WO2023116751A1

WO2023116751A1 - Anti-human angiopoietin-like 3 nanobody and use thereof

Info

Publication number: WO2023116751A1
Application number: PCT/CN2022/140639
Authority: WO
Inventors: 鞠佃文; 胡晓植; 韩磊; 范佳君; 巫涛; 南艳阳; 周楷程; 王翰麒
Original assignee: 复旦大学
Priority date: 2021-12-21
Filing date: 2022-12-21
Publication date: 2023-06-29
Also published as: CN116284377A

Abstract

The present invention relates to the biopharmaceutical field. The present invention discloses an anti-human ANGPTL3 nanobody. The present invention further discloses a method for preparing a fusion protein (anti-hANGPTL3 VHH-Fc) formed by linking the nanobody to a human IgG1 Fc fragment. In addition, the present invention further discloses the use of the anti-hANGPTL3 VHH-Fc fusion protein in non-alcoholic fatty liver disease and atherosclerosis. The fusion protein of the present invention maintains the biological activity of the VHH fragment of an alpaca anti-human ANGPTL3 heavy chain antibody, introduces the Fc fragment to prolong the half-life thereof, significantly alleviates the lipid deposition of non-alcoholic fatty liver disease, reduces the formation of atherosclerotic plaques, and has good application prospects.

Description

Anti-human angiopoietin 3 nanobody and application thereof

technical field

The invention belongs to the technical field of biopharmaceuticals, and relates to a nanobody specifically binding to human ANGPTL3; in particular to a preparation of an anti-human ANGPTL3 nanobody and a human immunoglobulin IgG1 Fc fragment fusion protein (anti-hANGPTL3 VHH-Fc); and the Use of the nanobody in the preparation of an ANGPTL3 inhibitor for preventing or treating non-alcoholic fatty liver and atherosclerosis.

Background technique

With economic growth and changes in living habits, non-alcoholic fatty liver disease (NAFLD) has become a new challenge in the field of global metabolic diseases and liver diseases. The latest epidemiological studies show that the global prevalence of NAFLD is as high as 25%, especially in developing countries, where the incidence is increasing rapidly. Hyperlipidemia is manifested as an abnormal increase in plasma lipids such as cholesterol and low-density lipoprotein cholesterol. When the body causes hyperlipidemia due to unhealthy diet, genetic defects or other metabolic diseases, abnormal lipids will accumulate in the liver and cause abnormal Alcoholic fatty liver. At the same time, abnormal lipid metabolism, especially low-density lipoprotein cholesterol, can lead to atherosclerosis or fibrous plaque formation in the vascular intima, eventually leading to stenosis of the vascular wall. Therefore, it is of great significance to study new therapeutic agents for NAFLD and atherosclerosis.

Angiopoietin-like protein 3 (ANGPTL3), also known as angiopoietin-5, is a member of the ANGPTL protein family. Studies have found that ANGPTL3 can be cleaved at Arg221 to Ala222 and Arg224 to Thr225, resulting in an N-terminal coiled-coil domain (CCD) and a C-terminal fibrinogen-like domain (FLD) ). Its N-terminal fragment can reversibly inhibit the catalytic activity of LPL, leading to an increase in plasma TG levels; the C-terminal fragment can bind to integrin-binding αvβ3 receptors and participate in angiogenesis. ANGPTL3 is mainly expressed in the liver and rarely expressed in other tissues and organs. More and more evidence shows that ANGPTL3 is related to most lipid metabolism-related diseases, especially hyperlipidemia, coronary heart disease and atherosclerosis very close. Recent studies have shown that ANGPTL3 is highly expressed in the liver of NAFLD patients, and the content of ANGPTL3 in serum is also higher than that of healthy people. Therefore, the levels of TG and LDL-C in plasma can be reduced by inhibiting ANGPTL3, thereby treating NAFLD and atherosclerosis. At present, the inhibitors of ANGPTL3 mainly include monoclonal antibodies, siRNA and antisense oligonucleotides, etc., all of which have played a very good role in reducing TG and LDL-C in plasma. The development of ANGPTL3 antibody drugs is mainly focused on mouse-derived traditional antibodies, and there are more other forms of monoclonal antibodies to be developed.

Since 1993, Hamers et al. discovered heavy chain antibodies naturally lacking light chains in the serum of camelids. This antibody only contains a heavy chain variable region (VHH) and two CH2 and CH3 regions, retaining a complete antigen-binding site , the VHH single domain antibody obtained by cloning and expression is also called nanobody (nanobody, Nb) because of its diameter of 2.5nm and height of 4nm. The size of nanobodies is only 1/10 of that of ordinary IgG. There are a large number of hydrophilic residues on the surface, disulfide bonds inside, and stable physical and chemical properties. Compared with murine antibodies, nanobodies have stronger resistance to heat and pH, and have the advantages of easy cloning and expression, good solubility, high affinity and easy labeling. In addition, through sequence homology analysis, it was found that the VHH germline gene sequence of Nanobodies is highly homologous to human VH3, but its CDR1 and CDR3 regions are slightly longer than those of humans, and the CDR3 region protrudes outward in the tertiary structure, so it is speculated that Nanobodies have higher specificity and affinity for antigen binding than murine antibodies. However, although the small size of nanobodies provides more advantages for their therapeutic functions, it has the disadvantage that small molecular proteins are easily cleared in vivo. To prolong its half-life, the C-terminus of Nanobodies can be linked directly or via a linker peptide to the N-terminus of an IgG Fc fragment of an immunoglobulin.

Based on the current state of the art, the inventors of the present application intend to provide nanobodies that specifically bind to human ANGPTL3; specifically relate to a fusion protein of anti-human ANGPTL3 nanobody and human immunoglobulin IgG1 Fc fragment (anti-hANGPTL3 VHH-Fc) and its uses.

Contents of the invention

The purpose of the present invention is based on the current state of the art, to provide a nanobody that specifically binds to human ANGPTL3; specifically relates to a fusion protein (anti-hANGPTL3 VHH-Fc) of an anti-human ANGPTL3 nanobody and human immunoglobulin IgG1 Fc segment and its use.

One of the objectives of the present invention is to provide a Nanobody specifically binding to ANGPTL3, and to provide the amino acid and nucleotide sequences of the Nanobody or its fragments.

The second object of the present invention is to provide a fusion protein of the above Nanobody and human IgG1 Fc fragment, and provide the amino acid and nucleotide sequence of the fusion protein.

The third object of the present invention is to provide recombinant vectors and recombinant cells for recombinantly expressing the fusion protein.

The fourth object of the present invention is to provide the preparation method and application of the above fusion protein.

The fifth object of the present invention is to verify the ability of the fusion protein to neutralize, inhibit, block, eliminate, reduce or interfere with at least one activity of ANGPTL3, especially human ANGPTL3, and the treatment of the fusion protein in NAFLD and atherosclerosis effect.

