Fully human anti-human NT-ANGPTL3 antibody, antigen-binding fragment thereof and applications thereof

Abstract

The present invention discloses a fully human anti-human NT-ANGPTL3 antibody, an antigen-binding fragment thereof and applications thereof. Using human NT-ANGPTL3 as an antigen, the present invention screens out a fully human anti-human NT-ANGPTL3 single-chain antibody by means of phage display technology. Then, a novel, highly active, fully human anti-human NT-ANGPTL3 full-length antibody is obtained by means of in vitro affinity maturation and antibody full-lengthization technology. This antibody can specifically bind to and inhibit the activity of NT-ANGPTL3, reduce serum TG and LDL-C, improve the hepatic tissue morphology, reduce hepatic lipid accumulation, and can be used for treating ANGPTL3-related diseases, such as hypertriglyceridemia, hypercholesterolemia, chylomicronemia, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a fully human anti-human NT-ANGPTL3 antibody, an antigen-binding fragment thereof, and their applications. Background Art

[0002] Cardiovascular disease (CVD) is one of the main causes of death globally. Clinically, the treatment of CVD mainly focuses on lifestyle modification, reduction of hypertension, control of diabetes, and treatment of dyslipidemia. Hyperlipidemia is characterized by elevated levels of total cholesterol (TC) and / or triglyceride (TG). Elevated plasma TG levels are an important risk factor for CVD, and many genetic polymorphisms involved in the lipolysis of TG-rich lipoproteins are associated with the risk of CVD.

[0003] Angiopoietin-like protein 3 (ANGPTL3) is a member of the ANGPTL family. Currently, 8 ANGPTLs have been discovered. Except for ANGPTL8, other ANGPTLs contain an amino-terminal-mediated coiled-coil domain (CCD) and a carboxyl-terminal-mediated fibrinogen-like domain (FLD). Patients with congenital loss-of-function mutations in ANGPTL3 exhibit hypolipidemia: low TG, low LDL-C, and low HDL-C. At the same time, the risk of coronary heart disease in these patients is reduced by 34% - 41%. Since then, the role of ANGPTL3 in lipid metabolism has attracted extensive attention and has become an important target for lowering blood lipids.

[0004] After ANGPTL3 is secreted and released into the blood, the N-terminal of ANGPTL3 (NT-ANGPTL3) functional domain is cleaved and released by proprotein convertase. NT-ANGPTL3 binds to lipoprotein lipase (LPL), causing a conformational change in LPL, inhibiting the hydrolysis of TG by LPL, leading to the accumulation of TG in the body and causing hypertriglyceridemia. Studies by Rene et al. have shown that ANGPTL3 acts independently of the low-density lipoprotein receptor (LDLR) in the absence of LDLR: inhibiting ANGPTL3 can de-inhibit endothelial lipase (EL), promote the clearance of very low-density lipoprotein (VLDL), limit the production of low-density lipoprotein (LDL) particles, and result in a decrease in low-density lipoprotein cholesterol (LDL-C). This suggests that ANGPTL3 inhibitors can effectively reduce LDL-C levels in patients with LDLR deficiency such as homozygous hypercholesterolemia. These studies indicate that ANGPTL3 function inhibitors can simultaneously reduce two independent risk factors for cardiovascular disease (CVD) - TG and LDL-C.

[0005] Currently, there are various lipid-lowering drugs used to control cardiovascular diseases, such as statins, fibrates, niacin, etc. Although statins are widely used, a large number of studies have found that most cardiovascular patients cannot achieve ideal effects when using conventional drug doses. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a fully human anti-human NT-ANGPTL3 antibody, its antigen-binding fragment, and their applications. The antibody and its antigen-binding fragment can specifically bind to the N-terminal helical domain of Angiopoietin Like Protein 3 (NT-ANGPTL3), de-inhibit LPL and EL, and restore the activities of LPL and EL, thereby reducing the serum TG and LDL-C levels.

[0007] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0008] A fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises at least one heavy-chain variable region selected from any of the amino acid sequences shown in SEQ ID NO: 1-14, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 1-14; and comprises at least one light-chain variable region selected from any of the amino acid sequences shown in SEQ ID NO: 15-28, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 15-28.

[0009] For the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof, the heavy-chain variable region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3; the light-chain variable region comprises complementarity-determining regions LCDR1, LCDR2, and LCDR3.

[0010] Wherein: HCDR1 comprises any of the amino acid sequences shown in SEQ ID NO: 40-53, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 40-53, and / or HCDR2 comprises any of the amino acid sequences shown in SEQ ID NO: 54-67, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 54-67, and / or HCDR3 comprises any of the amino acid sequences shown in SEQ ID NO: 68-81, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 68-81. LCDR1 comprises any of the amino acid sequences shown in SEQ ID NO: 82-95, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 82-95, and / or LCDR2 comprises any of the amino acid sequences shown in SEQ ID NO: 96-109, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 96-109, and / or LCDR3 comprises any of the amino acid sequences shown in SEQ ID NO: 110-123, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 110-123.

[0011] The full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is selected from the following structural forms: full-length antibody, Fab, F(ab’), F(ab’)2, Fv, scFv, diabody, microbody, bispecific antibody, multispecific antibody, chimeric antibody, CDR-grafted antibody, antibody functional fragment VH-L formed by the fusion of the heavy chain variable region and the complete light chain and the arrangement, tandem or combination of one or more CDRs.

[0012] A polynucleotide encoding the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof according to any one of the above.

[0013] A vector comprising the polynucleotide.

[0014] A host cell comprising the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof, the polynucleotide or the vector according to any one of the above.

[0015] A composition comprising the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof, the polynucleotide or the vector according to any one of the above and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0016] A conjugate comprising the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof according to any one of the above, wherein the conjugate is a detectable label, and the detectable label includes a fluorescent substance, a luminescent substance, a colored substance, a radioisotope or an enzyme.

