) total cholesterol in bile; (j) representative picture of gallbladder volume; (k) total cholesterol in feces; (l) bile acid concentration in bile; (m) total bile acid in bile; (n) body weight; (o) daily food intake; (p) liver-to-body weight ratio; (q) blood glucose; (r) serum alanine aminotransferase; (s) serum aspartate aminotransferase.

[0027] Figure 3. ASGR1 neutralizing antibody combined with atorvastatin shows synergistic lipid-lowering effect. 8-week-old Asgr1 knockout mice and wild-type mice from the same litter were randomly divided into 8 groups as shown in the figure, with 6 mice in each group. Free drinking water and high-fat, high-cholesterol and bile salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol and 0.5% bile salt) were fed. At the same time, mice were intraperitoneally injected with control antibody or ASGR1 neutralizing antibody 4B9 every other day at a dose of 10 mg / kg / day, and atorvastatin was gavaged at a dose of 30 mg / kg / day every day. After 14 days, mice were uniformly starved for 4 hours before being killed. All data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) total cholesterol in serum; (c) triglycerides in serum; (d) total cholesterol in liver; (e) triglycerides in liver; (f) cholesterol concentration in bile; (g) cholesterol amount in bile; (h) bile acid concentration in bile; (i) total bile acid amount in bile; (j) total cholesterol in feces; (k) body weight; (l) daily food intake; (m) liver-to-body weight ratio; (n) blood glucose; (o) serum alanine aminotransferase; (p) serum aspartate aminotransferase; (qr) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis and other pathways in mouse liver, with cyclophilin as an internal control; (s) hematoxylin-eosin staining of liver sections; (t) Oil red O staining of liver sections.

[0028] Figure 4. Neutralizing antibody 4B9 synergizes with ezetimibe. Eight-week-old Asgr1 knockout mice and wild-type mice from the same litter were randomly divided into 8 groups as shown in the figure, with 6 mice in each group. Free drinking water and high-fat, high-cholesterol and bile salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol and 0.5% bile salt) were fed. At the same time, mice were intraperitoneally injected with control antibody or ASGR1 neutralizing antibody 4B9 every other day at a dose of 10 mg / kg / day, and gavage with ezetimibe at a dose of 10 mg / kg / day every day. After 8 days, mice were uniformly starved for 4 hours before being killed. All data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001. (a) Western blot analysis of liver samples; (b) total cholesterol in serum; (c) triglycerides in serum; (d) total cholesterol in liver; (e) triglycerides in liver; (f) cholesterol concentration in bile; (g) total cholesterol in bile; (h) bile acid concentration in bile; (i) total bile acid in bile; (j) body weight; (k) daily food intake; (l) liver weight / body weight; (m) blood glucose; (n) serum alanine aminotransferase; (o) serum aspartate aminotransferase; (pq) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis and other pathways in mouse liver, with cyclophilin as an internal control; (r) hematoxylin-eosin staining of liver sections; (s) Oil red O staining of liver sections. DETAILED DESCRIPTION

[0029] definition In the present invention, "about" means that the value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in each practice in the art. Alternatively, "about" or "substantially including" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially including" should be assumed to be within the acceptable error range of the specific value.

[0030] Compositions or methods described herein as "comprising" one or more of the described elements or steps are open-ended, meaning that the elements or steps are required, but other elements or steps may be added within the scope of the composition or method. It should also be understood that any composition or method described as "comprising" one or more of the described elements or steps also describes a corresponding, more limited composition or method that "consists essentially of the described elements or steps," meaning that the composition or method includes the required elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0031] As used herein, the term "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signal transduction). "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0032] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for possible natural mutants that may be present in small amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include a variety of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and is not to be construed as requiring the preparation of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be prepared by hybridoma or recombinant DNA methods.

[0033] Monoclonal antibodies can include "chimeric" antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibody constitutes part of a larger biomolecule, such as a fusion protein or an antibody drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.

[0034] Thus, as used herein, examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), 2and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies; domain antibodies; and multispecific antibodies (such as bispecific antibodies), CAR-T and BiTE formed by antibody fragments. "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form F(ab') 2 molecule. "F(ab′) 2 A "fragment" contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F(ab') 2 The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant region. A "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment that contains the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, the Fv polypeptide contains an additional polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0035] "Fc" or "Fc segment" or "Fc region" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain, and in some cases, excluding part of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγR receptors or FcRn receptors.

[0036] As used herein, the term "epitope" refers to a portion of an antigen to which an antibody or antigen-binding fragment binds. In some embodiments, an epitope may be a conformational epitope, i.e., a portion of an antigen that is not covalently adjacent but is close to each other in three-dimensional space when the antigen is in a related conformation. For example, for ASGR1, a conformational epitope is an epitope that is contained in the extracellular domain of ASGR1 and is composed of multiple non-adjacent amino acid residues. In some embodiments, an epitope may be a linear epitope, i.e., an epitope that contains a sequence of amino acid residues that are adjacent in the primary structure in the extracellular domain of ASGR1. The exact sequence and / or particularly the amino acid residues for determining the epitope of ASGR1 are known in the literature, including competition with peptides from the antigen sequence for binding to ASGR1 sequences from different species, truncation and / or mutagenesis (e.g., by alanine scanning or other site-directed mutagenesis), phage display-based screening or (co) crystallography techniques.

[0037] As used herein, the term "humanized antibody" refers to an antibody comprising CDRs of an antibody derived from a mammal other than humans, and a framework region (FR) and constant region of a human antibody.

[0038] As used herein, the term "chimeric antibody" will be taken to refer to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified according to the disclosure) is obtained from a second species. In certain embodiments, the binding region or site of interest will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0039] An "equivalent" of an antibody or polypeptide refers to an antibody or polypeptide that has a certain degree of homology or sequence identity with the amino acid sequence of the antibody or polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some aspects, an equivalent has one, two, three, four or five additions, deletions, substitutions and combinations thereof compared to a reference antibody or polypeptide. In some aspects, an equivalent of an antibody or polypeptide retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0040] As used herein, a "variant" of a sequence refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0041] As used herein, "% identity" between two sequences refers to a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced when performing optimal alignment of the two sequences. Sequence comparison and determination of % identity between two sequences can be accomplished using a mathematical algorithm.

[0042] "Conservative substitutions" refer to amino acid substitutions known to those skilled in the art, which are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter or substantially change the biological activity. "No alteration or no substantial alteration" refers to no more than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% difference in one or more aspects compared to the object being compared when measured by the same or similar method.