In the present invention, a phage display library of anti-human ANGPTL3 nanobody is constructed, a high-affinity nanobody is screened out, and the nanobody is further connected with a human IgG Fc fragment to make a fusion protein to overcome the problem of rapid metabolism of the nanobody in vivo Disadvantages, experiments have verified the effect of the fusion protein on NAFLD and atherosclerosis models. Since ANGPTL3 inhibitors have significant blood lipid-lowering effects, and nanobodies have high specificity, affinity and stability, the fusion protein of the present invention is expected to become a new drug for the treatment of NAFLD and atherosclerosis, and has huge potential. prospects for clinical application.

Specifically, in order to achieve the above purpose, the present invention adopts the following technical solutions:

In a first aspect, the invention provides Nanobodies that specifically bind to and neutralize, inhibit, block, abolish, reduce or interfere with at least one activity of ANGPTL3, in particular human ANGPTL3. The activity of ANGPTL3 that can be neutralized, inhibited, blocked, eliminated, reduced or interfered with by the Nanobody includes but not limited to the inhibition of LPL catalytic activity, the effect of inducing angiogenesis and the like. In one embodiment, the Nanobodies of the invention are capable of neutralizing, inhibiting, blocking, abrogating, reducing or interfering with the activity of hANGPTL3 by binding to an epitope of hANGPTL3 directly involved in the targeting activity of hANGPTL3. In another embodiment, the Nanobodies of the invention are capable of neutralizing, inhibiting, blocking, abrogating, reducing or interfering with the activity of hANGPTL3 by binding to an epitope of hANGPTL3 that is not directly involved in the targeting activity of hANGPTL3, because it The bound antibody or fragment sterically or conformationally inhibits, blocks, abolishes, reduces or interferes with hANGPTL3 targeting activity. In another embodiment, the Nanobody of the invention binds to an epitope of hANGPTL3 that is not directly involved in hANGPTL3 targeting activity (e.g. inhibition of LPL activity, induction of angiogenesis, etc.) (i.e. a non-blocking antibody), but The bound Nanobody results in an increased clearance of hANGPTL3 from the circulation compared to the clearance of hANGPTL3 in the absence of the Nanobody, thereby indirectly inhibiting, blocking, eliminating, reducing or interfering with the activity of hANGPTL3. Combining two or more different non-blocking antibodies that do not compete with each other for specific binding to hANGPTL3 can, inter alia, increase the clearance of hANGPTL3 from the circulation.

Further, the present invention provides a Nanobody C44 specifically binding to ANGPTL3, comprising a heavy chain variable region (VHH), the amino acid sequence of which is shown in SEQ ID NO: 1, and the above VHH consists of framework regions (FR1, FR2, FR3 and FR4) and complementarity determining region (CDR1, CDR2 and CDR3), its FR1 amino acid sequence is as described in SEQ ID NO: 2, FR2 amino acid sequence is as described in SEQ ID NO: 3, and FR3 amino acid sequence is as described in SEQ ID NO: 4 The amino acid sequence of FR4 is as described in SEQ ID NO: 5, the amino acid sequence of CDR1 is as described in SEQ ID NO: 6, the amino acid sequence of CDR2 is as described in SEQ ID NO: 7, and the amino acid sequence of CDR3 is as described in SEQ ID NO: 8:

Further, the present invention also provides the coding nucleotide sequence of the above-mentioned Nanobody C44 such as SEQ ID NO: 9, the above-mentioned C44 Nanobody is composed of framework regions (FR1, FR2, FR3 and FR4) and complementarity determining regions (CDR1, CDR2 and CDR3 ), its FR1 nucleotide sequence is as described in SEQ ID NO: 10, the FR2 nucleotide sequence is as described in SEQ ID NO: 11, the FR3 nucleotide sequence is as described in SEQ ID NO: 12, and the FR4 nucleotide sequence The sequence is as described in SEQ ID NO: 13, the CDR1 nucleotide sequence is as described in SEQ ID NO: 14, the CDR2 nucleotide sequence is as described in SEQ ID NO: 15, and the CDR3 nucleotide sequence is as described in SEQ ID NO: 16 stated. The full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis.

In a second aspect, the invention provides Nanobodies and human IgG1 Fc fragment fusion proteins (Nanobody -Fc). The activity of ANGPTL3 that can be neutralized, inhibited, blocked, eliminated, reduced or interfered by the Nanobody-Fc fusion protein includes but not limited to the inhibition of LPL catalytic activity, the effect of inducing angiogenesis and the like. In one embodiment, the Nanobody-Fc fusion protein of the invention is capable of neutralizing, inhibiting, blocking, abrogating, reducing or interfering with the activity of hANGPTL3 by binding to an epitope of hANGPTL3 directly involved in the targeting activity of hANGPTL3. In another embodiment, the Nanobody-Fc fusion protein of the invention neutralizes, inhibits, blocks, abolishes, reduces or interferes with the activity of hANGPTL3 by binding to an epitope of hANGPTL3 that is not directly involved in the targeting activity of hANGPTL3 , because the antibody or fragment it binds to inhibits, blocks, eliminates, reduces or interferes with the targeting activity of hANGPTL3 in space or conformation. In another embodiment, the Nanobody-Fc fusion protein of the invention binds to an epitope of hANGPTL3 that is not directly involved in hANGPTL3 targeting activity (e.g. inhibition of LPL activity, induction of angiogenesis, etc.) (i.e. a non-blocking antibody), but the bound Nanobody-Fc fusion protein results in increased clearance of hANGPTL3 from the circulation compared to the clearance of hANGPTL3 in the absence of the Nanobody-Fc fusion protein, thereby indirectly inhibiting, blocking, Eliminates, reduces or interferes with the activity of hANGPTL3. Combining two or more different non-blocking antibodies that do not compete with each other for specific binding to hANGPTL3 can, inter alia, increase the clearance of hANGPTL3 from the circulation. The Fc used in the present invention includes but not limited to human IgG1-Fc, human IgG4-Fc, mouse IgG1-Fc, and mouse IgG4-Fc.

Further, the amino acid sequence of the Fc portion of the immunoglobulin according to the present invention is shown in SEQ ID NO: 17:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17, IgG1 Fc fragment)

The nucleotide sequence of the immunoglobulin Fc part of the present invention is shown in SEQ ID NO: 18:

GAGCCCAAGTCTTGCGACAAGACCCACACTTGTCCTCCTTGTCCAGCCCCAGAGCTGCTGGGAGGGCCCAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCTCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTA AGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCTCGGGAGGAGATGACCAAGAACCA GGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCTAGCGACATCGCCGTCGAGTGGGAATCTAACGGCCAGCCCGAGAACAACTACAAAGACCACCTCCAGTGCTGGATAGCGACGGAAGCTTCTTCCTGTACAGCAAGCTGA CCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTTTCTTGCAGCGTGATGCACGAGGCTCTGCACAAC CACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG (SEQ ID NO: 18, IgG1 Fc fragment)

Further, the fusion protein of the anti-human ANGPTL3 nanobody C44 and the human IgG1 Fc fragment provided by the present invention, the fusion protein has the nanobody C44 at the N-terminus and the IgG1 Fc fragment at the C-terminus, and its amino acid sequence is shown in SEQ ID NO: 19 Show:

The present invention also provides a nucleic acid molecule encoding the gene of the C44-Fc fusion protein, the nucleotide sequence of which is shown in SEQ ID NO:20.

