Antibodies or antigen-binding fragments thereof against vmat2 and compositions and uses thereof

Abstract

The present application provides an antibody or antigen-binding fragment thereof against VMAT2, and compositions and uses thereof. Specifically, an antibody or antigen-binding fragment thereof against VMAT2 is provided, which can effectively bind to the intracellular segment of human VMAT2 protein. The present application also provides an antibody drug conjugate comprising the antibody or antigen-binding fragment, which can be applied to the preparation of a medicament for treating diseases with overexpression of VMAT2, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, relates to an antibody or antigen-binding fragment thereof against VMAT2, and compositions and uses thereof. BACKGROUND

[0002] VMAT2 is a multi-pass transmembrane protein located on the synaptic vesicle membrane of neurons. Dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) and other monoamine neurotransmitters can be transported to synaptic vesicles for storage. Under external stimulation, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, bind to the corresponding target receptors on the postsynaptic membrane, and mediate downstream signaling. VMAT2 is involved in the regulation of movement, mood, sleep, reward, attention, addiction and many other functions.

[0003] Overexpression or overtransport of VMAT2 can lead to disorders of the monoamine neurotransmitter system, thereby inducing Huntington's disease, addictive diseases, tardive dyskinesia, hypertension and the like. To date, there is a severe lack of available treatment for such diseases.

[0004] Currently, small molecule inhibitors targeting VMAT2 as a drug target, such as respine, tetrabenazine, deutetrabenazine, valbenazine, GZ-793A and the like, are used to treat Huntington's disease, tardive dyskinesia and hypertension.

[0005] However, these inhibitors have the disadvantages of short half-life, low target specificity and easy to cause adverse reactions.

[0006] Therefore, there is an urgent need in the art to develop new VMAT2 drugs with long half-life and high specificity to make up for the shortcomings of small molecule drugs. SUMMARY

[0007] The present application provides a new VMAT2 drug with long half-life and high specificity.

[0008] In a first aspect of the present application, an anti-VMAT2 antibody or antigen-binding fragment thereof is provided, which has three complementarity determining regions CDR (HCDR) of a heavy chain variable region and three complementarity determining regions CDR (LCDR) of a light chain variable region selected from the group consisting of:

[0009] (A) three HCDRs and three LCDRs selected from group A:

[0010] (a1) HCDR1 as depicted in SEQ ID NO: 48,

[0011] HCDR2 as depicted in SEQ ID NO: 49,

[0012] HCDR3 as depicted in SEQ ID NO: 50,

[0013] LCDR1 as depicted in SEQ ID NO: 52,

[0014] LCDR2 as depicted in SEQ ID NO: 53,

[0015] LCDR3 as depicted in SEQ ID NO: 54;

[0016] (a2) HCDR1 as depicted in SEQ ID NO: 48,

[0017] HCDR2 as depicted in SEQ ID NO: 58,

[0018] HCDR3 as depicted in SEQ ID NO: 50,

[0019] LCDR1 as depicted in SEQ ID NO: 52,

[0020] LCDR2 as depicted in SEQ ID NO: 53,

[0021] LCDR3 as depicted in SEQ ID NO: 54;

[0022] (a3) HCDR1 as depicted in SEQ ID NO: 48,

[0023] HCDR2 as depicted in SEQ ID NO: 63,

[0024] HCDR3 as depicted in SEQ ID NO: 50,

[0025] LCDR1 as depicted in SEQ ID NO: 52,

[0026] LCDR2 as depicted in SEQ ID NO: 53,

[0027] LCDR3 as depicted in SEQ ID NO: 54;

[0028] (B) three HCDRs and three LCDRs selected from group B: (b1) HCDR1 as depicted in SEQ ID NO: 67,

[0029] HCDR2 as depicted in SEQ ID NO: 68,

[0030] HCDR3 as set forth in SEQ ID NO: 69,

[0031] LCDR1 as set forth in SEQ ID NO: 71,

[0032] LCDR2 as set forth in SEQ ID NO: 72,

[0033] LCDR3 as set forth in SEQ ID NO: 73;

[0034] (b2) HCDR1 as set forth in SEQ ID NO: 67,

[0035] HCDR2 as set forth in SEQ ID NO: 68,

[0036] HCDR3 as set forth in SEQ ID NO: 77,

[0037] LCDR1 as set forth in SEQ ID NO: 71,

[0038] LCDR2 as set forth in SEQ ID NO: 72,

[0039] LCDR3 as set forth in SEQ ID NO: 73;

[0040] (C) three HCDRs and three LCDRs selected from Group C: (c1) HCDR1 as set forth in SEQ ID NO: 82,

[0041] HCDR2 as set forth in SEQ ID NO: 83,

[0042] HCDR3 as set forth in SEQ ID NO: 84,

[0043] LCDR1 as set forth in SEQ ID NO: 86,

[0044] LCDR2 as set forth in SEQ ID NO: 87,

[0045] LCDR3 as set forth in SEQ ID NO: 88;

[0046] (c2) HCDR1 as set forth in SEQ ID NO: 82,

[0047] HCDR2 as set forth in SEQ ID NO: 92,

[0048] HCDR3 as set forth in SEQ ID NO: 93,

[0049] LCDR1 as set forth in SEQ ID NO: 95,

[0050] LCDR2 as set forth in SEQ ID NO: 96,

[0051] LCDR3 as set forth in SEQ ID NO: 97;

[0052] (D) three HCDRs and three LCDRs selected from Group D:

[0053] (d1) HCDR1 as set forth in SEQ ID NO: 101,

[0054] HCDR2 as set forth in SEQ ID NO: 102,

[0055] HCDR3 as set forth in SEQ ID NO: 103,

[0056] LCDR1 as set forth in SEQ ID NO: 71,

[0057] LCDR2 as set forth in SEQ ID NO: 72,

[0058] LCDR3 as set forth in SEQ ID NO: 105;

[0059] (d2) HCDR1 as set forth in SEQ ID NO: 109,

[0060] HCDR2 as set forth in SEQ ID NO: 110,

[0061] HCDR3 as set forth in SEQ ID NO: 111,

[0062] LCDR1 as set forth in SEQ ID NO: 71,

[0063] LCDR2 as set forth in SEQ ID NO: 72,

[0064] LCDR3 as set forth in SEQ ID NO: 105;

[0065] (d3) HCDR1 as set forth in SEQ ID NO: 116,

[0066] HCDR2 as set forth in SEQ ID NO: 117,

[0067] HCDR3 as set forth in SEQ ID NO: 118,

[0068] LCDR1 as set forth in SEQ ID NO: 120,

[0069] LCDR2 as set forth in SEQ ID NO: 72,

[0070] LCDR3 as set forth in SEQ ID NO: 105.

