Anti-ASGR1 monoclonal antibodies and uses thereof

Abstract

The present invention discloses an anti-ASGR1 monoclonal antibody and uses thereof. The present invention further discloses the use of an ASGR1 inhibitor, including an anti-ASGR1 monoclonal antibody, in combination with a second lipid-lowering drug to reduce total cholesterol and / or triglyceride levels in the blood and / or liver, or to treat and / or prevent cardiovascular disease. The present invention demonstrates that the combination of an ASGR1 inhibitor with a second lipid-lowering drug has a synergistic effect of lowering total cholesterol and triglycerides in serum and the liver.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to anti-ASGR1 monoclonal antibodies. The present invention also relates to the combination of an ASGR1 inhibitor (e.g., an anti-ASGR1 monoclonal antibody) with other lipid-lowering drugs.

[0002] [Background technology] Statins are commonly used to reduce low-density lipoprotein cholesterol (LDL-c) and are the first-line drug for cholesterol reduction. Currently, FDA-approved statins primarily include lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin, with atorvastatin and rosuvastatin being the most widely used. However, relevant studies have shown that long-term statin use can lead to statin resistance and liver toxicity in patients. Some patients are statin intolerant and experience side effects such as myasthenia, rhabdomyolysis, and diabetes. Moderate-intensity statins cannot meet the clinical needs for intensive lipid lowering. Doubling the dose of high-dose statins only increases efficacy by 6%, but doubles side effects, significantly limiting their clinical use.

[0003] Ezetimibe (EZ), the first cholesterol absorption inhibitor, primarily functions by blocking the absorption of cholesterol from food and bile by the small intestinal brush border, mediated by NPC1L1, without affecting the absorption of other lipid-soluble nutrients. It is primarily used for hypercholesterolemia. EZ treatment reduced hepatic lipid accumulation and blood cholesterol, triglycerides, and LDL-c levels, while simultaneously increasing high-density lipoprotein cholesterol (HDL-c). It had no significant effect on fat-soluble vitamins such as vitamin A, vitamin D, and vitamin E. However, because ezetimibe alone only weakly reduced plasma LDL-c levels (approximately 20%), it is often used in combination with statins in clinical trials. In four multicenter, double-blind, placebo-controlled, 12-week studies, 1187 patients with primary hypercholesterolemia were treated with 10 mg of ezetimibe alone or in combination with atorvastatin, simvastatin, pravastatin, or lovastatin. The degree of LDL-c reduction in patients receiving the combination is independent of the type and dosage of statins. The combination of ezetimibe with a minimum dose of a statin is more effective in lowering LDL-c than the use of a high-dose statin alone. Ezetimibe / simvastatin tablets are currently the only lipid-lowering combination formulation in China. They contain two lipid-lowering drugs widely used in clinical practice—simvastatin and ezetimibe—and are suitable for patients with hypercholesterolemia. Ezetimibe / simvastatin tablets have a dual mechanism of action: they inhibit cholesterol synthesis in the liver while also inhibiting cholesterol absorption in the small intestine, effectively reducing LDL-c by more than 50%.

[0004] Although the combined use of ezetimibe and statins has reduced the statin dosage and toxic side effects to a certain extent, the combination still has common side effects such as abnormal liver function tests such as elevated alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST), elevated blood creatine kinase (CK), rare elevated blood bilirubin, elevated blood uric acid, elevated gamma-glutamyl transpeptidase, elevated international normalized ratio, development of proteinuria, and weight loss.

[0005] WO2017058944A1, WO2022006327A1, and US20210163601A1 disclose methods for treating and preventing cardiovascular disease by administering an ASGR-1 antigen-binding protein, and disclose that anti-ASGR1 antibodies can reduce low-density lipoprotein cholesterol (LDL-c) and / or non-high-density lipoprotein cholesterol (Non-HDL-C) levels. However, no ASGR1 antibodies are currently approved for the treatment of hypercholesterolemia alone or other cardiovascular diseases.

[0006] [Summary of the Invention] 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, 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:

[0007] (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or an equivalent thereof, (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 10 to 12 or equivalents thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 13 to 15 or equivalents thereof, (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 16 to 18 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21 or an equivalent thereof, (d) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 22 to 24 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 25 to 27 or an equivalent thereof, (e) the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or an equivalent thereof; (f) the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31 or an equivalent thereof; (g) the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33 or an equivalent thereof; and (h) the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35 or an equivalent thereof.

[0008] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the above monoclonal antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier. In a third aspect, the present invention provides a method for lowering blood cholesterol and / or triglyceride levels 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.

[0009] In a fourth aspect, the present invention provides use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof or the above-mentioned pharmaceutical composition in the manufacture of a medicament for lowering cholesterol and / or triglyceride levels in the blood or for treating and / or preventing cardiovascular disease.

[0010] In a fifth aspect, the present invention provides the above-mentioned monoclonal antibody or antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition for use in lowering blood cholesterol and / or triglyceride levels, or for use in the treatment and / or prevention of cardiovascular disease.

[0011] In a sixth aspect, the present invention provides a nucleotide sequence encoding a monoclonal antibody or antigen-binding fragment thereof according to the present invention. In another aspect, the present invention further provides a vector comprising the nucleotide sequence. In another aspect, the present invention further provides a host cell comprising the vector. The present invention also provides a cell line 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 producing an antibody by culturing an antibody-producing cell line.

[0012] In any one of the above aspects, the monoclonal antibody is preferably a humanized antibody or a chimeric antibody. The inventors have also discovered that combined administration of an ASGR1 inhibitor with other lipid-lowering drugs results in significant synergistic lipid-lowering effects, significantly reducing both total cholesterol and triglyceride levels in the blood and / or liver.

[0013] 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 agent, and a pharmaceutically acceptable carrier. In an eighth aspect, the present invention provides a medicine chest containing a first pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a therapeutically effective amount of a second lipid-lowering drug and a pharmaceutically acceptable carrier.

[0014] In a ninth aspect, the present invention provides the use of a combination of an ASGR1 inhibitor and a second lipid-lowering drug in the manufacture of a medicament for lowering total cholesterol and / or triglyceride levels in the blood and / or liver or for treating and / or preventing cardiovascular disease.

[0015] In a tenth aspect, the present invention provides a combination of an ASGR1 inhibitor and a second lipid-lowering drug for use in lowering total cholesterol and / or triglyceride levels in the blood and / or liver or for the treatment and / or prevention of cardiovascular disease.

[0016] In an eleventh aspect, the present invention provides a method for lowering total cholesterol and / or triglyceride levels in the 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.

[0017] In any one of the above aspects, the ASGR1 inhibitor is preferably selected from an anti-ASGR1 monoclonal antibody or an antigen-binding fragment thereof, a nucleic acid encoding a nucleic acid targeting ASGR1, a nucleic acid aptamer targeting ASGR1, and combinations thereof.

[0018] In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence set forth 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, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 2. In some embodiments, the carbohydrate-binding region of ASGR1 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 3. In some embodiments, the ASGR1 monoclonal antibody binds to an epitope comprising 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 ASGR1 monoclonal antibody binds to an epitope comprising one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1. In some embodiments, the ASGR1 monoclonal antibody binds to an epitope comprising Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1.

[0019] In some embodiments, the anti-ASGR1 monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof described in the first aspect.