In a third aspect, the present invention relates to methods and combinations for preventing or attenuating non-alcoholic fatty liver disease in a subject. The methods of the invention comprise administering one or more doses of an angiopoietin-like protein-3 (ANGPTL3) inhibitor to a subject in need thereof. According to certain embodiments, the methods of the invention result in a reduction in non-alcoholic fatty liver lipid deposition and a reduction in liver damage in the subject.

In a fourth aspect, the present invention relates to methods and combinations for reducing atherosclerotic plaque in a subject. The methods of the invention comprise administering one or more doses of an angiopoietin-like protein-3 (ANGPTL3) inhibitor to a subject in need thereof. According to certain embodiments, the methods of the invention result in a decrease in atherosclerotic plaque area, an increase in plaque collagen content, and an increase in plaque stability in the subject.

The present invention also provides an expression vector and a host cell containing the gene encoding the above-mentioned anti-hANGPTL3 VHH-Fc fusion protein. The expression vector is a eukaryotic expression vector (pCHO 1.0, pFUSE-hIgG1e1-Fc2, pCDNA 3.1, PTT5, etc.), preferably a eukaryotic expression vector PTT5. The host cells are CHO cells, HEK-293 cells, etc., preferably CHO cells.

The present invention provides a preparation method of the above-mentioned ANGPTL3 nanobody: firstly, the ANGPTL3 antigen expressed by CHO cells is used to immunize alpacas, the peripheral blood cells of the immunized alpacas are collected, peripheral blood mononuclear cells (PBMCs) are separated, total RNA is extracted, and Nest -Clone the VHH region of the alpaca heavy chain antibody by PCR technology, insert it into the phage plasmid, construct the phage expression library, and then perform multiple rounds of screening on the ANGPTL3 antigen by phage display technology, and finally sequence the phage obtained from the screening to obtain the nanobody nucleic acid coding sequence.

The present invention provides a method for preparing the above-mentioned anti-hANGPTL3 VHH-Fc fusion protein: the method for preparing the anti-hANGPTL3 VHH-Fc fusion protein described in the present invention includes inserting a nucleic acid sequence encoding an anti-hANGPTL3 VHH-Fc fusion protein into into a suitable vector, obtain a corresponding suitable expression vector, and transfect a suitable host cell; and under suitable culture conditions, culture the transfected cell, and isolate and purify the expressed anti-hANGPTL3 VHH-Fc fusion protein therefrom, The affinity and binding constant of the obtained Nanobody-Fc fusion protein were verified by Biacore, and the high-affinity Nanobody-Fc fusion protein was screened out.

In the present invention, the conventionally used purification process in the art is adopted, including but not limited to: conventional treatment with protein precipitating agent (salting out method), centrifugation, osmotic bacterial destruction, ultracentrifugation, high performance liquid chromatography (HPLC) , Molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and other various liquid chromatography techniques and combinations of these methods. The purification process comprises the following steps: (a) collecting the cell culture supernatant; (b) separating Protein A; (c) further purifying with Superdex 200 molecular sieves. Purified by the process selected in the present invention, finally the anti-hANGPTL3 VHH-Fc fusion protein with a purity greater than 95% can be obtained.

In the first aspect of the present invention, an anti-human ANGPTL3 nanobody or an antigen-binding fragment thereof is provided, which comprises a determinant complementary region and a framework region, and the determinant complementary regions are all composed of CDR1, CDR2 and CDR3, characterized in that: The amino acid sequence of CDR1 is shown in SEQ ID NO: 6; the amino acid sequence of CDR2 is shown in SEQ ID NO: 7; the amino acid sequence of CDR3 is shown in SEQ ID NO: 8.

In another preferred example, the anti-human angiopoietin 3 Nanobody or its antigen-binding fragment further includes a framework region FR, and the framework region FR is selected from the following group:

Its FR1 amino acid sequence is described in SEQ ID NO:2, FR2 amino acid sequence is described in SEQ ID NO:3, FR3 amino acid sequence is described in SEQ ID NO:4, and FR4 amino acid sequence is described in SEQ ID NO:5.

In another preferred example, the amino acid sequence of the anti-human ANGPTL3 Nanobody or its antigen-binding fragment is shown in SEQ ID NO: 1.

In another preferred embodiment, the anti-human angiopoietin 3 nanobody or antigen-binding fragment thereof includes a monomer, a bivalent body (a bivalent antibody), a tetravalent body (a tetravalent antibody), and/or a multivalent body (multivalent antibody).

In another preferred example, the anti-human angiopoietin-3 nanobody or antigen-binding fragment thereof includes humanized antibody, camelid antibody, and chimeric antibody.

The second aspect of the present invention provides an anti-human ANGPTL3 Nanobody-Fc fragment fusion protein, which is composed of the C-terminus of the Nanobody or antigen-binding fragment described in the first aspect of the present invention directly or through a linker peptide and immunoglobulin IgG The N-terminals of the Fc fragments are connected; the Fc fragment is the Fc fragment of human immunoglobulin IgG1, and its amino acid is shown in SEQ ID NO: 17; the amino acid sequence of the fusion protein is shown in SEQ ID No: 19.

In another preferred example, the Fc fragment of IgG is selected from the group consisting of Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

The third aspect of the present invention provides a nucleic acid molecule encoding the anti-human ANGPTL3 nanobody described in the first aspect of the present invention or its antigen-binding fragment or its Fc fusion protein, the nucleic acid molecule sequence encoding CDR1 is as SEQ ID NO As shown in: 14, the nucleic acid molecule sequence encoding CDR2 is shown in SEQ ID NO: 15, and the nucleic acid molecule sequence encoding CDR3 is shown in SEQ ID NO: 16; encoding the anti-human angiopoietin 3 Nanobody or its antigen binding The nucleic acid molecule of the fragment is shown in SEQ ID NO:9.

In another preferred embodiment, a nucleic acid molecule encoding the anti-human ANGPTL3 Nanobody-Fc fragment fusion protein according to the second aspect of the present invention is provided; the nucleic acid sequence of the Fc portion of the fusion protein is as shown in SEQ ID NO: 18 Shown; Described fusion protein nucleic acid sequence is shown in SEQ ID No:20.

In another preferred example, the polynucleotide includes DNA or RNA.

The fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule according to the third aspect of the present invention.

In another preferred embodiment, the recombinant vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or combinations thereof.

Preferably, the recombinant vector includes a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

The fifth aspect of the present invention provides a recombinant cell into which the nucleic acid molecule according to the third aspect of the present invention is introduced, or transfected with the recombinant vector according to the fourth aspect of the present invention.

In another preferred example, the recombinant cells include prokaryotic cells or eukaryotic cells.

In another preferred embodiment, the recombinant cells are selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, phage, or combinations thereof.

In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, or combinations thereof.

The sixth aspect of the present invention provides a pharmaceutical composition, which comprises one or more of the above-mentioned antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors or recombinant cells, and optionally a pharmaceutical acceptable carrier or excipient.

In another preferred example, the pharmaceutical composition further comprises one or more drugs selected from HMG-CoA reductase inhibitors, cholesterol absorption inhibitors, bile acid reabsorption inhibitors or drugs that increase lipoprotein catabolism preparation.