[0071] In another preferred embodiment, the anti-VMAT2 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof against the cytoplasmic side of VMAT2 protein.

[0072] In another preferred embodiment, the anti-VMAT2 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof against the cytoplasmic side of VMAT2 protein.

[0073] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0074] In another preferred embodiment, the antigen-binding fragment comprises a Fab fragment, a F(ab')2 fragment, a Fv fragment.

[0075] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the anti-VMAT2 antibody or antigen-binding fragment thereof are selected from the group consisting of:

[0076] (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47, and a light chain variable region having the amino acid sequence of SEQ ID NO: 51;

[0077] (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57, and a light chain variable region having the amino acid sequence of SEQ ID NO: 59;

[0078] (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 62, and a light chain variable region having the amino acid sequence of SEQ ID NO: 51;

[0079] (d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66, and a light chain variable region having the amino acid sequence of SEQ ID NO: 70;

[0080] (e) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 76, and a light chain variable region having the amino acid sequence of SEQ ID NO: 78;

[0081] (f) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 81, and a light chain variable region having the amino acid sequence of SEQ ID NO: 85;

[0082] (g) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 91, and a light chain variable region having the amino acid sequence of SEQ ID NO: 94;

[0083] (h) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 100, and a light chain variable region having the amino acid sequence of SEQ ID NO: 104;

[0084] (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 108, and a light chain variable region having the amino acid sequence of SEQ ID NO: 112;

[0085] (j) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 115, and a light chain variable region having the amino acid sequence of SEQ ID NO: 119.

[0086] In another preferred embodiment, the heavy chain of the antibody or antigen binding fragment thereof further comprises a heavy chain constant region; and the light chain of the antibody or antigen binding fragment thereof further comprises a light chain constant region.

[0087] In another preferred embodiment, the antibody is a single chain antibody, a double chain antibody, or an antigen binding fragment.

[0088] In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

[0089] In another preferred embodiment, the heavy chain constant region is of human or murine origin.

[0090] In another preferred embodiment, the light chain constant region is of human or murine origin.

[0091] In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen binding fragment.

[0092] In another preferred embodiment, the antibody is a monoclonal antibody.

[0093] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0094] In another preferred embodiment, the antibody further comprises a linker peptide between the heavy chain variable region and the light chain variable region.

[0095] In a second aspect of the present application, there is provided a recombinant protein, the recombinant protein having:

[0096] (i) the anti-VMAT2 antibody or antigen binding fragment thereof of the first aspect of the present application; and

[0097] (ii) a tag sequence that optionally assists in expression and / or purification.

[0098] In another preferred embodiment, the tag comprises a Fc tag, a FLAG tag, a 6His tag, or a combination thereof.

[0099] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0100] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0101] In a third aspect of the present application, there is provided a nucleotide molecule encoding the anti-VMAT2 antibody or antigen-binding fragment thereof of the first aspect of the present application.

[0102] In another preferred embodiment, the nucleotide molecule is DNA, RNA, or cDNA.

[0103] In a fourth aspect of the present application, there is provided a vector comprising the nucleotide molecule of the third aspect of the present application.

[0104] In another preferred embodiment, the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0105] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0106] In a fifth aspect of the present application, there is provided a host cell comprising the vector of the fourth aspect of the present application, or having integrated into its genome the nucleotide molecule of the third aspect of the present application.

[0107] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0108] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0109] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell.

[0110] In another preferred embodiment, the prokaryotic cell is E. coli.

[0111] In another preferred embodiment, the cell is an immune cell, and the surface of which expresses a chimeric antigen receptor.

[0112] In another preferred embodiment, the immune cell is a T cell, a NK cell, or a combination thereof.

[0113] In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell).

[0114] In another preferred embodiment, the chimeric antigen receptor is an anti-VMAT2 antibody or antigen-binding fragment thereof.

[0115] In a sixth aspect of the present application, there is provided an antibody drug conjugate comprising:

[0116] (a) an antibody moiety comprising an anti-VMAT2 antibody or antigen-binding fragment thereof according to the first aspect of the application; and

[0117] (b) a conjugated moiety conjugated to the antibody or antigen-binding fragment thereof, the conjugated moiety selected from the group consisting of a detectable label, a drug, or a combination thereof.

[0118] In another preferred embodiment, the drug is a drug that inhibits the over-transport of monoamine neurotransmitters.

[0119] In another preferred embodiment, the monoamine neurotransmitters include dopamine (DA), serotonin (5-HT), norepinephrine (NE), and epinephrine (E).

[0120] In another preferred embodiment, the antibody drug conjugate is expressed as: mAb-(X-Y)n;

[0121] wherein,

[0122] mAb is the anti-VMAT2 antibody or antigen-binding fragment thereof;

[0123] X is a linker;

[0124] Y is a conjugated moiety, the conjugated moiety being a drug;

[0125] n is a positive integer ≤ 8;

[0126] The conjugated moiety is conjugated to the anti-VMAT2 antibody or antigen-binding fragment thereof via a linker.

[0127] In a seventh aspect of the application, there is provided a pharmaceutical composition comprising:

[0128] (i) an antibody or antigen-binding fragment thereof according to the first aspect of the application, a recombinant protein according to the second aspect of the application, a nucleotide molecule according to the third aspect of the application, a vector according to the fourth aspect of the application, or a host cell according to the fifth aspect of the application, or an antibody drug conjugate according to the sixth aspect of the application; and

[0129] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0130] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0131] In another preferred embodiment, the pharmaceutical composition is for use in the preparation of a medicament for treating a disease caused by over-expression or over-transport of VMAT2.

[0132] In another preferred embodiment, the disease is selected from the group consisting of Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0133] In an eighth aspect of the invention, antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, and antibody-drug conjugates as described in the sixth aspect of the invention are provided for the preparation of pharmaceuticals, reagents, detection plates, or kits.

[0134] The reagents, detection plates, or kits are used to detect VMAT2 protein in samples.

[0135] The agent is used to treat or prevent diseases that overexpress the VMAT2 protein.

[0136] In another preferred embodiment, the VMAT2 protein is the intracellular segment of the VMAT2 protein.

[0137] In another preferred embodiment, the VMAT2 protein is the VMAT2 cytoplasmic side protein.

