Anti-ANGPTL3 antibody and application thereof

Abstract

According to the anti-ANGPTL3 antibody and the application thereof, the antibody can be specifically combined with ANGPTL3, inhibition of the ANGPTL3 on lipoprotein lipase activity is effectively relieved, and lipid metabolism is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to anti-ANGPTL3 antibodies and applications thereof. Background Art

[0002] Angiopoietin-like protein 3 (ANGPTL3) is a member of the angiopoietin and angiopoietin-like protein family. It is named because it shares common structural features with members of the family, namely, an amino-terminal signal peptide associated with secretion, a helical domain that mediates the formation of homo-oligomers, a short connecting peptide and a fibrinogen-like domain at the carboxyl terminus that mediates ligand activity.

[0003] ANGPTL3 is a secretory protein whose mRNA is mainly expressed in the liver of humans and mice. Previous studies have shown that the biological effects of ANGPTL3 are manifested in two aspects: regulating angiogenesis and lipid metabolism. ANGPTL3 can reduce lipid metabolism by inhibiting the activity of lipoprotein lipase (LPL). Summary of the invention

[0004] The present invention provides anti-ANGPTL3 antibodies or antigen-binding fragments, which can specifically bind to ANGPTL3. In some embodiments, the anti-ANGPTL3 antibodies or antigen-binding fragments can specifically bind to the N-terminal helical-like domain of the ANGPTL3 antigen. In some embodiments, the anti-ANGPTL3 antibodies or antigen-binding fragments are inhibitory antibodies. In some embodiments, the anti-ANGPTL3 antibodies or antigen-binding fragments effectively alleviate the inhibition of ANGPTL3 on lipoprotein lipase activity and promote lipid metabolism.

[0005] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to ANGPTL3 and comprises one or more of the following amino acid sequences:

[0006] (a) HCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6, or consists of the same;

[0007] (b) HCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12, or consists of the same;

[0008] (c) HCDR3, which comprises the amino acid sequence as shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14, or consists of the same;

[0009] (d) LCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 15-17, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 15-17, or consists of the same;

[0010] (e) LCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-20, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 18-20, or consists of the same;

[0011] (f) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 21 or 22, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 21 or 22, or consists of the same.

[0012] In some embodiments, the antibody or antigen-binding fragment specifically binds ANGPTL3 and comprises:

[0013] (a) HCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6, or consists of the same; and

[0014] (b) HCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12, or consists of the same; and

[0015] (c) HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14, or consists of the same.

[0016] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:7, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0017] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:2, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:8, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0018] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:9, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:14.

[0019] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:14.

[0020] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:11, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:14.

[0021] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:6, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:12, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:14.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds ANGPTL3 and comprises:

[0023] (d) LCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 15-17, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 15-17, or consists of the same; and

[0024] (e) LCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-20, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 18-20, or consists of the same; and

[0025] (f) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 21 or 22, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 21 or 22, or consists of the same.

[0026] In some embodiments, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:15, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:18, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:21.

[0027] In some embodiments, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:16, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:19, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:22.

[0028] In some embodiments, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:17, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:20, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:21.

[0029] In some embodiments, the antibody or antigen-binding fragment specifically binds ANGPTL3 and comprises:

[0030] (a) HCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6, or consists of the same; and

[0031] (b) HCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12, or consists of the same; and

[0032] (c) HCDR3, which comprises the amino acid sequence as shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14, or consists of the same;

[0033] (d) LCDR1, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 15-17, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 15-17, or consists of the same; and

[0034] (e) LCDR2, which comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18-20, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 18-20, or consists of the same; and

[0035] (f) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 21 or 22, or an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 21 or 22, or consists of the same.

[0036] In some embodiments, the substitutions are conservative amino acid substitutions.

[0037] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:13, LCDR1 as shown in SEQ ID NO:15, LCDR2 as shown in SEQ ID NO:18 and LCDR3 as shown in SEQ ID NO:21.

[0038] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO:13, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:19 and LCDR3 as shown in SEQ ID NO:22.

[0039] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:9, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21.

[0040] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21.

[0041] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21.

[0042] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21.

[0043] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the antibody or antigen-binding fragment that specifically binds to ANGPTL3 of the present invention may respectively comprise any one or more amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 listed in Tables 1-2, or may be any combination of the amino acid sequences corresponding to the CDRs in Tables 1-2.

[0044] Table 1 Amino acid sequence of HCDR (using Kabat classification)

[0045]

[0046]

[0047] Table 2 Amino acid sequence of LCDR (using Kabat classification)

[0048]

[0049] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises, or consists of, an amino acid sequence as shown in any one of SEQ ID NOs: 40, 23-28, a sequence having at least 80% identity to a sequence as shown in any one of SEQ ID NOs: 40, 23-28, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 40, 23-28; and / or

[0050] The light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, a sequence having at least 80% identity with the sequence shown in SEQ ID NOs: 29-31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 29-31, or consists of the same.

[0051] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NOs: 40, 23-28, and / or the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NOs: 29-31.

[0052] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:23, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:29.

[0053] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:24, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:30.

[0054] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:25, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:31.

[0055] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:26, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:31.

[0056] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:27, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:31.

[0057] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:28, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:31.

[0058] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:40, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:30.

[0059] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0060] In some embodiments, Fc is a variant Fc region. In some embodiments, relative to the parent Fc region, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions. In some embodiments, relative to the parent Fc region activity, the amino acid modifications in the Fc region change the effector function activity. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization or cell binding. In some embodiments, relative to the parent Fc region, the Fc region amino acid modifications may change the affinity of the variant Fc region for FcγR (Fcγ receptor). In some embodiments, the variant Fc region may enhance the half-life of the antibody. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A, L234A or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).

[0061] In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, Fab' or F(ab) 2. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0062] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises, or consists of, an amino acid sequence as shown in any one of SEQ ID NOs: 34-36, or a sequence having at least 80% identity to a sequence shown in any one of SEQ ID NOs: 34-36, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence shown in any one of SEQ ID NOs: 34-36; and / or

[0063] The light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:37, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO:37, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:37, or consists of the same.

[0064] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 34, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 37. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 35, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 37. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 36, and the light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 37.