In another preferred embodiment, the pharmaceutical composition further comprises one or more other therapeutic agents selected from statins, niacin, fibrates, anti-hANGPTL4 antibodies and anti-PCSK9 antibodies .

The seventh aspect of the present invention provides one or more of the above-mentioned antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant cells or pharmaceutical compositions, which are used in the preparation of preventing, alleviating, improving or inhibiting Use in a preparation or medicine for a disease or disorder.

In another preferred example, the preparation or drug reduces or inhibits ANGPTL3 activity.

The eighth aspect of the present invention provides one or more of the above-mentioned antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant cells or pharmaceutical compositions used in the preparation and treatment of hyperlipidemia, non-alcoholic fatty Drug use in liver or atherosclerosis.

The ninth aspect of the present invention provides an anti-human ANGPTL3 antibody, said antibody comprising one or more anti-human ANGPTL3 nanobodies as described in the first aspect of the present invention.

In another preferred example, the anti-human ANGPTL3 antibody includes monomers, bivalents (bivalent antibodies), tetravalents (tetravalent antibodies), and/or multivalents (multivalent antibodies), or chimeric Antigen receptor antibody (CAR).

In another preferred example, the anti-human ANGPTL3 antibody includes one or more VHH chains having the amino acid sequence shown in SEQ ID NO:1.

A tenth aspect of the present invention provides a method for producing an anti-human ANGPTL3 Nanobody or its Fc fusion protein, comprising the steps of:

(a) culturing a recombinant cell as described above under conditions suitable for the production of a Nanobody or Fc fusion protein thereof, thereby obtaining a culture comprising said anti-human ANGPTL3 Nanobody or Fc fusion protein thereof;

(b) isolating or recovering said anti-human ANGPTL3 Nanobody or Fc fusion protein thereof from said culture; and

(c) Optionally, purifying and/or modifying the anti-human ANGPTL3 Nanobody or its Fc fusion protein obtained in step (b).

The eleventh aspect of the present invention provides a multispecific antibody, said multispecific antibody comprising: the anti-human ANGPTL3 nanobody as described in the first aspect of the present invention, or as described in the ninth aspect of the present invention anti-human ANGPTL3 antibody.

In another preferred example, the second antigen-binding region is a Nanobody.

In another preferred example, the multispecific antibody includes one or more second antigen-binding domains.

In another preferred example, the multispecific antibody further comprises an Fc segment of the antibody.

In the twelfth aspect of the present invention, a recombinant protein is provided, and the recombinant protein has:

(i) an anti-human ANGPTL3 Nanobody or its Fc fusion protein as described in the first aspect of the present invention, or an anti-human ANGPTL3 antibody as described in the ninth aspect of the present invention; and

(ii) Optional tag sequences to aid in expression and/or purification.

In another preferred example, the tag sequence includes Fc tag, HA tag and 6His tag.

In another preferred example, the recombinant protein specifically binds to human ANGPTL3 protein.

The twelfth aspect of the present invention provides a CAR construct, the antigen-binding region of the CAR construct comprises a determinant complementary region, and the determinant complementary region is composed of CDR1, CDR2 and CDR3, and the amino acid of CDR1 The sequence is shown in SEQ ID NO: 6; the amino acid sequence of CDR2 is shown in SEQ ID NO: 7; the amino acid sequence of CDR3 is shown in SEQ ID NO: 8

The thirteenth aspect of the present invention provides a recombinant immune cell expressing the exogenous CAR construct as described in the twelfth aspect of the present invention.

In another preferred example, the immune cells are selected from the group consisting of NK cells and T cells.

In another preferred example, the immune cells are from humans or non-human mammals (such as mice).

The fourteenth aspect of the present invention provides an immunoconjugate comprising:

(a) an anti-human ANGPTL3 Nanobody or its Fc fusion protein according to the first aspect of the present invention, or an anti-human ANGPTL3 antibody according to the ninth aspect of the present invention; and

(b) a coupling moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof .

In another preferred example, the radionuclides include:

(i) isotopes for diagnosis, the isotopes for diagnosis are selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or

(ii) isotope for treatment, the isotope for treatment is selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.

In another preferred example, the coupling moiety is a drug (such as a cytotoxic drug) or a toxin.

In another preferred example, the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

The fifteenth aspect of the present invention provides the anti-human ANGPTL3 Nanobody as described in the first aspect of the present invention, the anti-human ANGPTL3 Nanobody-Fc fragment fusion protein as described in the second aspect of the present invention, and the anti-human ANGPTL3 Nanobody-Fc fragment fusion protein as described in the first aspect of the present invention. Use of the anti-human ANGPTL3 antibody described in the ninth aspect, or the immunoconjugate as described in the fourteenth aspect of the present invention; (a) for the preparation of a medicament for preventing and/or treating diseases or disorders related to human ANGPTL3 (b) for preparing reagents, detection plates or kits for detecting human ANGPTL3.

In another preferred example, the detection includes flow detection and cell immunofluorescence detection.

In another preferred embodiment, the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.

The sixteenth aspect of the present invention provides a method for detecting human ANGPTL3 protein in a sample, the method comprising the steps of:

(1) Sample the anti-human ANGPTL3 nanobody described in the first aspect of the present invention, the anti-human ANGPTL3 nanobody-Fc fragment fusion protein described in the second aspect of the present invention, the anti-human ANGPTL3 nanobody described in the ninth aspect of the present invention Human ANGPTL3 antibody, or an immunoconjugate as described in the fourteenth aspect of the present invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of human ANGPTL3 protein in the sample.

In another preferred example, the method is a non-diagnostic and non-therapeutic method.

The seventeenth aspect of the present invention provides a human ANGPTL3 protein detection reagent, the detection reagent comprising:

(i) the anti-human ANGPTL3 Nanobody as described in the first aspect of the present invention, the anti-human ANGPTL3 Nanobody-Fc fragment fusion protein as described in the second aspect of the present invention, the anti-human ANGPTL3 as described in the ninth aspect of the present invention Antibody, or immunoconjugate as described in the fourteenth aspect of the present invention; and

(ii) Detection of a pharmaceutically acceptable carrier.

In another preferred example, the coupling moiety of the immunoconjugate is an isotope for diagnosis.

In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

In another preferred example, the detection reagent is one or more reagents selected from the group consisting of isotopic tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agent.

In another preferred example, the detection reagent is used for in vivo detection.

In another preferred example, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, freeze-dried powder, tablet, buccal preparation, aerosol).

The eighteenth aspect of the present invention provides a kit for detecting human ANGPTL3 protein, said kit containing the immunoconjugate described in the fourteenth aspect of the present invention or the detection reagent of the seventeenth aspect of the present invention, and instructions.

In another preferred example, the instructions describe that the kit is used to non-invasively detect the expression of human ANGPTL3 in the subject to be tested.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

The main advantages of the present invention include

The invention provides a fusion protein of anti-human ANGPTL3 VHH and anti-human ANGPTL3 VHH-Fc, which can specifically and efficiently bind human ANGPTL3, and can cross-react with mouse ANGPTL3, and has good antigen binding activity and blocking ANGPTL3 protein inhibits the in vitro activity of lipoprotein lipase, thereby effectively reducing the levels of TG, TC and LDL-C in the blood of hyperlipidemia model mice, effectively alleviating liver lipid deposition and liver damage in non-alcoholic fatty liver, effectively Reduces the formation of atherosclerotic plaques.