[0020] In any one 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 drug is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin. In some embodiments, the statin drug is atorvastatin. In some embodiments, the ACL inhibitor is bempedoic 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 (e.g., siRNA or shRNA) targeting the gene encoding PCSK9. In some embodiments, the PCSK9 inhibitor is evolocumab, alirocumab, or inclisiran.

[0021] Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0022] [Figure 1-1]Production of ASGR1 neutralizing antibodies. (a) Production steps for ASGR1 monoclonal neutralizing antibodies. Rabbits were immunized with purified ASGR1 protein as an antigen. After the first round of screening, four B cell monoclonal antibody candidates were obtained. The antibody variable region coding regions were then sequenced, cloned into antibody expression vectors, and transfected into mammalian cells. Their efficacy was confirmed by Western blotting and real-time quantitative PCR. Finally, the rabbit Fc fragment was replaced with a mouse Fc fragment. The neutralizing antibody 4B9 was selected for large-scale production and used in subsequent experiments. (b) ASGR1 protein purified from HEK293T cells was stained with Coomassie Brilliant Blue. (c) Huh7 cells were cultured with purified neutralizing antibodies for 72 hours, and LXRα protein expression was analyzed by Western blotting. (d) Primary hepatocytes were isolated from 8-week-old wild-type C57B / L6 mice. Primary hepatocytes were cultured with various concentrations of different monoclonal neutralizing antibodies for 72 hours, then harvested, lysed, and analyzed by Western blotting. (e) Primary hepatocytes were isolated from 8-week-old wild-type C57B / L6 mice. Primary hepatocytes were cultured with the ASGR1 neutralizing antibody 4B9 for 72 hours, then harvested, total RNA was extracted from the cells, and LXR target genes were analyzed by real-time quantitative PCR. [Figure 1-2] (f) Production of ASGR1 neutralizing antibodies. Huh7 cells were transfected with specific plasmids. 48 hours after transfection, the cells were harvested and lysed, and the expression of various proteins was analyzed by Western blotting. [Figure 2-1]Increased cholesterol efflux and reduced blood and liver lipids were observed with ASGR1 neutralizing antibodies. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into four groups of six mice each according to genotype, as shown in the figure. They were given a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, and 0.5% bile salts) with free access to water. Simultaneously, mice were intraperitoneally injected with a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day. After 14 days, mice were fasted for 4 hours before being euthanized. All data are presented 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 blotting analysis of liver samples. [Figure 2-2]Increased cholesterol efflux and reduced blood and liver lipids were observed with ASGR1 neutralizing antibodies. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into four groups of six mice each according to genotype, as shown in the figure. They were given a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, and 0.5% bile salts) with free access to water. Simultaneously, mice were intraperitoneally injected with a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day. After 14 days, mice were fasted for 4 hours before being euthanized. All data are presented 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. (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 (using cyclophilin as an internal standard). (c) Total cholesterol in serum. (d) Triglycerides in serum. (e) Total cholesterol in liver. (f) Triglycerides in liver. (g) Gallbladder volume. (h) Cholesterol concentration in bile. (i) Total cholesterol in bile. (j) Representative photographs 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 level. (r) Alanine transaminase in serum. (s) Aspartate transaminase in serum. [Figure 3-1]Synergistic lipid-lowering effects of the combination of ASGR1 neutralizing antibody and atorvastatin. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered atorvastatin at a dose of 30 mg / kg / day daily. After 14 days, mice were fasted for 4 hours before being euthanized. 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 blotting analysis of liver samples. [Figure 3-2]Synergistic lipid-lowering effects of the combination of ASGR1 neutralizing antibody and atorvastatin. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered atorvastatin at a dose of 30 mg / kg / day daily. After 14 days, mice were fasted for 4 hours before being euthanized. 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. (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 acids in bile, (j) total cholesterol in feces, (k) body weight, (l) daily food intake, (m) liver-to-body weight ratio, (n) blood glucose level, (o) alanine transaminase in serum, (p) aspartate transaminase in serum. [Figure 3-3]Synergistic lipid-lowering effects of the combination of ASGR1 neutralizing antibody and atorvastatin. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered atorvastatin at a dose of 30 mg / kg / day daily. After 14 days, mice were fasted for 4 hours before being euthanized. 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. (qr) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis, and genes in other pathways in mouse liver (cyclophilin used as an internal control). (s) Hematoxylin-eosin staining of liver sections. (t) Oil Red O staining of liver sections. [Figure 4-1] Synergistic effect of the neutralizing antibody 4B9 and ezetimibe. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered ezetimibe at a dose of 10 mg / kg / day daily. After 8 days, mice were fasted for 4 hours before being euthanized. 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 blotting analysis of liver samples. [Figure 4-2]Synergistic effect of the neutralizing antibody 4B9 and ezetimibe. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered ezetimibe at a dose of 10 mg / kg / day daily. After 8 days, mice were fasted for 4 hours before being euthanized. 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. (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 acids in bile, (j) body weight, (k) daily food intake, (l) liver-to-body weight ratio, (m) blood glucose level, (n) alanine transaminase in serum, (o) aspartate transaminase in serum. [Figure 4-3]Synergistic effect of the neutralizing antibody 4B9 and ezetimibe. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in the figure. They were allowed free access to water and fed a high-fat, high-cholesterol, and high-bile-salt (HF / HC / BS) diet (60% fat, 1.25% cholesterol, 0.5% bile salts). Simultaneously, mice received intraperitoneal injections of a control antibody or the ASGR1 neutralizing antibody 4B9 at a dose of 10 mg / kg / day every other day, and intragastrically administered ezetimibe at a dose of 10 mg / kg / day daily. After 8 days, mice were fasted for 4 hours before being euthanized. 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. (pq) Real-time quantitative PCR analysis of genes related to cholesterol efflux, cholesterol synthesis and absorption, lipid synthesis, bile acid synthesis, and genes in other pathways in mouse liver (cyclophilin used as an internal control). (r) Hematoxylin-eosin staining of liver sections. (s) Oil Red O staining of liver sections. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition In this disclosure, "about" refers to a numerical value within an acceptable error range for the particular numerical value, as determined by one of ordinary skill in the art, which will depend in part on the method of measurement or determination (i.e., the limitations of the measurement system). For example, "about" may mean a standard deviation of less than or more than 1, in accordance with practice in the art. Alternatively, "about" or "essentially comprising" may mean a range of up to 20%. Furthermore, the term may refer to a numerical value up to an order of magnitude or up to 5 times, particularly with respect to biological systems or processes. Unless otherwise stated, when a particular numerical value is described in this disclosure and claims, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range for the particular numerical value.

[0024] Any composition or method described herein as "comprising" one or more of the elements or steps is open-ended, meaning that the elements or steps are required, but that other elements or steps may be added within the scope of the composition or method. Any composition or method described as "comprising" one or more of the elements or steps also describes a corresponding, more limited composition or method "consisting essentially of those elements or steps," i.e., the composition or method includes the required elements or steps, and may include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method.

[0025] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity (e.g., inhibiting binding of a ligand to a receptor or inhibiting ligand-induced receptor signaling). "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding fragments of antibodies and antibody analogs, which generally contain at least a portion of the antigen-binding 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.

[0026] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies may be highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against only a single determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations which typically include multiple different antibodies directed against multiple different determinants (epitopes). The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be understood as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in the present invention may be produced by hybridoma technology or recombinant DNA technology.