[0017] Use of the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof according to any one of the above or the conjugate comprising the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof according to any one of the above in the preparation of a diagnostic reagent for detecting NT-ANGPTL3.

[0018] Use of the full human anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof according to any one of the above in the preparation of drugs for treating hypertriglyceridemia, hypercholesterolemia, chylomicronemia, atherosclerosis, cardiovascular and cerebrovascular diseases.

[0019] Construct the recombinant expression plasmid pET27b-NT-ANGPTL3 of human NT-ANGPTL3, then transform the plasmid into the E. coli expression strain E. coli BL21(DE3), add IPTG with a final concentration of 0.2 mM, induce expression at 16 °C for 20 h, and after extracting the periplasmic protein with arginine, separate and purify the recombinant human NT-AMGPTL3 protein by nickel column affinity chromatography and size exclusion chromatography.

[0020] Screening of anti-human NT-ANGPTL3 single-chain antibodies Sc-232, Sc-1519, Sc-2323, Sc-23183, Sc-4324, Sc-4429, Sc-4470, Sc-44107, Sc-44132, Sc-44362, Sc-44396, Sc-44448, Sc-44523, Sc-44566 from a high-capacity fully human phage antibody library using NT-ANGPTL3 as the target antigen. The amino acid sequences of their heavy variable regions are SEQ ID NO: 1-14 respectively, and the amino acid sequences of their light chain variable regions are SEQ ID NO: 15-28 respectively. The highly active anti-human NT-ANGPTL3 single-chain antibodies Sc-1519 and Sc-44132 were full-length cloned. The VH sequence of Sc-1519 was ligated into the heavy chain constant region (SEQ NO: 29) sequence in the form of IgG4, and the VL sequence was ligated into the lambda light chain constant region (SEQ NO: 30) sequence by conventional molecular biology operations; the VH sequence of Sc-44132 was ligated into the heavy chain constant region (SEQ NO: 29) sequence in the form of IgG4, and the VL sequence was ligated into the kappa light chain constant region (SEQ NO: 31) sequence, and named F1519 and F44132. Degenerate oligonucleotide primers were designed by combining KABAT database sequence alignment and mutation hot spot analysis, and mutants F1519-V90A, F1519-D95aA, F1519-H95bA, F1519-H95bD, F1519-H95bE, F1519-H95bK, F1519-H95bR, F1519-V90A / D95aA were obtained by alanine scanning site-directed mutagenesis. The amino acid sequence of their heavy chain variable regions is SEQ IDNO: 2, and the amino acid sequences of their light chain variable regions are SEQ ID NO: 32-39 respectively, and their activities of blocking NT-ANGPTL3 from inhibiting LPL in vitro were detected. On this basis, the in vivo lipid-lowering activities of F1519 and F1519-D95aA were verified.

[0021] The antibodies and antigen-binding fragments thereof obtained in the present invention can specifically bind to NT-ANGPTL3, effectively inhibit the interaction between NT-ANGPTL3 and LPL and EL, restore the activities of LPL and EL, and significantly reduce the serum TG and LDL-C levels.

[0022] More specifically, the monoclonal antibody or antigen-binding fragment described in the present invention comprises a heavy chain variable region and a light chain variable region. In some embodiments, the monoclonal antibody is a single-chain antibody (scFv), in which the heavy chain variable region and the light chain variable region are linked by a flexible peptide (GGGGSGGGGSGGGGS); in other embodiments, the monoclonal antibody is a full-length antibody, in which the heavy chain variable region and the light chain variable region are linked to an IgG4 constant region.

[0023] The complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain are represented by HCDR1, HCDR2, and HCDR3, respectively; the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain are represented by LCDR1, LCDR2, and LCDR3, respectively.

[0024] More specifically,

[0025] The fully human anti-human NT-ANGPTL3 antibody HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 40 to 53, or a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 40 to 53, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the said sequence;

[0026] HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 54 to 67, or a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 66 to 79, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the said sequence;

[0027] HCDR3 respectively comprises the amino acid sequence shown in any one of SEQ ID NOs: 68 to 81, or a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 68 to 81, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the said sequence;

[0028] LCDR1 comprises an amino acid sequence shown in any of SEQ ID NOs: 82 to 95, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 82 to 95, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence;

[0029] LCDR2 comprises an amino acid sequence shown in any of SEQ ID NOs: 96 to 109, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 96 to 109, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence;

[0030] LCDR3 respectively comprises an amino acid sequence shown in any of SEQ ID NOs: 110 to 123, or comprises a sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NOs: 110 to 123, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the said sequence.

[0031] A vector which comprises the above polynucleotide.

[0032] A host cell which comprises any of the above antibodies, antigen-binding fragments, polynucleotides, vectors. The host cell comprises prokaryotic cells, yeast, insects or mammalian cells.

[0033] A composition which comprises an antibody, antigen-binding fragment, polynucleotide or vector of any of the above and a pharmaceutically acceptable carrier, diluent or excipient thereof; or optionally in combination with another therapeutic agent.

[0034] A conjugate which comprises the above anti-NT-ANGPTL3 antibody or its antigen-binding fragment as a detectable label, and the detectable label includes a fluorescent substance, a luminescent substance, a colored substance, a radioisotope or an enzyme.

[0035] Use of the above anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof in the preparation of a reagent for detecting the presence of NT-ANGPTL3 in a sample. Contact the test sample with any one of the anti-human NT-ANGPTL3 antibodies or antigen-binding fragments thereof in the present invention, and then detect the presence of a complex of NT-ANGPTL3 or its skewness and the anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof, and the presence of the complex indicates the presence of NT-ANGPTL3.

[0036] Use of a conjugate of the above anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof in the preparation of a reagent for detecting the presence of NT-ANGPTL3 in a sample.