Description of drawings

Figure 1: Screening of anti-ANGPTL3 Nanobody and construction and purification of Nanobody-Fc fusion protein, wherein (A) ELISA screening; (B) structure of Nanobody-Fc fusion protein; (C) SDS-page analysis results.

Figure 2: C44-Fc fusion protein neutralizing antigens hANGPTL3(S17-K170)-mFc(A)hANGPTL3(S17-E460)-His10(B)mANGPTL3(S17-T206)-His6(C) and mANGPTL3(S17-T455 )-His10(D) inhibits the in vitro activity of lipoprotein lipase.

Figure 3: C44-Fc fusion protein significantly reduces the levels of TG (A and B), TC (C and D) and LDL (E and F) in the blood of hyperlipidemia model mice.

Figure 4: C44-Fc fusion protein significantly reduces the levels of TG (A), TC (B) and LDL (C) in the blood of non-alcoholic fatty liver model mice.

Figure 5: C44-Fc fusion protein significantly reduces liver lipid deposition and liver damage in non-alcoholic fatty liver model mice, in which (A) body weight; (B) liver morphology; (C) liver weight; (D) liver TG content; (E) liver H&E staining; (F) liver oil red staining; (G) oil red staining (H) ALT (G) AST.

Figure 6: C44-Fc fusion protein significantly reduces blood lipid levels in atherosclerosis model mice, wherein (A) TG; (B) TC; (C) LDL-C; (D) body weight.

Figure 7: C44-Fc fusion protein prevents the formation of atherosclerosis in the mouse model, in which (A) plaques at the aortic arch and branches of the mouse; (B) oil red staining in general; (C) aortic sinus Oil red staining of slices; (D) H&E staining of aortic sinus slices; (E) general oil red staining area statistics; (F) oil red staining area statistics of aortic sinus slices; (G) plaque area statistics of aortic sinus slices ( H) Masson staining of aortic sinus sections (I) statistics of plaque collagen content.

Detailed ways

After extensive and in-depth research and extensive screening, the inventors unexpectedly discovered a class of anti-human ANGPTL3 nanobody or its Fc fusion protein for the first time. The experimental results show that the nanobody of the present invention can specifically and efficiently bind to human ANGPTL3, and It can cross-react with mouse ANGPTL3, has good antigen binding activity and blocks the in vitro activity of ANGPTL3 protein to inhibit lipoprotein lipase. In addition, the Fc fusion protein of the nanobody of the present invention not only maintains the biological activity of the VHH fragment of the alpaca anti-human ANGPTL3 heavy chain antibody, but also has a prolonged half-life, and can significantly alleviate the lipid deposition of non-alcoholic fatty liver and reduce atherosclerotic plaque It has a good application prospect in the treatment of non-alcoholic fatty liver and atherosclerosis.

the term

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" can refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, the term "comprises" or "includes (comprising)" can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of", or "consisting of".

As used herein, the terms "Nanobody of the invention", "Nanobody of the invention", "anti-human ANGPTL3 Nanobody of the invention", "human ANGPTL3 Nanobody of the invention", "anti-human ANGPTL3 Nanobody", "human "ANGPTL3 Nanobody" has the same meaning and can be used interchangeably, both refer to Nanobodies that specifically recognize and bind to human ANGPTL3 (including human ANGPTL3).

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain . Specific amino acid residues form the interface between the variable domains of the light and heavy chains.

As used herein, the terms "single domain", "VHH", "nanobody", "heavy chain antibody" (single domain antibody, sdAb, or nanobody nanobody) have the same meaning and are used interchangeably, referring to Cloning the variable region of the antibody heavy chain to construct a Nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full functionality. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

As used herein, the term "variable" means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each contain four FR regions in a roughly b-sheet configuration connected by three CDRs that form connecting loops, which in some cases may form partial b-sheet structures. The CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form of conjugates. The present invention also includes cell surface markers or antigens that bind to the anti-human ANGPTL3 antibody or its fragments.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (CDR)", "determinant complementarity region".

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably, and all refer to a polypeptide that specifically binds to human ANGPTL3 protein, such as a protein with a heavy chain variable region or peptide. They may or may not contain starting methionine.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. Specifically, the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention The variable regions are identical or at least 90% homologous, preferably at least 95% homologous.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.

The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.

In addition, in addition to containing the HER2 Nanobody of the present invention, the fusion protein of the present invention also includes an optional tag sequence (such as 6His tag, GGGS sequence, FLAG tag) that assists expression and/or purification; or includes an optional therapeutic Functional polypeptide molecules or fragments; or optional protein functional domains that assist physicochemical or pharmaceutical (eg, molecules that can prolong the half-life of Nanobodies in vivo, such as Fc fragments, HLE, ABD).

As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The antibody of the present invention refers to a polypeptide that has human ANGPTL3 binding activity and includes the above-mentioned CDR region. The term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.

The invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof. In addition to substantially full-length polypeptides, the invention also includes fragments of the Nanobodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.

In the present invention, "conservative variants of the antibody of the present invention" refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention. An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

最初的残基initial residue	代表性的取代representative replacement	优选的取代preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Lys；ArgGln; His; Lys; Arg	GlnGln
Asp(D)Asp(D)	GluGlu	GluGlu
Cys(C)Cys(C)	SerSer	SerSer
Gln(Q)Gln(Q)	AsnAsn	AsnAsn
Glu(E)Glu(E)	AspAsp	AspAsp
Gly(G)Gly(G)	Pro；AlaPro;	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile (I)	Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe	LeuLeu
Leu(L)Leu(L)	Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr	LeuLeu
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe;	LeuLeu

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

A polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.

Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.

pharmaceutical composition

The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned Nanobody (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Labeled Nanobodies

In a preferred embodiment of the present invention, the Nanobody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.

Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the nanobody of human ANGPTL3 is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.

Reagent test kit

The present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention. In a preferred example of the present invention, the kit further includes a container, instructions for use, buffer and the like.

The present invention also provides a detection kit for detecting the level of human ANGPTL3, which includes an antibody that recognizes human ANGPTL3 protein, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffers , detection labels, detection substrates, etc. The test kit may be an in vitro diagnostic device.

The present invention will be explained below in conjunction with examples. As mentioned above, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. If specific conditions or techniques are not indicated in the following examples, it can be carried out according to the conventional conditions in the field such as described in "Molecular Cloning: A Laboratory Guide" (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the product manual. Percentages and parts are by weight unless otherwise indicated.

Example 1. Construction of a phage display library

(1) Immunization of alpaca with human ANGPTL3 protein

Mix 1 mg of human ANGPTL3 protein with an equal volume of Freund's adjuvant, and inject it at 3-5 points subcutaneously on the neck of the alpaca. Immunize once a month, 4 times in total, and take 10ml of alpaca peripheral blood each time. The collected blood was placed in EDTA anticoagulant tubes, mixed gently and thoroughly immediately, and placed on ice.