[0065] In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a heavy chain variable region and a heavy chain constant region, the heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 40, 23-28, a sequence having at least 80% identity with a sequence as shown in any one of SEQ ID NOs: 40, 23-28, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 40, 23-28, or consisting thereof; the heavy chain constant region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 34-36, or a sequence having at least 80% identity with a sequence as shown in any one of SEQ ID NOs: 34-36, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 34-36, or consisting thereof;

[0066] and / or

[0067] The light chain of the antibody or antigen-binding fragment comprises a light chain variable region and a light chain constant region, the light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 29-31, a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 29-31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 29-31; the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 37, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 37, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 37, or consists of the same.

[0068] In some embodiments, the heavy chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 40, 23-28, and the heavy chain constant region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 34-36;

[0069] The light chain variable region comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 29-31, and the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 37.

[0070] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:23 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0071] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:24 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0072] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:25 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0073] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:26 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0074] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:27 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0075] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:28 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0076] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:40 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:35; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0077] In some embodiments, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment that specifically binds to ANGPTL3 described in the present invention can be any combination of the amino acid sequences corresponding to the variable regions in Table 3.

[0078] Table 3 Amino acid sequences of variable regions

[0079]

[0080]

[0081] Table 4 Constant regions of anti-ANGPTL3 antibodies

[0082]

[0083]

[0084] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody or antigen-binding fragment (eg, a bispecific antibody or antigen-binding fragment).

[0085] In some embodiments, the antibody has two identical heavy chains and two identical light chains, and the Fc regions are paired to form disulfide bonds.

[0086] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

[0087] The present invention also provides a nucleic acid encoding the antibody or antigen binding fragment, or a portion thereof (such as a heavy chain variable region or a light chain variable region). In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, the nucleic acid sequence is selected from the nucleic acid sequences listed in Table 9.

[0088] The present invention also provides a vector comprising the nucleic acid. In some embodiments, the vector comprising the nucleic acid is a nucleic acid fragment, a plasmid, a phage or a virus. In some embodiments, the vector is an isolated vector.

[0089] The present invention also provides a host cell comprising the nucleic acid or vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a HEK cell (such as a HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell or a mouse L cell.

[0090] The present invention also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient.

[0091] The present invention also provides methods and uses for preventing or treating ANGPTL3-related diseases. In some embodiments, a method for treating an ANGPTL3-related disease is provided, the method comprising administering an effective dose of the anti-ANGPTL3 antibody or antigen-binding fragment to a patient. In some embodiments, use of the anti-ANGPTL3 antibody or antigen-binding fragment for treating an ANGPTL3-related disease is provided. In some embodiments, use of the anti-ANGPTL3 antibody or antigen-binding fragment for preparing a medicament for treating an ANGPTL3-related disease is provided.

[0092] In some embodiments, the ANGPTL3-related diseases include, but are not limited to, diseases involving lipid metabolism, such as hyperlipidemia, hyperlipoproteinemia and dyslipidemia (including atherogenic dyslipidemia, diabetic dyslipidemia), hypertriglyceridemia (including severe hypertriglyceridemia of TG>1000 mg / dL), hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), fat metabolism disorder, lipoatrophy, etc. The causes of the above diseases include, for example, decreased LPL activity and / or LPL deficiency, decreased LDL receptor (LDLR) activity and / or LDL receptor deficiency (for example, LDLR- / - homozygous familial hypercholesterolemia), ApoC2 changes, ApoE deficiency, ApoB increase, increased very low density lipoprotein (VLDL) production and / or decreased elimination, certain drug treatments (for example, dyslipidemia caused by glucocorticoid treatment), any genetic tendency, diet, lifestyle, etc.

[0093] In some embodiments, the ANGPTL3-related disease is a disease associated with or caused by hyperlipidemia, hyperlipoproteinemia and / or dyslipidemia, including but not limited to cardiovascular diseases or disorders, such as atherosclerosis, aneurysm, hypertension, angina pectoris, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, peripheral vascular disease, acute pancreatitis, non-alcoholic steatohepatitis (NASH), blood sugar disorders (such as diabetes), obesity, etc.

[0094] In some embodiments, the ANGPTL3-related disease is a cancer / tumor and / or non-tumor angiogenesis-related disease or condition, including ocular angiogenesis diseases or disorders (such as age-related macular degeneration, central retinal vein occlusion, branch retinal vein occlusion, diabetic retinopathy, retinopathy of prematurity, etc.), inflammatory diseases or disorders (such as arthritis, rheumatoid arthritis (RA), psoriasis, etc.).

[0095] The present invention also provides diagnostic methods and uses. In some embodiments, a method for detecting the expression of ANGPTL3 in a sample is provided, wherein the sample is contacted with the antibody or antigen-binding fragment, so that the antibody or antigen-binding fragment binds to ANGPTL3, and the binding thereof, i.e., the content of ANGPTL3 in the sample is detected. In some embodiments, the use of the antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing an ANGPTL3-related disease is provided. In some embodiments, a diagnostic or prognostic kit comprising the anti-ANGPTL3 antibody or antigen-binding fragment is provided.

[0096] The present invention provides an anti-ANGPTL3 antibody and its application. The antibody or antigen-binding fragment of the present invention can specifically bind to ANGPTL3, effectively alleviate the inhibition of ANGPTL3 on lipoprotein lipase activity, and promote lipid metabolism. The antibody or antigen-binding fragment of the present invention is used to treat ANGPTL3-related diseases, and is also used for the diagnosis and prognosis of ANGPTL3-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1A Schematic diagram of TG levels in mice within 19 days after administration;

[0098] Figure 1B Schematic diagram of the Chol level in mice 19 days after administration;

[0099] Figure 1C It shows the TG levels of mice on the 7th day after administration;

[0100] Figure 1D It shows the Chol level of mice on the 7th day after administration;

[0101] Figure 1E The figure shows the LDL-C level of mice on day 7 after administration.

[0102] the term

[0103] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0104] definition

[0105] It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0106] As used herein, the terms "comprising" or "including" mean that compositions and methods, etc. include the listed elements, such as components or steps, but do not exclude others. "Essentially consisting of" means that compositions and methods exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the composition or method. "Consisting of" means excluding elements not specifically listed.

[0107] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule formed by amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains, and the term "polypeptide" can be used to replace any of the above terms, or to be used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or non-natural amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.