[0043] Generally, HCDR3 and LCDR3 are considered to play a more important role than other CDRs in antigen recognition. Therefore, in the case of substitution, the present invention preferably performs conservative substitutions on CDRs other than HCDR3 and LCDR3. In some embodiments, HCDR1, HCDR3 and LCDR3 are not substituted. Preferred amino acid substitutions include, but are not limited to, substitutions that (1) reduce sensitivity to proteolysis, (2) reduce sensitivity to oxidation, (3) change binding affinity for forming protein complexes, and (4) provide or modify other physicochemical or functional properties of these analogs. Analogs may include mutations of various sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be performed in naturally occurring sequences (preferably in the polypeptide portion outside the region forming intermolecular contacts). Conservative amino acid substitutions should not substantially change the structural characteristics of the parent sequence (e.g., amino acid substitutions should not tend to destroy the helix present in the parent sequence, or characterize other secondary structure types that destroy the parent sequence).

[0044] It is contemplated that the binding domain of the monoclonal antibody or antigen-binding fragment thereof of the present invention may carry a signal peptide, which is usually located at the N-terminus of the secretory protein and generally consists of 15 to 30 amino acids. After the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), protein synthesis is paused or slowed down, and the signal recognition particle carries the ribosome to the endoplasmic reticulum, and protein synthesis restarts. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, and the signal peptide sequence is removed under the action of the signal peptidase. If the termination transport sequence is present at the C-terminus of the nascent peptide chain, it may not be removed by the signal peptidase, such as ovalbumin contains an internal signal peptide. Neither its precursor nor its mature form is removed by the signal peptidase.

[0045] When referring to ligand / receptor, antibody / antigen or other binding pairs, "specific" binding refers to the binding reaction of determining whether there is a protein in a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind to other proteins present in the sample in significant amounts. "Specific binding" means that the monoclonal antibody or its antigen-binding fragment of the present invention can specifically interact with at least two, three, four, five, six, seven, eight or more amino acids of each target molecule. The "specific binding" of an antibody is mainly characterized by two parameters: a qualitative parameter (binding epitope or antibody binding position) and a quantitative parameter (binding affinity or binding strength). Antibody binding epitopes can be determined by FACS, peptide point epitope mapping, mass spectrometry or peptide ELISA. Biacore and / or ELISA methods can determine the binding strength of antibodies to specific epitopes. The signal-to-noise ratio is usually used as a representative determination and calculation method for binding specificity. In such a signal-to-noise ratio, the signal represents the intensity of the antibody binding to the target epitope, while the noise represents the intensity of the antibody binding to other non-target epitopes. Preferably, the antibody being evaluated can be considered to bind to the target epitope in a specific manner, i.e., "specific binding", when the signal-to-noise ratio for the target epitope is about 50. An antigen binding protein (including an antibody) "specifically binds" to an antigen if it binds to the antigen with a high binding affinity as determined by an affinity constant (KD) value. In some embodiments, the affinity constant KD is less than 10 -9 M. As used herein, the term "KD" refers to the affinity constant for a specific antibody-antigen interaction.

[0046] As used herein, the term "patient" or "subject" refers to any organism to which the provided antibodies, antigen-binding fragments thereof, or pharmaceutical compositions are administered or can be administered for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the subject is a human. In some embodiments, the subject suffers from or is susceptible to one or more disorders or conditions. The patient may exhibit one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions. In some embodiments, the patient is receiving or has received a therapy for diagnosing and / or treating such diseases, disorders, or conditions.

[0047] In the present invention, the term "treatment" refers to therapeutic and preventive measures that prevent or slow the occurrence or progression of undesirable physiological changes or symptoms in a subject. Favorable or desired clinical effects include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, alleviation or alleviation of the disease state, and partial or complete cure of the disease, regardless of whether the above effects are detectable. "Treatment" may also refer to prolonged survival compared to no treatment. Subjects in need of treatment include subjects who already have the disease or condition, as well as subjects who are likely to have the disease or condition, or subjects who are to prevent the disease or condition.

[0048] "Administering" and "treating" when used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid contacts the cell. "Administering" and "treating" also mean in vitro and ex vivo treatment of cells, for example, by an agent, diagnostic agent, binding composition, or by other cells.

[0049] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of the monoclonal antibody or antigen-binding fragment thereof that binds to ASGR1 of the present invention that is effective in preventing or alleviating the disease or condition to be treated when it is administered alone or in combination with another therapeutic agent to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of the compound sufficient to cause a reduction in symptoms, such as treatment, cure, prevention or alleviation of a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the symptoms. When an active ingredient is administered to an individual alone, the therapeutically effective amount refers to the individual ingredient. When a combination is administered, the therapeutically effective amount refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether it is administered in combination, sequentially or simultaneously. The therapeutically effective amount will alleviate symptoms typically by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.

[0050] As used herein, "pharmaceutically acceptable carriers" include materials that, when combined with the active ingredients of the composition, allow the ingredient to maintain biological activity and do not cause a destructive reaction with the subject's immune system. These carriers may include stabilizers, preservatives, salt or sugar complexes or crystals, etc. "Pharmaceutically acceptable" refers to molecules and ingredients that do not produce allergic reactions or similar undesirable reactions when administered to the human body.

[0051] ASGR1 monoclonal antibody In one aspect of the present invention, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that binds to ASGR1, wherein the monoclonal antibody inhibits or blocks the binding of ASGR1 to its natural ligand and / or the internalization of ASGR1.

[0052] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 4-6 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 7-9 or the equivalents of each thereof.

[0053] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 10-12 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 13-15 or the equivalents of each thereof.

[0054] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 16-18 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 19-21 or the equivalents of each thereof.

[0055] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 22-24 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 25-27 or the equivalents of each thereof.

[0056] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 28 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29 or its equivalent.

[0057] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 30 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31 or its equivalent.

[0058] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 32 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33 or its equivalent.

[0059] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO: 34 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 35 or its equivalent.

[0060] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 4-6 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 7-9 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion.

[0061] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 10-12 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 13-15 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion.

[0062] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 16-18 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 19-21 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion.

[0063] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 22-24 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 25-27 or variants of each thereof, wherein the variants have a single substitution, deletion, or insertion.

[0064] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:28 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:28, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:29 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:29.

[0065] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:30 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:30, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:31 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:31.

[0066] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:32 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:32, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:33 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:33.

[0067] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:34 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:34, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:35 or a variant thereof, which has at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:35.

[0068] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs:4-6, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs:7-9.

[0069] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 10-12, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 13-15.

[0070] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 16-18, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 19-21.

[0071] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 22-24, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 25-27.