(2) Phage display library construction

Separation of lymphocytes in blood samples collected before immunization and after each immunization, separation of lymphocytes from blood samples collected before immunization and after each immunization, extraction of total RNA, cDNA synthesis, using the above synthesized cDNA as a template, amplified The V region (VHH) of the alpaca heavy chain antibody was increased, the VHH was connected to the vector and transformed into Escherichia coli and infected with M13K07 phage, amplified and purified by precipitation to obtain a phage display library of VHH.

Example 2, Phage Display Technology Screening Anti-ANGPTL3 Nanomab

(1) Panning of anti-ANGPTL3 antibody epitope phage library

Antigen hANGPTL3(S17-220P)-His was diluted with 0.5% NaHCO ₃ buffer solution (pH=9.6) to final concentrations of 5, 2 and 1 μg/mL respectively as coating concentrations for three pannings, and 100 μL of diluted Antigens were added to enzyme-labeled wells, coated overnight at 4°C, washed 3 times with PBS, added with 3% BSA-PBS blocking solution, and blocked at 37°C for 1 hour. After the blocking was completed, PBS was added to wash 3 times, the residual liquid was aspirated, and 100 μL of phage library was added, and incubated at 37°C for 1 h. Then, aspirate unbound phage from each well, wash 6 times with PBST, wash 2 times with PBS, add Gly-HCl eluent at the same time, incubate at 37°C for 8min, elute specifically bound phage, and Transfer the eluate to a 1.5 mL sterile centrifuge tube and quickly neutralize with 15 μL Tris-HCl neutralization buffer. The phages obtained in each round of panning were titered, amplified and purified for the next round of screening.

(2) Amplification of phage library

Mix the panning eluate with 20 mL of E.coli TG1 culture in the early logarithmic growth stage, let it stand at 37°C for 30 minutes, add 1 mL of 20% glucose and 4 μL of ampicillin sodium, and shake at 37°C at 180r/min Incubate for 30min. Then M13K07 helper phage was added according to the ratio of cell:phage=1:20, and after standing at 37°C for 30min, the shaking culture was continued for 30min under the condition of 180r/min at 37°C. Afterwards, the culture was divided into centrifuge tubes, centrifuged at 25°C and 5000r/min for 10min, and the cell pellet was collected and resuspended in 50mL 2×YT-AK liquid medium, and placed at 30°C and 230r/min Incubate overnight with shaking.

The overnight culture was centrifuged at 10,000 r/min at 4°C for 20 min, the supernatant was transferred to a new centrifuge tube, 1/5 volume of PEG/NaCl was added, mixed well, placed at 4°C and allowed to stand for 2 h. Then, at 4°C, centrifuge at 10,000r/min for 20min to remove the supernatant, resuspend the pellet in 1mL PBS, add 1/5 volume of PEG/NaCl, mix well and place at 4°C for 1h.

Drop the phages that have been panned and amplified for 3 times on the LB plate. After the colony grows, 10-20 clones are randomly selected and placed in 1 mL of culture medium for 8 hours, and then added according to the ratio of cell:phage=1:20 The M13K07 phage was left standing at 37°C for 15 minutes, and then shaken at 220r/min for 45 minutes. Then the culture was taken out, supplemented with 2×YT-AK in a volume of 800 μL, and cultured overnight at 30°C. On the second day, centrifuge at a centrifugal force of 12,000 g for 10 min at room temperature, and take the supernatant for monoclonal ELISA identification, so as to screen positive anti-ANGPTL3 nanobodies.

(3) Phage rescue

From the titer plate of the eluate from the third round of panning, 48 single clones were randomly picked and inoculated in 1 mL of 2×YT-A, and incubated at 37°C for 8 hours with shaking at 220 r/min. Take 200 μL of the above culture, add M13K07 phage at the ratio of cell:phage=1:20, let stand at 37°C for 15 minutes, and shake at 220 r/min for 45 minutes. Add 2×YT-AK in a volume of 800 μL, and culture overnight at 30° C. with vigorous shaking at 230 r/min. The next day, centrifuge at 12,000 rpm for 10 min at room temperature, and take the supernatant for monoclonal ELISA identification.

(4) Identification of positive phage clones

Dilute hANGPTL3(S17-220P)-His with carbonate buffer (pH 9.6) to a final concentration of 2 μg/mL, add 100 μL/well to the enzyme-labeled wells, and coat overnight at 4°C; discard the coating solution, Wash 3 times with 0.05% PBST, add 200 μL 2% skim milk to each well, block at 37°C for 1 hour; wash 3 times with 0.05% PBST, add 50 μL phage culture supernatant and 50 μL 2% skim milk to each well, incubate at 37°C for 1 hour; Wash 6 times with 0.05% PBST, add horseradish peroxidase-labeled anti-M13 antibody (1:5000 dilution with PBS), 100 μL/well, act at 37°C for 1 hour; wash the plate 6 times with 0.05% PBST. Add TMB for color development Liquid color development, 100 μL/well, 37°C, 7min, add stop solution to terminate the reaction, 50 μL/well, measure the optical density at 450nm. The OD ₄₅₀ of the supernatant well added to the culture solution is greater than 5 times the value of the blank control clone As positive clones, as shown in Figure 1A, 33 positive clones were obtained, sequenced, amino acid sequence alignment and phylogenetic tree analysis were performed, and 9 VHHs with different CDR regions were obtained.

Embodiment 3, construct and purify anti-hANGPTL3 VHHs-Fc fusion protein

In the present invention, the gene sequence of the Fc segment of human immunoglobulin is fused with the gene sequence of 9 anti-ANGPTL3 nanometer monoclonal antibodies screened in the above-mentioned Example 2 (as shown in FIG. 1B ), to obtain anti-ANGPTL3 nanometer monoclonal antibody and human IgG Fc segment The gene sequence of the fusion protein. Subsequently, through double enzyme digestion and T4 ligase ligation, the gene sequence of the bifunctional protein was reacted with the PTT5 plasmid at a molar ratio of 1:3, and it was constructed into the PTT5 plasmid. The ExpiFectamine ^TM CHO/plasmid DNA complex (plasmid DNA concentration: 1 μg/mL) was prepared using pre-cooled OptiPRO ^TM medium (4° C.), and incubated at room temperature for 5 min. After shaking and culturing at 37°C and 8% CO ₂ for 22 h, 24 mL of ExpiFectamine ^TM CHO enhancer and 24 mL of ExpiCHO ^TM adjuvant were added to the culture flask, and the culture was continued for 7 days. After being centrifuged at 3000 g for 10 min, the culture supernatant was collected by filtration with a 0.22 μm filter membrane, and its protein content was detected by the BCA method. After the fusion protein was obtained, its molecular weight was identified by SDS-PAGE (as shown in Figure 1C). It can be seen that the molecular weight of the fusion protein was 70kD, and it was 35kD after reduction, which was in line with the expected molecular weight, indicating that the fusion protein was successfully constructed.