[0108] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0109] "Conservative amino acid substitution" refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially change the functional properties of a protein. Examples of amino acid classes containing side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0110] The number of amino acids in “conservative amino acid substitutions of VL, VH” is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values ​​(including endpoints) or any value therein. The number of amino acids in the “conservative amino acid substitution in the heavy chain constant region, the light chain constant region, the heavy chain or the light chain” is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range (including endpoints) between any two of these values, or any value therein.

[0111] The term "isolated" used in the present invention for cells, nucleic acids, polypeptides, antibodies, etc., such as "isolated" DNA, RNA, polypeptides, antibodies refer to molecules separated from one or more of other components in the natural environment of the cell, such as DNA or RNA. The term "isolated" used in the present invention also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or cell culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in a natural state and will not exist in a natural state. The term "isolated" is also used in the present invention to refer to cells or polypeptides separated from other cell proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are usually prepared by at least one purification step. In some embodiments, the purity of isolated nucleic acids, polypeptides, antibodies, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including endpoints) between any two of these values ​​or any value therein.

[0112] The term "recombinant" in relation to a polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not occur in nature, and by way of non-limiting example, can be produced by combination of polynucleotides or polypeptides that do not normally occur.

[0113] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0114] "At least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0115] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" to another sequence means that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and identity percentage or sequence identity can be determined using visual inspection or software programs known in the art, such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage identities and encode polypeptides having the same or similar biological activity.

[0116] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) when the polynucleotide is RNA. A "polynucleotide sequence" can be represented by the alphabetic representation of the polynucleotide molecule. This alphabetic representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0117] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of unrestricted polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If the modification is present, the structural modification of the nucleotides can be performed before or after assembling the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of the present disclosure includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0118] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which, in its native state or when manipulated by methods well known to those skilled in the art, can produce the polypeptide and / or its fragments via transcription and / or translation.

[0119] The antibodies and antigen-binding fragments disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, single-chain Fv (scFv).

[0120] "Antibody", "antigen binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Antibodies and antigen binding fragments include but are not limited to the complementary determining region (CDR) of a heavy chain or light chain or its ligand binding portion, a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain constant region (CH), a light chain constant region (CL), a framework region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR region includes the CDR region (LCDR1-3) of the light chain and the CDR region (HCDR1-3) of the heavy chain. Antibodies and antigen binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes of one or more (such as two) antigens. Antibodies or antigen binding fragments that specifically recognize and bind to multiple (such as two) antigens can be referred to as multispecific (such as bispecific) antibodies or antigen binding fragments.

[0121] The term "antibody fragment" or "antigen binding fragment" refers to a portion of an antibody, and the composition of the antibody fragment of the present invention can be similar to F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. in a monospecific antibody fragment. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isomers, and bivalent antibodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0122] "Single chain variable fragment" or "scFv" refers to a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin. In some aspects, these regions are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to increase flexibility, as well as rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has been removed of the constant region and introduced with a linker, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, described in U.S. Patent No. 5,892,019.

[0123] The term "antibody" includes a wide variety of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that the categories of heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4) among them. The nature of the chain determines that the "type" of the antibody is IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been fully characterized and the functional specificity conferred is also known. All immunoglobulin species are within the scope of protection disclosed in the present invention. In some embodiments, the immunoglobulin molecule is an IgG species.

[0124] Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can be combined with a κ or λ light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light chains and heavy chains are bound by covalent bonds, and the "tail" parts of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is V λ .

[0125] The variable regions of the light (VL) and heavy (VH) chains determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant regions increases as they become more distal to the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0126] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-continuous amino acid sequences that specifically bind to the antigen, forming the antigen binding domain, assuming that the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, known as the "framework" region, show less intermolecular variability. The framework region mostly adopts a β-sheet conformation, and the CDRs form a loop structure connected to it, or in some cases form part of the β-sheet structure. Therefore, the framework region positions the CDRs in the correct orientation by forming a scaffold through interchain non-covalent interactions. The antigen binding domain with specifically positioned CDRs forms a surface complementary to the epitope on the antigen, which promotes non-covalent binding of the antibody and its antigenic epitope. For a given heavy chain or light chain variable region, one of ordinary skill in the art can identify the amino acids comprising the CDR and framework regions by known methods (see Kabat, E., et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1991) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0127] In the case where there are two or more definitions of a term used and / or accepted in the art, unless explicitly indicated to the contrary, the definition of the term used herein includes all of these meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1991) and Chothia et al. in J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference in their entirety.

[0128] The CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, the use of either definition to refer to the CDRs of antibodies or their variants is within the scope of the present invention. The exact residue numbering comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues a CDR comprises based on the variable region amino acid sequence of an antibody.

[0129] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. One of ordinary skill in the art can apply the "Kabat numbering" system to any variable region sequence independent of other experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1991). Antibodies can also use the EU or Chothia numbering system.

[0130] The antibodies disclosed in the present invention can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse or chicken antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

[0131] The "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., an upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another embodiment, a portion of the heavy chain can include a chimeric hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0132] "Light chain constant region" includes a portion of the amino acid sequence from the antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. "Light chain-heavy chain pair" refers to a collection of light chains and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are connected in a "Y" configuration by disulfide bonds, where the light chain starts from the "Y" mouth and continues through the variable region to surround the heavy chain.

[0133] The "VH domain" includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the "CH1 domain" includes the first (most amino-terminal) constant region of the immunoglobulin heavy chain. In the complete natural IgG molecule, N297 in each of the two CH2 domains is connected to a branched carbohydrate chain. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues. The "hinge region" includes a partial heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region contains about 25 residues and is tough, so that the two N-terminal antigen binding regions can move independently. The hinge region can be subdivided into three different domains: upper, middle and lower hinge domains.

[0134] "Disulfide bond" refers to a covalent bond formed between two sulfur atoms. A thiol group of cysteine ​​can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0135] "Chimeric antibody" refers to any antibody whose variable region is obtained or derived from a first species, and whose constant region (which may be complete, partial or modified) is derived from a second species. In certain embodiments, the variable region is from a non-human source (e.g., mouse or primate), and the constant region is from a human source.

