Binding protein of PCSK9 and angptl3, and use of binding protein

Abstract

Disclosed in the present invention are a binding protein of PCSK9 and ANGPTL3, and a use of the binding protein. Specifically, disclosed in the present invention are a bispecific antibody or an antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3, a pharmaceutical composition containing the antibody, a nucleic acid molecule encoding the antibody, and a use of the antibody in disease treatment.

Description

Binding proteins of pcsk9 and angptl3 and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine. In particular, the present application relates to antibodies that specifically bind PCSK9 and ANGPTL3, methods of making and uses thereof, for example for treating or preventing hyperlipidemia. BACKGROUND

[0002] Proprotein convertase subtilisin 9 (PCSK9) is a proprotein convertase of 74 kDa in size composed of 692 amino acids, which belongs to the proprotein convertase subtilisin family of members. The human PCSK9 gene is located on the short arm of chromosome 1, containing 12 exons and 11 introns, mostly expressed in hepatocytes, a small amount in kidney mesenchymal cells and intestinal epithelial cells, the encoded protein is synthesized as a soluble zymogen, and the concentration of PCSK9 in plasma is mainly regulated by the intracellular cholesterol level of hepatocytes. PCSK9 is a proteinase associated with familial hypercholesterolemia (FH) discovered in the past decade, PCSK9 inhibitors can effectively reduce the plasma low density lipoprotein cholesterol (LDL-C) level, PCSK9 promotes the degradation of low density lipoprotein (LDL) receptor, thereby increasing the content of LDL cholesterol in plasma, and LDL receptor mediates the endocytosis process of LDL in the liver, which is the main pathway for removing LDL from the circulatory system. Therefore, PCSK9 has become a very potential and new target for lowering blood lipids.

[0003] Angiopoietin like protein 3 (ANGPTL3) is a protein synthesized in the liver and secreted into the blood circulation system, which is composed of 460 amino acids, including a signal peptide, a coiled coil domain and a fibrinogen related domain. ANGPTL3 is an inhibitory protein of lipoprotein lipase (LPL) and endothelial lipase (EL), which can inhibit the enzyme activity of LPL or EL by binding to them, so that LPL cannot hydrolyze triglycerides in chylomicron and VLDL components, thereby significantly increasing the level of triglycerides in the blood circulation. If a blocking antibody is invented, which can block the binding of ANGPTL3 protein to LPL after binding to ANGPTL3 protein, thereby relieving the inhibition of LPL enzyme activity, then the triglycerides in the plasma can be degraded. Therefore, ANGPTL3 is also an important target for lowering triglycerides and cholesterol.

[0004] Hyperlipidemia has multiple types, including hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia, etc. Hypercholesterolemia mainly manifests as serum cholesterol higher than 6.2 mmol / L. Hypertriglyceridemia mainly manifests as serum triglyceride level higher than 2.3 mmol / L. Mixed hyperlipidemia manifests as both serum cholesterol and serum triglyceride levels significantly increased. Hyperlipidemia can also coexist with other metabolic disorders such as obesity, diabetes, hypertension, etc., which are usually referred to as metabolic syndrome.

[0005] Currently, for hypercholesterolemia, multiple monoclonal antibodies targeting PCSK9 have been marketed or are in clinical research phase, proving that antibodies targeting PCSK9 can effectively reduce the serum cholesterol level of patients. Antibodies targeting ANGPTL3 have also been proven in clinical experiments to have certain ability to reduce serum triglycerides and certain ability to reduce serum cholesterol levels. However, the approved antibodies of these two targets have problems in clinical efficacy, such as only reducing part of the indicators of serum cholesterol and serum triglyceride in mixed hyperlipidemia, or not reducing the indicators to a sufficient level.

[0006] Currently, there is still a lack of effective, sufficient, long-acting, and simultaneous treatment for reducing serum cholesterol and serum triglyceride. Therefore, it is urgent and necessary to develop a treatment that solves the above problems, which will provide patients with more medication options. SUMMARY

[0007] To solve the above technical problems and provide more effective antibody drugs for reducing serum cholesterol, triglyceride and low-density lipoprotein, the present application provides a new bispecific antibody.

[0008] In a first aspect of the present application, a bispecific antibody or an antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 is provided, comprising a first antigen-binding domain that specifically binds to PCSK9 and a second antigen-binding domain that specifically binds to ANGPTL3.

[0009] In some embodiments of the bispecific antibody or the antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 of the present application, wherein:

[0010] the first antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 9; and LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO: 10; and

[0011] the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region set forth in SEQ ID NO: 11; and LCDR1, LCDR2, and LCDR3 in a light chain variable region set forth in SEQ ID NO: 12.

[0012] In some embodiments, the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Abm, Chothia, or IMGT numbering system.

[0013] In some embodiments of the bispecific antibody of the present application, the first antigen binding domain comprises a heavy chain variable region 1 (VH1) and a light chain variable region 1 (VL1), wherein the VH1 comprises:

[0014] (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 13, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 14, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Kabat numbering system;

[0015] (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Abm numbering system;

[0016] (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 21, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 22, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Chothia numbering system; or

[0017] (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 23, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 24, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 25; wherein the CDRs are defined according to the IMGT numbering system; and / or

[0018] the VL1 comprises:

[0019] (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Kabat numbering system;

[0020] (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Abm numbering system;

[0021] (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Chothia numbering system; or

[0022] (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 27, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the IMGT numbering system.

[0023] In some embodiments of the bispecific antibody of the application, the second antigen binding domain comprises a heavy chain variable region 2 (VH2) and a light chain variable region 2 (VL2), wherein the VH2 comprises:

[0024] (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 28, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 29, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Kabat numbering system;

[0025] (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 34, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 35, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Abm numbering system;

[0026] (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 36, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 37, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Chothia numbering system; or

[0027] (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 38, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 39, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 40; wherein the CDRs are defined according to the IMGT numbering system; and / or

[0028] the VL2 comprises:

[0029] (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Kabat numbering system;

[0030] (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Abm numbering system;

[0031] (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Chothia numbering system; or

[0032] (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 42, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the IMGT numbering system.

[0033] In some embodiments, the present application provides a bispecific antibody or antigen binding fragment thereof that specifically binds to PCSK9 and ANGPTL3, comprising a first antigen binding domain that specifically binds to PCSK9 and a second antigen binding domain that specifically binds to ANGPTL3, wherein:

[0034] the first antigen binding domain comprises a heavy chain variable region 1 (VH1) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and a light chain variable region 1 (VL1) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10; and

[0035] the second antigen binding domain comprises a heavy chain variable region 2 (VH2) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; and a light chain variable region 2 (VL2) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12.

[0036] In some embodiments, the present application provides a bispecific antibody or antigen binding fragment thereof that specifically binds PCSK9 and ANGPTL3, comprising a first antigen binding domain that specifically binds PCSK9 and a second antigen binding domain that specifically binds ANGPTL3, wherein:

[0037] the first antigen binding domain comprises a VH1 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 and a VL1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and

[0038] the second antigen binding domain comprises a VH2 comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region VL2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12.

[0039] In some embodiments, the present application provides a bispecific antibody or antigen binding fragment thereof that specifically binds PCSK9 and ANGPTL3, comprising a first antigen binding domain that specifically binds PCSK9 and a second antigen binding domain that specifically binds ANGPTL3, wherein:

[0040] the first antigen binding domain comprises a VH1 consisting of the amino acid sequence of SEQ ID NO: 9 and a VL1 consisting of the amino acid sequence set forth in SEQ ID NO: 10; and

[0041] the second antigen binding domain comprises a VH2 consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region VL2 consisting of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the bispecific antibody or antigen binding fragment thereof provided herein is in the form of a bispecific F(ab')2, Fab-scFv, IgG-scFv, (scFv)2-Fc, bi-nanobody, Tandem diabody (TandAb), Dual- affinity retargeting (DART) antibody, DART-Fc, or a combination thereof.