[0027] Monoclonal antibodies may include "chimeric" antibodies, humanized antibodies, or fully humanized antibodies. In some embodiments, an antibody comprises part of a larger biomolecule, such as a fusion protein or antibody-drug conjugate. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent binding activity when activity is expressed on a molar basis. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity for the target.

[0028] Therefore, examples of antibody fragments used herein include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as sc-Fv, nanobodies, domain antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from antibody fragments, CAR-T, and BiTE. A "Fab fragment" is composed of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule. A "F(ab')2 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, an F(ab')2 fragment is composed of two Fab' fragments linked by disulfide bonds between the two heavy chains. The "Fv region" comprises the variable regions of both the heavy and light chains, but does not comprise the constant regions. A "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0029] "Fc" or "Fc fragment" or "Fc region" refers to the polypeptide of the constant region of an antibody minus the first constant region immunoglobulin domain, and optionally minus part of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and 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 to, for example, alter binding to one or more FcγR or FcRn receptors.

[0030] As used herein, the term "epitope" refers to the 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 the relevant conformation. For example, in the case of ASGR1, a conformational epitope is an epitope composed of multiple amino acid residues that are not contiguous within the extracellular domain of ASGR1. In some embodiments, an epitope may be a linear epitope, i.e., an epitope comprising a sequence of amino acid residues that are contiguous within the primary structure within the extracellular domain of ASGR1. Methods for determining the exact sequence and / or specific amino acid residues of an ASGR1 epitope are known in the literature and include competition with peptides of 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-crystallization techniques.

[0031] As used herein, the term "humanized antibody" refers to an antibody comprising CDRs of an antibody derived from a mammal other than a human and framework regions (FRs) and constant regions of a human antibody. As used herein, the term "chimeric antibody" is intended 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 in accordance with the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is from a human source.

[0032] An "equivalent" of an antibody or polypeptide refers to an antibody or polypeptide having a given 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, compared to a reference antibody or polypeptide, the equivalent has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof. In some aspects, an antibody or polypeptide equivalent retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0033] As used herein, a "variant" of a sequence refers to a sequence that differs from the sequence shown at one or more amino acid residue positions, but which retains the biological activity of the resulting molecule.

[0034] As used herein, "percent identity" between two sequences refers to a function of the number of identical positions shared by the sequences, taking into account the number of gaps that must be introduced to optimally align the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0035] "Conservative substitution" refers to an amino acid substitution known to those skilled in the art, and such substitutions typically do not alter the biological activity of the resulting molecule. It is generally recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide will generally not alter or substantially alter the biological activity. "Unaltered or substantially unchanged" refers to a difference of less 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% in one or more aspects compared to a comparator, when measured by the same or similar method.

[0036] In general, HCDR3 and LCDR3 are believed to play a more important role in antigen recognition than other CDRs. Therefore, in the present disclosure, conservative substitutions are preferably made for 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, (1) substitutions that reduce susceptibility to proteolysis, (2) substitutions that reduce susceptibility to oxidation, (3) substitutions that change binding affinity for forming protein complexes, and (4) substitutions that provide or modify other physicochemical or functional properties of these analogs. Analogs may include various mutations of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in a portion of the polypeptide outside the region forming intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid substitutions do not tend to disrupt helices present in the parent sequence or to characterize other secondary structure types that disrupt the parent sequence).

[0037] The binding domain of the monoclonal antibody or antigen-binding fragment thereof of the present disclosure is believed to be capable of carrying a signal peptide, which is typically located at the N-terminus of a secretory protein and generally consists of 15 to 30 amino acids. After synthesis, the signal peptide sequence is recognized by the signal recognition particle (SRP), halting or slowing down protein synthesis. The SRP then transports the ribosome to the endoplasmic reticulum, where protein synthesis resumes. Upon induction of the signal peptide, the newly synthesized protein enters the lumen of the endoplasmic reticulum, while the signal peptide sequence is cleaved by the action of a signal peptidase. If a stop transition sequence is present at the C-terminus of the nascent peptide chain, it may not be cleaved by a signal peptidase; for example, ovalbumin contains an internal signal peptide. Neither its precursor nor its mature form is cleaved by a signal peptidase.

[0038] "Specific" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, refers to a binding reaction that determines whether a protein is present in a heterogeneous population of proteins and / or other biological factors. Thus, under specific conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind significantly to other proteins present in a sample. "Specific binding" refers to the ability of a monoclonal antibody or antigen-binding fragment thereof of the present invention to specifically interact with at least 2, 3, 4, 5, 6, 7, 8, or more amino acids of each human target molecule. The "specific binding" of an antibody is characterized primarily by two parameters: a qualitative parameter (the binding epitope or the location to which the antibody binds) and a quantitative parameter (binding affinity or binding strength). The binding epitope of an antibody can be determined by FACS, peptide spot epitope mapping, mass spectrometry, or peptide ELISA. The binding strength of an antibody to a specific epitope can be determined by Biacore and / or ELISA. The signal-to-noise ratio is often calculated as a representative measure of binding specificity. In such a signal-to-noise ratio, the signal represents the strength of binding of the antibody to the target epitope, and the noise represents the strength of binding of the antibody to other non-target epitopes. Preferably, the evaluated antibody can be considered to specifically bind to the target epitope, i.e., "specifically bind," 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 when it binds to the antigen with high binding affinity, as determined by the affinity constant (KD) value. In some embodiments, the affinity constant KD is 10 -9 The term "KD" as used herein refers to the affinity constant of a particular antibody-antigen interaction.

[0039] As used herein, the term "patient" or "subject" refers to any organism to which a provided antibody, antigen-binding fragment thereof, or pharmaceutical composition is administered for experimental, diagnostic, prophylactic, 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 is suffering from or susceptible to one or more diseases or conditions. A patient may have been diagnosed with one or more diseases or conditions if they may exhibit one or more symptoms of the disease or condition. In some embodiments, a patient is undergoing or has undergone some therapy to diagnose and / or treat such disease, disease, or condition.

[0040] As used herein, the term "treatment" refers to therapeutic and preventative measures that inhibit or delay the occurrence of undesirable physiological changes or the onset or progression of a disease state in a subject. Beneficial or desirable clinical effects include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, alleviation or mitigation of the disease state, and partial or complete cure of the disease, regardless of whether the effect is detectable. "Treatment" can also refer to prolonging survival compared to the absence of treatment. Subjects in need of treatment include those already suffering from the disease or condition, as well as those at risk of contracting the disease or condition, or those in whom the disease or condition is to be prevented.

[0041] "Administration" and "treatment," when referring to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, treatment subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with a cell and contacting a reagent with a fluid, where the fluid contacts the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatments of cells, for example, with a reagent, diagnostic agent, binding composition, or other cells.

[0042] 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 ASGR1 of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective in preventing or ameliorating the disease or condition being treated. A therapeutically effective amount also refers to an amount of the compound sufficient to cause an improvement in symptoms, such as treatment, cure, prevention, or amelioration of the associated condition, or to increase the rate of treatment, cure, prevention, or amelioration of the condition. When an active ingredient is administered to an individual alone, a therapeutically effective amount refers to that ingredient alone. When administered in combination, a therapeutically effective amount refers to the combined amounts of the active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, sequentially, or simultaneously. A therapeutically effective amount generally alleviates symptoms by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0043] As used herein, "pharmaceutically acceptable carrier" includes substances that, when combined with an active ingredient of a composition, allow the ingredient to retain its biological activity without eliciting a destructive reaction in the subject's immune system. These carriers may include stabilizers, preservatives, salts, or sugar complexes or crystals, etc. "Pharmaceutically acceptable" refers to molecules and ingredients that do not elicit allergic or similar undesirable reactions when administered to the human body.