[0136] "Specific binding" or "specific for..." generally refers to the formation of a relatively stable complex between an antibody or antigen-binding fragment and a specific antigen through the complementary binding of its antigen-binding domain to the epitope. "Specificity" can be expressed by the relative affinity of the antibody or antigen-binding fragment to a specific antigen or epitope. For example, if antibody "A" has a greater relative affinity than antibody "B" for the same antigen, it can be considered that antibody "A" has a higher specificity for the antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant (KD), and a smaller KD means a tighter binding. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, biofilm optical interferometry, etc. "Specifically binding" antibodies to antigen a include antibodies with an equilibrium dissociation constant KD of less than or equal to about 100nM, less than or equal to about 10nM, less than or equal to about 5nM, and less than or equal to about 1nM with antigen a.

[0137] "Treatment" refers to both therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent, slow down, ameliorate or stop undesirable physiological changes or disorders, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or disappearance of the disease state (whether partial or complete), extension of the expected survival period compared to not receiving treatment, etc. Patients in need of treatment include patients who already have a disease or disorder, patients who are susceptible to a disease or disorder, or patients in need of prevention of the disease or disorder, and patients who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions disclosed in the present invention for detection, diagnostic procedures and / or treatment.

[0138] "Patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.

[0139] "About" refers to the normal error range of the corresponding numerical value that is easily known to those skilled in the relevant technical field. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0140] “EC 50 "Concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.

[0141] The "parent Fc region" in the present invention may be a naturally occurring Fc region, and the gene encoding the Fc region may be from humans, mice, rabbits, camels, monkeys, preferably humans and mice; for example, the parent Fc region is the Fc region in SEQ ID NO: 34, 35 or 36.

[0142] The relevant descriptions of publications mentioned herein are incorporated by reference in their entirety.

[0143] Anti-ANGPTL3 Antibodies

[0144] The present invention provides antibodies or antigen-binding fragments with high affinity for ANGPTL3 protein. The anti-ANGPTL3 antibodies or antigen-binding fragments of the present invention exhibit effective binding activity and biological activity and are used for treatment and diagnosis. For example, these antibodies or antigen-binding fragments can effectively alleviate the inhibition of ANGPTL3 on lipoprotein lipase activity and promote lipid metabolism.

[0145] In some embodiments, the antigen binding fragment is a scFv.

[0146] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in any one of SEQ ID NOs: 23-28 or 40, and the VL in the scFv comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29-31.

[0147] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:23, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:29.

[0148] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:24, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:30.

[0149] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:25, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:31.

[0150] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:26, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:31.

[0151] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:27, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:31.

[0152] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:28, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:31.

[0153] In some embodiments, the VH in the scFv comprises an amino acid sequence as shown in SEQ ID NO:40, and the VL in the scFv comprises an amino acid sequence as shown in SEQ ID NO:30.

[0154] In some embodiments, the linker connecting the heavy chain variable region and the light chain variable region in the scFv fragment is (G4S) n In some embodiments, n is 1, 2, 3, 4 or 5.

[0155] In some embodiments, the antibody contains two heavy chains with identical sequences and two light chains with identical sequences. In some embodiments, the antibody is of IgG type.

[0156] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:23 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0157] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:24 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0158] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:25 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0159] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:26 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0160] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:27 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0161] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:28 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0162] In some embodiments, the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:40 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:35; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:37.

[0163] It should also be understood by those skilled in the art that the antibody or antigen-binding fragment sequence disclosed in the present invention can be replaced, and its amino acid sequence after replacement is different from the naturally occurring amino acid sequence of the antibody. For example, the replaced amino acid sequence can be similar to the starting sequence, such as having a certain ratio of identity with the starting sequence, such as it can be about 80%, about 85%, about 90%, about 95%, about 98%, about 99% identical to the starting sequence, or a range (including endpoints) between any two of these values ​​or any value therein.

[0164] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence with one or more modification groups. For example, the antibody or antigen-binding fragment disclosed in the present invention may comprise a flexible linker sequence, or may be modified to add a functional group (e.g., PEG, a drug, a toxin, or a label).

[0165] The antibodies and antigen binding fragments disclosed in the present invention include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody or antigen binding fragment, wherein the covalent attachment does not prevent the antibody or antigen binding fragment from binding to the epitope. Including but not limited to the following examples, the antibody or antigen binding fragment can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytically cleaved, attached to cell ligands or other proteins, etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0166] In some embodiments, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0167] The antibody or antigen binding fragment can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody or antigen binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0168] Method for preparing antibody and polynucleotide encoding antibody

[0169] The present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention. The polynucleotides disclosed in the present invention may encode a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, a portion of a light chain variable region, a heavy chain or a light chain, etc. Methods for preparing antibodies are well known in the art and are described in the present invention.

[0170] In certain embodiments, the prepared antibodies do not cause harmful immune responses in the animal (e.g., human) to be treated. In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed in the present invention are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen binding properties of the parent antibody but have low immunogenicity in humans. It can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into the human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementary determining regions (CDRs) into the human framework and constant region, retaining or not retaining key framework residues; or (c) transplanting the entire non-human variable region, but "hiding" them by replacing surface residues with human-like parts. Usually the framework residues in the human framework region will be replaced by corresponding residues from the CDR donor antibody, such as residues that can improve antigen binding. These framework replacements can be identified by methods well known in the art, such as by simulating the interaction of CDR and framework residues to identify framework residues that play an important role in antigen binding and by sequence comparison to identify abnormal framework residues at specific positions. (See U.S. Pat. No. 5,585,089; the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (EP 239,400; WO 91 / 09967; U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596), and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0171] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes changing antibodies to modify T cell epitopes (see, for example, WO / 9852976A1 and WO / 0034317A2). For example, the heavy chain variable region sequence and the light chain variable region sequence from the starting antibody are analyzed, and a "map" of human T cell epitopes from each variable region is generated, showing the position of the epitope relative to the complementary determining region (CDRs) and other key residues in the sequence. Analyze a single T cell epitope from the T cell epitope map to identify selectable amino acid substitutions with a lower risk of changing antibody activity. Design a series of optional heavy chain variable region sequences and light chain variable region sequences containing amino acid substitution combinations, and then incorporate these sequences into a series of binding polypeptides. Then clone the genes of the complete heavy and light chains containing the modified variable region and human constant region into an expression vector, and then transfer the plasmid into a cell line to produce a complete antibody. Then compare antibodies in suitable biochemical and biological experiments to identify the best antibodies.