[0042] In some embodiments, the bispecific antibody or antigen binding fragment thereof provided herein further comprises a Fc region.

[0043] In some specific embodiments, the Fc region is selected from the Fc region of wild type human IgGl, human IgG2, human IgG3, and human IgG4.

[0044] In some embodiments, the Fc region is the Fc region of wild type human IgGl or IgG4.

[0045] In embodiments of the present application, the Fc region of the bispecific antibody of the present application comprises a first Fc region fragment (Fc1) and a second Fc region fragment (Fc2). The Fc1 and Fc2 can have the same or different amino acid sequences.

[0046] In some specific embodiments, the Fc region of the bispecific antibody provided herein further comprises point mutations introduced based on the knob-into-hole technology, wherein a knob mutation is introduced in Fc1 and a hole mutation is introduced in Fc2; or a knob mutation is introduced in Fc2 and a hole mutation is introduced in Fc1.

[0047] In some embodiments, the bispecific antibody provided herein comprises point mutations introduced based on the knob-into-hole technology in the CH3 of the Fc region, wherein a knob mutation is introduced in the CH3 of Fc1 and a hole mutation is introduced in the CH3 of Fc2; or a knob mutation is introduced in the CH3 of Fc2 and a hole mutation is introduced in the CH3 of Fc1. Amino acid substitutions that can be used to form a "knob" structure include, but are not limited to, S354C and T366W, wherein the numbering is according to the Eu numbering scheme. Amino acid substitutions that can be used to form a "hole" structure include, but are not limited to, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the Eu numbering scheme. Due to the mutual attraction between the "knob" structure and the "hole" structure, and the mutual repulsion between the "knob" structure and the "knob" structure, the knob-in-hole structure can effectively reduce the heavy chain mispairing in the bispecific antibody.

[0048] In some embodiments, to reduce the ADCC effect induced by the wild-type Fc polypeptide, the bispecific antibody provided herein has one or more mutations in the Fc region selected from the group consisting of D265A, N297A, L234A, L234F, L235A, L235E, and P329G; wherein the amino acid positions are numbered according to the EU index.

[0049] In some embodiments, the Fc region of the bispecific antibody provided herein has the following mutations: P329G, L234A, and L235A, to eliminate the binding to Fc gamma receptor.

[0050] In some embodiments, the Fc region of the bispecific antibody provided herein comprises Fc1 with a knob structure as set forth in SEQ ID NO: 43, and Fc2 with a hole structure as set forth in SEQ ID NO: 44.

[0051] In some embodiments, the Fc region of the bispecific antibody provided herein comprises Fc2 with a knob structure as set forth in SEQ ID NO: 43, and Fc1 with a hole structure as set forth in SEQ ID NO: 44.

[0052] Further, in some embodiments, the bispecific antibody provided herein further comprises a light chain constant region, optionally, the light chain constant region is a light chain constant region from a human kappa or lambda chain.

[0053] In some embodiments, the bispecific antibody provided herein further comprises a light chain constant region from a human kappa chain.

[0054] In some specific embodiments, the bispecific antibody provided herein further comprises a CH1 region and a hinge region from wild-type human IgGl, human IgG2, human IgG3, or human IgG4.

[0055] In particular, the bispecific antibody provided herein further comprises a CH1 region and a hinge region from wild-type human IgGl or human IgG4.

[0056] In some specific embodiments, the bispecific antibody provided herein further comprises a CH1 region and a hinge region from wild-type human IgGl.

[0057] In some embodiments, the bispecific antibody provided herein that specifically binds PCSK9 and ANGPTL3 comprises four polypeptide chains, wherein:

[0058] the first polypeptide chain has, from N-terminus to C-terminus, the following structure:

[0059] VL1-CL;

[0060] the second polypeptide chain has, from N-terminus to C-terminus, the following structure:

[0061] VH1-CH1-Fc1;

[0062] the third polypeptide chain has, from N-terminus to C-terminus, the following structure:

[0063] VH2-CL-Fc2; and

[0064] the fourth polypeptide chain has, from N-terminus to C-terminus, the following structure:

[0065] VL2-CH1.

[0066] In further embodiments:

[0067] (a) VH1 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and comprises: (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 13, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 14, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Kabat numbering system; (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Abm numbering system; (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 21, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 22, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Chothia numbering system; or (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 23, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 24, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 25; wherein the CDRs are defined according to the IMGT numbering system;

[0068] (b) VH2 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; and comprises: (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 28, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 29, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Kabat numbering system; (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 34, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 35, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Abm numbering system; (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 36, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 37, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Chothia numbering system; or (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 38, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 39, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 40; wherein the CDRs are defined according to the IMGT numbering system;

[0069] (c) VL1 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10; and comprises: (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Kabat numbering system; (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Abm numbering system; (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Chothia numbering system; or (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 27, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the IMGT numbering system;

[0070] (d) VL2 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; and comprises: (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Kabat numbering system; (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Abm numbering system; (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Chothia numbering system; or (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 42, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the IMGT numbering system;

[0071] (e) CL is a light chain constant region from a human kappa or lambda chain;

[0072] (f) CH1 is a CH1 region from wild-type human IgGl or human IgG4; and

[0073] (g) the Fc region is a variant of the Fc region of wild-type human IgGl or IgG4; which comprises, compared to the Fc region of wild-type human IgGl or IgG4

[0074] i) point mutations introduced based on the knob-into-hole technology; and

[0075] ii) point mutations comprising P329G, L234A and L235A, the amino acid positions being numbered according to the EU index.

[0076] In other embodiments, the present application provides a bispecific antibody that specifically binds to PCSK9 and ANGPTL3, comprising three polypeptide chains, wherein:

[0077] the first polypeptide chain has the following structure from N-terminus to C-terminus:

[0078] VL1-CL;

[0079] the second polypeptide chain has the following structure from N-terminus to C-terminus:

[0080] VH1-CH1-Fc1; and

[0081] a third polypeptide chain having the following structure from N-terminus to C- terminus:

[0082] VH2-VL2-Fc2.

[0083] In some embodiments, each component in the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain is optionally connected by or without a linker. In some embodiments, VH2 and VL2 in the third polypeptide chain are connected by a linker, e.g., a peptide linker. In some embodiments, the peptide linker can independently comprise GGGG (SEQ ID NO: 48). In some specific embodiments, the linker consists of a repeated GGGGS (SEQ ID NO: 47) amino acid sequence or a variant thereof. In some specific embodiments, the linker can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some specific embodiments, the linker is (GGGGS)4(SEQ ID NO: 45).

