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

Anti-ANGPTL3 antibodies and their applications 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 other 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 linker peptide, and a carboxyl-terminal fibrinogen-like domain that mediates ligand activity.

[0003] ANGPTL3 is a secreted protein whose mRNA is primarily expressed in the liver of humans and mice. Previous studies have shown that ANGPTL3's biological effects are manifested in two ways: regulating angiogenesis and lipid metabolism. ANGPTL3 can attenuate lipid metabolism by inhibiting lipoprotein lipase (LPL) activity.

[0004] Summary of the Invention

[0005] The present invention provides anti-ANGPTL3 antibodies or antigen-binding fragments that 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, thereby promoting lipid metabolism.

[0006] 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:

[0007] (a) HCDR1 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6;

[0008] (b) HCDR2 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12;

[0009] (c) HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14;

[0010] (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;

[0011] (e) LCDR2, which comprises or consists of 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;

[0012] (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.

[0013] In some embodiments, the antibody or antigen-binding fragment specifically binds ANGPTL3 and comprises:

[0014] (a) HCDR1 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6; and

[0015] (b) HCDR2 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12; and

[0016] (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14, or consisting of the same.

[0017] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13.

[0018] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:13.

[0019] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:14.

[0020] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:10, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:14.

[0021] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:11, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:14.

[0022] In some embodiments, HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:12, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:14.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds ANGPTL3 and comprises:

[0024] (d) LCDR1 comprising or consisting of 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; and

[0025] (e) LCDR2, which comprises or consists of 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; and

[0026] (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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In some embodiments, the antibody or antigen-binding fragment specifically binds ANGPTL3 and comprises:

[0031] (a) HCDR1 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 1-6; and

[0032] (b) HCDR2 comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 7-12, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in any one of SEQ ID NOs: 7-12; and

[0033] (c) HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13 or 14, or an amino acid sequence having a single substitution, deletion or insertion compared to the sequence shown in SEQ ID NO: 13 or 14;

[0034] (d) LCDR1 comprising or consisting of 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; and

[0035] (e) LCDR2, which comprises or consists of 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; and

[0036] (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.

[0037] In some embodiments, the substitutions are conservative amino acid substitutions.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the antibodies or antigen-binding fragments that specifically bind 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.

[0045] Table 1 Amino acid sequence of HCDR (using Kabat classification)

[0046] Table 2 Amino acid sequence of LCDR (using Kabat classification)

[0047] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 40, 23-28, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the sequence set forth 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 the sequence set forth in any one of SEQ ID NOs: 40, 23-28; and / or

[0048] 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 IgG, IgM, IgA, IgE, or IgD isotype. 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.

[0058] 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 receptors). 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).

[0059] In some embodiments, the antibody or antigen-binding fragment is a scFV, Fab, Fab' or F(ab) 2. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0060] 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to any one of SEQ ID NOs: 34-36, or an amino acid sequence having one or more conservative amino acid substitutions compared to any one of SEQ ID NOs: 34-36; and / or

[0061] 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to 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.

[0062] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 34, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 35, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 36, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 37.

[0063] In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 40, 23-28, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 40, 23-28, or an amino acid sequence having one or more conservative amino acid substitutions compared to any one of SEQ ID NOs: 40, 23-28, or consisting of a heavy chain constant region; and the heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 34-36, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 40, 23-28. a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 34-36, or an amino acid sequence that has one or more conservative amino acid substitutions compared to any one of SEQ ID NOs: 34-36;

[0064] and / or

[0065] 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 of any one of SEQ ID NOs: 29-31, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to any one of SEQ ID NOs: 29-31, or an amino acid sequence having one or more conservative amino acid substitutions compared to any one of SEQ ID NOs: 29-31; the light chain constant region comprises the amino acid sequence of SEQ ID NO: 37, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to any one of SEQ ID NOs: 29-31; NO:37 has a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in 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 consisting of the same.

[0066] 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;

[0067] and / or

[0068] 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.