[0037] Use of the above anti-human NT-ANGPTL3 antibody or antigen-binding fragment thereof in the preparation of a drug for preventing or treating diseases or symptoms that can be alleviated, improved, inhibited or prevented by an NT-ANGPTL3 antagonist;

[0038] wherein the diseases or symptoms include, but are not limited to: hypertriglyceridemia, mixed hyperlipidemia, atherosclerosis, cardiovascular and cerebrovascular diseases, etc.

[0039] The antibody or antigen-binding fragment thereof in the present invention is selected from the following structural forms: full-length antibody, Fab, F(ab’), F(ab’)2, Fv, scFv, diabody, microantibody, bispecific antibody, multispecific antibody, chimeric antibody, CDR-grafted antibody, antibody functional fragment formed by the arrangement, tandem or combination of the heavy chain variable region and the complete light chain fusion VH-L and one or more CDRs.

[0040] The antibody or antibody fragment of the present invention provides a new drug for the diagnosis, prevention and treatment of diseases such as lipid metabolism diseases.

[0041] Beneficial effects: (1) The present invention uses phage display technology to obtain antibodies and obtain fully human antibodies. (2) The present invention provides a highly active human anti-NT-ANGPTL3 single-chain antibody and its active functional fragments or variants or full-length antibodies, as well as their applications in the preparation of diagnostics, prevention, and treatment of hypertriglyceridemia, hypercholesterolemia, chylomicronemia, atherosclerosis, cardiovascular and cerebrovascular diseases, and other ANGPTL3-related diseases. (3) The Fc segment of the full-length antibody described in the present invention is of the IgG4 subclass. Functionally, IgG4 has a relatively weak affinity for most FcγR receptors, such as FcγRII and FcγRIII; moreover, it also lacks the ability to activate complement. For example, the affinity of IgG4 for C1q is lower than that of other subtypes. In vivo, due to Fab arm exchange, the typical feature of IgG4 is monovalent effectiveness, resulting in preventing the formation of immune complexes by antibodies. Therefore, during treatment, IgG4 can be selected as a blocking antibody, greatly avoiding the triggering of effector mechanisms. These properties make IgG4 exhibit relatively "non-inflammatory" characteristics and are more suitable for the production of therapeutic monoclonal antibodies. Brief Description of the Drawings

[0042] Figure 1 : Plasmid map of pET27b-ANGPTL3. Kan: Kanamycin resistance gene, NT-ANGPTL3: NT-ANGPTL3 coding gene, His tag: 6× histidine tag;

[0043] Figure 2 : Identification of purified recombinant human NT-ANGPTL3 protein by 12% (w / v) SDS-PAGE and Western Blot analysis;

[0044] A: Identification of purified recombinant human NT-ANGPTL3 protein by SDS-PAGE analysis, M: Protein Marker; Lanes 3 and 4: Purified recombinant human NT-ANGPTL3 protein. B: Identification of purified recombinant human NT-ANGPTL3 protein by Western Blot;

[0045] Figure 3 : Detection of the relative affinity of anti-human NT-ANGPTL3 single-chain antibody positive clones screened by Phage ELISA with NT-ANGPTL3. Among them, NT-ANGPTL3: Coated with 10 μg / mL recombinant human NT-ANGPTL3; BSA: 5 μg / mL BSA; M13K07: Helper phage M13K07 without any displayed single-chain antibody (n = 3, means ± SEM);

[0046] Figure 4 : Identification of single-chain antibody by 12% (w / v) SDS-PAGE;

[0047] M: Protein molecular weight standard; Lanes 1 - 14 are single-chain antibodies Sc-1519, Sc-232, Sc-2323, Sc-23183, Sc-4324, Sc-4429, Sc-4470, Sc-44107, Sc-44132, Sc-44362, Sc-44396, Sc-44448, Sc-44523, Sc-44566 respectively;

[0048] Figure 5 : Effects of anti-human NT-ANGPTL3 single-chain antibody on the triglyceride content in the serum of C57BL / 6 mice (n = 6, means ± SEM);

[0049] Figure 6 : pTT5-rhLPL plasmid map (A) and Western Blot analysis of rhLPL protein in the medium (B);

[0050] Figure 7 : pTT5-F1519 / F44132-Hc and pTT5-F1519 / F44132-Lc plasmid maps. AMP: Ampicillin resistance gene, F1519 / F44132-Hc: Heavy chain coding gene of F1519 or F44132, F1519 / F44132-Lc: Light chain coding gene of F1519 or F44132, CMV: CMV promoter;

[0051] Figure 8 : Effects of fully human anti-human NT-ANGPTL3 full-length antibodies F1519 and F44132 on the in vitro activity of LPL (n = 3, means ± SEM);

[0052] Figure 9 : Effects of fully human anti-human NT-ANGPTL3 full-length antibodies F1519-V90A, F1519-D95aA and F1519-H95aA on the in vitro activity of LPL (n = 3, means ± SEM);

[0053] Figure 10 ∶ Effects of fully human anti-human NT-ANGPTL3 full-length antibodies F1519-H95bD, F1519-H95bE, F1519-H95bK, F1519-H95bR and F1519-V90A / D95aA on the in vitro activity of LPL (n = 3, means ± SEM);

[0054] Figure 11:The TG-lowering activities of anti-human NT-ANGPTL3 antibodies F1519, F1519-V90A, and F1519-D95aA in C57BL / 6 mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Normal group (n = 8, means ± SEM);

[0055] Figure 12 :Effects of anti-human NT-ANGTL3 antibodies F1519 and F1519-D95aA on food intake (A) and body weight (B) of C57BL / 6 mice on a high-fat diet. Among them, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. HFD+vehicle group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. LFD+vehicle group. (n = 6, means ± SEM);

[0056] Figure 13 :Changes in serum TG (A) and LDL-C (B) levels of C57BL / 6 mice in each group during the administration of anti-human NT-ANGTL3 antibodies F1519 and F1519-D95aA. (n = 6, means ± SEM);