[0138] In a ninth aspect of the present invention, a method for preparing the anti-VMAT2 antibody or its antigen-binding fragment as described in the first aspect of the present invention is provided, the method comprising the following steps:

[0139] (a) Under expression conditions, host cells as described in the fifth aspect of the present invention are cultured to express the anti-VMAT2 antibody or an antigen-binding fragment thereof;

[0140] (b) Isolate and purify the anti-VMAT2 antibody or its antigen-binding fragment described in (a).

[0141] In a tenth aspect of the present invention, a method for detecting VMAT2 protein in a sample is provided, the method comprising the steps of:

[0142] (1) Contact the sample with an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0143] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of VMAT2 protein in the sample.

[0144] In another preferred embodiment, the sample includes: human or animal tissue samples, or exfoliated cell samples.

[0145] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0146] In another preferred embodiment, the method is an in vitro method.

[0147] In another preferred embodiment, the method further includes step (3) analyzing the affinity between the antibody and the antigen.

[0148] In an eleventh aspect of the present application, a detection plate is provided, which comprises a substrate (support plate) and a test strip, wherein the test strip comprises the antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody-drug conjugate of the sixth aspect of the present application.

[0149] In another preferred embodiment, the test strip further comprises an antigen spotting area.

[0150] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper in sequence.

[0151] In a twelfth aspect of the present application, a kit is provided, which comprises:

[0152] (1) a first container, which comprises the antibody or antigen-binding fragment thereof of the first aspect of the present application; and / or

[0153] (2) a second container, which comprises a secondary antibody against the antibody or antigen-binding fragment thereof of the first aspect of the present application; and / or

[0154] (3) a third container, which comprises a cell lysis reagent;

[0155] or,

[0156] The kit comprises the detection plate of the eleventh aspect of the present application.

[0157] In another preferred embodiment, the antibody in the first container is labeled with a detectable label.

[0158] In another preferred embodiment, the antibody in the second container is labeled with a detectable label.

[0159] In a thirteenth aspect of the present application, a method for treating a disease associated with VMAT2 overexpression or overtransport is provided, which comprises the step of administering to a subject in need thereof a therapeutically effective amount of the anti-VMAT2 antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the nucleotide molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the antibody-drug conjugate of the sixth aspect of the present application, or the pharmaceutical composition of the seventh aspect of the present application.

[0160] In another preferred embodiment, the disease associated with VMAT2 overexpression or overtransport is selected from the group consisting of Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0161] It should be understood that, in the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to constitute a new or preferred technical scheme. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0162] Figure 1 Results of sorting mouse IgG1 positive memory B cells after immunization by flow cytometry and control group sorting mouse IgG1 positive memory B cells are shown.

[0163] Figure 2 Results of gradient ELISA antigen-antibody binding experiments of culture supernatant of positive wells of ELISA experiments of antibody screening targeting VMAT2 in Example 3 are shown.

[0164] Figure 3 Results of gradient ELISA antigen-antibody binding experiments of culture supernatant of positive wells of ELISA experiments of antibody screening targeting VMAT2 in Example 3 are shown.

[0165] Figure 4 A schematic diagram showing intracellular expression and transport of VMAT2 protein is shown.

[0166] Figure 5 Results of supernatant of single B cell culture binding to cell surface hVMAT2 are shown.

[0167] Figure 6 Results of supernatant of single B cell culture not binding to cell surface hVMAT2 are shown.

[0168] Figure 7A -B shows the heavy chain vector of human IgG1 CH1 used for constructing the antibody; the light chain vector of human IgK used for constructing the antibody; the heavy chain vector of human IgG1 constant region used for constructing the antibody; and the ScFv vector.

[0169] Figure 8 Results of flow cytometry antigen-antibody binding experiments in Example 6 of the present application are shown.

[0170] Figure 9 Results of SPR antigen-antibody binding affinity detection in Example 7 of the present application are shown.

[0171] Figure 10 Results of verification of Fab antibody binding to different species of VMAT2 and VMAT1 in Example 8 of the present application are shown.

[0172] Figure 11 Results of experiments of antibody inhibiting transport of cells in Example 9 of the present application are shown. DETAILED DESCRIPTION

[0173] The present inventors have made extensive and in-depth studies, and unexpectedly obtained an anti-VMAT2 antibody through a large number of screenings. Experimental results show that the anti-VMAT2 antibody of the present application has high affinity and good biological activity. Moreover, the antibody of the present application can have cross-binding reactions with human VMAT2, murine VMAT2 and porcine VMAT2, and can also bind to VMAT1. The antibody of the present application can significantly inhibit the transport function of VMAT2. On this basis, the present application is completed.

[0174] Terms

[0175] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have meanings commonly understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be limiting, and the scope of the present application will only be limited by the appended claims.

[0176] As used herein, the term "about", when used in reference to a numerically recited value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0177] As used herein, the terms "comprising", "including", "containing", are interchangeable and are meant to encompass both the open- and the semi-closed-ended definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0178] As used herein, the term "pharmaceutically acceptable carrier" refers to a material that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0179] As used herein, the term "therapeutically effective amount" refers to an amount that produces functional or therapeutic effects for humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art will appreciate that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the use of other drugs, etc.

[0180] VMAT2

[0181] VMAT2 is a multi-pass transmembrane protein located on the synaptic vesicle membrane of neurons. Various monoamine neurotransmitters such as dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) are transported into synaptic vesicles for storage. Upon external stimuli, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, bind to the corresponding target receptors on the postsynaptic membrane, and mediate downstream signaling. VMAT2 is involved in the regulation of movement, mood, sleep, reward, attention, addiction, and many other functions.

[0182] The antibodies against the intracellular region of VMAT2 in the prior art only show binding activity to the intracellular region, but no transport inhibition activity. However, the antibodies against the intracellular region of VMAT2 obtained in the present application have not only binding activity to the intracellular region of VMAT2, but also good transport inhibition activity.

[0183] Antibodies

[0184] In the present application, the terms "antibody (Antibody, Ab for short)" and "immunoglobulin G (Immunoglobulin G, IgG for short)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region, and the heavy chain constant region is composed of three domains CH1, CH2, and CH3. One end of each light chain has a variable region (VL), and the other end has a constant region, and the light chain constant region includes one domain CL; the constant region of the light chain pairs with the CH1 domain of the constant region of the heavy chain, and the variable region of the light chain pairs with the variable region of the heavy chain. The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and the like. The heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes Kappa (Kappa) or Lambda (Lambda). The heavy chain and light chain of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by the inter-peptide disulfide bond formed between the hinge region.