Example 4, Screening and affinity determination of anti-hANGPTL3 VHHs-Fc fusion protein

All kinetic binding experiments were performed on a BIACORETM T200 label-free molecular interaction instrument (GE Healthcare) at 25°C using protein A or CM5 sensor chips, using HBS-EP (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) as electrophoresis buffer. The 9 VHHs-Fcs expressed and purified in Example 3 above were subjected to single-cycle affinity screening, as shown in Table 1, among which C27-Fc and C44-Fc have strong binding abilities to hANGPTL3(17-170)-Fc and are not easy Dissociate.

Table 1

Human and mouse fragments or full-length ANGPTL3 proteins were captured on the surface of the CM5 chip, and the captured recombinant proteins were: hANGPTL3 containing 17-170 amino acids of mouse IgG1-Fc [hANGPTL3(17-170)-Fc], containing Full-length mature human ANGPTL3 containing a C-terminal decahistidine tag [hANGPTL3(17-460)-His; R&D Systems, MN; Cat. No. 3829-AN], containing a C-terminal decahistidine tag [mANGPTL3( 17-455)-His; R&D Systems, MN; Cat. No. 136-AN] full-length mature ANGPTL3 derived from Mus musculus and containing a C-terminal hexahistidine tag [mANGPTL3(17-206)-His ; Novoprotein; catalog number Q9Y5C1] of ANGPTL3 derived from amino acids 17-206 of Mus musculus. To measure affinity, C27-Fc and C44-Fc were injected on the captured protein surface for 120 min at a flow rate of 30 μL/min, and the dissociation of the complex was observed for 480 s or 900 s. Use the Scrubber 2.0a version software (BioLogic Software) to process the binding data, multiply the value of kd by ka as the affinity K _D , as shown in Table 2, the affinity of C44-Fc is between 0.1402nM-0.3036nM, the affinity of C27-Fc The affinity is between 0.01811nM-8.202nM.

Table 2

Example 5. In vitro activity test of anti-hANGPTL3 VHH-Fc fusion protein neutralizing ANGPTL3 protein to inhibit lipoprotein lipase

Lipoprotein lipase (LPL) is an important enzyme in the process of lipid metabolism, which can hydrolyze triglycerides, and the N-terminal helical coil region of ANGPTL3 can inhibit its activity. According to the above example 4, the affinity of C44-Fc to human and mouse full-length ANGPTL3, and the N-terminal helical coil region of mouse ANGPTL3 is significantly higher than that of C27-Fc, so C44-Fc is selected to inhibit the LPL activity induced by ANGPTL3 Dropped cell-free experiments. Using a lipoprotein lipase fluorescence assay kit (Biovision, USA), four ANGPTL3 proteins were used to measure the inhibition of C44-Fc on four ANGPTL3 activities, including: hANGPTL3(17-170)-Fc, hANGPTL3(17-460 )-His, mANGPTL3(17-455)-His and mANGPTL3(17-206)-His.

Briefly, 0.2 nM bovine LPL, 0.1 μM human ApoC II, and 0.2 mg/mL BSA were premixed in PBS, and then serially diluted ANGPTL3 recombinant protein or ANGPTL3 recombinant protein and serially diluted C44-Fc fusion protein were added, After incubating at room temperature for 10 minutes, it was detected with the kit, and the fluorescence intensity was measured at 482nm/515nm (excitation/emission) with a BioTek multifunctional microplate reader, and the fluorescence intensity was proportional to the LPL activity. As shown in Figure 2 and Table 3, the IC ₅₀ of C44-Fc is between 1.6nM-5.4nM, and the affinity of C27-Fc is between 0.01811nM-8.202nM.

table 3

Example 6. Experiment of anti-hANGPTL3 VHH-Fc fusion protein alleviating diet-induced hyperlipidemia in mouse model

As described in Examples 4 and 5 above, C44-Fc exerts similar inhibitory effects on human and mouse ANGPTL3, so C57Bl/6 mice were selected for pharmacodynamic evaluation. C57Bl/6 mice were first fed with a high-fat and high-cholesterol diet (Teklad TD.90221, containing 15.8% fat, 1.25% cholesterol and 0.5% bile salt) for 4 weeks, fasted for 4 hours, and blood was taken to measure the total cholesterol in the serum Increased from 3.77mmol/L to 9.21mmol/L, LDL-C increased from 0.85mmol/L to 2.07mmol/L, blood lipid significantly increased more than 2 times. Afterwards, C44-Fc was injected subcutaneously at doses of 10 mg/kg and 25 mg/kg, blood was taken after fasting for 4 hours after administration as day 0, and blood was taken after fasting for 4 hours on the 1st, 4th, 7th, and 12th respectively , Determination of serum TG, TC, LDL-C content. As shown in Figure 3, after subcutaneous injection of C44-Fc at a dose of 25 mg/kg for 4 days, the serum TG content was significantly reduced by 44%, the serum TC content was significantly reduced by 36%, and the serum LDL-C content was significantly reduced by 50%.

Example 7. Experiment of anti-hANGPTL3 VHH-Fc fusion protein alleviating NAFLD in mouse model

C57Bl/6 mice were first fed with a high-fat and high-cholesterol diet (Teklad TD.90221, containing 15.8% fat, 1.25% cholesterol and 0.5% bile salt) for 8 weeks, and blood was taken after 4 hours of fasting to measure the Blood lipids increased significantly, in which total cholesterol increased from 4.3mmol/L to 10.6mmol/L, and LDL-C increased from 0.41mmol/L to 2.3mmol/L. Afterwards, the mice were subcutaneously injected with C44-F at a dose of 25 mg/kg every week and their body weight was recorded. After 4 hours of fasting, the contents of TG, TC and LDL-C in the serum of the mice were determined. As shown in Figure 4, injection of C44-Fc significantly decreased the levels of TG, TC and LDL-C in mouse serum. After 6 weeks of administration, the mice were euthanized, the liver was dissected, the weight was recorded and its TG content was determined, and the serum of the mice was collected to determine the ALT and AST content. As shown in Figure 5, the liver weight of the mice in the high-fat and high-cholesterol diet group significantly increased by 73%, the liver was significantly enlarged, and the color was whitish. H&E showed obvious lipid droplet vacuoles, and oil red staining showed that the lipid content was significantly The content of TG in the liver also increased significantly, and the content of ALT and AST increased significantly, indicating that there was obvious liver damage. The above conclusions proved that the non-alcoholic fatty liver model was successfully established. In the C44-Fc treatment group, serum TG, TC and LDL-C levels were significantly reduced, the liver size and weight were significantly reduced, and the pale liver color was significantly improved. H&E showed that lipid droplet vacuoles were significantly reduced, and oil red staining showed lipid The content of C44-Fc is significantly reduced, and the determination of liver TG content is also significantly reduced. The significant reduction of ALT and AST content indicates that liver damage has been significantly improved. The above conclusions prove that C44-Fc has a significant improvement effect on NAFLD, reducing lipid deposition in the liver of mice, and alleviating liver damage.