[0084] In further embodiments:

[0085] (a) VH1 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and comprises: (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 13, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 14, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Kabat numbering system; (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Abm numbering system; (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 21, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 22, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Chothia numbering system; or (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 23, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 24, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 25; wherein the CDRs are defined according to the IMGT numbering system;

[0086] (b) VH2 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; and comprises: (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 28, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 29, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Kabat numbering system; (2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 34, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 35, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Abm numbering system; (3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 36, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 37, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Chothia numbering system; or (4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 38, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 39, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 40; wherein the CDRs are defined according to the IMGT numbering system;

[0087] (c) VL1 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10; and comprises: (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Kabat numbering system; (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Abm numbering system; (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Chothia numbering system; or (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 27, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the IMGT numbering system;

[0088] (d) VL2 comprises or consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; and comprises: (1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Kabat numbering system; (2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Abm numbering system; (3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Chothia numbering system; or (4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 42, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the IMGT numbering system;

[0089] (e) CL is a light chain constant region from a human kappa or lambda chain;

[0090] (f) CH1 is a CH1 region from wild-type human IgGl or human IgG4; and

[0091] (g) the Fc region is a variant of the Fc region of wild-type human IgGl or IgG4; which comprises, compared to the Fc region of wild-type human IgGl or IgG4

[0092] i) point mutations introduced based on the knob-into-hole technology; and

[0093] ii) point mutations comprising P329G, L234A and L235A, the amino acid positions being numbered according to the EU index.

[0094] In some specific embodiments, the point mutations introduced based on the knob-into-hole technology comprise:

[0095] a S354C and a T366W mutation on the “knob” chain, and

[0096] a Y349C, T366S, L368A and Y407V mutation on the “hole” chain.

[0097] wherein the amino acid positions are numbered according to the EU index.

[0098] In some embodiments, the CH1 region has the sequence as set forth in SEQ ID NO: 49. In some embodiments, the CL region has the sequence as set forth in SEQ ID NO: 50.

[0099] In some embodiments, the bispecific antibody provided herein that specifically binds PCSK9 and ANGPTL3 comprises four polypeptide chains, wherein:

[0100] the first polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2,

[0101] the second polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5,

[0102] the third polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, and

[0103] the fourth polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0104] In some embodiments, the bispecific antibody provided herein that specifically binds PCSK9 and ANGPTL3 comprises four polypeptide chains, wherein:

[0105] the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 2,

[0106] the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 5,

[0107] the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 6, and

[0108] the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0109] ​​In some embodiments, the bispecific antibodies provided herein that specifically bind to PCSK9 and ANGPTL3 comprise three polypeptide chains, wherein:

[0110] the first polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2,

[0111] the second polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, and

[0112] the third polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0113] In some embodiments, the bispecific antibodies provided herein that specifically bind to PCSK9 and ANGPTL3 comprise three polypeptide chains, wherein:

[0114] the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 2,

[0115] the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 5, and

[0116] the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0117] In some embodiments, the bispecific antibodies provided herein that specifically bind to PCSK9 and ANGPTL3 have one or more, or a combination thereof, of the following characteristics:

[0118] i) binds to human PCSK9 with high binding affinity;

[0119] ii) binds to human ANGPTL3 with high binding affinity;

[0120] iii) effectively blocks the binding of PCSK9 to the LDLR receptor;

[0121] iv) effectively blocks the PCSK9 protein's inhibition of LDL-pHrodo uptake;

[0122] v) effectively blocks the ANGPTL3's inhibition of LPL enzyme activity;

[0123] vi) significantly reduces the levels of LDL-C and / or TC in serum of high-fat diet mice.

[0124] In another aspect, the present application provides an isolated nucleic acid molecule or a set of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody that specifically binds to PCSK9 and ANGPTL3 provided by the present application.

[0125] In specific embodiments of a bispecific antibody comprising four polypeptide chains, the isolated nucleic acid molecule or set of nucleic acid molecules described herein comprises a nucleotide sequence encoding a first polypeptide chain of a bispecific antibody provided by the present application, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain. In some specific embodiments, the set of nucleic acid molecules includes a plurality of nucleic acid molecules, each of which comprises at least one of the nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain.

[0126] In specific embodiments of a bispecific antibody comprising three polypeptide chains, the isolated nucleic acid molecule or set of nucleic acid molecules described herein comprises a nucleotide sequence encoding a first polypeptide chain of a bispecific antibody provided by the present application, a nucleotide sequence encoding a second polypeptide chain, and a nucleotide sequence encoding a third polypeptide chain. In some specific embodiments, the set of nucleic acid molecules includes a plurality of nucleic acid molecules, each of which comprises at least one of the nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, and a nucleotide sequence encoding a third polypeptide chain.

[0127] In another aspect, the present application provides a vector or set of vectors (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule or set of nucleic acid molecules of the present application.

[0128] In specific embodiments of a bispecific antibody comprising four polypeptide chains, the vector or set of vectors described herein comprises a nucleotide sequence encoding a first polypeptide chain of a bispecific antibody provided by the present application, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain. In some specific embodiments, the set of vectors includes a plurality of vectors, each of which comprises at least one of the nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain.

[0129] In specific embodiments of bispecific antibodies comprising three polypeptide chains, the vector or vector set described herein comprises a nucleotide sequence encoding a first polypeptide chain of a bispecific antibody provided by the present application, a nucleotide sequence encoding a second polypeptide chain, and a nucleotide sequence encoding a third polypeptide chain. In some specific embodiments, the vector set comprises a plurality of vectors, each of which comprises at least one of the nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, and a nucleotide sequence encoding a third polypeptide chain.

[0130] In some specific embodiments, the vector or vector set of the present application is, for example, a plasmid, a cosmid, a bacteriophage, and the like. In some specific embodiments, the vector or vector set is capable of expressing a bispecific antibody or antigen-binding fragment thereof of the present application in vivo in a subject, for example, a mammal, for example, a human.

[0131] In another aspect, the present application provides a host cell comprising an isolated nucleic acid molecule, a nucleic acid molecule set, a vector, or a vector set of the present application. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, for example, mouse cells, human cells, and the like). In some specific embodiments, the host cell of the present application is a mammalian cell, for example, a CHO (e.g., CHO-K1, CHO-S, CHO DG44) or HEK, HEK293, HEK-293F, Expi293F, PER.C6, NSO cell, or a lymphocyte.

[0132] In another aspect, the present application provides a method of making a bispecific antibody of the present application, comprising, culturing a host cell of the present application under conditions permitting expression of a bispecific antibody of the present application, and recovering a bispecific antibody of the present application from the cultured host cell culture.

[0133] In specific embodiments of bispecific antibodies comprising four polypeptide chains, the method of making a bispecific antibody of the present application comprises:

[0134] (1) constructing an expression vector or an expression vector set comprising a nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain, wherein the expression vector set comprises a plurality of vectors, each of which comprises at least one of the nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, a nucleotide sequence encoding a third polypeptide chain, and a nucleotide sequence encoding a fourth polypeptide chain;

[0135] (2) transforming the expression vector or expression vector set described in step (1) to a host cell;

[0136] (3) culturing the host cell of step (2) under conditions that allow the expression of the bispecific antibody of the application; and

[0137] (4) recovering the bispecific antibody from the cultured host cell culture.

[0138] In a specific embodiment of the bispecific antibody comprising three polypeptide chains, the method of making the bispecific antibody of the application comprises:

[0139] (1) constructing an expression vector or a set of expression vectors comprising a nucleotide sequence encoding a first polypeptide chain, a nucleotide sequence encoding a second polypeptide chain, and a nucleotide sequence encoding a third polypeptide chain, wherein the set of expression vectors comprises a plurality of vectors, each of the plurality of vectors comprising at least one of the nucleotide sequence encoding a first polypeptide chain, the nucleotide sequence encoding a second polypeptide chain, and the nucleotide sequence encoding a third polypeptide chain;

[0140] (2) transforming the expression vector of step (1) to a host cell;

[0141] (3) culturing the host cell of step (2) under conditions that allow the expression of the bispecific antibody of the application; and

[0142] (4) recovering the bispecific antibody from the cultured host cell culture.