[0172] The binding specificity of the antibodies or antigen-binding fragments disclosed in the present invention can be detected by in vitro assays, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0173] The preparation of scFv can refer to the technology of producing single chain units (U.S. Patent No. 4,694,778). The heavy chain and light chain fragments of the Fv region are bridged by amino acids to form a single chain unit, producing a single chain fusion peptide. The technology of assembling functional Fv fragments in E. coli can also be used.

[0174] Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498. For certain uses including the use of antibodies in humans and in vitro detection experiments, chimeric antibodies, humanized antibodies, or fully human antibodies can be used. Chimeric antibodies are a class of molecules in which different parts of an antibody are derived from different animal species, such as antibodies with variable regions of a mouse monoclonal antibody and constant regions of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.

[0175] In addition, techniques for generating primate antibodies containing monkey variable region and human constant region sequences are described in US Pat. Nos. 5,658,570, 5,693,780, and 5,756,096, the entire contents of each of which are incorporated herein by reference.

[0176] Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. Reference may also be made to U.S. Patents 4,444,887 and 4,716,111, and PCT Publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO91 / 10741, the entire contents of each of which are incorporated herein by reference.

[0177] In another embodiment, using conventional methods (e.g., using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains), DNA encoding the desired monoclonal antibody can be isolated and sequenced. Isolated and subcloned hybridoma cells can be used as the source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a prokaryotic or eukaryotic host cell such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce other immunoglobulins. The isolated DNA (which can be synthetic as described herein) can also be used to prepare sequences of constant and variable regions of antibodies, as described in U.S. Patent No. 5,658,570, the entire contents of which are incorporated herein by reference. The method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR technology using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Patent No. 5,658,570.

[0178] In addition, using conventional recombinant DNA technology, one or more CDRs of the antibodies of the present invention can be inserted into the framework region, for example, into the human framework region to construct a humanized non-fully human antibody. The framework region can be a naturally occurring or shared framework region, preferably a human framework region. Some polynucleotides can encode antibodies that specifically bind to at least one epitope of the target antigen produced by the combination of the framework region and the CDR. One or more amino acid substitutions can be made in the framework region, and amino acid substitutions that can improve the binding of the antibody to its antigen can be selected. In addition, this method can be used to replace or delete cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other changes to polynucleotides within the technical scope of the art are also encompassed in the present invention.

[0179] Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines that produce antibodies can be selected, constructed and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

[0180] In some embodiments, the DNA encoding the antibody can be designed and synthesized according to the antibody amino acid sequence described herein in a conventional manner, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium to produce a monoclonal antibody. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoters of cytomegalovirus, and promoters of other cells such as the actin promoter can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1 (+ / -), pcDNA / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0181] In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein.

[0182] In addition, mutations can be introduced into the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations that result in amino acid substitutions. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid replacements, less than 30 amino acid replacements, less than 25 amino acid replacements, less than 20 amino acid replacements, less than 15 amino acid replacements, less than 10 amino acid replacements, less than 5 amino acid replacements, less than 4 amino acid replacements, less than 3 amino acid replacements, or less than 2 amino acid replacements relative to the original heavy chain variable region and light chain variable region. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutations, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity.

[0183] Anti-ANGPTL3 antibodies can be screened by various methods known in the art. For example, a fully human phage display library is used to screen scFvs that bind to the ANGPTL3 antigen, and the screened VH and VL are assembled with the heavy chain constant region and light chain constant region of human IgG, respectively, to obtain anti-ANGPTL3 antibodies, which are fully human antibodies. Another example is to immunize mice with ANGPTL3 antigen, fuse the spleen cells of the immunized mice with SP2 / 0 myeloma cells, obtain hybridoma cells, screen positive clones that bind to the ANGPTL3 antigen, obtain anti-ANGPTL3 antibodies, and assemble the VH and VL of the antibody with the heavy chain constant region and light chain constant region of human IgG, respectively, to obtain anti-ANGPTL3 chimeric antibodies.

[0184] Treatment

[0185] The present invention also provides methods and uses of treatment. In some embodiments, a method for treating an ANGPTL3-related disease is provided, the method comprising administering an effective dose of an anti-ANGPTL3 antibody or antigen-binding fragment to a patient in need thereof. In some embodiments, use of an anti-ANGPTL3 antibody or antigen-binding fragment for treating an ANGPTL3-related disease is provided. In some embodiments, use of the anti-ANGPTL3 antibody or antigen-binding fragment for preparing a medicament for treating an ANGPTL3-related disease is provided.

[0186] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody or derivative used, the patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are subject to the judgment of a medical care provider, who is within the scope of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the anti-ANGPTL3 antibody is administered to a patient at a dosage of 0.01 mg / kg to 20 mg / kg of the patient's body weight, daily to once every six months.

[0187] The methods of administration of antibodies, Fabs or derivatives thereof include, but are not limited to, intradermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal, epidural and oral injection. The pharmaceutical composition can be administered by any convenient route, such as by infusion or bolus, absorbed by epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Therefore, the pharmaceutical composition containing the antibodies, Fabs or derivatives of the present invention can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneal, externally (e.g., by powder, ointment, drops or transdermal patch), orally or by oral or nasal spray.

[0188] The term "parenteral" used in the present invention refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The administration method can be systemic or local.

[0189] In some embodiments, the compositions of the invention comprise nucleic acids or polynucleotides encoding antibodies or antigen-binding fragments thereof, which can be administered in vivo to promote expression of the protein encoded by them by constructing them as part of an appropriate nucleic acid expression vector, and then administering the above-mentioned part of the vector to make it intracellular, for example, by using retroviral vectors (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection agents, or by administering them in conjunction with homeobox peptides known to enter the nucleus, etc. Alternatively, the nucleic acid can be introduced into the cell and integrated into the host cell DNA for expression by homologous recombination.

[0190] Various known delivery systems can be used to administer the antibodies, antigen-binding fragments or derivatives thereof of the present invention or polynucleotides encoding the same, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of nucleic acids as part of a retrovirus or other vector, etc.