[0069] 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.

[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: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.

[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: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.

[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: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.

[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: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.

[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: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.

[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: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.

[0076] 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 herein can be any combination of the amino acid sequences corresponding to the variable regions in Table 3.

[0077] Table 3 Amino acid sequences of variable regions

[0078] Table 4 Constant regions of anti-ANGPTL3 antibodies

[0079] 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).

[0080] In some embodiments, the antibody has two identical heavy chains and two identical light chains, with the Fc regions pairing to form disulfide bonds.

[0081] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

[0082] The present invention also provides nucleic acids 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.

[0083] 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.

[0084] 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.

[0085] The present invention also provides a conjugate comprising the antibody or antigen-binding fragment and a conjugated portion, wherein the conjugated portion is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0086] In some embodiments, the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.

[0087] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment or the conjugate, and pharmaceutically acceptable excipients.

[0088] 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, comprising administering to a patient an effective dose of the anti-ANGPTL3 antibody or antigen-binding fragment, the conjugate, or the pharmaceutical composition. In some embodiments, use of the anti-ANGPTL3 antibody or antigen-binding fragment, the conjugate, or the pharmaceutical composition for treating an ANGPTL3-related disease is provided. In some embodiments, use of the anti-ANGPTL3 antibody or antigen-binding fragment, the conjugate, or the pharmaceutical composition for preparing a medicament for treating an ANGPTL3-related disease is provided.

[0089] 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 and diabetic dyslipidemia), hypertriglyceridemia (including severe hypertriglyceridemia with TG>1000 mg / dL), hypercholesterolemia, chylomicronemia, mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.), lipodystrophy, 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 (e.g., LDLR- / - homozygous familial hypercholesterolemia), ApoC2 changes, ApoE deficiency, ApoB increase, increased very low density lipoprotein (VLDL) production and / or decreased elimination, certain drug treatments (e.g., dyslipidemia caused by glucocorticoid treatment), any genetic predisposition, diet, lifestyle, etc.

[0090] 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.

[0091] 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.).

[0092] The present invention also provides diagnostic methods and uses. In some embodiments, a method for detecting ANGPTL3 expression in a sample is provided, comprising contacting the sample with the antibody or antigen-binding fragment, allowing the antibody or antigen-binding fragment to bind to ANGPTL3, and detecting the binding, i.e., the ANGPTL3 content in the sample. In some embodiments, use of the antibody or antigen-binding fragment in preparing 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.

[0093] The present invention provides anti-ANGPTL3 antibodies and their uses. The antibodies or antigen-binding fragments of the present invention can specifically bind to ANGPTL3, effectively alleviating ANGPTL3's inhibition of lipoprotein lipase activity and promoting lipid metabolism. The antibodies or antigen-binding fragments of the present invention are used to treat ANGPTL3-related diseases and are also used for the diagnosis and prognosis of ANGPTL3-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1A shows the TG levels of mice within 19 days after administration;

[0095] Figure 1B shows the Chol levels in mice 19 days after administration;

[0096] Figure 1C shows the TG levels of mice on day 7 after administration;

[0097] FIG1D shows the Chol levels in mice on day 7 after administration;

[0098] FIG1E shows the LDL-C levels of mice on day 7 after administration.

[0099] the term

[0100] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0101] definition

[0102] It should be noted that "a" entity refers to one or more of the entity, for example, "an antibody" should be understood as one or more antibodies, and therefore, "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0103] "Comprising" or "including" means that compositions and methods, etc. include the listed elements, such as components or steps, but do not exclude others. "Consisting essentially 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.

[0104] "Polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule formed by amino acid monomers linearly linked by amide bonds (also known as peptide bonds). "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. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and "polypeptide" may be used in place of or interchangeably with any of the foregoing terms. "Polypeptide" is also intended to refer to products of polypeptides that have been modified after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it need not be translated from a specified nucleic acid sequence and may be produced by any means, including chemical synthesis.