[0044] 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 endocytosis of ASGR1.

[0045] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or their equivalents.

[0046] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 10 to 12 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 13 to 15 or their equivalents.

[0047] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 16 to 18 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21 or their equivalents.

[0048] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 22 to 24 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 25 to 27 or their equivalents.

[0049] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or an equivalent thereof.

[0050] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31 or an equivalent thereof.

[0051] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33 or an equivalent thereof.

[0052] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35 or an equivalent 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, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or their respective mutants, wherein the mutants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or their respective mutants, wherein the mutants have a single substitution, deletion, or insertion.

[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, each comprising the sequences set forth in SEQ ID NOs: 10 to 12 or their respective mutants, the mutants having a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 13 to 15 or their respective mutants, the mutants having a single substitution, deletion, or insertion.

[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, each comprising the sequences set forth in SEQ ID NOs: 16 to 18 or their respective mutants, wherein the mutants have a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21 or their respective mutants, wherein the mutants have a single substitution, deletion, or insertion.

[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 LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 22 to 24 or their respective mutants, the mutants having a single substitution, deletion, or insertion, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 25 to 27 or their respective mutants, the mutants having a single substitution, deletion, or insertion.

[0057] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 28, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 29.

[0058] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 30, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 31.

[0059] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 32, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 33.

[0060] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 34, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35 or a variant thereof, wherein the variant has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 35.

[0061] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences shown in SEQ ID NOs: 4 to 6, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences shown in SEQ ID NOs: 7 to 9.

[0062] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences shown in SEQ ID NOs: 10 to 12, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences shown in SEQ ID NOs: 13 to 15.

[0063] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 16 to 18, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21.

[0064] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences shown in SEQ ID NOs: 22 to 24, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences shown in SEQ ID NOs: 25 to 27.

[0065] In any of the above embodiments, the LCDR1 to LCDR3 and HCDR1 to HCDR3 are defined according to any one of the definition schemes of IMGT, Kabat, Chothia, Contact, and Martin. In some embodiments, the LCDR1 to LCDR3 and HCDR1 to HCDR3 are defined according to the definition scheme of IMGT.

[0066] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29.

[0067] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31.

[0068] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33.

[0069] In some embodiments, the ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35.

[0070] In some embodiments, the ASGR1 monoclonal antibody is of the IgG type, for example, IgG1, IgG2, IgG3, or IgG4 type. In some embodiments, the ASGR1 monoclonal antibody is of the IgG1 type.

[0071] In some embodiments, the substitutions described herein are conservative substitutions. A "conservative (amino acid) substitution" refers to the replacement of an amino acid residue with an amino acid having a similar side chain. Families of amino acid residues having 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), nonpolar 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 replaced with another amino acid residue from the same side chain family. In another embodiment, the string of amino acids may be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0072] It should also be understood by those skilled in the art that the antibodies disclosed herein may be modified so that their amino acid sequence differs from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar to the starting sequence, e.g., have a certain percent identity, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% identity to the starting sequence, or a range between any two of these values.

[0073] In some embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may include flexible linker sequences or may be modified to add a functional moiety (e.g., polyethylene glycol (PEG), a drug, a toxin, or a label).

[0074] The antibodies, variants, or derivatives of the present disclosure include modified derivatives in which any type of molecule is covalently attached to the antibody, provided that the covalent attachment does not interfere with the binding of the antibody to the epitope. For example, the antibody may be modified by, but is not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like. Any of a number of chemical modifications can be performed using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody may contain one or more non-classical amino acids.

[0075] In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, drug, or PEG. The antibody may be conjugated or fused to a therapeutic agent, including a detectable label (e.g., a radioactive label), an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound-enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0076] 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 arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0077] Additionally, fluorescent metals (e.g., 152 Antibodies can also be detectably labeled using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for attaching various moieties to antibodies are well known.

[0078] Humanized and chimeric antibodies In any one aspect of the invention, the monoclonal antibody is preferably a humanized or chimeric antibody.

[0079] Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to a desired antigen and have one or more complementarity-determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Typically, framework residues in the human framework regions are replaced with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison, thereby identifying unusual framework residues at specific positions. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting, veneering or resurfacing, and chain shuffling.

[0080] Completely human antibodies are particularly ideal for treating human patients. Human antibodies can be made by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0081] Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen (e.g., all or part of a desired target polypeptide). Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutation. Therefore, using this technology, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies.

[0082] Fully human antibodies that recognize a selected epitope can also be generated using a technique called "guided selection," in which a non-human monoclonal antibody, such as a murine antibody, is selected to guide the selection of fully human antibodies that recognize the same epitope.

[0083] DNA encoding the desired monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a mouse antibody). Isolated and subcloned hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins. More specifically, the isolated DNA can be used to clone constant and variable region sequences used in antibody production. Essentially, this requires extracting RNA from the selected cells, converting it to cDNA, and amplifying it by PCR using Ig-specific primers. As described herein, transformed cells expressing the desired antibody can be grown in relatively large quantities to provide clinical and commercial supplies of immunoglobulins.

[0084] Furthermore, conventional recombinant DNA techniques can be used to insert one or more CDRs of an antigen-binding polypeptide of the present disclosure into framework regions, e.g., human framework regions, to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions. Preferably, the polynucleotide generated from the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions may be made within the framework regions, and preferably, the amino acid substitutions increase binding of the antibody to its antigen. Furthermore, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds to generate antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to polynucleotides are encompassed by this disclosure and within the skill of the art.

[0085] Furthermore, techniques developed to produce "chimeric antibodies" by splicing the genes for a mouse antibody molecule with the appropriate antigen specificity and 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 those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

[0086] Another efficient method for producing recombinant antibodies is to generate primate antibodies containing monkey variable domains and human constant sequences. Thus, 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:

[0087] (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or an equivalent thereof, (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 10 to 12 or equivalents thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 13 to 15 or equivalents thereof, (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 16 to 18 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21 or their equivalents, and (d) The light chain variable region comprises LCDR1, LCDR2, and LCDR3, each of which comprises the sequences set forth in SEQ ID NOs: 22 to 24 or equivalents thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each of which comprises the sequences set forth in SEQ ID NOs: 25 to 27 or equivalents thereof.

[0088] In some embodiments, the humanized ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or their equivalents, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or their equivalents.

[0089] Accordingly, the present invention provides a chimeric ASGR1 monoclonal antibody, which comprises a light chain variable region, a heavy chain variable region and a human constant region, and is selected from any one of the following antibodies:

[0090] (e) the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or an equivalent thereof; (f) the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31 or an equivalent thereof; (g) the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33 or an equivalent thereof; and (h) the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35 or an equivalent thereof.

[0091] In some embodiments, the chimeric ASGR1 monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or an equivalent thereof.

[0092] Either of the above chimeric ASGR1 monoclonal antibodies or humanized ASGR1 monoclonal antibodies may comprise a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is, for example, a human IgG1 heavy chain constant region shown in SEQ ID NO: 37, and the light chain constant region is, for example, a human Igκ light chain constant region shown in SEQ ID NO: 38.