[0143] The bispecific antibodies of the application can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Typically, derivatization of the antibody does not adversely affect its binding to ANGPTL3 and PCSK9. Thus, the bispecific antibodies of the application also include such derivatized forms. For example, the bispecific antibodies of the application can be functionally linked (by chemical

[0144] Thus, in one aspect, the application provides conjugates comprising a bispecific antibody as described above and a conjugating moiety linked thereto.

[0145] In some embodiments, the conjugating moiety is selected from a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin, or a radionuclide).

[0146] In some embodiments, the conjugating moiety is selected from a substance capable of improving the biological properties of the bispecific antibody, e.g. increasing the serum half-life, e.g. can be a chemical group, e.g. polyethylene glycol (PEG), a Formazan group or an ethyl group, or a sugar group. In another aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody or antigen binding fragment thereof of the present application, or the isolated nucleic acid molecule or set of nucleic acid molecules of the present application, or the vector or set of vectors of the present application, or the host cell of the present application, or the conjugate of the present application, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0147] In some embodiments, the pharmaceutical composition contains 0.01 to 99 % by weight of the aforementioned bispecific antibody or antigen binding fragment thereof of the present application in a unit dose.

[0148] In some embodiments, the pharmaceutical composition contains 0.1-2000 mg, more preferably 1-1000 mg of the bispecific antibody or antigen binding fragment thereof of the present application in a unit dose.

[0149] In another aspect, the present application provides the use of the bispecific antibody or antigen binding fragment thereof according to the present application, the isolated nucleic acid molecule or set of nucleic acid molecules of the present application, the vector or set of vectors of the present application, the host cell of the present application, the conjugate of the present application or the pharmaceutical composition of the present application for the manufacture of a medicament for the treatment of a PCSK9 and / or ANGPTL3 related disease.

[0150] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is associated with high cholesterol and / or high triglycerides.

[0151] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis or a cardiovascular disease.

[0152] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipidemia or atherosclerotic disease.

[0153] In another aspect, the present application provides a method of treating a PCSK9 and / or ANGPTL3 related disease, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody or antigen binding fragment thereof of the present application, the isolated nucleic acid molecule or set of nucleic acid molecules of the present application, the vector or set of vectors of the present application, the host cell of the present application, the conjugate of the present application, or the pharmaceutical composition of the present application.

[0154] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis, or a cardiovascular disease.

[0155] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis, or a cardiovascular disease.

[0156] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hyperlipidemia, or atherosclerotic disease.

[0157] In some embodiments, the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, or atherosclerotic disease.

[0158] In another aspect, the present application provides a method of reducing cholesterol levels and / or triglyceride levels in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody or antigen binding fragment thereof of the present application, the isolated nucleic acid molecule or set of nucleic acid molecules of the present application, the vector or set of vectors of the present application, the host cell of the present application, the conjugate of the present application, or the pharmaceutical composition of the present application. In some embodiments, the method reduces cholesterol levels and / or triglyceride levels in the blood of the subject. In some embodiments, the method reduces cholesterol levels and / or triglyceride levels in the serum of the subject.

[0159] In another aspect, the bispecific antibody or antigen binding fragment thereof of the present application, the isolated nucleic acid molecule or set of nucleic acid molecules of the present application, the vector or set of vectors of the present application, the host cell of the present application, the conjugate of the present application, or the pharmaceutical composition of the present application provided by the present application can also be used in prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications based on those well known and standard to the person skilled in the art as described herein.

[0160] In some embodiments, the bispecific antibodies of the present application can be used for predicting, diagnosing and / or monitoring a subject having or suspected of having a cancer or other disease and / or an intended or desired treatment response by assaying and / or detecting PCSK9 and / or ANGPTL3 protein levels in a biological sample in vitro. In some embodiments, the bispecific antibodies of the present application can be used for applications for non-diagnostic purposes, such as detecting PCSK9 and / or ANGPTL3 protein levels in a cell sample, and the cell sample is not derived from a subject.

[0161] In another aspect, the present application provides a kit comprising a bispecific antibody or antigen binding fragment thereof of the present application, or an isolated nucleic acid molecule or set of nucleic acid molecules of the present application, or a vector or set of vectors of the present application, or a host cell of the present application, or a conjugate of the present application, or a pharmaceutical composition of the present application, and optionally instructions for use and / or a device for administration.

[0162] In some embodiments, the kit can be used for in vitro assessment and evaluation of a disease associated with PCSK9 and / or ANGPTL3. For example, the kit can be used for predicting, diagnosing and monitoring to assess a patient sample, including a patient sample known to have or suspected of having a disease associated with PCSK9 and / or ANGPTL3, or to evaluate an intended or desired treatment response.

[0163] In some embodiments, the kit can be used for treating a disease associated with PCSK9 and / or ANGPTL3.

[0164] In some embodiments, the disease associated with PCSK9 and / or ANGPTL3 is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis or a cardiovascular disease. In some embodiments, the disease associated with PCSK9 and / or ANGPTL3 is selected from hypercholesterolemia, hypertriglyceridemia or atherosclerotic disease. BRIEF DESCRIPTION OF DRAWINGS

[0165] Figure 1 A shows a schematic diagram of the structure of Bs-1 bispecific antibody;

[0166] Figure 1 B shows a schematic diagram of the structure of Bs-2 bispecific antibody;

[0167] Figure 2 shows that the antibodies block the binding of PCSK9 to the LDLR receptor;

[0168] Figure 3 shows that the antibodies restore the uptake of fluorescent-low density lipoprotein by Huh7 cells;

[0169] Figure 4 shows that the antibody blocks the inhibition of LPL enzymatic activity by ANGPTL3;

[0170] Figures 5A-5B show the changes in serum biochemical indicators in each group of DIO model mice. Compared with the model group, ** indicates P<0.01. DETAILED DESCRIPTION

[0171] TERMS

[0172] The terms used herein are merely for the purpose of describing the embodiments and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0173] In this document, when referring to "first" and "second", it is merely for the purpose of distinguishing two domains or two chains, and does not indicate the position of the two domains in any way.

[0174] The term "antibody" as used herein refers to an immunoglobulin, and a naturally intact antibody is a four polypeptide chain structure connected by interchain disulfide bonds, which is composed of two identical heavy chains and two identical light chains. According to the difference in the amino acid composition and the arrangement order of the constant region of the immunoglobulin heavy chain, the immunoglobulin can be divided into five types, or called the isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same type of Ig can be divided into different subtypes according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0175] The sequence of about 110 amino acids near the N-terminus of the heavy chain and light chain of the antibody varies greatly, which is the variable region; the remaining amino acid sequence near the C-terminus is relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions, and the sequence arranged from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain are LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2 and HCDR3.

[0176] An "antibody fragment" or "antigen binding fragment" is a molecule other than a whole antibody that comprises a portion of an intact antibody that retains the antigen binding ability of the intact antibody. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibody, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0177] The term "Fab" fragment includes a heavy chain variable domain and a light chain variable domain, and also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.