[0072] In any of the above embodiments, the LCDR1 to LCDR3 and HCDR1 to HCDR3 are defined based on any one of the definitions of IMGT, Kabat, Chothia, Contact or Martin. In some embodiments, the LCDR1 to LCDR3 and HCDR1 to HCDR3 are defined based on the IMGT definition.

[0073] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:28, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:29.

[0074] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:30, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:31.

[0075] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:32, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:33.

[0076] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the sequence shown in SEQ ID NO:34, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:35.

[0077] In some embodiments, the ASGR1 monoclonal antibody is of IgG type, such as IgG1, IgG2, IgG3 or IgG4 type. In some embodiments, the ASGR1 monoclonal antibody is of IgG1 type.

[0078] In some embodiments, the substitutions described herein are conservative substitutions.

[0079] "Conservative (amino acid) substitutions" refer to substitutions of amino acid residues with amino acids having similar side chains. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably substituted with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids may be substituted with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0080] It will also be appreciated by those skilled in the art that the antibodies disclosed herein can be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein can be similar to the starting sequence, such as having a certain percentage of identity, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or a range between any two of these values ​​identical to the starting sequence.

[0081] In some embodiments, the antibody comprises an amino acid sequence or one or more portions not typically associated with an antibody. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may comprise a flexible linker sequence, or may be modified to add a functional portion (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0082] Antibodies, variants or derivatives thereof of the present invention include modified derivatives, i.e., covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibody may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications may be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-classical amino acids.

[0083] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0084] The antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels (e.g., radioactive labels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents (which may be drugs or toxins), ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0085] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0086] Fluorescent emitting metals (e.g. 152The antibody can be detectably labeled with Eu or other metals of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The techniques for conjugating various moieties to antibodies are well known.

[0087] Humanized and chimeric antibodies In any aspect of the present invention, the monoclonal antibody is preferably a humanized antibody or a chimeric antibody.

[0088] Humanized antibodies are antibody molecules derived from non-human species antibodies, which bind to the desired antigens with one or more complementary determining regions (CDRs) from non-human species and from the framework regions of human immunoglobulin molecules. Typically, the framework residues in the human framework region will be replaced by the corresponding residues from the CDR donor antibody to change, preferably improve antigen binding. These framework replacements are identified by methods well known in the art, for example, by modeling the interaction of CDR and framework residues to identify framework residues that are important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting, veneer or surface remodeling and chain shuffling, etc.

[0089] Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0090] Transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can also be used to produce human antibodies. For example, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be introduced into human immunoglobulin loci separately or simultaneously by homologous recombination. In particular, the homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized with selected antigens in a normal manner, such as all or part of the desired target polypeptide. Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations. Therefore, using this technology, it is possible to generate therapeutically useful IgG, IgA, IgM and IgE antibodies.

[0091] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection." In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope.

[0092] Conventional procedures can be used (for example, by using oligonucleotide probes that can be specifically bound to the genes encoding mouse antibody heavy and light chains) to easily separate and sequence the DNA encoding the desired monoclonal antibody. The hybridoma cells separated and subcloned are used as the preferred source of such DNA. After separation, the DNA can be placed in an expression vector, which is then transfected into a prokaryotic or eukaryotic host cell, such as an Escherichia coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins. More specifically, the DNA separated can be used to clone constant region and variable region sequences for the manufacture of antibodies. Essentially, this requires extracting RNA from selected cells, converting it into cDNA, and amplifying it by PCR using Ig-specific primers. As described herein, the transformed cells expressing the desired antibody can be grown in relatively large quantities to provide clinical and commercial supply of immunoglobulins.

[0093] In addition, using conventional recombinant DNA techniques, one or more CDRs of the antigen-binding polypeptides of the present disclosure can be inserted into the framework region, for example, into the human framework region to humanize non-human antibodies. The framework region can be a naturally occurring or shared framework region, and preferably a human framework region. Preferably, the polynucleotide encoding produced by the combination of the framework region and the CDR specifically binds to an antibody of at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made in the framework region, and preferably, the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods can be used to carry out amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotides are included in the present disclosure and are within the technical scope of the art.

[0094] In addition, techniques developed for producing "chimeric antibodies" by splicing genes from a mouse antibody molecule with the appropriate antigenic specificity and genes from a human antibody molecule with the appropriate biological activity can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a molecule having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0095] Another efficient method for generating recombinant antibodies is to generate primate antibodies containing monkey variable domains and human constant sequences.

[0096] Accordingly, the present invention provides a humanized ASGR1 monoclonal antibody, which comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NO: 4-6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NO: 7-9 or an equivalent thereof; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 10-12 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 13-15 or an equivalent thereof; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 16-18 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 19-21 or an equivalent thereof; and (d) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 22-24 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 25-27 or the equivalents of each thereof.

[0097] In some embodiments, the humanized ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively comprising the sequences shown in SEQ ID NOs: 4-6 or the equivalents of each thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively comprising the sequences shown in SEQ ID NOs: 7-9 or the equivalents of each thereof.

[0098] Accordingly, the present invention provides a chimeric ASGR1 monoclonal antibody, which comprises a light chain variable region and a heavy chain variable region and a human constant region, and is selected from any one of the following antibodies: (e) the light chain variable region comprises the sequence shown in SEQ ID NO: 28 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29 or its equivalent; (f) the light chain variable region comprises the sequence shown in SEQ ID NO: 30 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31 or its equivalent; (g) the light chain variable region comprises the sequence shown in SEQ ID NO: 32 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33 or its equivalent; and (h) the light chain variable region comprises the sequence shown in SEQ ID NO: 34 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 35 or its equivalent.

[0099] In some embodiments, the chimeric ASGR1 monoclonal antibody comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 28 or its equivalent, and a heavy chain variable region comprising the sequence shown in SEQ ID NO: 29 or its equivalent.

[0100] Any of the chimeric ASGR1 monoclonal antibodies or humanized ASGR1 monoclonal antibodies described above may comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is, for example, the human IgG1 heavy chain constant region shown in SEQ ID NO:37, and the light chain constant region is, for example, the human Igκ light chain constant region shown in SEQ ID NO:38.

[0101] ASGR1 inhibitors Asialoglycoprotein receptor 1 (ASGR1) is mainly located in liver cells. ASGR1 is a single transmembrane protein, including the cytoplasmic end, transmembrane segment, hinge region and carbohydrate binding region. After being processed by neuraminidase, glycoproteins in serum form desialylated glycoproteins, which then bind to ASGR1 on the cell membrane, initiate endocytosis, and enter the endosomal pathway. In the acidic environment of the endosomal, the receptor dissociates from the ligand, and these proteins are transported to the lysosome for degradation, while ASGR1 circulates to the cell surface for reuse.