Example 8. Experiment of anti-hANGPTL3 VHH-Fc fusion protein alleviating atherosclerosis in ApoE ^-/- mouse model

ApoE ^-/- mice were first fed with a high-fat and high-cholesterol diet (Teklad TD.90221, containing 15.8% fat, 1.25% cholesterol and 0.5% bile salts) for 7 weeks, and blood was taken after fasting for 4 hours to measure blood lipids Significantly increased, in which total cholesterol increased from 24.9mmol/L to 56.9mmol/L, and LDL-C increased from 17.4mmol/L to 22.7mmol/L. After that, C44-Fc was subcutaneously injected weekly at a dose of 25 mg/kg and the body weight of the mice was recorded. After weekly subcutaneous injection of C44-Fc for 4 days, they were fasted for 4 hours and the contents of TG, TC and LDL-C in the mouse serum were determined. As shown in Figure 6, the injection of C44-Fc significantly reduced the serum levels of ApoE ^-/- mice The content of TG, TC and LDL-C. After 5 weeks of administration, the mice were euthanized, the mice were dissected, and the aortic arch and three branches were separated, washed with saline and carefully stripped of the fat on the outside. As shown in Figure 7A, the injection of C44-Fc significantly reduced plaques form. Afterwards, the abdominal aorta was dissected, and the full-length sample of the aorta was fixed in 4% paraformaldehyde fixative solution for more than 24 hours, and then stained with Oil Red O. As shown in Figure 7B and 7E, injection of C44-Fc significantly reduced abdominal Aortic plaque formation. At the same time, oil red O staining, H&E staining and Masson staining were performed on the sections of the aortic sinus to study the vascular stenosis caused by atherosclerotic plaque and the collagen content and stability of the plaque, as shown in Figure 8C-D As shown in and 8F-I, the injection of C44-Fc significantly reduced the formation of aortic sinus plaques, alleviated vascular stenosis caused by atherosclerotic plaques, and the collagen content in the plaques was significantly increased, which is conducive to plaque stability.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

An anti-human angiopoietin 3 nanobody or its antigen-binding fragment, which comprises a determinant complementary region and a framework region, and the determinant complementary region is composed of CDR1, CDR2 and CDR3, characterized in that: the amino acid sequence of CDR1 is as SEQ ID NO: 6; the amino acid sequence of CDR2 is shown in SEQ ID NO: 7; the amino acid sequence of CDR3 is shown in SEQ ID NO: 8.
The anti-human angiopoietin 3 nanobody or its antigen-binding fragment according to claim 1, whose amino acid sequence is shown in SEQ ID NO: 1.
An anti-human ANGPTL3 nanobody-Fc fragment fusion protein, characterized in that: the C-terminal of the nanobody or antigen-binding fragment described in claim 1 or 2 directly or through the N-terminal of the connecting peptide and the immunoglobulin IgG Fc fragment connected; the Fc fragment is the Fc fragment of human immunoglobulin IgG1, and its amino acid is shown in SEQ ID NO: 17; the amino acid sequence of the fusion protein is shown in SEQ ID No: 19.
A nucleic acid molecule encoding the anti-human angiopoietin 3 nanobody or an antigen-binding fragment thereof according to claim 1 or 2; it is characterized in that the nucleic acid molecule sequence encoding CDR1 is as shown in SEQ ID NO: 14, and the encoding CDR2 The nucleic acid molecule sequence is shown in SEQ ID NO: 15, the nucleic acid molecule sequence encoding CDR3 is shown in SEQ ID NO: 16; the nucleic acid molecule encoding the anti-human angiopoietin 3 nanobody or its antigen-binding fragment is shown in SEQ ID NO :9.
A nucleic acid molecule encoding the anti-human ANGPTL3 Nanobody-Fc fragment fusion protein according to claim 3; the Fc part nucleic acid sequence of the fusion protein is as shown in SEQ ID NO: 18; the fusion protein nucleic acid sequence is as shown in SEQ ID NO: 18 ID No: 20.
A recombinant vector, characterized in that it comprises the nucleic acid molecule according to claim 4 or 5.
A recombinant cell, characterized in that the nucleic acid molecule according to claim 4 or 5 is introduced into the recombinant cell, or the recombinant vector according to claim 6 is transfected. 8. A pharmaceutical composition, characterized in that: the composition comprises one or more antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors or recombinant antibodies described in any one of claims 1-7 cells, and optionally a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition according to claim 8, further comprising one or more compounds selected from HMG-CoA reductase inhibitors, cholesterol absorption inhibitors, bile acid reabsorption inhibitors or increasing lipoprotein catabolism Pharmaceutical preparations.
The pharmaceutical composition according to any one of claims 8 or 9, further comprising one or more drugs selected from the group consisting of statins, niacin, fibrates, anti-hANGPTL4 antibodies and Other therapeutic agents for anti-PCSK9 antibodies.
One or more antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant cells or pharmaceutical compositions according to any one of claims 1-10, used in the preparation of prevention, alleviation, improvement or inhibition Use in a preparation or medicine for a disease or disorder.
The use according to claim 11, characterized in that the preparation or drug reduces or inhibits ANGPTL3 activity.
One or more antibodies, antigen-binding fragments, fusion proteins, nucleic acid molecules, recombinant vectors, recombinant cells or pharmaceutical compositions as described in any one of claims 1-10 are used in the preparation and treatment of hyperlipidemia, non-alcoholic Use in medicine for fatty liver or atherosclerosis.
An anti-human ANGPTL3 antibody, characterized in that the antibody comprises one or more anti-human ANGPTL3 nanobodies as claimed in claim 1.
A multispecific antibody, characterized in that the multispecific antibody comprises: the anti-human ANGPTL3 nanobody as claimed in claim 1, or the anti-human ANGPTL3 antibody as claimed in claim 14.
A kind of recombinant protein, it is characterized in that, described recombinant protein has:

(i) the anti-human ANGPTL3 nanobody as claimed in claim 1, the anti-human ANGPTL3 nanobody-Fc fragment fusion protein as claimed in claim 3, or the anti-human ANGPTL3 antibody as claimed in claim 14; and

(ii) Optional tag sequences to aid in expression and/or purification.
A CAR construct, characterized in that the antigen-binding region of the CAR construct comprises a determinant complementary region, and the determinant complementary region is composed of CDR1, CDR2 and CDR3, and the amino acid sequence of CDR1 is as SEQ ID NO: 6; the amino acid sequence of CDR2 is shown in SEQ ID NO: 7; the amino acid sequence of CDR3 is shown in SEQ ID NO: 8
A recombinant immune cell, characterized in that the immune cell expresses an exogenous CAR construct according to claim 17.
An immunoconjugate, characterized in that, the immunoconjugate contains:

(a) the anti-human ANGPTL3 nanobody as claimed in claim 1, the anti-human ANGPTL3 nanobody-Fc fragment fusion protein as claimed in claim 3, or the anti-human ANGPTL3 antibody as claimed in claim 14; and

(b) a coupling moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof .
The anti-human ANGPTL3 nanobody as claimed in claim 1, the anti-human ANGPTL3 nanobody-Fc segment fusion protein as claimed in claim 3, or the anti-human ANGPTL3 antibody as claimed in claim 14, or as claimed in claim 19 The purposes of the immunoconjugate; It is characterized in that, (a) is used for preparing the medicine that prevents and/or treats the disease or disease relevant with human ANGPTL3; (b) is used for preparing the reagent, detection plate or reagent of detecting human ANGPTL3 box.