[0185] In the present invention, the terms "Fab" and "Fc" mean that papain can cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., fragment crystallizable (Fc), consists of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen binding activity and is the site of interaction of the antibody with effector molecules or cells.

[0186] In the present invention, the term "scFv" is a single chain antibody fragment (scFv) in which the variable region of the heavy chain and the variable region of the light chain of an antibody are connected by a linker of 15 to 25 amino acids.

[0187] In the present invention, the term "variable" means that certain portions of the variable regions of antibodies differ in sequence among various antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions that are located in the variable region of the heavy chain and the variable region of the light chain. The more conserved portions of variable regions are called framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, joined by three CDRs, that are generally β-sheet in conformation, and that typically form a structurally distinct site of antibody-antigen binding. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0188] As used herein, the term "framework region" (FR) refers to the amino acid sequences that are interposed between the CDRs, i.e., those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated L-FR1, L-FR2, L-FR3, L-FR4, and H-FR1, H-FR2, H-FR3, H-FR4, respectively. Accordingly, the light chain variable domain can thus be referred to as (L-FR1)-(LCDR1)-(L-FR2)-(LCDR2)-(L-FR3)-(LCDR3)-(L-FR4) and the heavy chain variable domain can thus be referred to as (H-FR1)-(HCDR1)-(H-FR2)-(HCDR2)-(H-FR3)-(HCDR3)-(H-FR4). Preferably, the FRs of the present application are human antibody FRs or derivatives thereof that are substantially identical to naturally occurring human antibody FRs, i.e., have a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0189] Given the amino acid sequences of the CDRs, one skilled in the art can readily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0190] As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to a framework region of a naturally occurring human antibody.

[0191] As used herein, the term "linker" refers to one or more amino acid residues that are inserted into an immunoglobulin domain to provide sufficient mobility to the domains of the light and heavy chains to fold into an exchangeable dual variable domain immunoglobulin. In the present application, preferred linkers refer to Linker 1 and Linker 2, wherein Linker 1 connects the VH and VL of a single chain antibody (scFv) and Linker 2 is used to connect the scFv to the heavy chain of another antibody.

[0192] Examples of suitable linkers include a single glycine (Gly) or serine (Ser) residue, the identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure element that is desired to be achieved in the linker.

[0193] In the present application, the antibodies of the present application also include conservative variants thereof, i.e., polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or identical amino acids as compared to the amino acid sequence of the specific antibodies of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table A.

[0194] Table A

[0195] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0196] In the present application, the terms "anti-", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. Typically, an antibody binds to an antigen with a dissociation constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less. In the present application, the term "KD" refers to the dissociation constant of a particular antibody-antigen interaction, which is used to describe the affinity of an antibody for an antigen. The smaller the dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody for an antigen can be determined using Surface Plasmon Resonance (abbreviated as SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of an antibody for an antigen.

[0197] In the present application, the term "epitope" refers to a polypeptide determinant that is specifically bound by an antibody. The epitope of the present application is the region of an antigen that is bound by an antibody.

[0198] Polynucleotides, vectors and host cells

[0199] The present application also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0200] The polynucleotides encoding the mature polypeptides of the present application include: only the coding sequence encoding the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0201] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only the polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0202] The present application also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0203] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment of the sequence can be obtained by synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0204] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually achieved by cloning the sequence into a vector, transforming the vector into a cell, and then isolating the sequence from the proliferated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules in isolated form.

[0205] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0206] The present application also relates to vectors comprising the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0207] The host cell can 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 include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0208] 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 prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, the transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, or lipofection.

[0209] The resulting transformant can be cultured in conventional nutrient media using standard procedures known in the art. The culture medium used will vary depending on the host cell selected. In general, however, the medium will include a nutrient and a surfactant. The culture conditions, such as temperature, pH and the like, can be selected by the skilled artisan to facilitate the growth of the host cells.

[0210] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0211] The antibodies of the present application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these.

[0212] 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 enzymes capable of producing detectable products.

[0213] Therapeutic agents that can be combined or conjugated with the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2 and the like; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolyzing enzyme-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0214] Pharmaceutical compositions and uses

[0215] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection), intracranial injection, or intracavity injection. In the present application, the term "pharmaceutical composition" means that the specific antibody of the present application can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to more stably exert the therapeutic effect, which can ensure the conformational integrity of the amino acid core sequence of the specific antibody disclosed in the present application, and also protect the multi-functional groups of the protein from degradation (including but not limited to condensation, deamination or oxidation). The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the specific antibody (or its conjugate) described above in the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using normal saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as needle and solution should be manufactured under sterile conditions. The administration amount of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the specific antibody of the present application can also be used with other therapeutic agents.

[0216] When using the pharmaceutical composition, a safe and effective amount of the specific antibody or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider the administration route, the health status of the patient, and other factors, which are within the skill of a skilled physician.

[0217] Antibody-drug conjugate (ADC)

[0218] The present application also provides an antibody-drug conjugate (ADC) based on the antibody of the present application.

[0219] Typically, the antibody conjugate comprises the antibody, and an effector molecule, which is conjugated, and preferably chemically conjugated, to the antibody. The effector molecule is preferably a therapeutically active agent. In addition, the effector molecule can be one or more of a toxin, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0220] The antibody of the present application can be conjugated to the effector molecule via a conjugating agent. Examples of the conjugating agent can be any one or more of a non-selective conjugating agent, a carboxyl group utilizing conjugating agent, a peptide chain, and a disulfide bond utilizing conjugating agent. The non-selective conjugating agent is a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde or the like. The carboxyl group utilizing conjugating agent can be any one or more of aconitic anhydride conjugating agent (such as aconitic anhydride), and an acylhydrazone conjugating agent (with an acylhydrazone as a conjugation site).

[0221] Certain residues on the antibody, such as Cys or Lys, are used to attach a variety of functional groups, including imaging agents (e.g., chromophoric and fluorescent groups), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection agent, a stabilizing agent) is conjugated (covalently linked) to the antibody. The functional agent can be linked to the antibody directly, or indirectly via a linker.

[0222] The antibody can be conjugated to a drug to form an antibody drug conjugate (ADC). Typically, the ADC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. A degradable linker is typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, e.g., enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases (e.g., lysosomal or endosomal proteases), or saccharide linkers, e.g., glucuronide-containing linkers that are degradable by glucuronidases. Peptide-based linkers can include, e.g., dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, e.g., pH-sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that are susceptible to reduction (e.g., disulfide linker). A non-degradable linker is typically susceptible to release of the drug under conditions in which the antibody is hydrolyzed by proteases.