[0102] Human ASGR1 contains 291 amino acids, with a molecular weight of 33,186Da, and the amino acid sequence is shown in SEQ ID NO:1 (UniProtKB / Swiss-Prot:P07306.2). ASGR1 can bind to the ligand in the blood, namely asialoglycoprotein, and then enter the lysosome for degradation through clathrin-mediated endocytosis. The cytoplasmic end of human ASGR1 is short (1-40aa), the transmembrane region is 40-60aa, and the extracellular region is divided into the stem region (62-141aa) and the carbohydrate binding region (142-291, SEQ ID NO:2). The gene ID of the human ASGR1 gene in NCBI is 432, and the coding region sequence is shown in SEQID NO:36 (NCBI Reference Sequence:NM_001671.5).

[0103] Herein, "ASGR1 inhibitor" refers to a substance that can reduce or block the binding of ASGR1 to its natural ligand and / or the internalization of ASGR1, and a substance that can reduce or block the expression of the gene encoding ASGR1.

[0104] In some embodiments, the ASGR1 inhibitors herein may include: (1) inhibitors that bind to ASGR1; (2) inhibitors that bind to the ligand of ASGR1 (e.g., asialoglycoprotein); (3) inhibitors that reduce or block the binding of ASGR1 to its ligand; (4) inhibitors that reduce or block the endocytosis of ASGR1; (5) inhibitors that reduce the protein level of ASGR1; (6) inhibitors that reduce or block the protein activity of ASGR1; and (7) inhibitors that reduce or block the expression of the gene encoding ASGR1.

[0105] In some embodiments, the ASGR1 inhibitor of the present invention can be a small molecule compound, a nucleic acid targeting a gene encoding ASGR1, a nucleic acid aptamer targeting ASGR1, an anti-ASGR1 antibody, or a combination thereof.

[0106] In some embodiments, the ASGR1 inhibitor of the present invention is an ASGR1 antibody. In some embodiments, the ASGR1 antibody is an ASGR1 monoclonal antibody or an antigen-binding fragment thereof.

[0107] In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence shown in SEQ ID NO: 1, and inhibits the binding of human ASGR1 to its natural ligand (eg, asialoglycoprotein) and / or the internalization of human ASGR1.

[0108] In some embodiments, the ASGR1 monoclonal antibody binds to the carbohydrate binding region of ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or the endocytosis of human ASGR1. In some embodiments, the carbohydrate binding region of ASGR1 comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 2. In some embodiments, the carbohydrate binding region of ASGR1 comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 3.

[0109] In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, D266, D267, R237, N209, H257, T259, and Y273 in SEQ ID NO: 1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1. In some embodiments, the epitope to which the ASGR1 monoclonal antibody binds comprises one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1.

[0110] In some embodiments, the ASGR1 monoclonal antibody is a fully human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the ASGR1 monoclonal antibody is a fully human antibody or a humanized antibody. In some embodiments, the ASGR1 monoclonal antibody is a chimeric antibody.

[0111] In some embodiments, the ASGR1 monoclonal antibody is the antibody described in the above “ASGR1 monoclonal antibody” section, for example, the monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NO: 4-6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NO: 7-9 or an equivalent thereof; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 10-12 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 13-15 or an equivalent thereof; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 16-18 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 19-21 or an equivalent thereof; (d) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising a sequence as shown in SEQ ID NOs: 22-24 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising a sequence as shown in SEQ ID NOs: 25-27 or an equivalent thereof; (e) the light chain variable region comprises the sequence shown in SEQ ID NO: 28 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29 or its equivalent; (f) the light chain variable region comprises the sequence shown in SEQ ID NO: 30 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31 or its equivalent; (g) the light chain variable region comprises the sequence shown in SEQ ID NO: 32 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33 or its equivalent; and (h) the light chain variable region comprises the sequence shown in SEQ ID NO: 34 or its equivalent, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 35 or its equivalent.

[0112] Other exemplary ASGR1 monoclonal antibodies include those described in WO 2017058944 A1, WO 2022006327 A1, or US20210130473A1, the entire disclosures of which are incorporated herein by reference.

[0113] In some embodiments, the ASGR1 inhibitor of the present invention is a nucleic acid that targets the DNA or mRNA of ASGR1 and inhibits the expression of ASGR1.

[0114] In some embodiments, the nucleic acid is selected from antisense oligonucleotides (ASOs), siRNAs, shRNAs, and gRNAs.

[0115] In some embodiments, the nucleic acid is an ASO, which can be modified on the backbone, sugar or base to resist in vivo degradation. Suitable modifications include, but are not limited to, phosphorothioate (PSP), phosphorodiamidate morpholino oligonucleotide (PMO), 2'-O-methoxyethyl modification (2'-MOE), 5-methylcytosine (5mC).

[0116] In some embodiments, the nucleic acid is siRNA or shRNA, and the siRNA can be delivered via a suitable carrier. The suitable carrier is, for example, GalNAc, LNP or AAV.

[0117] In some embodiments, the nucleic acid targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the gene encoding ASGR1.

[0118] In some embodiments, the nucleic acid is a gRNA, and the gRNA and the CRISPR / Cas enzyme (e.g., CRISPR / Cas9) constitute a gene editing system to inhibit or block the expression of the gene encoding ASGR1.

[0119] On the premise that the gene encoding ASGR1 is known, those skilled in the art can obtain and screen the sequence of the above nucleic acid by conventional techniques.

[0120] In some embodiments, the nucleic acid (such as ASO, siRNA, shRNA or gRNA) targets the sequence shown in SEQ ID NO: 36 and inhibits the expression of the gene encoding ASGR1.

[0121] In some embodiments, the ASGR1 inhibitor of the present invention is a nucleic acid aptamer targeting human ASGR1. In some embodiments, the nucleic acid aptamer binds to human ASGR1 and inhibits the binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or the endocytosis of human ASGR1.

[0122] Treatment methods and applications One aspect of the present invention provides a method for lowering blood cholesterol levels, the method comprising administering to a subject a therapeutically effective amount of any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof.

[0123] Another aspect of the present invention provides a method for reducing triglyceride levels in blood, the method comprising administering to a subject a therapeutically effective amount of any one of the above monoclonal antibodies or antigen-binding fragments thereof.

[0124] In another aspect, the present invention provides a method for treating and / or preventing cardiovascular diseases such as atherosclerosis, stroke, myocardial infarction or coronary artery disease, which comprises administering to a subject a therapeutically effective amount of any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof.