[0191] Combination therapy

[0192] In some embodiments, the anti-ANGPTL3 antibodies or antigen-binding fragments of the present invention can be combined with other treatment or prevention regimens, including administration of one or more antibodies or antigen-binding fragments of the present invention, and one or more other therapeutic agents or methods to treat ANGPTL3-related diseases. In some embodiments, other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, immunotherapy, and surgical therapy. For combined treatment, the anti-ANGPTL3 antibody can be administered simultaneously or separately with the other therapeutic agent. When administered separately, the anti-ANGPTL3 antibody or antigen-binding fragment can be administered before or after administration of another other therapeutic agent.

[0193] In some embodiments, the therapeutic agent is a non-antibody drug. In some embodiments, the therapeutic agent is a drug that reduces the level of one or more serum lipids. In some embodiments, the therapeutic agent includes, but is not limited to, cholesterol synthesis inhibitors (statins), such as HMG-CoA reductase inhibitors (such as lovastatin, simvastatin, pravastatin and fluvastatin); bile sequestrants, such as cholestyramine and other resins; VLDL secretion inhibitors, such as nicotinic acid; lipophilic antioxidants, such as probucol; acyl-CoA cholesterol acyltransferase inhibitors; farnesoid X receptor antagonists; sterol-regulated binding protein cleavage activation protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; and ApoE-related peptides. In some embodiments, the therapeutic agent is a drug that increases high-density lipoprotein (HDL). In some embodiments, cholesterol ester transfer protein (CETP) inhibitors.

[0194] Pharmaceutical composition

[0195] The present invention also provides a pharmaceutical composition. Such a composition comprises an effective dose of an anti-ANGPTL3 antibody or antigen-binding fragment, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1%-90% of an anti-ANGPTL3 antibody or antigen-binding fragment. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0196] In some embodiments, the term "pharmaceutically acceptable" refers to substances for animals, particularly for humans, that are approved by a government regulatory agency or listed in a generally recognized pharmacopoeia. In addition, "pharmaceutically acceptable excipients" generally refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, etc.

[0197] The term "excipient" refers to a diluent, adjuvant, excipient or carrier that can be applied to a patient together with the active ingredient. This type of pharmaceutical carrier can be a sterile liquid, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tension such as dextran are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain clinically effective doses of antibodies or antigen-binding fragments, preferably in purified form, together with an appropriate amount of adjuvants to provide a dosage form suitable for the patient. The preparation should be suitable for administration mode. The parent preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.

[0198] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection in human body according to conventional steps. The composition for intravenous administration is usually a solution in a sterile isotonic aqueous buffer. The composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally speaking, the active ingredient is supplied alone or mixed together in a unit dose form, such as in a sealed container (such as an ampoule or a pouch) that indicates the amount of active agent in the form of a dry lyophilized powder or anhydrous concentrate. In the case of administering the composition by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be used so that the active ingredient can be mixed before administration.

[0199] The antibodies described herein may be neutral, i.e., substantially free of net charge, such as when the pH of the antibody-containing composition is the isoelectric point of the antibody, the antibody is electrically neutral. The antibodies described herein may exist in the form of positive ions, such as when the pH is below the isoelectric point, the antibody molecule as a whole exhibits a positive charge. The antibodies described herein may exist in the form of negative ions, such as when the pH is above the isoelectric point, the antibody molecule as a whole exhibits a negative charge. DETAILED DESCRIPTION

[0200] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications or adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0201] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0202] Example 1 scFv preparation

[0203] In MagicMedia containing ampicillin (BBI) TM Phage-infected Escherichia coli TG1 expressing scFv(12-12), scFv(12-21), scFv(34-12), scFv(34-15), scFv(34-20), scFv(34-25), and scFv(12-24) was cultured in Escherichia coli expression medium (Invtrogen).

[0204] The scFv isolated from E. coli strain 12-12 is scFv (12-12), the scFv isolated from strain 12-21 is scFv (12-21), the scFv isolated from strain 34-12 is scFv (34-12), the scFv isolated from strain 34-15 is scFv (34-15), the scFv isolated from strain 34-20 is scFv (34-20), the scFv isolated from strain 34-25 is scFv (34-25); the scFv isolated from strain 12-24 is scFv (12-24). In scFV, from the N-terminus to the C-terminus, VH is connected to VL through a linker (GGGGS) 2. The composition of scFv is shown in Table 5, and the amino acid sequences of CDR, VH and VL are shown in Tables 1-3.

[0205] Table 5 Composition of scFv

[0206] serial number VH sequence number (SEQ ID NO) VL Serial Number scFv(12-12) 23 29 scFv(12-21) 24 30 scFv(34-12) 25 31 scFv(34-15) 26 31 scFv(34-20) 27 31 scFv(34-25) 28 31 scFv(12-24) 40 30

[0207] After the above-mentioned E. coli strain was cultured at 37°C to the logarithmic phase, the culture temperature was adjusted to 28°C for overnight culture; the next day, the supernatant was centrifuged at 4000 rpm and 4°C for 30 minutes (min), and the diluted supernatant (the diluent was PBST containing 1% BSA) was transferred to the ELISA plate coated with hANGPTL3-His (the coating process was: the concentration of hANGPTL3-His was 2.5 μg / ml, 100 μl / well, the coating buffer was PBS (pH7.2-7.4), and the coating was performed at 4°C overnight; PBS containing 3% BSA was used for blocking for 2 hours), and incubated at 37°C for 1 hour (h); unbound antibodies were washed away with PBST (containing 1% BSA), and anti-myc HRP (Abcam), placed at 37℃ for 1 hour (h); unbound antibodies were washed with PBST, TMB colorimetric solution was added, placed at 37℃ for 5-10 minutes (min), and then 0.1M sulfuric acid was added to terminate the reaction; the absorbance value was read at a wavelength of 450nm on an ELISA reader. Positive was judged as an OD value higher than 1.0. The ELISA test results are shown in Table 6.