[0057] Figure 14 :Changes in serum TC (A) and HDL-C (B) levels of C57BL / 6 mice in each group 8 weeks after the administration of anti-human NT-ANGTL3 antibodies F1519 and F1519-D95aA (n = 6, means ± SEM) and changes in APOB (C) (n = 3, means ± SEM);

[0058] Figure 15 :Effects of anti-human NT-ANGTL3 antibodies F1519 and F1519-D95aA on liver morphology (A) and lipid accumulation (B) of C57BL / 6 mice fed a high-fat diet;

[0059] Figure 16 :Effects of anti-human NT-ANGTL3 antibodies F1519 and F1519-D95aA on liver TG (A), TC (B), and LDL-C (C) levels of C57BL / 6 mice fed a high-fat diet. (n = 6, means ± SEM). Detailed implementation manners

[0060] The following further illustrates the present invention in combination with specific examples.

[0061] Example 1: Expression, Purification and Identification of Recombinant Human NT-ANGPTL3 Protein

[0062] 1.1 Construction of Prokaryotic Expression Vector of Recombinant Human NT-ANGPTL3

[0063] The coding region sequence of human NT-ANGPTL3 was derived from the GenBank database (accession number: NP_055310), and the amino-terminal active domain (114bp - 734bp) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After PCR amplification of the human NT-ANGPTL3 gene fragment, it was subcloned into the prokaryotic expression vector pET27b using conventional molecular biological methods, and this recombinant plasmid was named pET27b-NT-ANGPTL3( Figure 1 ). For the convenience of purifying the expression product, a 6×histidine tag (6×His-tag) was introduced at the 5' end of the coding gene.

[0064] 1.2 Expression and Purification of Recombinant Human NT-ANGPTL3 Protein

[0065] The above plasmid was transformed into the Escherichia coli expression strain E. coli BL21(DE3). This expression strain was inoculated into 2×YT-K (containing 50 μg / mL kanamycin) medium and cultured until OD 600 nm = 0.6, then IPTG (Sangon Biotech, cat # A600168) with a final concentration of 0.2 mM was added, and induction was carried out at 16 °C for 20 h. Centrifugation was performed at 4640 rpm and 4 °C for 10 min to collect the bacterial cells; the periplasmic space protein was extracted by incubating with 0.4 mol / L arginine buffer (pH 8.0) at 4 °C for 45 min (Patent US19960594469.3, 1 Jan 1996); centrifugation was performed at 12000 rpm and 4 °C for 30 min to collect the supernatant; after the supernatant was filtered through a 0.22 μm filter membrane, the target protein was purified using a nickel ion affinity column (GE Healthcare, cat#17-5318-01). The impurity proteins were eluted with PBS buffer containing 30 mmol / L, 50 mmol / L, and 100 mmol / L imidazole, and the target protein was eluted with PBS buffer containing 500 mmol / L imidazole. The collection tubes containing the target protein were ultrafiltered and concentrated, and then further purified by Superdex75 size exclusion chromatography (GE Healthcare, cat # 17-5174-01), and the collected samples were detected by 12% SDS-PAGE( Figure 2 A). The purified fusion protein was filtered through a 0.22 μm sterile filter head to remove bacteria, then aliquoted and stored in a -80 °C refrigerator.

[0066] 1.3 Western Blot Identification of Recombinant Human NT-ANGPTL3 Protein

[0067] The obtained protein was identified by Western Blot using Anti-6×His rabbit polyclonal antibody (Sangon Biotech, cat # D110002) ( Figure 2 B). After subjecting the purified NT-ANGPTL3 protein to SDS-PAGE electrophoresis (separating gel concentration 12%), it was transferred onto a PVDF membrane (Roche, cat # 03010040001) at 4°C and a constant voltage of 100V for 100 min. The membrane was blocked in 5% MTBST (TBS containing 5% skim milk and 0.1% Tween 20) at room temperature for 1 h. The Anti-6×His rabbit polyclonal antibody (Sangon Biotech, cat # D110002) was diluted 1:2000 with 5% MTBST and incubated at room temperature for 2 h, followed by washing 3 times with TBST, 10 min each time. The HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (Sangon Biotech, cat # D110058) was diluted 1:5000 with 5% MTBST and incubated at room temperature for 1 h, then washed 3 times with TBST, 10 min each time, and developed with ECL.

[0068] Example 2: Screening of Human Anti-Human NT-ANGPTL3 Single-Chain Antibodies by Phage Display Technology

[0069] 2.1 Solid-Phase Panning and Enrichment of Anti-Human NT-ANGPTL3 Single-Chain Antibodies

[0070] Dilute NT-ANGPTL3 protein to a concentration of 80 μg / mL with coating buffer (50 mM NaHCO 3 , pH 9.6), take 2 mL and add it to an immunotube (Beijing Baikewei Food Safety Biotechnology Co., Ltd., cat #(444202), the rotary shaker was incubated overnight at 4 °C (the coating antigen concentrations in the second to fifth rounds were 40, 20, 10, and 5 μg / mL respectively). The next day, the supernatant was discarded, and the immunotubes were quickly washed 3 times with PBS. PBS buffer containing 5% BSA was added and incubated at 37 °C for 2 h for blocking. The blocking solution was discarded, and the immunotubes were washed 3 times with PBS. The activated phage antibody library (constructed in our laboratory previously, Chinese patent document, CN111620950A) was suspended in 4 mL of 5% BSA buffer and added to the immunotubes, and incubated on a rotary shaker at room temperature for 1 h, and then left standing at room temperature for 2 h (the standing times in the second to fifth rounds were 100, 80, 60, and 60 min respectively). Then the immunotubes were washed 5 times with PBST and PBS respectively (the washing times of PBST and PBS in the second to fifth rounds were 10, 15, 20, and 30 times respectively). 1 mL of 100 mM Gly-HCl (pH 2.2) was added, and incubated with rotation at room temperature for 10 min. The eluted phages were aspirated, and immediately 0.5 mL of 1 M Tris-HCl (pH 7.4) was added and mixed for neutralization. The neutralized phages were used to infect 50 mL of exponentially growing Escherichia coli TG1 and cultured to 200 mL. The corresponding amount of helper phage M13KO7 was added according to the ratio of TG1 to helper phage M13KO7 of 1:20 for infection, and cultured overnight at 30 °C; the next day, the culture supernatant was collected by centrifugation, and after PEG precipitation, it was used for the next round of panning, and a total of 5 rounds of enrichment were performed.