[0125] Accordingly, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the preparation of a medicament for lowering blood cholesterol levels.

[0126] Accordingly, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the preparation of a medicament for reducing the level of triglycerides in blood.

[0127] Accordingly, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the preparation of a medicament for treating and / or preventing cardiovascular disease.

[0128] Accordingly, another aspect of the present invention provides any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof for use in lowering blood cholesterol levels.

[0129] Accordingly, another aspect of the present invention provides any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof for use in reducing triglyceride levels in blood.

[0130] Accordingly, another aspect of the present invention provides any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof for use in treating and / or preventing cardiovascular diseases.

[0131] On the other hand, the inventors found that the combined administration of ASGR1 inhibitors and lipid-lowering drugs produced a significant synergistic lipid-lowering effect, with total cholesterol levels and triglyceride levels in serum and liver significantly reduced.

[0132] In some embodiments, the present invention provides a method of reducing total cholesterol levels in the blood and / or liver, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering drug.

[0133] In some embodiments, the present invention provides a method of reducing triglyceride levels in the blood and / or liver, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor described herein and a therapeutically effective amount of a second lipid-lowering drug.

[0134] In some embodiments, the present invention provides a method for treating and / or preventing cardiovascular disease, the method comprising administering to a subject a therapeutically effective amount of an ASGR1 inhibitor as described herein and a therapeutically effective amount of a second lipid-lowering drug.

[0135] Accordingly, in some embodiments, the present invention provides the use of a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering drug in the preparation of a medicament for lowering total cholesterol levels in the blood and / or liver.

[0136] Accordingly, in some embodiments, the present invention provides the use of a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering drug in the preparation of a medicament for lowering triglyceride levels in the blood and / or liver.

[0137] Accordingly, in some embodiments, the present invention provides use of a combination of an ASGR1 inhibitor and a second lipid-lowering drug as described herein in the preparation of a medicament for treating and / or preventing cardiovascular disease.

[0138] Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering agent for use in lowering total cholesterol levels in the blood and / or liver.

[0139] Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering agent for use in reducing triglyceride levels in the blood and / or liver.

[0140] Accordingly, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor as described herein and a second lipid-lowering drug for use in treating and / or preventing cardiovascular disease.

[0141] In any of the above embodiments, the second lipid-lowering drug is preferably an HMGCR inhibitor, an ACL inhibitor, an NPC1L1 inhibitor and / or a PCSK9 inhibitor.

[0142] In any of the above embodiments, the second lipid-lowering drug can be administered simultaneously with the ASGR1 inhibitor or administered at intervals. For example, the second lipid-lowering drug can be administered to the subject before the ASGR1 inhibitor is administered, or the second lipid-lowering drug can be administered to the subject after the ASGR1 inhibitor is administered.

[0143] In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pitavastatin. In some embodiments, the statin is atorvastatin.

[0144] In some embodiments, the ACL inhibitor is bepedic acid.In some embodiments, the NPC1L1 inhibitor is ezetimibe.

[0145] In some embodiments, the PCSK9 inhibitor is a PCSK9 antibody or a nucleic acid (such as siRNA or shRNA) targeting a PCSK9 encoding gene. In some embodiments, the PCSK9 antibody is Evolocumab, Alirocumab or Inclisiran. Other exemplary PCSK9 inhibitors include PCSK9 inhibitors described in WO2017220701A1, WO2012088313A1, WO2009026558A1, WO2009102427A2 or WO2017035340A1, and all of their disclosures are incorporated herein by reference.

[0146] Suitable administration routes include parenteral administration (e.g., intramuscular, intravenous or subcutaneous administration) and oral administration. Other conventional administration methods include administration via endotracheal intubation, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, intraarterial injection.

[0147] The appropriate dosage is determined by a clinician based on parameters or factors known or suspected to affect treatment or expected to affect treatment in the art. Typically, the initial dose is slightly lower than the optimal dose, and then increased in small amounts until the desired or optimal effect relative to any adverse side effects is achieved. Important monitoring indicators include measuring, for example, inflammatory symptoms or the levels of inflammatory cytokines produced.

[0148] Pharmaceutical composition One aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.

[0149] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the ASGR1 inhibitor described herein, a therapeutically effective amount of a second lipid-lowering drug, and a pharmaceutically acceptable carrier.

[0150] In any of the above aspects, the pharmaceutical composition is used to reduce total cholesterol levels in the blood and / or liver. In some embodiments, the pharmaceutical composition is used to reduce triglyceride levels in the blood and / or liver. In some embodiments, the pharmaceutical composition is used to treat and / or prevent cardiovascular diseases, such as atherosclerosis, stroke, myocardial infarction or coronary artery disease.

[0151] Another aspect of the present invention provides a drug kit comprising a first pharmaceutical composition and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a therapeutically effective amount of the ASGR1 inhibitor described herein and a pharmaceutically acceptable carrier, and the second pharmaceutical composition comprises a therapeutically effective amount of a second lipid-lowering drug and a pharmaceutically acceptable carrier.

[0152] In any of the above aspects, the second lipid-lowering drug is preferably an HMGCR inhibitor, an ACL inhibitor, an NPC1L1 inhibitor and / or a PCSK9 inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pravastatin. In some embodiments, the statin is atorvastatin. In some embodiments, the ACL inhibitor is bepedoic acid. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody or a nucleic acid targeting a PCSK9 encoding gene (such as siRNA or shRNA). In some embodiments, the PCSK9 antibody is Evolocumab, Alirocumab or Inclisiran.

[0153] To prepare a pharmaceutical composition or sterile composition, the drug is mixed with a pharmaceutically acceptable carrier or excipient. Formulations in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers. Pharmaceutically acceptable carriers are well known in the art. It is known in the art how to prepare aqueous compositions containing as active ingredients. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions; solid forms suitable for formulation into solutions or suspensions prior to injection or spraying can also be prepared.

[0154] Sequence Listing Example

[0155] Materials and Methods Mouse CRISPR / Cas9-mediated Asgr1 whole-body knockout mice were constructed by Chengdu Jicui Pharmaceutical Co., Ltd. The sgRNA-mediated cas9 endonuclease cuts between the 2nd and 3rd introns and between the 8th and 9th introns. The part between exons 3 to 8 was deleted through homologous recombination, thereby obtaining whole-body Asgr1 knockout mice.