[0223] The linker has a reactive group capable of reacting with certain amino acid residues prior to attachment to the antibody, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodo, bromo, or chloro); haloesters (e.g., iodo, bromo, or chloro); halomethylketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl)dioxane, while the counterion is acetate, chloride, or nitrate; and polymethylenedimethylthioether sulfonate. The linker can include, for example, a maleimide for attachment to the antibody via a thio succinimide.

[0224] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker attaches the antibody to the drug, and the drug has a functional group that can bond to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group that can bond to the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0225] Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present application, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two- or multi-step process. For example, a cysteine residue is reacted with the reactive portion of the linker in a first step, and in a subsequent step, the functional group on the linker is reacted with the drug to form the ADC.

[0226] Typically, the functional group on the linker is chosen to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkyne. Other useful functional groups include, for example, ketones and aldehydes (for reaction with hydrazides and alkoxylamines), phosphines (for reaction with azides); isocyanates and isothiocyanates (for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (for reaction with amines and alcohols). These and other ligation strategies, such as those described in Bioconjugate Techniques, 2ndEdition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of the drug moiety and the linker, each member of a complementary pair of reactive functional groups can be used on either the linker or the drug when the complementary pair is chosen.

[0227] The application also provides methods of making an ADC, which can further comprise: combining the antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0228] In certain embodiments, the methods of the application comprise: combining the antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the application further comprise: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0229] In some embodiments, the antibody drug conjugate (ADC) is of the following formula:

[0230]

[0231] wherein:

[0232] Ab is an antibody,

[0233] LU is a linker;

[0234] D is a drug;

[0235] and subscript p is a value selected from 1 to 8.

[0236] Detection uses and kits

[0237] The antibodies of the application can be used in detection applications, for example for detecting a sample to provide diagnostic information.

[0238] In the present application, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present application shall include all kinds of biopsies known to those skilled in the art. Therefore, the biopsy used in the present application can include, for example, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0239] The sample used in the present application includes a fixed or preserved cell or tissue sample.

[0240] The present application also provides a kit comprising the antibody (or fragment thereof) of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present application can be immobilized on a detection plate.

[0241] Applications

[0242] The present application provides the use of the antibody of the present application, for example, for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and / or treatment of a WAMT2-related disease.

[0243] In a preferred embodiment, the WAMT2-related disease is a disease related to overexpression or overtransport of WAMT2. In a preferred embodiment, the disease includes Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0244] The main advantages of the present application include:

[0245] (a) The anti-VMAT2 antibody of the present application has good specificity and binding activity to human VMAT2, and has the ability to inhibit the overexpression or overtransport of VMAT2 to store various monoamine neurotransmitters such as dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) in synaptic vesicles, thereby improving the disorder of the monoamine neurotransmitter system.

[0246] (b) The VMAT2 antibody of the present application has cross-binding activity to humans, rats, mice and pigs.

[0247] (c) Compared with VMAT2 small molecule inhibitors, the antibody of the present application has a long half-life and high specificity.

[0248] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0249] Example 1 Obtaining human VMAT2 protein mimicking native conformation

[0250] In this example, the human full-length VMAT2 gene sequence (EBI: ENST00000644641.2) is constructed into a mammalian cell expression vector, such as pCAG vector, using HEK293 cells for exogenous expression to obtain a large amount of VMAT2 protein sample, by using the conventional techniques in the art.

[0251] The expressed protein will be purified using the conventional biochemical techniques in the art, and the purification conditions will be optimized to maintain the native conformation of the protein as much as possible. Subsequently, the purified VMAT2 protein will be packaged with Amphipol reagent to maximize the simulation of the native conformation of VMAT2 protein on the cell membrane surface.

[0252] Example 2 Mouse immunization, mouse B cell sorting and culture

[0253] The VMAT2 protein obtained in Example 1 is used to immunize mice according to the conventional known techniques in the art. The immunization strategy is as follows: 10-12 week old C57BL / 6 mice are selected, and an immunization group and a control group are set up. The mice in the immunization group are injected intraperitoneally and intraplantarly with 30 μg of the VMAT2 protein (antigen) prepared in Example 1 above and 20 μg of CpG ODN2395 (5'-tcgtcgttttcggcgcgcgccg-3') (SEQ ID NO: 123) and an equal volume of AddaVax TM (InvivoGen) are injected intraperitoneally and intraplantarly, and the mice in the control group are injected with 20 μg of CpG ODN2395 and an equal volume of AddaVax TM The immunization is performed once every 3-4 days, and the immunization is performed 7 times.

[0254] Sorting of mouse B cells: The experimental and control groups of mice were sacrificed 3-7 days after the last immunization, and the spleen and lymph node tissues were obtained. Single cell suspensions were prepared from the spleen and lymph nodes (popliteal and inguinal), and red blood cells were removed using red blood cell lysis buffer (Sangon Biotech Cat. b541001-0100). After washing twice with 2% FBS in PBS (FACS buffer), the cells were prepared for staining, and the Cytoflex SRT (Beckman Coulter) cell sorter was used to sort the staining ring gate strategy for obtaining the target cells as follows:

[0255] DAPI-CD19+CD38+GL7-IgG1+;

[0256] The reagents used are as follows:

[0257] DAPI (live / dead; Invitrogen Cat. D1306),

[0258] anti-mouse CD19 (APC / Cyanine7; Biolegend Cat. 115530),

[0259] anti-mouse CD38 (PE / Cyanine7; Biolegend Cat. 102718),

[0260] anti-mouse / human GL7 Antigen (T and B cell activation marker), 488; Biolegend Cat. 144612);

[0261] anti-mouse IgG1 (APC; Biolegend Cat. 406610).

[0262] IgG1-positive memory B cells were sorted by flow cytometry after immunization.

[0263] The results of flow sorting are shown in Figure 1 . As can be seen from Figure 1 , a large number of IgG1-positive memory B cells can be sorted from the experimental group mice after using the immunization method in the scheme, and the proportion of IgG1-positive memory B cells in the total B cells is higher. The proportion of IgG1-positive memory B cells in the control group is much less than that in the experimental group.