[0105] "Amino acids" refer to organic compounds containing both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in the form of precursors. Individual amino acids are encoded by nucleic acids 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).

[0106] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of amino acid classes with chemically similar side chains 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; and 6) acidic side chains: aspartic acid and glutamic acid.

[0107] 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, light chain constant region, heavy chain or 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 between any two of these values ​​(including endpoints) or any value therein.

[0108] As used herein with respect to cells, nucleic acids, polypeptides, antibodies, and the like, the term "isolated" refers to a molecule that has been separated from one or more of the other components of a cell's natural environment, such as DNA or RNA. "Isolated," as used herein, also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their natural state and do not exist in their natural state. "Isolated" is also used herein to refer to cells or polypeptides that have been separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, the purity of an isolated nucleic acid, polypeptide, antibody, and the like is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including the endpoints) between any two of these values, or any value therein.

[0109] "Recombinant" in reference 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 produce a polynucleotide or polypeptide that does not normally exist by combination.

[0110] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between the sequences is a function of the number of matching or homologous positions shared by the sequences.

[0111] 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, that percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and percent identity or sequence identity can be determined visually or using software programs known in the art, such as those 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 of which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expectation=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.

[0112] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, in the case of RNA, thymine replaced by uracil (U). A "polynucleotide sequence" can be represented by an alphabetic representation of the polynucleotide molecule. This alphabetic representation can be entered into a database on a computer with a central processing unit and used in bioinformatics applications, such as functional genomics and homology searches.

[0113] "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 non-limiting examples of 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 may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A 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 specified or required, any polynucleotide embodiment of the present invention includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0114] "Encoding" when applied to a polynucleotide refers to a polynucleotide that "encodes" a polypeptide and, 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.

[0115] The antibodies and antigen-binding fragments provided by 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).

[0116] "Antibody" and "antigen-binding fragment" refer to polypeptides or polypeptide complexes that specifically recognize and bind to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof. Therefore, "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs) of a heavy chain or light chain or its ligand-binding portion, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the framework regions (FRs), or any portion thereof, or at least a portion of a binding protein. CDR regions include the CDR regions (LCDR1-3) of the light chain and the CDR regions (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 (e.g., two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (e.g., two) antigens can be referred to as multispecific (e.g., bispecific) antibodies or antigen-binding fragments.

[0117] "Antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody. Antibody fragments of the present invention may be structured in forms similar to monospecific antibody fragments, such as F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. "Antibody fragments" include aptamers, Spiegelmers, and diabodies. "Antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0118] "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 with 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 to the C-terminus of VL, or vice versa. Although the constant region has been removed and a linker has been introduced into the protein, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0119] "Antibody" includes a wide variety of polypeptides that can be distinguished biochemically. It will be understood 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). The properties of this chain determine the "class" of the antibody, which 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 classes are within the scope of protection of the present invention. In some embodiments, the immunoglobulin molecule is an IgG class.

[0120] Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be combined with a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are bound by covalent bonds, and the "tail" portions 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 V κ ; The variable region of the immunoglobulin λ light chain is V λ .

[0121] 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, and complement fixation. By convention, the numbering of the constant regions increases as they become more distal to the antibody's antigen-binding site, or amino terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0122] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that form the antigen binding domain, assuming the antibody assumes its three-dimensional configuration in an aqueous environment, and that specifically bind to the antigen. The remaining amino acids in the antigen binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework regions largely adopt a β-sheet conformation, with the CDRs forming loops connected thereto, or in some cases forming part of the β-sheet structure. Thus, the framework regions, by forming a scaffold, position the CDRs in the correct orientation through interchain non-covalent interactions. The antigen binding domain, with its specifically positioned CDRs, forms a surface complementary to an epitope on the antigen, which promotes non-covalent binding between the antibody and its antigenic epitope. For a given heavy or light chain variable region, one of ordinary skill in the art can identify the amino acids comprising the CDRs 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)).