[0071] 2.2 Phage ELISA Screening for Anti-human NT-ANGPTL3 Single-chain Antibodies

[0072] The phage particles eluted in the 3rd to 5th rounds were used to infect Escherichia coli TG1 and then spread on 2×YT-AG (containing 100 μg / mL ampicillin and 1% (w / v) glucose) plates. Single colonies were picked with sterile toothpicks into a 96-well deep-well plate (Corning, cat # 3590). 500 μL of 2×YT-A (containing 100 μg / mL ampicillin) medium was added to each well and cultured overnight at 37 °C. 50 μL of the overnight culture was transferred to a new 96-well deep-well plate per well, 500 μL of 2×YT-AG medium was added to each well, and cultured to the exponential phase at 37 °C. The corresponding amount of M13KO7 was added according to the ratio of TG1 to M13KO7 of 1:20. After standing at 37 °C for 30 min, it was cultured with shaking (220 rpm) for 30 min. After centrifugation at 2000 g at low temperature for 20 min, the supernatant was completely aspirated. Each well was resuspended with 500 μL of 2×YT-AK (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin) medium and cultured with shaking at 30 °C and 220 rpm for 16 h. The next day, the deep-well plate was centrifuged at 2000 rpm for 10 min, and the obtained supernatant was the displayed phage antibody, which was used for phage ELISA screening.

[0073] Dilute human NT-ANGPTL3 protein with coating buffer to 10 μg / mL, add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4 °C. Wash the ELISA plate 3 times with PBS, add 300 μL of PBS buffer containing 5% BSA to each well, and block at 37 °C for 2 h. Centrifuge to remove the blocking solution, add the monoclonal phage supernatant obtained above to the 96-well ELISA plate coated with antigen, and incubate at 37 °C for 2 h. Wash the ELISA plate 3 times with PBST and 1 time with PBS. Dilute HRP-conjugated mouse anti-M13phage mAb (Beijing Sino Biological, cat # 11973-MM05T-H) at 1:5000, add 100 μL / well to the ELISA plate, and incubate at 37 °C for 2 h. Wash the ELISA plate 3 times with PBST and 1 time with PBS, add the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) for color development, and measure the OD 450 at nm. Use the M13KO7 well as a negative control, and use values three times higher than the negative control value as positive clones. Sequence the positive clones with higher affinity to obtain 14 wild-type positive clones, which express single-chain antibodies Sc-232, Sc-1519, Sc-2323, Sc-23183, Sc-4324, Sc-4429, Sc-4470, Sc-44107, Sc-44132, Sc-44396, Sc-44362, Sc-44448, Sc-44523, Sc-44566( Figure 3 ). The heavy chain variable region sequences are shown in SEQ ID NO: 1-14, and the light chain variable region sequences are shown in SEQ ID NO: 15-28.

[0074] 2.3 Soluble expression, isolation and purification of anti-human NT-ANGPTL3 single-chain antibody

[0075] According to conventional molecular cloning operations, subclone the gene encoding the positive single-chain antibody onto the expression vector pET 27b and transform it into the E. coli expression strain E. coli BL21(DE3). Inoculate the expression strain into 2×YT-K (containing 50 μg / mL kanamycin) medium, culture until OD 600 at nm = 0.6, add IPTG at a final concentration of 0.2 mM, and induce expression at 16 °C for 20 h. Centrifuge at 4640 rpm at 4 °C for 10 min to collect the bacteria; incubate with 0.4 mol / L arginine buffer (pH 8.0) at 4 °C for 45 min to extract the periplasmic space protein; centrifuge at 12000 rpm at 4 °C for 30 min to collect the supernatant; after filtering the supernatant through a 0.22 μm filter membrane, use a nickel ion affinity column (GE, cat #(17-5318-01) was separated and purified, and imidazole with different concentrations was used to elute the miscellaneous proteins and the target protein; 12% SDS-PAGE was used to detect the collected samples; the eluate containing the target protein was ultrafiltered and concentrated, and then further purified by Superdex 75 size exclusion chromatography (GE Healthcare, cat # (17-5174-01). Finally, the purified protein was identified by 12% SDS-PAGE ( Figure 4 ).

[0076] Example 3. In vivo activity detection of anti-human NT-ANGPTL3 single-chain antibody

[0077] Single-chain antibodies Sc-1519, Sc-4470, Sc-44132, and Sc-44566 with relatively high affinity for NT-ANGPTL3 were selected for in vivo activity detection in mice. Referring to the literature (J Lipid Res. 2015, 56(7): 1308-1317), 42 C57BL / 6 mice (Qinglongshan Animal Farm, Jiangning District, Nanjing City, license number: SCXK (Su) 2017-0001, certificate number: 201927998) were randomly divided into seven groups, with six mice in each group. During the experiment, the room temperature in the animal house was maintained at 22°C - 24°C, the humidity was 40% - 60%, and the light condition was 12 h of light and 12 h of darkness. During the experiment, the mice had free access to water and were given sufficient regular feed. After one week of adaptive feeding, the experiment was carried out. The experimental model establishment and drug administration doses are shown in Table 1. In the experiment, except for the blank control group, mice in other groups were intraperitoneally injected with 20% fat emulsion injection (Sichuan Kelun Pharmaceutical Co., Ltd., cat # B18100905CW-2) to form a hypertriglyceridemia model, and then the drug was immediately administered by subcutaneous injection to each drug administration group. After fasting for 18 h without water deprivation, the mice were anesthetized and blood was collected by eye socket puncture, and then the mice were sacrificed by cervical dislocation.