[0264] Culture of mouse B cells: The IgGl positive memory B cells obtained by sorting above were cultured, the culture method can refer to Haniuda and Kitamura, (2019). Induced Germinal Center B Cell Culture System, Bio-protocol 9(4): e3163. DOI: 10.21769 / BioProtoc.3163, and the feeder culture medium is: RPMI-1640, (Gibco, Cat. 11875-093), 10% Fetal Bovine Serum (FBS, VivaCell, Cat. C04002-500), 55 mM 2-mercaptoethanol (ThermoFisher, Cat. 21985), 100 units / ml Penicillin (Biosharp), 100 pg / ml Streptomycin (Biosharp), 10 mM HEPES (ThermoFisher, Cat. 15630-080), 1 mM Sodium Pyruvate (ThermoFisher, Cat. 11360-070) and 0.1 mM MEM nonessential amino acid (ThermoFisher, Cat. 11140-050), 1 day before sorting, feeder cells expressing CD40L and BAFF were seeded into 96-well plates, 1000 cells per well; single IgGl positive B cells were sorted into each well, 2-10 ng / ml IL-4 was added to the feeder culture medium for two days, 4-10 ng / ml IL-21 was added for continuous culture for 6-8 days, the liquid was changed every day, the supernatant was collected on the 10th day, stored at 4°C, and the 96-well plate cells were stored at -80°C for subsequent lysis to obtain the RNA of single B cells.

[0265] ELISA experiment, results and analysis of antibody screening targeting VMAT2

[0266] The VMAT2 protein obtained in Example 1 was coated in a 96-well ELISA plate, incubated at 4°C overnight, after the coating buffer was discarded, 100-120 μl blocking buffer (4% BSA in 1xPBS) was added to the plate, incubated at room temperature for 2 hours, after the blocking buffer was removed, 20-60 μl of single cell culture supernatant collected in Example 2 was added to each well, incubated at 4°C overnight, after washing 3 times, 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat. 1030-04) was added, incubated at room temperature for 2 hours, after washing 4 times, 20-30 ul of color developing solution containing 4-nitrophenyl phosphate disodium hexahydrate (CSNpharm, Cat. CSN66207) was added to each well. OD405 was measured using MDSpectraMax iD3.

[0267] The results are shown in Tables 1-4 below: part of the detection data of the ELISA detection of the 96-well plate is shown.

[0268] Tables 1-4 show plate 1 antigen detection and plate 1 secreted IgG1 antibody detection; plate 2 antigen detection and plate 2 secreted IgG1 antibody detection, respectively.

[0269] Table 1 Plate 1 antigen specificity detection

[0270] 1 2 3 4 5 6 7 8 9 10. 11 12 A 0.10 0.10 0.12 0.11 0.12 0.11 0.14 0.11 0.10 0.11 0.10 0.09 B 0.10 0.10 0.12 0.65 0.12 0.10 0.10 0.10 0.10 0.13 0.10 0.09 C 0.10 0.11 0.11 0.11 0.10 0.10 0.10 0.10 0.10 0.11 0.11 0.09 D 0.10 3.59 0.11 0.11 0.10 0.10 0.18 0.11 0.11 0.10 0.13 0.10 E 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 1.60 0.11 0.62 0.09 F 0.10 0.10 0.10 0.10 0.12 0.09 0.10 0.10 0.10 0.10 0.10 0.09 G 0.09 0.09 0.10 0.10 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.09 H 0.08 0.09 0.09 0.09 0.08 0.08 0.09 0.09 0.09 0.09 0.09 0.08

[0271] Table 2 Plate 1 cell secreted IgG1 antibody detection

[0272] 1 2 3 4 5 6 7 8 9 10 11 12 A 3.59 0.38 3.64 0.51 0.45 0.44 3.62 2.97 0.51 3.60 3.55 0.78 B 0.35 0.51 0.48 3.57 0.87 1.27 0.49 0.44 0.64 3.57 0.44 0.38 C 3.45 3.53 0.43 0.78 0.70 3.55 3.59 0.47 0.59 0.48 0.57 0.45 D 0.37 3.59 0.46 0.45 0.68 3.42 3.33 0.46 0.46 0.47 0.68 3.55 E 0.36 0.43 0.40 0.52 1.45 0.51 0.54 1.06 1.61 0.45 1.60 0.75 F 0.31 0.40 0.57 0.42 3.55 3.19 0.43 0.41 3.60 0.40 1.70 0.34 G 0.26 3.15 0.32 0.33 0.48 3.59 0.44 0.35 3.26 0.36 0.32 0.28 H 0.23 0.73 0.25 0.26 0.31 0.28 0.29 0.29 0.29 0.27 0.28 0.25

[0273] Table 3 Plate 2 antigen specificity detection

[0274]

[0275]

[0276] Table 4 Plate 2 cell secreted IgG1 antibody detection

[0277] 1 2 3 4 5 6 7 8 9 10 11 12 A 3.28 0.46 0.55 1.83 1.37 1.02 0.51 0.57 0.79 0.46 0.47 0.40 B 0.60 0.91 0.50 1.08 0.50 0.49 0.53 0.56 3.57 0.70 0.83 0.48 C 3.62 3.58 3.64 0.88 0.51 1.58 3.23 0.71 0.50 1.50 3.60 3.58 D 0.63 0.92 3.58 0.53 0.62 3.62 3.58 0.53 0.92 0.61 3.60 0.43 E 3.57 0.51 0.50 0.56 1.37 3.57 3.55 1.00 0.52 0.51 0.50 0.67 F 0.49 0.46 3.55 0.95 0.47 1.32 0.48 0.51 0.66 3.02 0.45 0.36 G 1.45 0.46 0.44 0.54 0.42 0.76 0.45 0.72 0.44 0.42 0.41 0.34 H 1.20 0.87 2.34 0.35 0.35 3.57 1.02 3.66 0.45 3.58 0.33 0.28

[0278] The ELISA detection results show that the supernatant of the IgG1+ positive B cell culture screened contains antibodies specifically bound to VMAT2 protein, and the color development is different because of the different antibody binding force or antibody concentration in the culture supernatant. Further ELISA concentration gradient detection shows that the above antibodies all have antigen-antibody binding activity.