[0123] In the case where there are two or more definitions of a term used and / or accepted in the art, the definition of the term used herein includes all of these meanings unless explicitly stated to the contrary. 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., US Pat. of Health and Human Services, Sequences of Proteins of Immunological Interest (1991) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference in their entirety.

[0124] The CDRs defined according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, the application of either definition to refer to the CDRs of an antibody or its 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 generally determine which specific residues a CDR comprises based on the variable region amino acid sequence of an antibody.

[0125] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence independently of experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Department of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1991). Antibodies may also use the EU or Chothia numbering system.

[0126] The antibodies of 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. In another embodiment, the variable region can be of condricthoid origin (e.g., from shark).

[0127] The "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., 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.

[0128] A "light chain constant region" comprises a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer via disulfide bonds between the light chain CL domain and the heavy chain CH1 domain. These four chains are connected by disulfide bonds in a "Y" configuration, with the light chain beginning at the mouth of the "Y" and continuing through the variable region to surround the heavy chain.

[0129] 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 the partial heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region contains about 25 residues and is resilient, allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: upper, middle and lower hinge domains.

[0130] A "disulfide bond" refers to a covalent bond formed between two sulfur atoms. A thiol group of a 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.

[0131] A "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.

[0132] "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 in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a specific antigen or epitope. For example, if antibody "A" has a greater relative affinity for the same antigen than antibody "B", it can be considered that antibody "A" has a higher specificity for the antigen than antibody "B". Specific binding can be described by an equilibrium dissociation constant (KD), with a smaller KD meaning 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, and the like. Antibodies that "specifically bind" to antigen a include antibodies with an equilibrium dissociation constant KD of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, or less than or equal to about 1 nM.

[0133] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow, ameliorate or halt an undesirable physiological change or disorder, 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 elimination of the disease state (whether partial or complete), prolongation of life compared to that expected in the absence of treatment, etc. Patients in need of treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those in need of prevention of the condition or disorder, and those who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions of the invention for detection, diagnostic procedures and / or treatment.

[0134] "Patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.

[0135] "About" refers to the conventional error range of the corresponding numerical value that is easily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0136] “EC 50 "Concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.

[0137] In the present invention, the "parent Fc region" can be a naturally occurring Fc region, and the gene encoding the Fc region can 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.

[0138] The relevant descriptions of publications mentioned herein are incorporated by reference in their entirety.

[0139] Anti-ANGPTL3 antibodies

[0140] The present invention provides antibodies or antigen-binding fragments with high affinity for the ANGPTL3 protein. These anti-ANGPTL3 antibodies or antigen-binding fragments exhibit effective binding and biological activity and are useful for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively alleviate the inhibition of ANGPTL3 on lipoprotein lipase activity, thereby promoting lipid metabolism.

[0141] In some embodiments, the antigen binding fragment is a scFv.

[0142] 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.

[0143] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:23, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:29.

[0144] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:24, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:30.

[0145] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:25, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:31.

[0146] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:26, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:31.

[0147] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:27, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:31.

[0148] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:28, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:31.

[0149] In some embodiments, the VH in the scFv comprises the amino acid sequence shown in SEQ ID NO:40, and the VL in the scFv comprises the amino acid sequence shown in SEQ ID NO:30.

[0150] 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.

[0151] In some embodiments, the antibody comprises two heavy chains with identical sequences and two light chains with identical sequences.In some embodiments, the antibody is of IgG type.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[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: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.

[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: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.

[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: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.

[0159] It will also be understood by those skilled in the art that the antibody or antigen-binding fragment sequence of the present invention can be substituted, and the amino acid sequence after substitution is different from the naturally occurring amino acid sequence of the antibody. For example, the substituted amino acid sequence can be similar to the starting sequence, such as having a certain ratio of identity with the starting sequence, such as about 80%, about 85%, about 90%, about 95%, about 98%, about 99% identity with the starting sequence, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0160] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence with one or more modifying groups. For example, the antibody or antigen-binding fragment of the 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).