[0078] Table 1. Mouse grouping and drug administration

[0079]

[0080] The mouse blood samples were placed in centrifuge tubes and left to stand in a 4°C refrigerator for 4 h, then centrifuged at 4000 rpm for 20 min at 4°C, and the upper serum was aspirated. A triglyceride test kit (Nanjing Jiancheng Bioengineering Institute, cat # A110-1-10) was used to measure the triglyceride (TG) content in the serum samples ( Figure 5) After injecting 20% fat emulsion into the abdominal cavity of mice, the triglyceride content in the model group increased by 124.6% compared with the blank control group. After modeling, single-chain antibodies Sc-Evinacumab (positive control), Sc-1519, Sc-4470, Sc-44132, and Sc-44566 were respectively injected subcutaneously into the mice. The data showed that the concentration of triglyceride in the serum of the drug administration groups decreased to a certain extent compared with the model group. Among them, the triglyceride-lowering effects of the Sc-Evinacumab group, Sc-1519 group, and Sc-44132 group were the most significant, and they decreased by 40.7%, 39.2%, and 41.0% respectively compared with the model group. The results indicated that the single-chain antibodies Sc-1519 and Sc-44132 screened by a series of methods in this experiment had the same in vivo activity of lowering triglyceride as the single-chain form of the positive control drug Evinacumab (a marketed drug of Regeneron Pharmaceuticals, Inc., USA).

[0081] Example 4. Establish a detection method for the anti-NT-ANGPTL3 antibody to block the inhibition of LPL activity by NT-ANGPTL3

[0082] Lipoprotein lipase (LPL) plays a key role in the lipid metabolism process of humans. LPL catalyzes the hydrolysis of triglycerides and releases free fatty acids. NT-ANGPTL3 can inhibit the activity of LPL. Therefore, to block the inhibition of LPL activity by NT-ANGPTL3, we established a cell-free in vitro bioactivity detection method to determine the ability of the anti-NT-ANGPTL3 antibody to block the inhibition of LPL activity by NT-ANGPTL3 (Chinese Patent CN103732624 A; Chinese Patent CN101855241 A; Patent WO2012174178 A1).

[0083] To obtain the LPL conditioned medium, the pTT5-rhLPL expression vector was constructed. The amino acid sequence of rhLPL was derived from the GeneBank database (accession number: P06858, SEQ ID NO: 124). It was synthesized by Sangon and subcloned into the eukaryotic expression vector pTT5, and this recombinant expression plasmid was named pTT5-rhLPL( Figure 6 A). To facilitate the detection of the expression product, a 6× histidine tag (6×His-tag) was introduced at the 3' end of the rhLPL coding gene. The above expression plasmid pTT5-rhLPL was transfected into CHO 3E7 cells using PEI for transient protein expression. Hyclone TMAfter 7 days of suspension culture in Hycell CHO Medium serum-free medium, the cell culture supernatant was collected by centrifugation. After 10% (w / v) SDS-PAGE electrophoresis, it was transferred to a PVDF membrane. The membrane was blocked in 5% MTBST (TBS containing 5% skim milk and 0.1% Tween 20) at room temperature for 1 h. Anti-6×His rabbit polyclonal antibody (Sangon Biotech, cat # D110002) antibody was diluted 1:2000 with 5% MTBST and incubated at room temperature for 2 h, followed by washing 3 times with TBST for 10 min each time. HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (Sangon Biotech, cat # D110058) was diluted 1:5000 with 5% MTBST and incubated at room temperature for 1 h, followed by washing 3 times with TBST for 10 min each time, and then developed with ECL ( Figure 6 B).

[0084] The test samples were prepared as shown in Table 2, incubated at 37 °C for 2 h, and the free fatty acid level in the test samples was measured using a free fatty acid detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A042-2-1).

[0085] Table 2. Detection of the inhibition of LPL activity by anti-NT-ANGPTL3 antibody blocking NT-ANGPTL3

[0086]

[0087]

[0088] Example 5. Full-lengthization of anti-NT-ANGPTL3 single-chain antibody and in vitro activity detection of blocking NT-ANGPTL3 inhibition of LPL

[0089] 5.1 Preparation of full-length antibody in IgG form

[0090] To further evaluate the activity of the full-length antibody, after analyzing the subtypes and subgroups of the variable regions of the single-chain antibody (http: / / www.abysis.org / ), the VH sequence of the dominant single-chain antibody Sc-1519 screened in Example 3 was ligated into the sequence of the heavy-chain constant region in the form of IgG4 (SEQ NO: 29) by conventional molecular biology operations, and the VL sequence was ligated into the sequence of the lambda-type light-chain constant region (SEQ NO: 30); the VH sequence of the dominant single-chain antibody Sc-44132 was ligated into the sequence of the heavy-chain constant region in the form of IgG4 (SEQ NO: 29), and the VL sequence was ligated into the sequence of the kappa-type light-chain constant region (SEQ NO: 31). They were respectively subcloned into the eukaryotic expression vector pTT5 ( Figure 7 ). A Kozak sequence (GCCGCCACC) and a signal peptide sequence (heavy chain: MDWTWRFLFVVAAATGVQS; light chain: MDMRVPAQLLGLLLLWLSGARC) were introduced at the N-terminus of VH and VL.