[0279] Example 4 Antibody binding detection to cell surface VMAT2 antigen (antibody detection of single B cell culture supernatant), screening for antibodies binding to the cytosolic side of hVMAT2

[0280] HEK293T cells were seeded at a density of 1 x 10 7 cells in a 10 cm cell culture dish and incubated in a carbon dioxide incubator (37 °C, 5% CO2) for 12 hours. The cells were co-transfected with Lipofectamine 3000 and a plasmid expressing full-length human VAMT2 (the VAMT2 plasmid was constructed using conventional techniques in the art) and a pMax GFP plasmid. After 24 hours, the fresh culture medium was replaced and the cells were incubated. After 48 hours of transfection, the cells were treated with 2 mM EDTA-PBS buffer and then washed with 2% FBS-containing PBS buffer. The single cell suspension was filtered and placed in a 96-well plate. The transfection cells were incubated with the supernatant of the single B cell culture for 1 hour, and then washed twice with FACS. The cells were incubated with DAPI (live / dead; Invitrogen Cat. D1306) and anti-mouse IgG1 (APC; Biolegend Cat. 406610) for 15 minutes. All staining processes were completed on ice. A CytoFLEX LX (Beckman Coulter) flow cytometer was used to analyze the DAPI-GFP+IgG1+cells.

[0281] As shown in Figure 4 , after the ribosome synthesizes the VMAT2 polypeptide chain, it is transferred to the endoplasmic reticulum and Golgi for further synthesis, processing, and modification. Finally, in vivo, VMAT2 is localized to the synaptic vesicle membrane in cells. In the antibody screening experiment, human VMAT2 (hVMAT2) is overexpressed on the membrane surface of HEK293T cells. In this process, VMAT2 is flipped to the extracellular region (i.e., the cell membrane surface region) from the vesicle membrane lumen side region. After flipping, the vesicle membrane cytosolic side is still the plasma membrane cytosolic side. Therefore, in the preliminary screening of antibodies, the present application excludes antibodies binding to cell surface VMAT2. The remaining antibodies (i.e., anti-VMAT2 cytosolic side antibodies) that are positive in ELISA screening will be cloned and further verified.

[0282] ELISA experiment, VMAT2 protein is a whole protein, including the lumen side and the cytoplasm side, but the VMAT2 protein is not on the cell membrane in the ELISA experiment, both the lumen side and the cytoplasm side of the protein are exposed. When the VMAT2 protein encounters the antibody, both sides can detect the antibody. When doing cell flow experiment, VAMT2 is expressed on the cell membrane, so VMAT2 will have a lumen side and a cytoplasm side, and at this time the detection can only detect the antibody bound to the cell surface, and the antibody bound to the cytoplasm side cannot be detected. Therefore, it is considered that the ELISA positive sample, except for the sample combined with the overexpressed cell surface, the remaining ELISA positive sample is the sample combined with the antibody on the cytoplasm side.

[0283] Specifically, the flow results are as shown in Figures 5-6 The cell surface binding detection shows that the mouse monoclonal antibody binds to the overexpressed hVMAT2 on the cell surface, which corresponds to the hVMAT2 in the protruding vesicle, that is, it is combined on one side of the cavity, and such antibodies will be excluded in the screening.

[0284] Further screening of antibodies combined on the cytoplasm side of hVMAT2, therefore, the antigen-antibody binding positive antibody finally screened in this embodiment is the antibody combined on the cytoplasm side of hVMAT2. The antibody on the cytoplasm side of hVMAT2 can be used as a marker antibody of VMAT2 antigen, and also as a functional antibody for inhibiting its transport.

[0285] Example 5 ELISA gradient detection of single B cell culture supernatant

[0286] The VMAT2 protein obtained in Example 1 was coated in a 96-well ELISA plate, incubated at 4°C under humid conditions overnight, after the coating buffer was discarded, 100-120 μl of blocking buffer (4% BSA in 1xPBS) was added to the plate, incubated at room temperature for 2 hours, after the blocking buffer was removed, the single cell culture supernatant showing antigen-antibody binding in Example 3 and Example 4 was added to each well, the single cell culture supernatant was gradient diluted, i.e. 4-fold, 16-fold, 64-fold, 256-fold, 20-60 μl of single cell culture supernatant collected on the 10th day in Example 2 was added to each well, incubated at 4°C under humid conditions overnight, washed 3 times, 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat. 1030-04) was added, incubated at room temperature for 2 hours, washed 4 times, and 20-30 μl of color developing liquid containing 4-nitrophenyl phosphate disodium hexahydrate (CSNpharm, Cat. CSN66207) was added to each well.

[0287] The OD405 results were measured using MDSpectraMax iD3, and the results are as shown in Figures 2-3The results are shown in the figure. As can be seen from the figure, each sample in the figure shows strong antigen-antibody binding activity.

[0288] Molecular cloning method and VDJ / VJ sequence of monoclonal antibody obtained in Example 6

[0289] Based on the results of the ELISA of Example 3 and Example 5 and the antigen-antibody cell surface binding detection of Example 4, the cells in the positive wells (antigen-antibody binding) of the 96-well cell culture plate previously frozen in the -80°C refrigerator were selected for RNA extraction for subsequent molecular cloning to obtain VDJ / VJ sequences.

[0290] The total RNA of the B cells in the 96-well cell culture plate detected as positive (antigen-antibody binding) was extracted by TRIzol Reagent (Thermo Fisher). Reverse transcription and PCR were performed according to the paper (Cloning and expression of murine Ig genes from single B cells, Tiller et al, Journal of Immunological Methods, 2009). Briefly, cDNA synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher) under the following program: 42°C for 5 minutes, 25°C for 10 minutes, 50°C for 60 minutes and 94°C, and then two rounds of semi-nested PCR were performed using HotStar DNA Polymerase (Qiagen) to enrich heavy chains and light chains. The PCR products were purified and sequenced. The sequencing results were analyzed using the IgBlast tool and the IMGT database. The VDJ / VJ fragments were amplified using gene-specific primers (Table 5), and the VDJ fragment heavy chain and VJ fragment light chain vectors were cloned by homologous recombination or T4 ligase ligation.

[0291] Table 5 Gene-specific primers for amplifying VDJ / VJ

[0292]

[0293] Two rounds of semi-nested PCR used primers as shown in Table 6: PCR program: 95°C for 15 minutes, 95°C for 30 seconds, 50-65°C for 30 seconds, and 72°C for 5 minutes.

[0294] Table 6 Semi-nested PCR primers

[0295]

[0296] The VDJ / VJ specific primers in the present embodiment include the homologous arm part of the heavy / light chain vector and the specific part of the VDJ / VJ fragment, and the specific primers as a whole will use different homologous arms according to the different cloning vectors used. The present application is not limited to the specific primers used in the embodiments.