[0161] The antibodies and antigen-binding fragments of 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. Examples include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0162] In some embodiments, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0163] 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 can then be 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.

[0164] Methods for preparing antibodies and polynucleotides encoding antibodies

[0165] The present invention also provides polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention. The polynucleotides of the present invention can 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. Methods for preparing antibodies are well known in the art and are described herein.

[0166] In certain embodiments, the prepared antibodies do not cause a harmful immune response in the animal to be treated (e.g., a human). In some embodiments, the antibodies, antigen-binding fragments, or derivatives of 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, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into a 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 a 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 portions. Typically, 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 modeling the interactions of CDRs and framework residues to identify framework residues that play an important role in antigen binding and by sequence comparison to identify unusual framework residues at specific positions. (See U.S. Patent 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. Patents 5,225,539, 5,530,101 and 5,585,089), repair or resurfacing (EP 592,106; EP 519,596), and chain rearrangement (U.S. Patent No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0167] Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, "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 light chain variable region sequence from the starting antibody are analyzed, and a human T cell epitope "map" 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. A single T cell epitope from the T cell epitope map is analyzed to identify optional amino acid substitutions with a lower risk of changing the antibody activity. A series of optional heavy chain variable region sequences and light chain variable region sequences containing amino acid substitution combinations are designed, and these sequences are subsequently incorporated into a series of binding polypeptides. The genes for the complete heavy and light chains containing the modified variable regions and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibodies.

[0168] The binding specificity of the antibodies or antigen-binding fragments of the present invention can be tested by in vitro assays, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0169] The preparation of scFv can be seen in the technology for producing single-chain units (U.S. Patent 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.

[0170] Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include those described in U.S. Patents 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. Patents 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.

[0171] Additionally, techniques for generating primate antibodies containing monkey variable region and human constant region sequences are described in US Patents 5,658,570, 5,693,780, and 5,756,096, the entire contents of each of which are incorporated herein by reference.

[0172] Antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived 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 WO 91 / 10741, the entire contents of each of which are incorporated herein by reference.

[0173] In another embodiment, using conventional methods (such as using oligonucleotide probes that can specifically bind to the genes encoding mouse antibody heavy and light chains), the DNA encoding the desired monoclonal antibody can be isolated and sequenced. Isolation and subcloning of hybridoma cells can be used as the source of this type of DNA. Once isolated, DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. Isolation of DNA (which can be synthetic as described herein) can also be used to prepare the sequence of the 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 is converted 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.

[0174] 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 within 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 this art are also encompassed in the present invention.

[0175] Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors and cell lines can be selected, constructed, and cultured using techniques well 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 by reference in their entirety.

[0176] In some embodiments, DNA encoding the antibody can be designed and synthesized according to conventional methods based on the antibody amino acid sequence described herein, inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector comprises 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 either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1 (+ / -), pcDNA3.1 / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), pcDNA3.1 / G418 (+ / -), 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.

[0177] In some embodiments, the inserted gene fragment needs to contain a selection marker. Common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and other selection markers to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.

[0178] In addition, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the antibodies of the present invention, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis 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, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

[0179] Anti-ANGPTL3 antibodies can be screened using various methods known in the art. For example, a fully human phage display library can be used to screen for scFvs that bind to the ANGPTL3 antigen. The screened VH and VL fragments are then assembled with the heavy and light chain constant regions of human IgG, respectively, to obtain fully human anti-ANGPTL3 antibodies. Another example is the use of an ANGPTL3 antigen to immunize mice, fusing spleen cells from the immunized mice with SP2 / 0 myeloma cells to obtain hybridoma cells, and screening for positive clones that bind to the ANGPTL3 antigen to obtain anti-ANGPTL3 antibodies. The VH and VL fragments are then assembled with the heavy and light chain constant regions of human IgG, respectively, to obtain chimeric anti-ANGPTL3 antibodies.