[0156] In the experiment, wild-type littermates were used as controls. All mice were housed in an SPF (specific pathogen free) animal room and maintained at 12 hours of light / 12 hours of darkness. Male or female mice aged 8 weeks were fed a high-fat, high-cholesterol bile salt diet (Research Diets, D12109C) for the required time in the experiment. In antibody neutralization or antibody and statin or ezetimibe, mice were intraperitoneally injected or gavaged with antibodies according to the specified amount. All animals were starved for 4 hours before being killed. All animal experiments strictly abide by the relevant regulations on experimental animal welfare ethics and protection of the country and Wuhan University.

[0157] Materials and plasmids Lovastatin (purity ≥ 98%, HPLC) was purchased from Shanghai Pharm Vally. Sodium mevalonate (#4667), anti-FLAG M2 beads (#A2220), anti-MYC beads (E6654), Fetuin A (SRP6217), D-Galactose (G5388), N-acetyl-D-galactosamine (A-2795), Phenylmethanesulfonylfluoride (PMSF, #P7626), Protease inhibitor cocktail (#P8340) and β-mercaptoethanol (#M3148) were purchased from Sigma. Dil (1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanineperchlorate)-LDL (#20614ES76) was purchased from Shanghai Yisheng. Lipofectamine RNAiMAX (#13778150) was purchased from Theromo Fisher. MG132 (#I-130) was purchased from Boston Biochem. Puromycin (#BS111) was purchased from Biosharp. G418 (#345810), Pepstatin A (#516481) and ALLN (N-acetyl-leu-leu-norleucinal, #208719) were purchased from Calbiochem. Ni-NTA Agarose (#30230) was purchased from Qiagen. LPEI (Linearpolyethylenimine, #23966-1) was purchased from Polysciences. FuGENE HD (#E2311) and M-MLV RTase (#M1701) were purchased from Promega. Leupeptin (#11034626001) was purchased from Roche. DTT (DL-Dithiothreitol, #A100281) and NP-40 (A100109) were purchased from Shanghai Biotech. Phosphatase inhibitor (P1082) was purchased from Biotech. The culture medium and fetal bovine serum (FBS) used to culture cells were purchased from Life Technology. Taq enzyme was purchased from Tiangen. KOD Hot Start DNA polymerase (#KOD-401; TOYOBO) was purchased from Takara; RNA duplex was synthesized by Guangzhou Ruibo Company, and Q-PCR 2×MIX was purchased from Mona.Total cholesterol (TC) kit, total triglyceride (TG) kit, and bile acid kit were purchased from Nanjing Kehua Biological Company. NEFA kit (294-63601) and Phospholipid kit (292-63901) were purchased from WAKO. ALT, AST, and AKP kits were purchased from Nanjing Jiancheng Biological Company. Blood glucose test strips and blood glucose meters were purchased from Wenhao. Lipoprotein-deficient serum (d>1.215g / mL), i.e., LPPS, was prepared from newborn calf serum by ultracentrifugation.

[0158] The following plasmids were constructed by standard molecular cloning techniques: Human and mouse Asgr1, Asgr2, Lxrα, and Lxrβ gene fragments were derived from cDNAs formed by reverse transcription of RNA from Huh7 and mouse liver tissue. The human BARD1 gene fragment was amplified from Huh7 and cloned into p3×Flag-CMV14, pEGFP-C1, and pcDNA3-C-5×Myc vectors, respectively. pDEST-FRT / T0-GFP-BRCA1 (#71116) was purchased from Addgene. Various truncations and point mutations of ASGR1 were constructed by point mutation.

[0159] Huh7 and HEK293T cells were cultured at 37°C and 5% CO 2 The cells were grown as monolayers in an environment of . The cells were maintained in medium A (DMEM containing 100 units / mL penicillin and 100 mg / mL streptomycin sulfate) supplemented with 10% fetal bovine serum (FBS), and cholesterol-deficient medium B was obtained by supplementing medium A with 5% lipoprotein-free serum (LPPS), 1 μM lovastatin, and 10 μM mevalonic acid. Primary mouse hepatocytes were cultured in medium D (M199) supplemented with 5% FBS, 100 units / mL penicillin, and 100 mg / mL streptomycin sulfate.

[0160] Immunoblotting The collected cells or tissues were lysed with RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. RIPA lysis buffer contains 50 mM Tris-HCl (pH = 8.0), 150 mM NaCl, 2 mM MgCl 2, 1.5% NP-40, 0.1% SDS and 0.5% sodium deoxycholate. Protease inhibitors included 10 μM MG-132, 10 μg / ml leupeptin, 1 mM PMSF, 5 μg / ml pepstatin, 25 μg / ml ALLN, 1 mM DTT. The protein concentration of the lysate was determined using the BCA method (Thermo Fisher Scientific). The protein samples were incubated with membrane lysis buffer (62.5 mM Tris-HCl (pH = 6.8), 15% SDS, 8 M urea, 10% glycerol and 100 mM DTT) and 4× loading buffer (150 mM Tris-HCl (pH = 6.8), 12% SDS, 30% glycerol, 6% 2-mercaptoethanol and 0.02% bromophenol blue) at 37°C for 30 minutes. The protein samples were separated by SDS-PAGE gel and then transferred to PVDF membranes, which were blocked for 1 hour with TBS containing 0.075% Tween 20 and 5% skim milk (3% BSA for phosphorylation experiments), i.e., TBST. The membranes were incubated with the designated primary antibodies overnight at 4°C, followed by washing with TBST three times. Finally, the membranes were detected with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

[0161] Primary antibodies used in the experiments Anti-β-actin (#A5441) and anti-FLAG (#F3165) were purchased from Sigma. anti-AMPK (10929-2-AP), anti-ACC (67373-I-Ig), anti-FASN (10624-2-AP), anti-GAPDH (60004-1-Ig), and anti-ASGR1 (11739-1-AP) were purchased from ProteinTech. Anti-CYP7A1 (sc-518007), anti-SREBP1 (sc-13551), and anti-BRCA1 (sc-6954) were purchased from Santa Cruz. Anti-c-Myc and anti-HMGCR were prepared and purified from hybridoma cell lines (ATCC) 9E10 and A9, respectively. Anti-LDLR and anti-H2 (HMGCR) were obtained by selecting soluble segments and immunizing rabbits to obtain antiserum and affinity-purified antibodies. Anti-EGFP was obtained by expressing and purifying EGFP protein in E. coli and immunizing rabbits. Anti-LXRα (ab41902) was purchased from Abcam. Anti-LXRβ (NB100-74457), anti-ABCG8 (NBP1-71706F), and anti-ABCA1 (NB400-105) were purchased from Novus. Anti-BARD1 (A300-263A) was purchased from Bethy. Anti-phos ACC (118187) and anti phos-AMPK (#2535) were purchased from Cell signaling Tech. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories.