[0093] ASGR1 inhibitors Asialoglycoprotein receptor 1 (ASGR1) is primarily found in hepatocytes. ASGR1 is a single transmembrane protein containing a cytoplasmic tail, a transmembrane segment, a hinge region, and a carbohydrate-binding domain. Serum-resident glycoproteins are processed by neuraminidase to generate desialylated glycoproteins, which then bind to ASGR1 on the plasma membrane to initiate endocytosis and enter the endosomal pathway. In the acidic environment of the endosome, the receptor dissociates from the ligand, and these proteins are transported to lysosomes for degradation, while ASGR1 is recycled to the cell surface for reuse.

[0094] Humanized ASGR1 contains 291 amino acids with a molecular weight of 33,186 Da. The amino acid sequence is shown in SEQ ID NO: 1 (UniProtKB / Swiss-Prot: P07306.2). After binding to its ligand, asialoglycoprotein, in the blood, ASGR1 enters the lysosome via clathrin-mediated endocytosis and is ultimately degraded. Human ASGR1 has a short cytoplasmic tail (aa 1-40), a transmembrane domain (aa 40-60), and an extracellular domain (e.g., aa 62-141) and a carbohydrate-binding domain (aa 142-291, SEQ ID NO: 2). The NCBI gene ID for the humanized ASGR1 gene is 432, and the coding sequence is shown in SEQ ID NO: 36 (NCBI Reference Sequence: NM_001671.5).

[0095] As used herein, the term "ASGR1 inhibitor" refers to a substance that can reduce or inhibit the binding of ASGR1 to its natural ligand and / or the endocytosis of ASGR1, and a substance that can reduce or inhibit the expression of the gene encoding ASGR1.

[0096] In some embodiments, ASGR1 inhibitors described herein may include (1) inhibitors that bind to ASGR1, (2) inhibitors that bind to a ligand of ASGR1 (e.g., asialoglycoprotein), (3) inhibitors that reduce or inhibit the binding of ASGR1 to its ligand, (4) inhibitors that reduce or inhibit the endocytosis of ASGR1, (5) inhibitors that reduce the protein level of ASGR1, (6) inhibitors that reduce or inhibit the protein activity of ASGR1, and (7) inhibitors that reduce or inhibit the expression of the gene encoding ASGR1.

[0097] In some embodiments, the ASGR1 inhibitor of the present invention may be a small molecule compound, a nucleic acid targeting the gene encoding ASGR1, a nucleic acid aptamer targeting ASGR1, an anti-ASGR1 antibody, or a combination thereof.

[0098] In some embodiments, the ASGR1 inhibitor of the present invention is an ASGR1 antibody, hi some embodiments, the ASGR1 antibody is an ASGR1 monoclonal antibody or an antigen-binding fragment thereof.

[0099] In some embodiments, the ASGR1 monoclonal antibody binds to human ASGR1 comprising the sequence set forth in SEQ ID NO: 1 and inhibits the binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or the endocytosis of human ASGR1.

[0100] In some embodiments, the ASGR1 monoclonal antibody binds to the carbohydrate-binding region of ASGR1 and inhibits binding of human ASGR1 to its natural ligand (e.g., asialoglycoprotein) and / or endocytosis of human ASGR1. In some embodiments, the carbohydrate-binding region of ASGR1 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 2. In some embodiments, the carbohydrate-binding region of ASGR1 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 3.

[0101] In some embodiments, the epitope that binds to the ASGR1 monoclonal antibody 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 that binds to the ASGR1 monoclonal antibody comprises one or more of Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1. In some embodiments, the epitope that binds to the ASGR1 monoclonal antibody comprises Q240, D242, W244, E253, N265, and D266 in SEQ ID NO: 1.

[0102] 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.

[0103] In some embodiments, the ASGR1 monoclonal antibody is an antibody described in the "ASGR1 Monoclonal Antibody" section above, 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:

[0104] (a) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 4 to 6 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 7 to 9 or an equivalent thereof, (b) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 10 to 12 or equivalents thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 13 to 15 or equivalents thereof, (c) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 16 to 18 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 19 to 21 or an equivalent thereof, (d) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each comprising the sequences set forth in SEQ ID NOs: 22 to 24 or an equivalent thereof, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each comprising the sequences set forth in SEQ ID NOs: 25 to 27 or an equivalent thereof, (e) the light chain variable region comprises the sequence set forth in SEQ ID NO: 28 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29 or an equivalent thereof; (f) the light chain variable region comprises the sequence set forth in SEQ ID NO: 30 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31 or an equivalent thereof; (g) the light chain variable region comprises the sequence set forth in SEQ ID NO: 32 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33 or an equivalent thereof; and (h) the light chain variable region comprises the sequence set forth in SEQ ID NO: 34 or an equivalent thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 35 or an equivalent thereof.

[0105] Other exemplary ASGR1 monoclonal antibodies include those described in WO2017058944A1, WO2022006327A1, or US20210130473A1, the contents of which are incorporated herein by reference in their entireties.

[0106] In some embodiments, the ASGR1 inhibitor of the present invention is a nucleic acid that is DNA or mRNA that targets ASGR1 and inhibits the expression of ASGR1. In some embodiments, the nucleic acid is selected from an antisense oligonucleotide (ASO), an siRNA, an shRNA, and a gRNA.

[0107] In some embodiments, the nucleic acid is an ASO, and the ASO can be modified on the backbone, glycosyl, or base to resist degradation in vivo. Suitable modifications include, but are not limited to, phosphorothioate (PSP), phosphorodiamidate morpholino oligomer (PMO), 2'-O-methoxyethyl (2'-MOE), and 5-methylcytosine (5mC).

[0108] In some embodiments, the nucleic acid is an siRNA or shRNA, and the siRNA can be delivered by a suitable vector, such as GalNAc, LNP, or AAV.

[0109] In some embodiments, the nucleic acid targets the sequence set forth in SEQ ID NO: 36 and inhibits expression of the gene encoding ASGR1. In some embodiments, the nucleic acid is a gRNA, and the gRNA and a CRISPR / Cas enzyme (e.g., CRISPR / Cas9) form a gene editing system to inhibit or block expression of the gene encoding ASGR1.

[0110] Given that the gene encoding ASGR1 is known, one skilled in the art can obtain and screen the above nucleic acid sequences using conventional techniques. In some embodiments, the nucleic acid (e.g., ASO, siRNA, shRNA, or gRNA) targets the sequence set forth in SEQ ID NO: 36 and inhibits expression of the gene encoding ASGR1.

[0111] In some embodiments, the ASGR1 inhibitor of the present invention is a nucleic acid aptamer that targets 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.

[0112] Treatment Methods and Uses One aspect of the present invention provides a method for lowering blood cholesterol levels, comprising administering to a subject a therapeutically effective amount of any one of the above monoclonal antibodies or antigen-binding fragments thereof.

[0113] Another aspect of the present invention provides a method for lowering blood triglyceride levels, comprising administering to a subject a therapeutically effective amount of any one of the above monoclonal antibodies or antigen-binding fragments thereof.

[0114] A further aspect of the present invention provides a method for treating and / or preventing cardiovascular diseases such as atherosclerosis, stroke, myocardial infarction or coronary artery disease, comprising administering to a subject a therapeutically effective amount of any one of the above-mentioned monoclonal antibodies or antigen-binding fragments thereof.

[0115] Thus, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the manufacture of a medicament for lowering blood cholesterol levels.

[0116] Thus, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the manufacture of a medicament for lowering blood triglyceride levels.