[0180] Treatment

[0181] The present invention also provides treatment methods and uses. In some embodiments, a method for treating an ANGPTL3-related disease is provided, 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 treating an ANGPTL3-related disease is provided.

[0182] 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 timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors are within the discretion of a healthcare provider, who is within the skill of the art. The dosage will also depend on the individual patient being 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 employed 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.

[0183] Methods of administration of antibodies, Fabs, or derivatives thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. Pharmaceutical compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed by epithelial or mucocutaneous membranes (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Thus, pharmaceutical compositions containing antibodies, Fabs, or derivatives thereof of the present invention can be administered orally, rectally, parenterally, intracisternal, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, drops, or transdermal patches), orally, or by oral or nasal spray.

[0184] As used herein, "parenteral" refers to administration routes including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion. Administration routes may be systemic or local.

[0185] 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 encoded protein by constructing them into a suitable nucleic acid expression vector, which is then administered intracellularly by, for example, using retroviral vectors (see U.S. Patent No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection reagents, or by administering them in conjunction with a homeobox-like peptide known to enter the cell nucleus, etc. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

[0186] 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 retroviral or other vectors, etc.

[0187] Combination therapy

[0188] In some embodiments, the anti-ANGPTL3 antibodies or antigen-binding fragments of the present invention may 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 an ANGPTL3-associated disease. In some embodiments, the other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, immunotherapy, and surgery. For combination therapy, the anti-ANGPTL3 antibody and the other therapeutic agent may be administered simultaneously or separately. When administered separately, the anti-ANGPTL3 antibody or antigen-binding fragment may be administered before or after administration of the other therapeutic agent.

[0189] 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 niacin; 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.

[0190] Pharmaceutical composition

[0191] The present invention also provides pharmaceutical compositions. Such compositions comprise 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% to 90% of the 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).

[0192] "Pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. In some embodiments, "pharmaceutically acceptable" refers to substances approved by a national government or listed in a recognized pharmacopoeia for use in animals, particularly humans.

[0193] "Excipients" refer to diluents, adjuvants, excipients, or carriers that can be administered to a patient along with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition can also contain a small amount of a wetting agent or emulsifier, or a 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 regulating tension such as dextrose are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories with conventional binders 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 a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of excipients to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The parent formulation can be packaged in an ampoule, disposable syringe, or a multi-dose vial made of glass or plastic.

[0194] 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 generally a solution in a sterile isotonic aqueous buffer. The composition may also include a solubilizing agent 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.

[0195] The antibodies described herein can be neutral, i.e., have substantially no net charge, such as when the pH of the composition containing the antibody is at the antibody's isoelectric point, the antibody is electrically neutral. The antibodies described herein can exist in a positive ionic form, such as when the pH is below the isoelectric point, the antibody molecule as a whole exhibits a positive charge. The antibodies described herein can exist in a negative ionic form, such as when the pH is above the isoelectric point, the antibody molecule as a whole exhibits a negative charge. DETAILED DESCRIPTION

[0196] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications or adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0197] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0198] Example 1 Antibody Screening

[0199] 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).

[0200] 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), and the scFv isolated from strain 34-25 is scFv(34-25); the scFv isolated from strain 12-24 is scFv(12-24). In the scFv, from the N-terminus to the C-terminus, the VH is connected to the VL via the linker GGGGSGGGGS. The composition of the scFv is shown in Table 5, and the amino acid sequences of the CDRs, VH, and VL are shown in Tables 1-3.