[0178] The term "single-chain antibody," "single-chain Fv" or "scFv" as used herein means a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS (SEQ ID NO: 47) amino acid sequences or variants thereof, for example, using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0179] As used herein, the term "bispecific" antibody is an antibody having two different antigen binding domains that bind to two different antigens. Bispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetravalent antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single-chain antibodies or linked single-chain Fv (scFv), camelized antibodies, affybodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fv, and disulfide-linked Fv (sdFv). Bispecific antibodies can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) In certain embodiments, the bispecific antibodies described herein are IgG antibodies, or a class thereof (e.g., human IgGl, IgG2, or IgG4) or a subclass thereof. In certain embodiments, the bispecific antibodies described herein comprise two polypeptides, optionally comprising the same polypeptide, each polypeptide comprising, in order from amino-terminus to carboxy-terminus, a first scFv antigen binding domain, a linker (optionally wherein the linker is a hinge region), an immunoglobulin constant region, and a second scFv antigen binding domain.

[0180] As used herein, the term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to a pyroglutamate or pyroglutamic acid. Thus, in compositions comprising bispecific antibodies disclosed herein, the bispecific antibodies therein can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or an N-terminal amino acid cyclized to a pyroglutamic acid.

[0181] As used herein, the "Fc region" of an antibody is the terminal end of the antibody heavy chain comprising domains CH2, CH3. The "CH2 domain" of a human IgG Fc region generally extends from about amino acid 231 to about amino acid 340. The "CH3 domain" comprises the sequence of residues C-terminal to the CH2 domain in the Fc region, i.e., about amino acid residue 341 to about amino acid residue 447 of IgG.

[0182] Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0183] The term "Fc region of an antibody" as used herein includes an Fc region having an amino acid sequence identical to that of a naturally occurring human antibody Fc region. Naturally occurring human antibody Fc regions include human IgGl Fc region, human IgG2 Fc region, IgG3 Fc region, human IgG4 Fc region, and naturally occurring variants thereof.

[0184] As used herein, a "variant of an Fc region" includes an amino acid sequence that differs from that of a "native" or "wild-type" Fc region by at least one amino acid substitution, deletion, or insertion. For example, a variant Fc region has at least one amino acid substitution, deletion, or insertion compared to a "native" or "wild-type" Fc region.

[0185] As used herein, "knob-in-hole" refers to the introduction of mutations in a first Fc polypeptide and a second Fc polypeptide of an Fc region, respectively, using "knob-in-hole" technology, to form a knob on the interface of the first Fc polypeptide and a complementary hole on the interface of the second Fc polypeptide.

[0186] As used herein, CrossMab technology is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, either the exchange of the complete Fab domain (CrossMAb Fab) or the exchange of only the variable region (CrossMAb V H -V L ) or only the constant region (CrossMAb C H 1-C L ) of the Fab domain. Bispecific antibodies with the CrossMab format are also described in the literature Klein C, et al. The use of CrossMAb technology for the generation of bi- and multi-specific antibodies. MAbs. 2016 Aug-Sep;8(6):1010-20.

[0187] "Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than approximately 10-1. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0188] In this invention, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., the antibodies of this invention) have a dissociation equilibrium constant of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D The antigen (e.g., HBsAg) is bound to the antigen, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR). The term "k" is used in this context. d (sec) -1 ) refers to the dissociation rate constant of a specific antibody-antigen interaction, also known as the k-off value.

[0189] "ADCC" stands for antibody-dependent cell-mediated cytotoxicity. It refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc fragment of an antibody. The ADCC effector function of antibodies can be reduced or eliminated by modifying the Fc fragment of IgG. This modification refers to mutations in the constant region of the antibody's heavy chain, such as N297A, L234A, L235A, and P329G selected from IgG1; and F234A / L235A mutations selected from IgG2 / 4chimera and IgG4.

[0190] "Complement-dependent cytotoxicity" or "CDC" refers to a form of cellular toxicity that is mediated by the activation of the complement cascade by binding of complement component Clq to the Fc of an antibody. Methods of testing for ADCC, CDC activity of antibodies are known in the art, e.g., CDC can be evaluated by assaying for binding activity between the antibody of interest and an Fc receptor, e.g., Clq.

[0191] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.

[0192] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

[0193] An "isolated" nucleic acid molecule is one which has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from that of normal.

[0194] The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "operably linked" when they are functionally connected. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0195] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein can be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell and can replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0196] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the genera Escherichia, e.g., strains of Escherichia coli or Salmonella; Bacillaceae, e.g., Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells, and NSO cells.

[0197] A "pharmaceutical composition" denotes a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism and to facilitate absorption of the active ingredient to thereby elicit a biological activity.

[0198] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering an antibody or antigen-binding fragment. The carrier can be an anti-adherent, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, an antibacterial or an antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifying agent, a buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0199] "Administering," "administered," and "treatment" when applied to a subject (e.g., an animal, a human), a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid. "Administering," "administered," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administering," "administered," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0200] "Treatment" means the administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the disclosure, to a patient having one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on the symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") can vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test method used by a physician or other professional health care provider to assess the severity or progression of the symptom. Although an embodiment of the disclosure (e.g., a method of treatment or article of manufacture) can not be effective in alleviating each target disease symptom, it is determined that the target disease symptom should be alleviated in a statistically significant number of patients according to any statistical test method known in the art, such as the Student t-test, the chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0201] The present disclosure will now be described with reference to the following examples, which are intended to illustrate the present disclosure (and not to limit it).

[0202] Unless otherwise indicated, the molecular biology experimental methods and immunoassays used in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples describe the present application by way of example only and are not intended to limit the scope of the application as claimed.

[0203] Sequence information

[0204] The description of the sequences involved in the present application is provided in Table 1 below.

[0205] Table 1 Sequence information table

[0206] Example 1: Expression and preparation of monoclonal antibodies and bispecific antibodies

[0207] 1.1 Structure and sequence of monoclonal antibodies and bispecific antibodies

[0208] According to the published patents US8080243B2 and WO2012 / 174178, the sequences of the antibody Bococizumab against PCSK9 and the antibody Evinacumab against ANGPTL3 are determined, respectively. Specifically, the heavy chain sequence of the antibody Bococizumab is SEQ ID NO: 1; the light chain sequence is SEQ ID NO: 2; the heavy chain sequence of the antibody Evinacumab is SEQ ID NO: 3; the light chain sequence is SEQ ID NO: 4.

[0209] On the basis of the antibody Bococizumab against PCSK9 and the antibody sequence against ANGPTL3, the bispecific antibodies Bs-1 and Bs-2 provided by the present application, which can simultaneously bind to PCSK9 and ANGPTL3, adopt the "1+1 IgG crossmab" and "1+1 Ybody" structures, respectively. The specific description is as follows.

[0210] Bispecific antibody Bs-1

[0211] The bispecific antibody Bs-1 employs a "1+1 IgG crossmab" structure antibody, as shown in FIG. 1A, which consists of 4 chains, including 2 different heavy chains and two different light chains. Point mutations of Knob-into-hole were introduced into the CH3 domains of the two heavy chains to prevent mispairing of the two heavy chains. Specifically, the amino acid mutations S354C and T366W were employed in Bs-1 to form the "knob" structure; and the amino acid mutations Y349C, T366S, L368A, and Y407V were employed to form the "hole" structure. According to the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant regions of the Knob and hole heavy chains to eliminate binding to Fcy receptors. Among them, the positions of the above-mentioned amino acids are according to the EU numbering. The CH1 and CL domains in the PCSK9-binding Fab retain the original order of the antibody (Fab with VH-CH1 and VL-CL structure), while the CH1 and CL domains in the ANGPTL3-binding Fab are exchanged to obtain Fab with VH-CL and VL-CH1 structure, in order to facilitate the correct assembly of the two different light chains.