[0091] PEI and the constructed mammalian cell expression plasmids of the heavy and light chains were co-transfected into CHO3E7 cells at a ratio of 5:1 and cultured in serum-free cell culture medium. After 7 days, the supernatant of the expressed cell culture fluid was collected, and the anti-human NT-ANGPTL3 full-length antibodies were purified using a Protein A-Agarose (Roche, cat#11134515001) affinity chromatography pre-packed column (column volume 1 mL, sample loading flow rate 0.5 mL / min), and named F1519 and F44132 respectively.

[0092] 5.2 In vitro activity detection of the full human anti-NT-ANGPTL3 full-length antibody blocking NT-ANGPTL3 from inhibiting LPL

[0093] The in vitro activities of the full human anti-human NT-ANGPTL3 full-length antibodies F1519 and F44132 were detected according to Example 4. The results showed that F1519 could effectively neutralize NT-ANGPTL3 and maintain the hydrolytic activity of LPL against TG ( Figure 8 ).

[0094] Example 6. Screening of high-activity mutants of F1519

[0095] Identifying key amino acid residue sites that affect antibody-antigen interaction is crucial for antibody affinity maturation (Plos One. 2015; 10: e0134600). In terms of the degree of participation in antibody-antigen interaction, generally CDR3 > CDR2 > CDR1. Therefore, after analyzing the CDR3 mutation hotspots, alanine scanning is preferentially performed on amino acid residues other than the conserved amino acids in LCDR3. Using the F1519 light chain plasmid as a template, the full-length plasmid was amplified by site-directed mutagenesis using one-step PCR. The mutation site statistics table is shown in Table 3, and the primers for site-directed mutagenesis at the designated sites are shown in Table 4.

[0096] Table 3. Statistics table of LCDR3 mutation sites of full-length antibody F1519

[0097]

[0098] Table 4. Table of primers for site-directed mutagenesis of LCDR3 of full-length antibody F1519

[0099]

[0100] After expression and purification according to the method described in Example 5, the mutants were obtained: F1519-V90A (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 32), F1519-D95aA (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 33), F1519-H95bA (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 34), and their in vitro activities were detected according to the method described in Example 4. The results showed that Evinacumab, F1519, F1519-V90A, and F1519-D95aA could all effectively neutralize NT-ANGPTL3 and maintain the hydrolysis activity of LPL on TG; while the neutralization effect of F1519-H95bA on NT-ANGPTL3 was not obvious ( Figure 9 ). It indicates that the amino acid residue at position 95b is a key amino acid residue affecting antibody-antigen binding. Therefore, it was mutated to other charged polar amino acid residues (Asp, Glu, Lys, Arg); and positions 90 and 95a were simultaneously mutated to alanine. The primers for site-directed mutagenesis at the designated sites are shown in Table 5.

[0101] Table 5. Table of primers for site-directed mutagenesis at positions 95a and 95b of full-length antibody F1519

[0102]

[0103]

[0104] After expression and purification according to Example 5, the mutants were obtained: F1519-H95bD (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 35), F1519-H95bE (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 36), F1519-H95bK (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 37), F1519-H95bR (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 38), F1519-V90A / D95aA (the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 39). The in vitro activity results showed that F1519-H95bD, F1519-H95bE, and F1519-H95bK had no obvious neutralizing effect on NT-ANGPTL3; the in vitro activities of F1519-H95bR and F1519-V90A / D95aA were both weaker than that of F1519( Figure 10 ). In summary, the in vivo TG-lowering activities of F1519-V90A and F1519-D95aA with higher in vitro activities were preferentially studied.

[0105] Example 7. Study on the Hypolipidemic Activity of a Fully Human Anti-human NT-ANGPTL3 Antibody in C57BL / 6 Mice

[0106] C57BL / 6 mice were adaptively fed for one week and randomly divided into a normal control group and four drug administration groups (Evinacumab group, F1519 group, F1519-V90A and F1519-D95aA group), with n = 8. Each drug administration group was intraperitoneally injected with Evinacumab, F1519, F1519-V90A, and F1519-D95aA at 10 mg / kg, and the normal control group was intraperitoneally injected with an equal volume of PBS. Blood was collected from the fundus venous plexus on the 1st, 4th, 7th, and 12th days after drug administration to detect the serum TG level. The results showed that the fully human antibodies F1519, F1519-V90A, and F1519-D95aA all had in vivo TG-lowering activities. Among them, F1519-D95aA had a longer time to reduce the serum TG level than Evinacumab( Figure 11 ).

[0107] Example 8. Study on the Hypolipidemic Activity of an Anti-NT-ANGPTL3 Antibody in a C57BL / 6 Hyperlipidemic Mouse Model

[0108] 8.1 Detection of the lipid-lowering activity of anti-NT-ANGPTL3 antibody in C57BL / 6 hyperlipidemic mice

[0109] C57BL / 6 mice were adaptively fed for one week and randomly divided into a normal control group, a model group, and three drug administration groups (Evinacumab group, F1519 group, and F1519-D95aA group), with n = 6. The normal control group was given a low-fat control diet; the model group, Evinacumab group, F1519 group, and F1519-D95aA group of mice were given a high-fat diet. Three days after modeling, drug administration began: the Evinacumab group, F1519 group, and F1519-D95aA group were respectively given subcutaneous injection of Evinacumab, F1519, and F1519-D95aA (25 mg / kg, in 200 μL PBS), and the normal control group and the model group were given an equal volume of PBS. The drug was administered once a week for 8 consecutive weeks.