[0201] Table 5 Composition of scFv

[0202] After the above-mentioned E. coli strain was grown to the logarithmic phase at 37°C, the culture temperature was adjusted to 28°C and cultured overnight. The next day, the supernatant was centrifuged at 4000 rpm and 4°C for 30 minutes. The diluted supernatant (diluent: PBST containing 1% BSA) was transferred to an ELISA plate coated with hANGPTL3-His (coating process: hANGPTL3-His concentration was 2.5 μg / ml, 100 μl / well, coating buffer: PBS (pH 7.2-7.4), coating was performed overnight at 4°C, and blocking was performed with PBS containing 3% BSA for 2 hours). The plate was incubated at 37°C for 1 hour. Unbound antibody was washed away with PBST (containing 1% BSA), and anti-myc was added. HRP (Abcam) was incubated at 37°C for 1 hour. Unbound antibody was washed with PBST, and TMB colorimetric solution was added. The reaction was incubated at 37°C for 5-10 minutes, followed by termination with 0.1 M sulfuric acid. The absorbance was read at 450 nm on a microplate reader. A positive result was determined by an OD value greater than 1.0. The ELISA test results are shown in Table 6.

[0203] Table 6 ELISA test results

[0204] Example 2 Antibody Preparation Method

[0205] 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 regions are shown in Table 3, the constant regions are shown in Table 4 (the Fc region is underlined), and the nucleic acid sequences of antibodies 12-24 are shown in Table 9. The nucleic acid sequences encoding the heavy and light chains were inserted into plasmids using molecular cloning techniques to generate recombinant plasmids for expressing the full antibody. These recombinant plasmids were then transformed into HEK-293F cells. After 6 days of culture, the supernatant was collected and purified to obtain the antibody protein.

[0206] Table 7 Composition of anti-ANGPTL3 antibodies

[0207] Table 8 Variable region amino acid sequence of Evinacumab

[0208] Table 9 Nucleic acid sequences of antibodies 12-24

[0209] Example 3 ELISA detection of antibody-antigen binding

[0210] 3.1 ELISA detection of the binding of antibodies 12-12, 12-21, 34-12, 34-15, 34-20, and 34-25 to antigens

[0211] hANGPTL3-His or murine mANGPTL3-His (Sino Biological, Catalog No. 50003-M08B1) was coated onto a 96-well ELISA plate at 2.5 μg / ml, 100 μl / well, one day in advance, overnight at 4°C. The next day, the plate was blocked with 3% BSA-containing PBS at 300 μl / well for 2 hours at 37°C. After blocking, serially diluted antibodies (starting at an initial concentration of 10 μg / ml, followed by 3-fold serial dilutions) were added and allowed to bind at 37°C for 1 hour. After washing five times with PBST, goat anti-human Fc-HRP (Abcam) was added and allowed to bind at 37°C for 1 hour. Unbound antibody was washed away with PBST, and TMB colorimetric solution was added. The plate was incubated at 37°C for 5-10 minutes before being terminated by the addition of 0.1 M sulfuric acid. The absorbance was read at 450 nm on a microplate reader.

[0212] As shown in Tables 10 and 11, anti-ANGPTL3 antibodies bound to hANGPTL3 and mANGPTL3.

[0213] Table 10 EC of anti-ANGPTL3 antibodies binding to hANGPTL3-His 50 (μg / ml)

[0214] Table 11 EC of anti-ANGPTL3 antibodies binding to mANGPTL3-His 50 (μg / ml)

[0215] 3.2 ELISA detection of the binding of antibody 12-24 to antigen

[0216] 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 for 2 hours the next day. The supernatant was discarded and the antibody dilutions were added (starting from 8 μg / mL, diluted in half, for a total of 11 gradients, 100 μL / well was added to the corresponding wells) and blocked at 37°C for 2 hours. The supernatant was discarded and the secondary antibody (Anti-Human Kappa light chain (Bound and Free)-Peroxidase antibody produced in goat (Sigma, A7164-1ml), 1:5000 dilution), 100 μL / well were added to the corresponding wells, and the cells were blocked in a 37°C incubator for 1 hour; the supernatant was discarded, and 100 μL / well of TMB substrate were added, and the cells were incubated at 37°C for 5-10 minutes before adding 0.1 M H2SO4 stop solution; the absorbance was read at a wavelength of 450 nm on a microplate reader.