[0208] Table 6 ELISA test results

[0209]

[0210] Example 2 Antibody Preparation Method

[0211] The anti-ANGPTL3 antibodies prepared in this example include antibodies 12-12, 12-21, 34-12, 34-15, 34-20, 34-25, and 12-24. The composition of the antibodies is shown in Table 7, the variable region is shown in Table 3, the constant region is shown in Table 4 (the Fc region is underlined), and the nucleic acid sequence of antibodies 12-24 is shown in Table 9. The nucleic acid sequences encoding the heavy chain and the light chain were respectively inserted into the plasmid by molecular cloning technology to obtain a recombinant plasmid for expressing the whole antibody, and then the recombinant plasmid was transferred into HEK-293F cells; after culturing for 6 days, the supernatant was collected, and finally the antibody protein sample was obtained by purification.

[0212] Table 7 Composition of anti-ANGPTL3 antibodies

[0213]

[0214]

[0215] Table 8 Variable region amino acid sequence of Evinacumab

[0216]

[0217] Table 9 Nucleic acid sequences of antibodies 12-24

[0218]

[0219]

[0220] Example 3 ELISA to detect the binding of antibody and antigen

[0221] 3.1 ELISA to detect the binding of antibodies 12-12, 12-21, 34-12 to antigens

[0222] hANGPTL3-His or mouse mANGPTL3-His (Sino Biological, catalog number 50003-M08B1) was coated on a 96-well ELISA plate at 2.5 μg / ml and 100 μl / well one day in advance, and coated overnight at 4°C; the next day, PBS containing 3% BSA was used to block at 37°C for 2 hours (h); after blocking, gradient dilutions of antibodies (starting from an initial concentration of 10 μg / ml, 3-fold gradient dilution) were added and bound at 37°C for 1 hour (h); after washing with PBST for 5 times, goat anti human Fc-HRP (Abcam) was added and bound at 37°C for 1 hour (h); unbound antibodies were washed away with PBST, TMB colorimetric solution was added, and the reaction was terminated by adding 0.1 M sulfuric acid after being placed at 37°C for 5-10 minutes (min); the absorbance value was read at a wavelength of 450 nm on an enzyme reader.

[0223] As shown in Tables 10 and 11, anti-ANGPTL3 antibodies bound to hANGPTL3 and mANGPTL3.

[0224] Table 10 EC of anti-ANGPTL3 antibodies binding to hANGPTL3-His 50 (μg / ml)

[0225] Antibody <![CDATA[EC 50 ]]> Antibody <![CDATA[EC 50 ]]> 12-12 0.03 34-15 0.015 12-21 0.025 34-20 0.013 34-12 0.011 34-25 0.017 Evinacumab 0.012 / /

[0226] Table 11 EC of anti-ANGPTL3 antibodies binding to mANGPTL3-His 50 (μg / ml)

[0227] Antibody <![CDATA[EC 50 ]]> Antibody <![CDATA[EC 50 ]]> 12-12 0.007 34-15 0.004 12-21 0.007 34-20 0.006 34-12 0.004 Evinacumab 0.005 / 0.006

[0228] 3.2 ELISA to detect the binding of antibody 12-24 to antigen

[0229] h-ANGPTL3-220-His (Sino Biological, 10770-H08H2) was coated on a 96-well ELISA plate at 4 μg / ml and 100 μl / well one day in advance, and coated overnight at 4°C; the supernatant was discarded, and the plates were blocked with PBS containing 3% BSA at 200 μl / well at 37°C incubator for 2 hours (h) the next day; the supernatant was discarded, and the antibody dilutions were added respectively (starting from 8 μg / mL, diluted by half, a total of 11 gradients, 100 μL / well were added to the corresponding wells), and blocked at 37°C incubator for 2 hours; the supernatant was discarded, and the secondary antibodies (Anti-Human Kappa light chain (Bound and Free)-Peroxidase antibody produced ingoat (Sigma, A7164-1ml), 1:5000 dilution), 100 μL / well were added to the corresponding wells, and the wells were sealed at 37°C for 1 hour; the supernatant was discarded, and 100 μL / well of TMB substrate were added, and the wells were placed at 37°C for 5-10 minutes (min), and then 0.1M H2SO4 stop solution was added; the absorbance value was read at a wavelength of 450 nm on an ELISA reader.

[0230] Table 12 EC binding of antibodies 12-24 to hANGPTL3-His 50 (μg / ml)

[0231] Antibody <![CDATA[EC 50 ]]> 12-24 0.151 Evinacumab 0.197

[0232] Example 4 Blood lipid determination

[0233] 4.1 Blood lipid determination (antibodies 12-12, 12-21, 34-12, 34-15, 34-20, 34-25)

[0234] After 7-week-old C57BL / 6J (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were adaptively fed for 3 days, the subsequent experiments were started; 7 days before the formal administration, the blood of the mice was collected from the orbits after fasting for 4 hours (h), and the plasma was separated to detect the levels of TG (triglycerides), Chol (cholesterol) and LDL-C (low-density lipoprotein cholesterol). The mice were randomly divided into 8 groups with 6 mice in each group, based on TG and Chol as the main grouping basis and LDL-C as the secondary grouping basis. After each group of mice recovered for 7 days, the corresponding solvent or drug was given once by subcutaneous injection (sc). The day of injection of the drug or solvent was defined as Day 0. On Day 1, Day 7, Day 14, and Day 19, they were fasted for 4 hours, and then the orbital blood was collected from the mice in each group, and the plasma was separated to detect the levels of TG, chol and LDL-C.

[0235] 1) After grouping, the mice in each group were raised conventionally, and there was no significant difference in the body weight of the mice in each group during the drug efficacy period of Day 0, Day 7, Day 14, and Day 19, indicating that the mice had good resistance to antibodies 12-12, 12-21, 34-12, 34-15, 34-20, and 34-25.

[0236] 2) If Figures 1A-1E As shown, in the G4 group, the TG level decreased significantly on Day 1, reached the lowest point after Day 7, and then rose slowly; the Chol level decreased on Day 1, reached the lowest point on Day 7, and then rose slowly; the LDL-C level did not change significantly; in the G3, G5, G6, G7 and G8 groups, the TG level decreased significantly on Day 1 of administration, reached the lowest point, and then rose slowly; the Chol level and LDL-C did not change significantly.