[0117] Thus, another aspect of the present invention provides the use of any one of the above monoclonal antibodies or antigen-binding fragments thereof in the manufacture of a medicament for treating and / or preventing cardiovascular disease.

[0118] Thus, another aspect of the present invention provides any one of the above monoclonal antibodies or antigen-binding fragments thereof for use in lowering blood cholesterol levels.

[0119] Thus, another aspect of the present invention provides any one of the above monoclonal antibodies or antigen-binding fragments thereof for use in lowering blood triglyceride levels.

[0120] Thus, another aspect of the present invention provides any one of the above monoclonal antibodies or antigen-binding fragments thereof for use in the treatment and / or prevention of cardiovascular disease. In another aspect, the inventors have also found that combined administration of an ASGR1 inhibitor and a lipid-lowering drug results in a significant synergistic lipid-lowering effect, significantly reducing both total cholesterol and triglyceride levels in serum and liver.

[0121] In some embodiments, the present invention provides a method for lowering total cholesterol levels in the blood and / or liver, 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.

[0122] In some embodiments, the present invention provides a method for lowering triglyceride levels in the blood and / or liver, 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.

[0123] In some embodiments, the present invention provides a method for treating and / or preventing cardiovascular disease, 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.

[0124] Thus, in some embodiments, the present invention provides the use of a combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug in the manufacture of a medicament for lowering total cholesterol levels in the blood and / or liver.

[0125] Thus, in some embodiments, the present invention provides the use of a combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug in the manufacture of a medicament for lowering triglyceride levels in the blood and / or liver.

[0126] Thus, in some embodiments, the present invention provides the use of a combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug in the manufacture of a medicament for treating and / or preventing cardiovascular disease.

[0127] Thus, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug for use in lowering total cholesterol levels in the blood and / or liver.

[0128] Thus, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering drug for use in lowering triglyceride levels in the blood and / or liver.

[0129] Thus, in some embodiments, the present invention provides a combination of an ASGR1 inhibitor described herein and a second lipid-lowering agent for use in the treatment and / or prevention of cardiovascular disease.

[0130] 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. In any of the above embodiments, the second lipid-lowering drug can be administered simultaneously with or sequentially to the ASGR1 inhibitor. For example, the second lipid-lowering drug can be administered to a subject before or after the ASGR1 inhibitor.

[0131] In some embodiments, the HMGCR inhibitor is a statin drug. In some embodiments, the statin drug is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin. In some embodiments, the statin drug is atorvastatin.

[0132] In some embodiments, the ACL inhibitor is bempedoic acid. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is a PCSK9 antibody or a nucleic acid (e.g., siRNA or shRNA) targeting a gene encoding PCSK9. In some embodiments, the PCSK9 antibody is evolocumab, alirocumab, or inclisiran. Other exemplary PCSK9 inhibitors include the PCSK9 inhibitors described in WO2017220701A1, WO2012088313A1, WO2009026558A1, WO2009102427A2, or WO2017035340A1, the contents of which are incorporated herein by reference in their entireties.

[0133] Suitable routes of administration include parenteral administration (e.g., intramuscular, intravenous, or subcutaneous) and oral administration. Other conventional methods of administration include intratracheal administration, ingestion, inhalation, topical administration, or transdermal, subcutaneous, intraperitoneal, or intraarterial injection.

[0134] The appropriate dosage is determined by the clinician based on parameters or factors known, suspected, or expected to affect treatment in the art. Usually, the dosage starts at a dose slightly lower than the optimal dose and is then increased by small increments until the desired or optimal effect, taking into account side effects, is achieved. Important monitoring indicators include measurements of inflammatory symptoms or inflammatory cytokine production.

[0135] Pharmaceutical Composition One aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above monoclonal antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier.

[0136] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor described herein, a therapeutically effective amount of a second lipid-lowering agent, and a pharmaceutically acceptable carrier. In any one of the above aspects, the pharmaceutical composition is used to lower total cholesterol levels in the blood and / or liver. In some embodiments, the pharmaceutical composition is used to lower 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.

[0137] Another aspect of the present invention provides a medicine chest containing a first pharmaceutical composition comprising a therapeutically effective amount of an ASGR1 inhibitor described herein and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a therapeutically effective amount of a second lipid-lowering drug and a pharmaceutically acceptable carrier.

[0138] In any one 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 drug. In some embodiments, the statin drug is selected from lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, and pitavastatin. In some embodiments, the statin drug is atorvastatin. In some embodiments, the ACL inhibitor is bempedoic acid. In some embodiments, the NPC1L1 inhibitor is ezetimibe. In some embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody or a nucleic acid (e.g., siRNA or shRNA) targeting the gene encoding PCSK9. In some embodiments, the PCSK9 antibody is evolocumab, alirocumab, or inclisiran.

[0139] To prepare a pharmaceutical composition or a sterile composition, the drug is mixed with a pharmaceutically acceptable carrier or excipient. For example, a formulation in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension can be prepared by mixing with a physiologically acceptable carrier, excipient, or stabilizer. Pharmaceutically acceptable carriers are well known in the art. Methods for preparing aqueous compositions containing active ingredients are known in the art. Typically, these compositions are prepared as injections or sprays, such as liquid solutions or suspensions, and can also be prepared as solid forms suitable for formulation into solutions or suspensions before injection or spraying.

[0140] Sequence Listing

[0141] [Table 1] JPEG0007792564000002.jpg229170JPEG0007792564000003.jpg225170JPEG0007792564000004.jpg230170

[0142] Example Materials and Methods mouse CRISPR / Cas9-mediated Asgr1 whole-body knockout mice were constructed by Chengdu GemPharmatech Co., Ltd., and sgRNA-mediated Cas9 endonuclease cleaved between the second and third introns and between the eighth and ninth introns. Partial deletion of exons between the third and eighth exons was achieved by homologous recombination, resulting in Asgr1 whole-body knockout mice.

[0143] Wild-type littermates were used as controls in the experiments. All mice were housed in specific pathogen-free (SPF) animal rooms and maintained under a 12-hour light / 12-hour dark regime. Eight-week-old male or female mice were treated with a high-fat and high-cholesterol cholate diet (Research Diets, D12109C) for the duration required for the experiment. For treatments involving antibody neutralization or antibody in combination with statins or ezetimibe, mice were given the specified amount of antibody by intraperitoneal or intragastric injection. All animals were fasted for 4 hours before being euthanized. All animal experiments strictly adhered to the ethical review guidelines for laboratory animal welfare at Wuhan University, China.

[0144] Materials and plasmids Lovastatin (purity ≥98%, HPLC) was purchased from Shanghai Pharma Valley. Sodium mevalonate (#4667), anti-FLAG M2 beads (#A2220), anti-MYC beads (E6654), fetuin A (SRP6217), D-galactose (G5388), N-acetyl-D-galactosamine (A-2795), phenylmethanesulfonyl fluoride (PMSF, #P7626), Protail (#P8340), and β-mercaptoethanol (#M3148) were all purchased from Sigma. Dil (1,1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine perchlorate)-LDL (#20614ES76) was purchased from Shanghai YEASEN. Lipofectamine RNAiMAX (#13778150) was purchased from Thermo Fisher Scientific. 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 (Linear polyethylenimine, #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 Sangon. Phosphatase inhibitor (P1082) was purchased from Beyotime. Fetal bovine serum (FBS), a cell culture medium, was purchased from Life Technology.Taq was purchased from TIANGEN. KOD Hot Start DNA polymerase (#KOD-401, TOYOBO) was purchased from Takara. RNA duplex was synthesized from Guangzhou RiboBio. Q-PCR 2x MIX was purchased from Mona. Total cholesterol (TC) kit, total triglyceride (TG) kit, and bile acid kit were all purchased from Nanjing Kehua Bio-engineering. NEFA kit (294-63601) and Phospholipid kit (292-63901) were all purchased from WAKO. ALT, AST, and AKP kits were all purchased from Nanjing Jiancheng Bioengineering. Blood glucose test strips and blood glucose meters were purchased from OneTouch. Lipoprotein-deficient serum (d>1.215 g / mL), i.e., LPPS, was prepared from newborn calf serum by ultracentrifugation.