[0212] The bispecific antibody Bs-1 includes 4 polypeptide chains, wherein:

[0213] (1) a first polypeptide chain (SEQ ID NO: 2) having the following structure from N-terminus to C-terminus: VL1-CL;

[0214] (2) a second polypeptide chain (SEQ ID NO: 5) having the following structure from N-terminus to C-terminus: VH1-CH1-Fc1;

[0215] (3) a third polypeptide chain (SEQ ID NO: 6) having the following structure from N-terminus to C-terminus: VH2-CL-Fc2; and

[0216] (4) a fourth polypeptide chain (SEQ ID NO: 7) having the following structure from N-terminus to C-terminus: VL2-CH1.

[0217] The bispecific antibody Bs-2

[0218] Bs-2 is a bispecific antibody using a "1+1 Ybody" antibody structure, consisting of 3 chains, including 2 different heavy chains and 1 different light chain, as shown in Figure 1B. Point mutations of Knob-into-hole were introduced into the CH3 domains of the two heavy chains to prevent mispairing of the two heavy chains. Specifically, the amino acid mutations used in Bs-2 to form the "knob" structure are S354C and T366W; the amino acid mutations used to form the "hole" structure are Y349C, T366S, L368A, and Y407V. According to the method described in WO 2012 / 130831, Pro329Gly, Leu234Ala, and Leu235Ala mutations were introduced into the constant region of the Knob and hole heavy chains to eliminate binding to Fc gamma receptors. Among them, the positions of the above amino acids are according to the EU numbering. At the N-terminus of the hole chain is the variable region single-chain Fv (scFv) of the Evinacumab antibody, which contains a peptide linker with the sequence (GGGGS)4(SEQ ID NO: 45).

[0219] Bs-2 bispecific antibody comprises three polypeptide chains, wherein:

[0220] (1) the first polypeptide chain (SEQ ID NO: 2) has the following structure from N-terminus to C-terminus: VL1-CL;

[0221] (2) the second polypeptide chain (SEQ ID NO: 5) has the following structure from N-terminus to C-terminus: VH1-CH1-Fc1; and

[0222] (3) the third polypeptide chain (SEQ ID NO: 8) has the following structure from N-terminus to C-terminus: VH2-VL2-Fc2.

[0223] 1.2 Expression and obtaining of monoclonal antibodies and bispecific antibodies

[0224] According to the sequences of the monoclonal antibodies and bispecific antibodies described above, after codon optimization by Genscript, the antibody heavy and light chain nucleotide sequences were synthesized and cloned into pTT5 vectors. After plasmid extraction, the corresponding pTT5 plasmids of the antibody heavy and light chains were transfected into CHOS-EBNA cells at the same time. After centrifugation to collect the supernatant, the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain the internally prepared Bococizumab, Evinacumab, Bs-1 and Bs-2 antibodies.

[0225] Example 2: Binding activity of bispecific antibodies

[0226] 2.1 Binding of bispecific antibodies to different species of PCSK9 antigens

[0227] The protein dynamic affinity of the bispecific antibodies and control mAbs to human full length PCSK9 (Peprotech, PC9-H5223), cynomolgus monkey full length PCSK9 (Peprotech, PC9-C5223), mouse full length PCSK9 (Peprotech, PC9-M5228) was detected by ForteBio (Pall life sciences) instrument.

[0228] The specific method is as follows: the antibody to be tested is diluted to 5 mg / ml with PBST (0.02% Tween-20), and each antigen protein is gradient-diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, then the antibody to be tested is captured with Protein A Sensor (Pall life sciences) for 60 s in PBST (0.02% Tween-20) solution, after equilibration in buffer for 30 s, then combined with the above-mentioned proteins for 60 s respectively, and then dissociated for 200 s, the results are opened in Data Analysis 11.0 software, 1:1 mode and global fitting are selected, and the results are analyzed to obtain the affinity constant.

[0229] The results are shown in Table 2. The results show that the Bs-1 and Bs-2 bispecific antibodies both bind to human, cynomolgus monkey and mouse full length PCSK9 proteins, and Bs-1 and Bs-2 both have high affinity dynamic activity to antigen proteins.

[0230] Table 2. Binding of bispecific antibodies to different species of PCSK9 antigens

[0231] 2.2 Binding of bispecific antibodies to different species of ANGPTL3 antigens

[0232] The protein dynamic affinity of the bispecific antibodies and control mAbs to human full length ANGPTL3 (Sino Biological, 10770-H08B), cynomolgus monkey N-terminal ANGPTL3 (Sino Biological, 90027-C08B) and mouse full length ANGPTL3-His (Sino Biological, 50003-M08B) was detected by ForteBio (Pall life sciences) instrument.

[0233] The specific method is as follows: the antibody to be tested is diluted to 5 mg / ml with PBST (0.02% Tween-20), and each antigen protein is gradient-diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, then the antibody to be tested is captured for 60 s with Protein A Sensor (Pall life sciences) in a PBST (0.02% Tween-20) solution, after equilibration in the buffer for 30 s, then combined with the above protein for 60 s, then dissociated for 200 s, and the results are processed by Data Analysis 11.0 software, 1:1 mode is selected, global fitting is used, and the results are analyzed to obtain the affinity constant.

[0234] The results are shown in Table 3, and the results show that the Bs-1 and Bs-2 bispecific antibodies both bind to the full-length ANGPTL3 protein of human, cynomolgus monkey and mouse, and Bs-1 and Bs-2 both have high affinity dynamic activity to the antigen protein, and the affinity is equivalent to the affinity of Evinacumab.

[0235] Table 3. Binding of bispecific antibodies to ANGPTL3 antigens of different species

[0236] Example 3: Bispecific antibodies block the binding of PCSK9 to the LDLR receptor

[0237] PCSK9 protein was labeled according to the instruction of Biotin Labeling Kit (Thermo\A39257) to obtain human PCSK9-Biotin protein. To detect the blocking activity of bispecific antibodies on the binding of human PCSK9 to LDLR receptor, ELISA method was used to detect the blocking activity. The specific experimental method is as follows: 3 mg / ml human LDLR-hFc fusion protein was diluted with CBS buffer, 100 μl / well, coated at 4°C overnight. 300 μl PBST (0.05% Tween-20) was used for washing once, 100 ml of 2% BSA in PBS was added to each well, and incubated at 37°C for 1 hour. Bispecific antibodies, control antibodies were diluted with PBST (pH 5.5, 0.05% Tween-20) + 0.5% BSA. Human full-length PCSK9-Biotin was diluted with PBST (pH 5.5, 0.05% Tween-20) + 0.5% BSA to a concentration of 200 ng / ml. After dilution, 60 μl of each of the antibody and PCSK9-biotin were mixed and incubated at 37°C for 30 min. 100 μl of the mixture was added to the enzyme-labeled plate and incubated at 37°C for 1 hour. Discard the solution, wash the enzyme-labeled plate with PBST (pH 5.5, 0.05% Tween-20) for 3 times, 320 μl per well. Dry the enzyme-labeled plate, add 100 ml of HRP-conjugated-SA secondary antibody (Biolegend) diluted 10000 times with PBST (pH 5.5, 0.05% Tween-20) + 0.5% BSA to each well, and incubate at 37°C for 1 hour. Discard the solution, wash the enzyme-labeled plate with PBST (pH 5.5, 0.05% Tween-20) for 5 times. Dry the enzyme-labeled plate, add 100 ml of TMB to each well and develop the color in the dark, then add 50 ml of 2N concentration of sulfuric acid to stop the color developing reaction, and read the absorbance at 450 nm with an enzyme-labeled instrument.