[0217] Table 12 EC values ​​of antibodies 12-24 binding to hANGPTL3-His 50 (μg / ml)

[0218] Example 4 Blood lipid determination

[0219] 4.1 Lipid analysis (antibodies 12-12, 12-21, 34-12, 34-15, 34-20, 34-25)

[0220] After 7-week-old C57BL / 6J mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were adapted to feeding for 3 days, subsequent experiments began. 7 days before formal administration, blood was collected from the mice orbitally after fasting for 4 hours (h), and plasma was separated to detect TG (triglycerides), Chol (cholesterol), and LDL-C (low-density lipoprotein cholesterol) levels. TG and Chol were used as the primary grouping basis, and LDL-C was used as the secondary grouping basis. The mice were randomly divided into 8 groups, with 6 mice in each group. After 7 days of recovery, each group of mice was given a subcutaneous injection (sc) of the corresponding solvent or drug. The day of drug or solvent injection was defined as Day 0. On Day 1, Day 7, Day 14, and Day 19, the mice were fasted for 4 hours, and then orbital blood was collected from the mice in each group, and plasma was separated to detect TG, chol, and LDL-C levels.

[0221] 1) After grouping, the mice in each group were housed conventionally. 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 drug resistance to antibodies 12-12, 12-21, 34-12, 34-15, 34-20, and 34-25.

[0222] 2) As shown in Figures 1A-1E, in the G4 group, TG levels decreased significantly on Day 1, reached the lowest point after Day 7, and then slowly increased. Chol levels decreased on Day 1, reached the lowest point on Day 7, and then slowly increased. LDL-C levels did not change significantly. In the G3, G5, G6, G7, and G8 groups, TG levels decreased significantly on Day 1 of administration, reached the lowest point, and then slowly increased. Chol levels and LDL-C did not change significantly.

[0223] Table 13 Dosage regimen

[0224] 4.2 Blood lipid measurement (antibody 12-24)

[0225] (1) Model: T015765B6-hPCSK9 mice (Jiangsu Jicui Pharmaceutical Kang 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 establish a hyperlipidemia mouse model.

[0226] (2) Grouping: After fasting for 4-5 hours, blood samples were collected to measure TG, Chol, and LDL-C levels. Based on LDL-C as the main grouping factor, mice were randomly divided into 10 groups, with 6 mice in each group.

[0227] (3) Administration: The first administration day 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.

[0228] Table 14 Mouse grouping and dosing regimen

[0229] (4) Observation: On Days 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. All animals were fed a Western diet.

[0230] The test results are shown in Tables 15-17:

[0231] Table 15 LDL-C levels in mice within 14 days after administration (mmol / L)

[0232] Table 16 Chol levels in mice within 14 days after administration (mmol / L)

[0233] Table 17 TG levels in mice within 14 days after administration (mmol / L)

Claims

1. An antibody or antigen-binding fragment that specifically binds to ANGPTL3, the antibody or antigen-binding fragment comprising or consisting of 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 or consisting of 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, or a HCDR3 as shown in SEQ ID NOs: 13 or 14.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising or consisting of 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 or consisting of 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 or consists of 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 or consists of 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 or consists of 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 or consists of 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 or consists of 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 consists of 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 consists of the sequence; 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 consists of 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 consists of 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 consists of 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 consists of 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, or consists of it.

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. Biomaterials, for (1) a nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 8, or a portion thereof; or (2) a vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 8 or a portion thereof; or (3) A host cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 8 or a portion thereof.

10. A conjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 8 and a conjugated moiety, wherein: The coupling part is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme or a combination thereof; or, the coupling part is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol or an enzyme.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 8 or the conjugate according to claim 10; or further comprising a pharmaceutically acceptable excipient.

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

13. A method and use for preventing or treating a disease, comprising administering to a patient a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 8, the conjugate of claim 10, or the pharmaceutical composition of claim 11, 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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