[0145] The following plasmids were constructed using standard molecular cloning techniques: Human and mouse Asgr1, Asgr2, Lxrα, and Lxrβ gene fragments were derived from cDNA reverse-transcribed from RNA of Huh7 and mouse liver tissues. Human BARD1 gene fragments were amplified from Huh7 cells 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 mutagenesis.

[0146] Huh7 and HEK293T cells were cultured in monolayer at 37°C in a 5% CO environment. Cells were maintained in medium A (DMEM containing 100 units / mL penicillin and 100 mg / mL streptomycin) supplemented with 10% fetal bovine serum (FBS). Cholesterol-deficient medium B was prepared by adding 5% lipoprotein-depleted serum (LPPS), 1 μM lovastatin, and 10 μM mevalonate to medium A. Primary mouse hepatocytes were cultured in medium D (M199) supplemented with 5% FBS, 100 units / mL penicillin, and 100 mg / mL streptomycin.

[0147] Western blotting Harvested cells or tissues were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. RIPA lysis buffer contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2, 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, and 1 mM DTT. Protein concentrations of the lysates were measured using the BCA method (Thermo Fisher Scientific). Protein samples were incubated with membrane lysis buffer (62.5 mM Tris-HCl (pH 6.8), 15% SDS, 8 M uronium, 10% glycerol, and 100 mM DTT) and 4x 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 min. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membranes, and blocked for 1 h with TBS containing 0.075% Tween 20 and 5% skim milk (or 3% BSA for phosphorylation experiments), i.e., TBST. The membranes were incubated overnight with the indicated primary antibodies at 4°C and then washed three times with TBST. Finally, proteins were detected with Pierce ECL Plus Western blotting substrate (Thermo Fisher Scientific).

[0148] Primary antibodies used in the experiment 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 produced and purified from hybridoma cell lines (ATCC) 9E10 and A9, respectively. Anti-LDLR and anti-H2 (HMGCR) antisera and affinity-purified antibodies were obtained by selecting soluble segments and immunizing rabbits. Anti-EGFP was obtained by expressing purified 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.

[0149] Blood and liver chemistry analysis Blood was collected from the eyeballs to measure total cholesterol and triglyceride levels, and serum was obtained. Livers were homogenized, the supernatant collected, and lipids extracted to obtain total hepatic cholesterol. Liver triglyceride (TG) and total cholesterol (TC) levels were measured using a detection kit (Kehua, China). Phospholipid levels were measured using a kit (Phospholipid, WAKO, Japan). Serum ALT and AST levels were detected using kits from Nanjing Jiancheng Bioengineering (China).

[0150] Results and Analysis Example 1: The ASGR1 neutralizing antibody 4B9 exhibited therapeutic effects of promoting the excretion of cholesterol into bile and feces and lowering lipids.

[0151] ASGR1 can bind to a large number of desialylated glycoproteins. To better simulate the ameliorative effects of Asgr1 knockout on high-fat, high-cholesterol-induced metabolic syndrome, we purified ASGR1 protein from HEK293T cells and immunized rabbits to obtain neutralizing antibodies that inhibit ASGR1 function. The specific process is shown in Figure 1a: ASGR1 monoclonal neutralizing antibody production steps. Rabbits were immunized with purified ASGR1 protein as an antigen, and after the first round of screening, four B cell monoclonal antibody strains were selected as candidates. The antibody variable region coding regions were then sequenced, cloned into antibody expression vectors, transfected into mammalian cells, and the proteins were purified. The efficacy was confirmed by Western blotting and real-time quantitative PCR. Finally, the rabbit Fc fragment was replaced with a mouse Fc fragment. The most effective neutralizing antibody, 4B9, was selected for large-scale production and used in subsequent experiments. Experimental results demonstrated that 4B9 has potential lipid-lowering effects. 4B9 and the other three ASGR1 monoclonal neutralizing antibodies all significantly increased LXRα protein stability (Figures 1c and 1d), and cholesterol efflux-related genes were significantly upregulated (Figure 1e). Further analysis revealed that 4B9 primarily recognizes sequences between 182 and 274 aaa (Figure 1f).

[0152] Furthermore, we investigated whether 4B9 could fully mimic the hyperlipidemia and hypercholesterolemia caused by Asgr1 deletion. Eight-week-old Asgr1 knockout mice and wild-type littermates were selected and randomly divided into four groups of six mice each according to genotype, as shown in Figure 2. Mice were allowed free access to water and fed a high-fat, high-cholesterol, and bile salt diet (60% fat, 1.25% cholesterol, 0.5% bile salts, HF / HC / BS). Simultaneously, mice were intraperitoneally injected with a control antibody or the ASGR1-neutralizing antibody 4B9 at a dose of 10 mg / kg every other day. After 14 days, the mice were fasted for 4 hours and then euthanized. Experimental results showed that wild-type mice injected with 4B9 showed results consistent with Asgr1 deletion, i.e., cholesterol efflux-related proteins such as LXR, ABCG8, ABCA1, and CYP7A1 were significantly increased, and p-ACC was significantly upregulated. Lipid synthesis-related proteins SREBP1 and FASN were significantly decreased, while LDLR was unaffected. BARD1 was significantly decreased, and there was no effect of 4B9 injection in Asgr1 knockout mice, indicating that 4B9 specifically targets ASGR1 (Figure 2a). Real-time quantitative PCR also showed results consistent with changes in protein levels (Figure 2b). Biochemical parameters showed that compared with the control group, mice injected with the 4B9 antibody showed significant decreases in serum total cholesterol and triglycerides (Figure 2c, d), liver total cholesterol and triglycerides (Figure 2e, f), gallbladder volume (Figure 2g, j), bile total cholesterol concentration and total amount (Figure 2h, i), fecal total cholesterol (Figure 2k), and bile total bile acid concentration and total amount (Figure 2l, m). Meanwhile, there were no significant changes in body weight, daily food intake, liver-to-body weight ratio, blood glucose, alanine aminotransferase, and aspartate aminotransferase (Figure 2n-s). Based on the above results, the ASGR1 neutralizing antibody 4B9 exhibited highly significant lipid-lowering effects and could significantly alleviate metabolic syndrome induced by a high-fat, high-cholesterol diet.

[0153] Example 2: The combined use of antibody 4B9 and atorvastatin showed a synergistic lipid-lowering effect. Previous experimental results showed that 4B9 significantly improved the metabolic syndrome phenotype induced by a high-fat, high-cholesterol diet (Figure 2). The inventors further investigated whether the combination of 4B9 and atorvastatin had a synergistic lipid-lowering effect. Eight-week-old Asgr1 knockout mice and wild-type littermate mice were randomly divided into eight groups of six mice each according to genotype, as shown in Figure 3. Mice were allowed free access to water and were fed a high-fat, high-cholesterol, and bile salt diet (60% fat, 1.25% cholesterol, and 0.5% bile salts, HF / HC / BS).