[0238] The results are shown in Figure 2 and Table 4 below. Bispecific antibodies Bs-1 and Bs-2 have the activity of blocking the binding of PCSK9 to LDLR receptor, with an IC50 of about 0.4 nM.

[0239] Table 4. Blocking of the binding of PCSK9 antigen to LDLR-hFc by bispecific antibodies

[0240] Example 4: Inhibition activity of bispecific antibodies on LDL-pHrodo uptake blocked by PCSK9 protein

[0241] To fully investigate the activity of bispecific antibodies against PCSK9 protein, an experiment of antibody restoring LDL-C uptake of hepatocyte was performed. The specific protocol was as follows, Huh7 cells in exponential growth phase were digested and resuspended in FluroBrite TM DMEM medium containing 0.3% BSA, and counted and plated at 6x10^4 cells per well in 96-well plates, so that the cells were cultured in FluroBrite TM DMEM medium containing 0.3% BSA, and counted and plated at 6x10^4 cells per well in 96-well plates, so that the cells were cultured in FluroBrite TM DMEM + 0.3% BSA medium was used to dilute the bispecific antibodies and control antibodies, and human full-length PCSK9 protein was diluted to a protein concentration of 40 mg / ml. 60 ul of gradient bispecific antibodies and 60 ml of PCSK9 protein were mixed in equal proportions and incubated at 37°C for 30 min. The cell culture medium in the 96-well plate was discarded and 100 ml of antibody-antigen pre-incubation solution was added to the 96-well plate, which was incubated at 37°C, 5% CO2 for 4 h. 75 ml of 96-well plate incubation solution was discarded, and 25 ml of pHrodo Green-LDL (Thermo fisher) with a working concentration of 10 mg / ml was added to the well plate, which was incubated at 37°C, 5% CO2 for 3 h. After digestion and resuspension in PBS, the cells were subjected to flow cytometry detection.

[0242] The results are shown in Figure 3 and Table 5, and the bispecific antibodies Bs-1 and BS-2 both have good activity of blocking human PCSK9 protein to inhibit LDL uptake of Huh7 cells.

[0243] Table 5. Activity of bispecific antibodies in restoring low-density lipoprotein fluorescence uptake of Huh7 cells

[0244] Example 5: Experiment of bispecific antibodies blocking ANGPTL3 inhibiting the activity of LPL enzyme

[0245] The following method was used to determine the activity effect of Bs-1 and Bs-2 bispecific antibodies on LPL enzyme. Dilute LPL (sigma, L2254-1KU) to 100 units / ml with dilution buffer (PBS + 2 mg / mL delipidated BSA); dilute human full-length ANGPTL3 with dilution buffer to a concentration of 40 μg / mL; dilute bispecific antibodies with dilution buffer, 100 ug / ml start, then 3-fold gradient dilution of 10 concentrations. Take 20 μL, 20 ul, 50 ul of the diluted LPL, human full-length ANGPTL3 and bispecific antibodies, respectively, into a black non-bottom transparent 96-well plate (Corning). After mixing, incubate in a 37°C incubator for 30 minutes. Dilute DGGR (lipase substrate) with dilution buffer to a concentration of 100 mM, take 10 mL / well into the 96 black bottom plate, mix well and incubate in a 37°C incubator for 30 minutes. Set up control well 1: LPL enzyme + DGGR substrate + ANGPTL3 antigen group (Ag + LPL + DGGR); control well 2: LPL enzyme + DGGR substrate group (LPL + DGGR); control group 3: only substrate group (DGGR). Read Ex540 / Em620 fluorescence on the enzyme marker.

[0246] The results of the enzyme activity test are shown in Figure 4 and Table 6. Both bispecific antibodies Bs-1 and Bs-2 substantially maintain the inhibitory activity of Evinacumab on LPL enzyme to inhibit the activity of ANGPTL3 protein.

[0247] Table 6. Antibody blocking ANGPTL3 activity inhibition of LPL enzyme results

[0248] Example 6: In vivo efficacy study of bispecific antibodies on DIO model mice

[0249] 5-week-old C57BL / 6 mice were used to induce DIO model mice for 10 weeks using high-fat diet (Research diet, item number: D12492), and high-fat feed was continued during the experiment. The day of grouping and administration was designated as D0, and random grouping was performed according to the body weight and serum biochemical LDL content on D0. The model group (n = 6), Bococizumab group, Evinacumab group, Bs-1 group and Bs-2 group were administered at a dose of 20 mg / kg (n = 6). Tail vein administration was performed on D0, D7, D10, D14, a total of 4 times. On D0, D2, D6, D9, D12, D16, D19, D23, blood was collected from the eye orbit of each group of animals to separate serum, and the content of low-density cholesterol (LDL-C) and total triglyceride (TC) in the serum was detected. The specific results are shown in Figures 5A-5B.

[0250] The results showed that Bs-1 and Bs-2 significantly reduced serum LDL-C and TC levels (p<0.01). The dual anti-Bs-1 and Bs-2 regimen had a synergistic effect, demonstrating better simultaneous reduction of both LDL cholesterol and triglycerides than using Bococizumab or Evinacumab alone.

[0251] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3, comprising a first antigen-binding domain that specifically binds to PCSK9 and a second antigen-binding domain that specifically binds to ANGPTL3.

2. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 of claim 1, wherein: the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region set forth in SEQ ID NO: 9; and LCDR1, LCDR2, and LCDR3 in a light chain variable region set forth in SEQ ID NO: 10; and the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region set forth in SEQ ID NO: 11; and LCDR1, LCDR2, and LCDR3 in a light chain variable region set forth in SEQ ID NO: 12; preferably, the amino acid sequences of the HCDRs and the LCDRs are determined according to the Kabat, Abm, Chothia, or IMGT numbering system.

3. The bispecific antibody or antigen binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of claim 1 or 2, wherein, the first antigen-binding domain comprises a heavy chain variable region 1 (VH1) and a light chain variable region 1 (VL1), wherein the VH1 comprises: 1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 13, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 14, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Kabat numbering system; 2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Abm numbering system; 3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 21, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 22, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; wherein the CDRs are defined according to the Chothia numbering system; or 4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 23, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 24, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 25; wherein the CDRs are defined according to the IMGT numbering system; and / or the VL1 comprises: 1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Kabat numbering system; 2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Abm numbering system; 3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the Chothia numbering system; or 4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 27, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18; wherein the CDRs are defined according to the IMGT numbering system.

4. The bispecific antibody or antigen binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of any one of claims 1-3, wherein, the second antigen binding domain comprises a heavy chain variable region 2 (VH2) and a light chain variable region 2 (VL2), wherein the VH2 comprises: 1) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 28, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 29, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Kabat numbering system; 2) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 34, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 35, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Abm numbering system; 3) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 36, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 37, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 30; wherein the CDRs are defined according to the Chothia numbering system; or 4) HCDR1 of the amino acid sequence set forth in SEQ ID NO: 38, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 39, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 40; wherein the CDRs are defined according to the IMGT numbering system; and / or the VL2 comprises: 1) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Kabat numbering system; 2) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Abm numbering system; 3) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the Chothia numbering system; or 4) LCDR1 of the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 of the amino acid sequence set forth in SEQ ID NO: 42, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 33; wherein the CDRs are defined according to the IMGT numbering system.

5. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-4, wherein the first antigen binding domain comprises a heavy chain variable region 1 (VH1) and a light chain variable region 1 (VL1), the VH1 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; the VL1 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10; and the second antigen binding domain comprises a heavy chain variable region 2 (VH2) and a light chain variable region 2 (VL2), the VH2 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11; the VL2 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; Preferably, the first antigen binding domain comprises a heavy chain variable region 1 (VH1) and a light chain variable region 1 (VL1), the VH1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9; the VL1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and the second antigen binding domain comprises a heavy chain variable region 2 (VH2) and a light chain variable region 2 (VL2), the VH2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11; the VL2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

12.

6. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-5, wherein the bispecific antibody or antigen-binding fragment thereof is in the form of a bispecific F(ab')2, Fab-scFv, IgG-scFv, (scFv)2-Fc, bi-nanobody, Tandem Diabody (TandAb), Dual Affinity Retargeting (DART) antibody, DART-Fc, or a combination thereof.

7. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-6, wherein the bispecific antibody or antigen-binding fragment thereof further comprises an Fc region; Preferably, the Fc region is selected from the Fc region of wild-type human IgGl, human IgG2, human IgG3, and human IgG4.

8. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-6, wherein the bispecific antibody or antigen-binding fragment thereof further comprises an Fc region; Preferably, the Fc region is a variant of the Fc region of wild-type human IgGl or human IgG4; Preferably, the Fc region comprises point mutations introduced based on the knob-into-hole technology compared to the Fc region of wild-type human IgGl or human IgG4, and / or the Fc region comprises point mutations P329G, L234A, and L235A compared to the Fc region of wild-type human IgGl or human IgG4; Further preferably, the point mutations introduced based on the knob-into-hole technology comprise: a S354C and a T366W mutation on the "knob” chain, and a Y349C, a T366S, a L368A, and a Y407V mutation on the "hole” chain; wherein the amino acid positions are numbered according to the EU index.

9. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to claim 8, wherein the Fc region comprises a first Fc region fragment (Fc1) and a second Fc region fragment (Fc2), the bispecific antibody or antigen-binding fragment thereof comprising four polypeptide chains: a first polypeptide chain having the following structure from N-terminus to C-terminus: VL1-CL; a second polypeptide chain having the following structure from N-terminus to C-terminus: VH1-CH1-Fc1; a third polypeptide chain having the following structure from N-terminus to C-terminus: VH2-CL-Fc2; and a fourth polypeptide chain having the following structure from N-terminus to C-terminus: VL2-CH1; Preferably, the VH1, VL1, VH2, and VL2 are as defined in any one of claims 3-5; the CH1 region is a CH1 region from wild-type human IgGl, human IgG2, human IgG3, or human IgG4; the CL is a light chain constant region from a human kappa or lambda chain; and the Fc1 and Fc2 are Fc regions from wild-type human IgGl, human IgG2, human IgG3, or human IgG4. the Fc region is a variant of the Fc region of wild-type human IgGl or IgG4, and the Fc region comprises, compared to the Fc region of wild-type human IgGl or IgG4 i) point mutations introduced based on the knob-into-hole technology; and ii) point mutations comprising P329G, L234A and L235A; Further preferably, the point mutations introduced based on the knob-into-hole technology comprise: a S354C and a T366W mutation on the "knob" chain, and a Y349C, a T366S, a L368A and a Y407V mutation on the "hole" chain; wherein the amino acid positions are numbered according to the EU index.

10. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to claim 9, wherein: the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, and the fourth polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; Preferably, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 5, the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 6, and the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

7.

11. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to claim 8, wherein the Fc region comprises a first Fc region fragment (Fc1) and a second Fc region fragment (Fc2), the bispecific antibody or antigen-binding fragment thereof comprising three polypeptide chains: a first polypeptide chain having the following structure from N-terminus to C-terminus: VL1-CL; a second polypeptide chain having the following structure from N-terminus to C-terminus: VH1-CH1-Fc1; and a third polypeptide chain having the following structure from N-terminus to C-terminus: VH2-VL2-Fc2, Preferably, the VH1, VL1, VH2 and VL2 are as defined in any one of claims 3-5; the CH1 region is a CH1 region from wild-type human IgGl, human IgG2, human IgG3 or human IgG4; the CL is a light chain constant region from a human kappa or lambda chain; and the Fc region is a variant of the Fc region of wild-type human IgGl or IgG4, and the Fc region comprises, compared to the Fc region of wild-type human IgGl or IgG4 i) point mutations introduced based on the knob-into-hole technology; and ii) point mutations comprising P329G, L234A and L235A; ii) point mutations comprising P329G, L234A and L235A; Further preferably, the point mutations introduced based on the knob-into-hole technology comprise: a S354C and a T366W mutation on the "knob" chain, and a Y349C, a T366S, a L368A and a Y407V mutation on the "hole" chain; wherein the amino acid positions are numbered according to the EU index.

12. The bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to claim 11, wherein: the first polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, and the third polypeptide chain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; Preferably, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 5, and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

13. An isolated nucleic acid molecule or a set of nucleic acid molecules comprising a nucleotide sequence encoding the bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-12.

14. A vector or a set of vectors comprising the isolated nucleic acid molecule or a set of nucleic acid molecules according to claim 13.

15. A host cell comprising the isolated nucleic acid molecule or a set of nucleic acid molecules according to claim 13, or the vector or a set of vectors according to claim 14.

16. A conjugate comprising the bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-12 and a conjugating moiety linked thereto; Preferably, the conjugating moiety is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin or a radionuclide).

17. A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof that specifically binds to PCSK9 and ANGPTL3 according to any one of claims 1-12, or the isolated nucleic acid molecule or a set of nucleic acid molecules according to claim 13, or the vector or a set of vectors according to claim 14, or the host cell of claim 15, or the conjugate of claim 16, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

18. A diagnostic or therapeutic kit comprising the bispecific antibody or antigen-binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of any one of claims 1-12, or the isolated nucleic acid molecule or set of nucleic acid molecules of claim 13, or the vector or set of vectors of claim 14, or the host cell of claim 15, or the conjugate of claim 16, or the pharmaceutical composition of claim 17, and optionally instructions for use and / or a device for administration.

19. Use of the bispecific antibody or antigen-binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of any one of claims 1-12, or the isolated nucleic acid molecule or set of nucleic acid molecules of claim 13, or the vector or set of vectors of claim 14, or the host cell of claim 15, or the conjugate of claim 16, or the pharmaceutical composition of claim 17, for the manufacture of a medicament for the prevention and / or treatment of a PCSK9 and / or ANGPTL3 related disease, preferably the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis or a cardiovascular disease; preferably the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia or atherosclerotic disease.

20. A method of preventing and / or treating a PCSK9 and / or ANGPTL3 related disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody or antigen-binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of any one of claims 1-12, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 13, the vector or set of vectors of claim 14, the host cell of claim 15, the conjugate of claim 16 or the pharmaceutical composition of claim 17; preferably the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, dyslipidemia, nephrotic syndrome, hypothyroidism, obesity, diabetes, lipodystrophy, nonalcoholic steatohepatitis (NASH), atherosclerosis or a cardiovascular disease; preferably the PCSK9 and / or ANGPTL3 related disease is selected from hypercholesterolemia, hyperlipidemia or atherosclerotic disease.

21. A method of reducing a cholesterol level and / or a triglyceride level in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific antibody or antigen binding fragment thereof that specifically binds PCSK9 and ANGPTL3 of any one of claims 1-12, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 13, the vector or set of vectors of claim 14, the host cell of claim 15, the conjugate of claim 16, or the pharmaceutical composition of claim 17; Preferably, the method reduces a cholesterol level and / or a triglyceride level in the blood of the subject; Preferably, the method reduces a cholesterol level and / or a triglyceride level in the serum of the subject.

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