[0110] During the drug administration period, the food intake and body weight changes of the mice were recorded weekly. After 3 weeks of high-fat diet feeding, the body weight of the mice in the model group was significantly higher than that in the normal control group, while there was no significant difference in the body weight of the mice in each drug administration group compared with the model group. Evinacumab, F1519, and F1519-D95aA did not affect the body weight of C57BL / 6 mice fed a high-fat diet ( Figure 12 ). In addition, mouse serum was collected at 2, 4, 6, and 8 weeks after drug administration, and the TG detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A110-1) and the LDL-C detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A113-1) were used to measure the changes in serum TG and LDL-C levels during drug administration. F1519 and F519-D95aA could significantly reduce the serum TG and LDL-C levels of C57BL / 6 mice fed a high-fat diet during drug administration ( Figure 13 ). At the same time, the TC detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A111-1) and the HDL-C kit (Nanjing Jiancheng Bioengineering Institute, cat # A112-1) were used to detect the serum TC and HDL-C levels of the mice 8 weeks after drug administration, and Western Blot was used to detect the APOB level in the mouse serum. The results showed that F1519-D95aA had a certain effect on reducing TC and had no significant effect on HDL-C; F1519 had no significant effect on TC and HDL-C; the serum APOB protein level of mice fed a high-fat diet was significantly increased, and the APOB protein levels in each drug administration group were significantly decreased, with the strongest reducing effect of F1519-D95aA, followed by F1519. Figure 14 ).

[0111] 8.2 Improvement effect of anti-NT-ANGPTL3 antibody on liver lipid deposition in C57BL / 6 hyperlipidemic mice

[0112] After 8 weeks of administration, the livers of mice were taken for photography to observe the liver morphology. The results showed that the livers of mice in the normal control group (LFD+vehicle) were bright red; the livers of the model control group (HFD+vehicle) were pale in color and greasy and rough on the surface; the liver morphology of the administration groups was restored to varying degrees ( Figure 15 A). Another fresh liver was embedded with OCT embedding medium (SAKURA, cat # 4583), frozen embedded and made into 10μm frozen sections, and stained with Oil Red O staining solution (Beijing Solarbio Science & Technology Co., Ltd., cat # G1261) to observe the liver lipid deposition in mice. The results showed that the livers of mice in the normal control group (LFD+vehicle) were bright red, and almost no orange-red lipid droplets were visible; the livers of the model control group (HFD+vehicle) were pale in color, and a large number of orange-red lipid droplets accumulated; the orange-red lipid droplets in the livers of the administration groups were all reduced ( Figure 15 B). The above results indicate that Evinacuman, F1519 and F1519-D95aA can all significantly improve the liver morphology of mice fed with high-fat diet and reduce liver lipid deposition. At the same time, after the mouse liver was homogenized and treated with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., cat#R0020), TG detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A110-1), TC detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A111-1) and LDL-C detection kit (Nanjing Jiancheng Bioengineering Institute, cat # A113-1) were used to detect the levels of TG, TC and LDL-C in the mouse liver. The results showed that F1519 and F1519-D95aA had no significant effect on liver TG and TC; they had different degrees of reduction on LDL-C ( Figure 16 ).

[0113] Example 9. Determination of the affinity constant (K D ) of anti-NT-ANGPTL3 antibody

[0114] ForteBio Octet QK was used eDetermination of the affinity constant between the anti-NT-ANGPTL3 antibody and NT-ANGPTL3 using a biomolecular interaction analyzer: Mix NT-ANGPTL3 protein and biotin at a molar ratio of 1:5, incubate at room temperature for 2 h, and remove unreacted biotin using a desalting column to obtain biotinylated NT-ANGPTL3. Turn on the instrument at least 45 min in advance before measurement, and hydrate the biosensor in 1×SD Buffer for at least 10 min. Add 200 μL of 1×SD Buffer to each well in the first and third columns of a black 96-well plate; dilute biotinylated NT-ANGPTL3 to 50 μg / mL with SD Buffer and add 200 μL to each well in the second column; dilute the antibody to be tested with SD Buffer in 4 concentration gradients: 800 nM, 400 nM, 200 nM, 100 nM, and add 200 μL to each well in the fourth column; add SD Buffer to the last well as a control well. Place the 96-well ELISA plate into the OctetQK e biomolecular interaction analyzer; in the ForteBio Octet QK e system, set the program. After the streptavidin biosensors are sequentially equilibrated in SD buffer for 60 s, loaded in biotinylated NT-ANGPTL3 for 300 s, and equilibrated in SD buffer for 120 s, they are allowed to bind to the antibody for 300 s and dissociate in SD buffer for 600 s. Use the ForteBio Octet QK e data analysis software to calculate the equilibrium dissociation constant K D value and other kinetic parameters. The experimental results are shown in Table 6.

[0115] Table 6 Kinetic parameters of the binding between the anti-NT-ANGPTL3 antibody and NT-ANGPTL3

[0116]

[0117]

[0118] *N / A indicates not detected.

Claims

1. A fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof, wherein, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2; and comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:

33.

2. The fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1, wherein, the antibody or the antigen-binding fragment is selected from the following structural forms: full-length antibody, Fab, F(ab'), F(ab’)2, Fv, scFv, diabody, microbody, bispecific antibody, multispecific antibody, chimeric antibody, and a functional fragment of an antibody in which a heavy chain variable region is fused with a complete light chain, VH-L.

3. A polynucleotide, wherein, it encodes the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1.

4. A vector, wherein, it contains the polynucleotide according to claim 3.

5. A host cell, wherein, it contains the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1, the polynucleotide according to claim 3, or the vector according to claim 4.

6. A composition, wherein, it contains the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1, the polynucleotide according to claim 3, or the vector according to claim 4, and a pharmaceutically acceptable carrier, diluent or excipient thereof.

7. A conjugate comprising the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1, wherein, the conjugate is a detectable label, and the detectable label includes a fluorescent substance, a colored substance, a radioisotope or an enzyme.

8. Use of the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1, or the conjugate comprising the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 7 in the preparation of a diagnostic reagent for detecting NT-ANGPTL3.

9. Use of the fully human anti-human NT-ANGPTL3 antibody or an antigen-binding fragment thereof according to claim 1 in the preparation of a medicament for treating hypertriglyceridemia, hypercholesterolemia, chylomicronemia, and atherosclerosis.

Images