[0237] Table 13 Dosage regimen

[0238] Group Drug group Dosage (mg / kg) way Actual number of medications G1 Solvent control group - sc 1 time G2 Evinacumab 15 sc 1 time G3 Antibody 12-12 15 sc 1 time G4 Antibody 12-21 15 sc 1 time G5 Antibody 34-12 15 sc 1 time G6 Antibody 34-15 15 sc 1 time G7 Antibody 34-20 15 sc 1 time G8 Antibody 34-25 15 sc 1 time

[0239] 4.2 Blood lipid measurement (antibody 12-24)

[0240] (1) Model: T015765B6-hPCSK9 mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were adaptively raised for 3-5 days and then switched to a Western diet (compared to a conventional diet, a Western diet is mainly high in fat, sugar, and cholesterol) for 5-6 weeks to construct a hyperlipidemia mouse model.

[0241] (2) Grouping: After fasting for 4-5 hours, blood was collected to detect TG, Chol and LDL-C levels. Based on LDL-C as the main grouping basis, the mice were randomly divided into 10 groups, with 6 mice in each group.

[0242] (3) Administration: The first day of administration was defined as Day 0. Two subcutaneous administrations were performed on Day 0 and Day 7. The administration volume was 5 μL / g × mouse body weight (g). The mice were fed a Western diet throughout the entire period.

[0243] Table 14 Mouse grouping and dosing regimen

[0244] Group Test drug Dose (mpk) Dosing frequency Route of administration Number of animals Group1 Normal saline — QW*2weeks sc 6 Group2 Evinacumab 10 QW*2weeks sc 6 Group3 Antibody 12-24 10 QW*2weeks sc 6

[0245] (4) Observation: On Day 1, 8, and 14, the animals were fasted for 4-5 hours, 70-80 μl of blood was collected, and plasma was separated to measure TG, Chol, and LDL-C levels. The animals were fed a Western diet throughout the entire period.

[0246] The test results are shown in Tables 15-17:

[0247] Table 15 LDL-C levels in mice within 14 days after administration (mmol / L)

[0248] LDL-C (mmol / L) Day-2 Day 1 Day 8 Day 14 Normal saline 1.09±0.213 1.427±0.305 1.56±0.371 1.233±0.297 Antibody 12-24 1.1±0.161 1.39±0.277 1.387±0.252 1.213±0.265 Evinacumab 1.08±0.328 1.347±0.96 1.143±0.139 0.937±0.113

[0249] Table 16 Chol levels in mice within 14 days after administration (mmol / L)

[0250] Chol (mmol / L) Day-2 Day 1 Day 8 Day 14 Normal saline 5.383±0.441 5.953±0.873 6.403±0.999 5.613±0.682 Antibody 12-24 5.207±0.55 5.243±0.678 5.117±0.854 5.233±0.645 Evinacumab 5.143±0.786 5.547±0.638 4.653±0.478 4.807±0.854

[0251] Table 17 TG levels in mice within 14 days after administration (mmol / L)

[0252] TG (mmol / L) Day-2 Day 1 Day 8 Day 14 Normal saline 0.317±0.079 0.243±0.061 0.543±0.136 0.497±0.125 Antibody 12-24 0.41±0.037 0.107±0.027 0.33±0.085 0.497±0.059 Evinacumab 0.383±0.122 0.223±0.034 0.660±0.068 0.64±0.099

Claims

1. An antibody or antigen-binding fragment that specifically binds to ANGPTL3, the antibody or antigen-binding fragment comprising one or more of HCDR1 as shown in any one of SEQ ID NOs: 1-6, HCDR2 as shown in any one of SEQ ID NOs: 7-12, HCDR3 as shown in SEQ ID NOs: 13 or 14, LCDR1 as shown in any one of SEQ ID NOs: 15-17, LCDR2 as shown in any one of SEQ ID NOs: 18-20, and LCDR3 as shown in SEQ ID NOs: 21 or 22.

2. The antibody or antigen-binding fragment of claim 1, comprising a HCDR1 as shown in any one of SEQ ID NOs: 1-6, a HCDR2 as shown in any one of SEQ ID NOs: 7-12, and a HCDR3 as shown in SEQ ID NOs: 13 or 14.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising LCDR1 as shown in any one of SEQ ID NOs: 15-17, LCDR2 as shown in any one of SEQ ID NOs: 18-20, and LCDR3 as shown in SEQ ID NO:

21.

4. An antibody or antigen-binding fragment, comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 7, HCDR3 as shown in SEQ ID NO: 13, LCDR1 as shown in SEQ ID NO: 15, LCDR2 as shown in SEQ ID NO: 18 and LCDR3 as shown in SEQ ID NO: 21; or The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO:13, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:19 and LCDR3 as shown in SEQ ID NO:22; or The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:9, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20, and LCDR3 as shown in SEQ ID NO:21; or The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20 and LCDR3 as shown in SEQ ID NO:21; or The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20 and LCDR3 as shown in SEQ ID NO:21; or The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO:14, LCDR1 as shown in SEQ ID NO:17, LCDR2 as shown in SEQ ID NO:20 and LCDR3 as shown in SEQ ID NO:

21.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 40, 23-28, a sequence having at least 80% identity to a sequence as shown in any one of SEQ ID NOs: 40, 23-28, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 40, 23-28; and / or The light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NOs: 29-31, a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 29-31, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 29-31, or consists of the same.

6. An antibody or antigen-binding fragment, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 23, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 29; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 24, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 30; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 25, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 31; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 26, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 31; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 27, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 31; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 28, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO: 31; or The heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:40, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:

30.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the heavy chain constant region of the antibody or antigen-binding fragment comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 34 to 36, a sequence having at least 80% identity to a sequence as shown in any one of SEQ ID NOs: 34 to 36, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence as shown in any one of SEQ ID NOs: 34 to 36; and / or The light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:37, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:37, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:37, or consists of the same.

8. An antibody, wherein the heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 23 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 29 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 24 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 25 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 26 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 27 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 28 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 31 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 37; or The heavy chain of the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:40 and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:35; the light chain of the antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO:30 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

37.

9. A nucleic acid or nucleic acid fragment encoding the antibody or antigen-binding fragment of any one of claims 1 to 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-8.

11. A diagnostic or prognostic kit comprising the antibody or antigen-binding fragment according to any one of claims 1 to 8.

12. A method and use for preventing or treating a disease, comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 10, wherein the disease is a disease involving lipid metabolism or a disease related to cancer / tumor and / or non-neoplastic angiogenesis.

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