[0154] Mice were simultaneously injected intraperitoneally with a control antibody or the ASGR1-neutralizing antibody 4B9 at a dose of 10 mg / kg every other day, and intragastrically administered atorvastatin at a dose of 30 mg / kg / day. After 14 days, the mice were fasted for 4 hours and then euthanized. Experimental results showed that 4B9 alone increased the levels of cholesterol efflux-related genes and proteins and inhibited the levels of lipid synthesis-related genes and proteins, without affecting the levels of cholesterol synthesis or absorption-related genes or proteins. However, the combination of atorvastatin and 4B9 did not affect the stimulatory effect of 4B9 on the expression of cholesterol efflux-related genes and proteins, such as LXR, ABCG8, ABCA1, and CYP7A1, but more significantly inhibited the expression of lipid synthesis-related genes and proteins, such as SREBP1 and FASN (Figures 3a, 3q-r). While 4B9 or atorvastatin alone effectively reduced serum and liver total cholesterol and triglyceride levels, the combination of 4B9 and atorvastatin showed a more significant lipid-lowering effect (Figures 3b-e, 3s-t). While 4B9 alone increased total cholesterol and bile acids in bile, atorvastatin had little effect on these levels (Figures 3g, i). While 4B9 alone significantly increased fecal total cholesterol efflux, atorvastatin had no effect on fecal cholesterol (Figure 3j). There were no significant effects of 4B9 or atorvastatin alone or in combination on mouse body weight, daily food intake, liver-to-body weight ratio, blood glucose, or alanine aminotransferase and aspartate aminotransferase activity in the eight groups (Figures 3k-p). In summary, the combination of 4B9 and atorvastatin can achieve a better lipid-lowering effect than either of them used alone.

[0155] Example 3: The combination of antibody 4B9 and ezetimibe showed synergistic lipid-lowering effects. 4B9 was confirmed to have a certain lipid-lowering effect in mice (Figure 2). Therefore, we predicted that the combined use of 4B9 and EZ would further enhance the lipid-lowering effect. To test this hypothesis, we designed an experiment as shown in Figure 4. Mice were randomly divided into eight groups (n = 6) according to genotype and fed a HF / HC / BS diet. EZ was administered intragastrically at 10 mg / kg daily and 4B9 (10 mg / kg) was injected intraperitoneally every other day. The mice were euthanized on day 7 and tissues were collected. The results showed that when 4B9 was used alone, the levels of cholesterol efflux-related genes and their proteins, such as LXRs, ABCG8, ABCA1, and CYP7A1, increased, and the levels of lipid synthesis-related genes and their proteins, such as SREBP1 and FASN, were inhibited, while the levels of cholesterol synthesis or absorption-related genes and their proteins were not affected. In contrast, when ezetimibe was administered alone or in combination with 4B9, the levels of cholesterol efflux-related genes and their proteins and lipid synthesis-related genes and their proteins were significantly reduced (Figures 4a, 4p-q). While 4B9 or ezetimibe alone effectively reduced serum and liver total cholesterol and triglyceride levels, the combination of 4B9 and ezetimibe showed a more significant lipid-lowering effect (Figures 4b-e, 4r-s). While 4B9 alone increased total cholesterol and bile acid levels in bile, ezetimibe alone or in combination with 4B9 significantly inhibited these levels (Figures 4g, i). Meanwhile, 4B9 or ezetimibe alone or in combination had no significant effects on mouse body weight, daily food intake, liver-to-body weight ratio, blood glucose, and alanine aminotransferase and aspartate aminotransferase activity among the eight groups (Figures 4j-o). In summary, the combination of 4B9 and ezetimibe achieved a more lipid-lowering effect than either of these drugs alone.

[0156] While the present invention has been specifically disclosed by preferred embodiments and selective features, it will be understood that those skilled in the art may make modifications, improvements, and variations to the invention disclosed herein which are considered to be within the scope of the invention. The materials, methods, and examples provided herein are representative and illustrative of preferred embodiments and are not intended to limit the scope of the invention.

Claims

1. A monoclonal antibody that binds to ASGR1, 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; (a) an antibody in which the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each having the sequences set forth in SEQ ID NOs: 4 to 6, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each having the sequences set forth in SEQ ID NOs: 7 to 9; and (b) an antibody wherein the light chain variable region has the sequence set forth in SEQ ID NO: 28 and the heavy chain variable region has the sequence set forth in SEQ ID NO: 29; and The monoclonal antibody is a humanized antibody or a chimeric antibody, and comprises a human IgG1 heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO: 37, and a human Igκ light chain constant region consisting of the amino acid sequence shown in SEQ ID NO:

38.

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

3. 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; The pharmaceutical composition, wherein the second lipid-lowering drug is atorvastatin or ezetimibe.

4. a first pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising a therapeutically effective amount of a second lipid-lowering agent and a pharmaceutically acceptable carrier; The second lipid-lowering drug is atorvastatin or ezetimibe.

5. Use of the monoclonal antibody described in claim 1 in the manufacture of a medicament for lowering cholesterol and / or triglyceride levels in the blood or for the treatment and / or prevention of cardiovascular disease.

6. The use described in claim 5, wherein the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

7. 10. Use of a combination of the monoclonal antibody of claim 1 and a second lipid-lowering drug in the manufacture of a medicament for lowering blood cholesterol and / or triglyceride levels or for the treatment and / or prevention of cardiovascular disease, comprising: The use wherein said second lipid-lowering drug is atorvastatin or ezetimibe.

8. The use described in claim 7, wherein the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

9. Use of a monoclonal antibody or antigen-binding fragment thereof that binds to ASGR1 in the manufacture of a medicament for the treatment of cardiovascular disease, wherein the monoclonal antibody inhibits or blocks binding of ASGR1 to its natural ligand and / or endocytosis of ASGR1, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region; (a) an antibody in which the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each having the sequences set forth in SEQ ID NOs: 4 to 6, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each having the sequences set forth in SEQ ID NOs: 7 to 9; and (b) an antibody wherein the light chain variable region has the sequence set forth in SEQ ID NO: 28 and the heavy chain variable region has the sequence set forth in SEQ ID NO: 29; The use of any one of the antibodies.

10. The use described in claim 9, wherein the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

11. Use of a monoclonal antibody or antigen-binding fragment thereof that binds to ASGR1 in combination with a second lipid-lowering drug in the manufacture of a medicament for the treatment of cardiovascular disease, wherein the second lipid-lowering drug is atorvastatin or ezetimibe, the monoclonal antibody inhibits or blocks binding of ASGR1 to its natural ligand and / or endocytosis of ASGR1, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region; (a) an antibody in which the light chain variable region comprises LCDR1, LCDR2, and LCDR3, each having the sequences set forth in SEQ ID NOs: 4 to 6, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, each having the sequences set forth in SEQ ID NOs: 7 to 9; and (b) an antibody wherein the light chain variable region has the sequence set forth in SEQ ID NO: 28 and the heavy chain variable region has the sequence set forth in SEQ ID NO: 29; The use of any one of the antibodies.

12. The use of claim 11, wherein the cardiovascular disease is selected from atherosclerosis, stroke, myocardial infarction and coronary artery disease.

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