[0162] Blood and liver chemistry analysis Blood was collected from the eyeball to obtain serum for testing total cholesterol and triglycerides. The liver was homogenized and the supernatant was collected for lipid extraction to obtain liver total cholesterol, and the liver triglyceride (TG) and total cholesterol (TC) levels were determined using a test kit (Kehua, China). Phospholipid levels were determined using a kit (Phospholipid, WAKO, Japan). ALT and AST levels in serum were tested using a kit from Nanjing Jiancheng (China).

[0163] Results and Analysis Example 1: ASGR1 neutralizing antibody 4B9 promotes cholesterol excretion into bile and feces, showing lipid-lowering therapeutic effect.

[0164] ASGR1 can bind to a large number of desialylated glycoproteins. In order to better simulate the improvement effect of Asgr1 knockout on metabolic syndrome induced by high fat and high cholesterol, we purified ASGR1 protein from HEK293T cells and immunized rabbits to obtain neutralizing antibodies that block the function of ASGR1. The specific process is shown in Figure 1a: Steps for preparing ASGR1 monoclonal neutralization. Purified ASGR1 protein was used as an antigen to immunize rabbits. After the first round of screening, 4 B cell monoclonal clones were obtained as candidates. Then the coding region of the variable region of the antibody was sequenced, constructed into an antibody expression vector, and transfected into mammalian cells to purify the protein. Its effect was confirmed by protein immunoblotting and real-time quantitative PCR, and finally the rabbit Fc fragment was replaced with the mouse Fc fragment. Finally, the neutralizing antibody 4B9 with the best effect was selected for large-scale production and used in subsequent experiments. The experimental results verified that 4B9 has a potential lipid-lowering effect. 4B9 and the other three ASGR1 monoclonal neutralizing antibodies can significantly increase the protein stability of LXRα (Figure 1c, 1d), and cholesterol efflux genes are also significantly upregulated ( Figure 1e ). In a more detailed step, we transfected ASGR1 with different truncated plasmids and detected the segment recognized by 4B9. We found that 4B9 mainly recognized the sequence between 182-274aa ( Figure 1f ).

[0165] Further, we tested whether 4B9 can completely mimic the improvement of Asgr1 deficiency on high fat and high cholesterol. Eight-week-old Asgr1 knockout mice and wild-type mice from the same litter were randomly divided into four groups as shown in Figure 2 according to genotype, with 6 mice in each group. Free drinking water, high fat, high cholesterol and bile salt diet (60% fat, 1.25% cholesterol and 0.5% bile salt, HF / HC / BS) were fed. At the same time, mice were intraperitoneally injected with control antibodies or ASGR1 neutralizing antibodies 4B9 every other day at a dose of 10 mg / kg / day. After 14 days, the mice were uniformly starved for 4 hours before being killed. The experimental results showed that wild-type mice injected with 4B9 antibodies showed consistent results with Asgr1 deficiency, that is, cholesterol efflux-related proteins such as LXR, ABCG8, ABCA1, and CYP7A1 increased significantly, and p-ACC was also significantly upregulated, while lipid synthesis-related proteins SREBP1 and FASN decreased significantly, and LDLR was not affected. BARD1 was significantly decreased; and there was no effect of injecting or not injecting 4B9 in Asgr1 knockout mice, indicating that 4B9 specifically targets ASGR1 ( Figure 2a). Real-time quantitative PCR also showed results consistent with the changes in protein levels (Figure 2b). Biochemical indicators showed that compared with the control group, the total cholesterol and triglycerides in the serum of mice injected with 4B9 antibodies decreased significantly (Figure 2c, d), the total cholesterol and triglycerides in the liver decreased significantly (Figure 2e, f), the volume of the gallbladder increased significantly (Figure 2g, j), the concentration and total amount of total cholesterol in bile increased significantly (Figure 2h, i), the total cholesterol in feces increased significantly (Figure 2k), and the concentration and total amount of total bile acid in bile increased significantly (Figure 2l, m). However, there were no significant changes in body weight, daily food intake, liver-to-body weight ratio, blood sugar, alanine aminotransferase and aspartate aminotransferase (Figure 2n-s). Based on the above results, the ASGR1 neutralizing antibody 4B9 showed a very significant lipid-lowering effect and can greatly alleviate the metabolic syndrome induced by high-fat and high-cholesterol diet.

[0166] Example 2: Antibody 4B9 combined with atorvastatin showed a synergistic lipid-lowering effect.

[0167] 4B9 has been shown in previous experimental results to significantly improve the metabolic syndrome phenotype induced by a high-fat, high-cholesterol diet (Figure 2). Further, we wanted to test whether the combination of 4B9 and atorvastatin has a synergistic lipid-lowering effect. Eight-week-old Asgr1 knockout mice and wild-type mice from the same litter were randomly divided into 8 groups as shown in Figure 3 according to genotype, with 6 mice in each group. They were fed with free drinking water and a high-fat, high-cholesterol and bile salt diet (60% fat, 1.25% cholesterol and 0.5% bile salt, HF / HC / BS).

[0168] At the same time, mice were intraperitoneally injected with control antibodies or ASGR1 neutralizing antibodies 4B9 at a dose of 10 mg / kg / day every other day, and gavage with atorvastatin at a dose of 30 mg / kg / day every day. After 14 days, the mice were starved for 4 hours before being killed. The experimental results showed that when 4B9 was used alone, it still increased the levels of genes and proteins related to cholesterol efflux and inhibited the levels of genes and proteins related to lipid synthesis, but did not affect the levels of genes or proteins related to cholesterol synthesis or absorption. The combination of atorvastatin and 4B9 did not affect the expression of 4B9 in promoting cholesterol efflux-related genes and proteins such as LXRs, ABCG8, ABCA1, and CYP7A1, but was able to more significantly inhibit the expression of lipid synthesis-related genes and proteins such as SREBP1 and FASN ( Figure 3a ,3q-r). When 4B9 or atorvastatin was used alone, it could effectively reduce the total cholesterol and triglyceride levels in serum and liver. When 4B9 and atorvastatin were used together, it showed a more significant lipid-lowering effect ( Figure 3b-e, Figure 3s-t), 4B9 alone can increase the total amount of total cholesterol and total bile acid in bile, while atorvastatin has almost no effect on total cholesterol and total bile acid in bile (Figure 3g,i). 4B9 alone can significantly increase the excretion of total cholesterol in feces, while atorvastatin has no effect on cholesterol in feces (Figure 3j). Whether 4B9 or atorvastatin is used alone or in combination, there is no significant effect on the body weight, daily food intake, liver-to-body weight ratio, blood sugar, and alanine aminotransferase and aspartate aminotransferase of mice among the 8 groups (Figure 3k-p). In summary, the combination of 4B9 and atorvastatin can achieve better lipid-lowering effects than when either is used alone.

[0169] Example 3: Antibody 4B9 combined with ezetimibe showed a synergistic lipid-lowering effect.

[0170] 4B9 has been shown to have a certain lipid-lowering effect in mice (Figure 2). Therefore, will the combination of 4B9 and EZ increase the lipid-lowering effect to a greater extent? Therefore, we designed an experiment as shown in Figure 4 to verify this hypothesis. Mice were randomly divided into 8 groups (n=6) according to genotype, fed HF / HC / BS feed, EZ was gavaged at 10 mg / kg per day, and 4B9 (10 mg / kg) was injected intraperitoneally every other day. The mice were collected on the 7th day and the mouse tissues were taken. The results showed that when 4B9 was used alone, it still increased the levels of genes and proteins related to cholesterol efflux, such as LXRs, ABCG8, ABCA1, and CYP7A1, and inhibited the levels of genes and proteins related to lipid synthesis, such as SREBP1 and FASN. It did not affect the levels of genes and proteins related to cholesterol synthesis or absorption. When ezetimibe was used alone or in combination with 4B9, the genes and proteins related to cholesterol efflux and lipid synthesis were significantly decreased ( Figure 4a ,4p-q). When 4B9 or ezetimibe was used alone, it could effectively reduce the total cholesterol level and triglyceride level in serum and liver, and when 4B9 and ezetimibe were used together, it showed a more significant lipid-lowering effect ( Figure 4b- e, Figure 4r-s), 4B9 alone can increase the total amount of cholesterol and total bile acid in bile, while ezetimibe alone or in combination with 4B9 significantly inhibited the total amount of total cholesterol and total bile acid in bile (Figure 4g,i). Whether 4B9 or ezetimibe is used alone or in combination, there is no significant effect on the body weight, daily food intake, liver-to-body weight ratio, blood sugar, and alanine aminotransferase and aspartate aminotransferase of mice among the 8 groups (Figure 4j-o). In summary, the combination of 4B9 and ezetimibe can achieve a better lipid-lowering effect than when either is used alone.

[0171] It should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may make modifications, improvements and variations to the present invention disclosed herein, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein are representative and exemplary of preferred embodiments, and are not intended to be limiting of the scope of the present invention.

Claims

1. A monoclonal antibody that binds to ASGR1, wherein the monoclonal antibody inhibits or blocks the binding of ASGR1 to its natural ligand and / or the internalization of ASGR1, wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 10-12, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 13-15, respectively; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 16-18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 19-21, respectively; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 22-24, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 25-27, respectively; (d) the light chain variable region is the sequence shown in SEQ ID NO: 30, and the heavy chain variable region is the sequence shown in SEQ ID NO: 31; (e) the light chain variable region is the sequence shown in SEQ ID NO: 32, and the heavy chain variable region is the sequence shown in SEQ ID NO: 33; and (f) the light chain variable region is the sequence shown in SEQ ID NO: 34, and the heavy chain variable region is the sequence shown in SEQ ID NO: 35; And wherein the monoclonal antibody is a humanized antibody or a chimeric antibody and comprises a human IgG1 heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:37 and a human Igκ light chain constant region with an amino acid sequence as shown in SEQ ID NO:

38.

2. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody according to claim 1, and a pharmaceutically acceptable carrier.

3. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1, a therapeutically effective amount of a second lipid-lowering drug, and a pharmaceutically acceptable carrier, wherein the second lipid-lowering drug is atorvastatin or ezetimibe.

4. A drug kit comprising a first pharmaceutical composition and a second pharmaceutical composition, wherein the first pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier, and the second pharmaceutical composition comprises a therapeutically effective amount of a second lipid-lowering drug and a pharmaceutically acceptable carrier, wherein the second lipid-lowering drug is atorvastatin or ezetimibe.

5. Use of the monoclonal antibody according to claim 1 in the preparation of a medicament for lowering the level of cholesterol and / or triglycerides in the blood or for treating and / or preventing cardiovascular diseases; preferably, the cardiovascular diseases are selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

6. Use of the combination of the monoclonal antibody of claim 1 and a second lipid-lowering drug in the preparation of a medicament for lowering the level of cholesterol and / or triglycerides in the blood or for treating and / or preventing cardiovascular disease, wherein the second lipid-lowering drug is atorvastatin or ezetimibe; preferably, the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

7. Use of a monoclonal antibody or an antigen-binding fragment thereof that binds to ASGR1 in the preparation of a medicament for treating cardiovascular disease, wherein the monoclonal antibody inhibits or blocks the binding of ASGR1 to its natural ligand and / or the endocytosis of ASGR1, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 10-12, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 13-15, respectively; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 16-18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 19-21, respectively; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 22-24, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 25-27, respectively; (d) the light chain variable region is the sequence shown in SEQ ID NO: 30, and the heavy chain variable region is the sequence shown in SEQ ID NO: 31; (e) the light chain variable region is the sequence shown in SEQ ID NO: 32, and the heavy chain variable region is the sequence shown in SEQ ID NO: 33; and (f) the light chain variable region is the sequence shown in SEQ ID NO:34, and the heavy chain variable region is the sequence shown in SEQ ID NO:35; preferably, the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

8. Use of a monoclonal antibody or an antigen-binding fragment thereof that binds to ASGR1 in combination with a second lipid-lowering drug in the preparation of a medicament for treating cardiovascular disease, wherein the second lipid-lowering drug is atorvastatin or ezetimibe, and the monoclonal antibody inhibits or blocks the binding of ASGR1 to its natural ligand and / or the endocytosis of ASGR1, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region and is selected from any one of the following antibodies: (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 10-12, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 13-15, respectively; (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 16-18, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 19-21, respectively; (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are sequences shown in SEQ ID NOs: 22-24, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are sequences shown in SEQ ID NOs: 25-27, respectively; (d) the light chain variable region is the sequence shown in SEQ ID NO: 30, and the heavy chain variable region is the sequence shown in SEQ ID NO: 31; (e) the light chain variable region is the sequence shown in SEQ ID NO: 32, and the heavy chain variable region is the sequence shown in SEQ ID NO: 33; and (f) the light chain variable region is the sequence shown in SEQ ID NO: 34, and the heavy chain variable region is the sequence shown in SEQ ID NO: 35; preferably, the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

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