[0297] The VDJ / VJ variable region nucleotide sequence and amino acid sequence of the antibody after reverse transcription and two rounds of semi-nested PCR are shown in Table 7.

[0298] Table 7

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305] The human heavy chain constant region CH1 vector and light chain vector with strep II tag and His tag (expressing Fab) and human full-length constant region heavy and light chain vector (expressing human IgG1) and SCFV vector are constructed respectively. Finally, the antibody plasmid is extracted, the antibody expression is carried out in Expi293F suspension cells, the full-length antibody is separated and purified by rProtein A Beads, and the Fab is separated and purified by strep II tag agarose resin. The Fab vector, full-length human IgG1 heavy and light chain vector and scFV vector are used to prepare the antibody of VMAT2 in the present application, and the vector maps are shown in Figure 7A -B.

[0306] Example 7 Antigen-antibody binding experiment

[0307] Polystyrene Protein A (Spherotech) microspheres were resuspended in 1x PBS at room temperature, centrifuged, and then resuspended in 10 μg / ml anti-flag (hIgG1) antibody. The mixture was incubated for 15 minutes with stirring, washed twice with 1x PBS, and incubated for 1-2 hours with 2 μg / ml hVMAT2-2% FACS solution. After washing twice with 2% FACS solution, 50 μl of Fab antibody diluted in 2% FACS solution (expressed as JPF) was added, and the mixture was incubated for 1 hour. After washing twice with 1x PBS, 50 μl of secondary antibody (Monoclonal Mouse Strep Tag II Antibody, LSBio, Cat.LS-C203631) was added, and the mixture was incubated on ice for 15 minutes. All procedures were performed on ice. Flow cytometry analysis and MFI were performed using a CytoFLEX LX (Beckman) system.

[0308] The results are as follows Figure 8 As shown, the antigen-antibody binding experiment of polystyrene Protein A showed that antibodies JPF0514, JPF1217, JPF1254, JPF1258, JPF1026, JPF1206, JPF1212, JPF0568, JPF1039, and JP0503 all had antigen-binding activity.

[0309] Example 8: SPR Experiment, Results, and Analysis

[0310] Surface plasmon resonance (SPR) binding experiments were performed using a Biacore 8K+ (Cytiva) sensor at 25°C, with 10 mM HEPES sodium salt (pH 7.4), 150 mM NaCl, and 0.05% (v / v) Tween 20. In short, the anti-flag antibody (hIgG1) was immobilized on a Cytiva Protein A sensor chip, followed by sequential binding of the hVMAT2 protein and the Fab fragment. The KD value was calculated using Cytiva's Biacore Insight Evaluation software.

[0311] The results are as follows Figure 9 As shown in Table 8, the antibody binding and dissociation constants obtained by the SPR antigen-antibody binding affinity test were obtained, and all the screened antibodies had antigen binding activity.

[0312] Table 8

[0313] mAb ID Ka (1 / Ms) Kd (1 / s) KD (M) JPF1217 2.16e+05 4.24e-04 1.96e-09 JPF1254 1.48e+05 7.18e-04 4.86e-09 JPF0514 1.93e+05 6.36e-04 3.29e-09 JPF1026 1.32e+05 6.13e-04 4.65e-09 JPF1258 3.58e+06 7.69e-03 2.15e-09 JPF1039 3.09e+05 4.19e-03 1.36e-08 JPF1206 4.98e+05 1.27e-02 2.56e-08 JPF1212 1.30e+05 9.14e-03 7.03e-08 JPF0503 1.97e+05 9.22e-03 4.67e-08 JPF0568 1.58e+05 2.35e-02 1.49e-07

[0314] Example 9: Verification of the binding of Fab antibody to VMAT2 and VMAT1 from different species

[0315] hVMAT2 (human VMAT2, already constructed in Example 1), hVMAT1 (human VMAT1, Ensembl: ENSG00000036565), rVMAT2 (rat VMAT2, Ensembl:

[0316] ENSRNOG00000008890), rVMAT1 (rat VMAT1, Ensembl: ENSRNOG00000011992), mVMAT2 (mouse VMAT2, Ensembl: ENSMUSG00000025094), mVMAT1 (mouse VMAT1, Ensembl: ENSMUSG00000036330), pVMAT2 (pig VMAT2, Ensembl:

[0317] ENSSSCG00000020671), pVMAT1 (pig VMAT1, Ensembl: ENSSSCG00000009601), plasmids (human, rat, mouse and pig VAMT2 or VAMT1 plasmids were constructed using routine techniques in the art), HEK293 T cells were seeded at 1 x 10 7The cells were seeded at a density of one cell per 10 cm cell culture dish and incubated in a carbon dioxide incubator (37℃, 5% CO2) for 12 hours. The cells were co-transfected with Lipofectamine 3000 and plasmids expressing hVMAT2 (human VMAT2), rVMAT2 (rat VMAT2), rVMAT1 (rat VMAT1), mVMAT1 (mouse VMAT1), pVMAT2 (pig VMAT2), and pVMAT1 (pig VMAT1) and pMax GFP plasmid, respectively, according to conventional techniques in the art. After 24 hours, the medium was replaced with fresh medium and the cells were incubated. After 48 hours of transfection, the cells were detached with 2 mM EDTA in PBS and then washed with PBS. The single-cell suspension was filtered and placed in a 96-well plate. The transfected cells were incubated with live / dead eflour 450 dye (Thermo Fisher scientific; cat. no. 65-0863-14) for 15 minutes, washed once with PBS, and then incubated with fixation buffer (Thermo Fisher scientific; cat. no. 00-8222-49) for 20 minutes. After centrifugation, the supernatant was removed and permeabilization buffer (Thermo Fisher scientific; cat. no.; 00-8333-56) was added. After centrifugation, the supernatant was removed and Fab antibody was added for incubation for 1 hour. Anti-human Ig light chain kappa antibody (Invitrogen, Cat. 2806744) was then added for co-incubation for 15 minutes. All staining processes were completed on ice. A CytoFLEX LX (Beckman Coulter) flow cytometer was used to analyze DAPI-GFP+IgK+cells. The detection principle was the same as that of Example 4.

[0318] The results are shown in Table 1. Figure 10 As shown in Table 1, it was found that, under the condition of an antibody concentration of 10 μg / ml, JPF1026, JPF1217, JPF1254, JPF1258, JPF0514, JPF0568, JPF0503, JPF1212, JPF1039, and JPF1206 could all bind to hVMAT2. JPF1026, JPF1217, JPF1258, JPF0568, JPF1212, and JPF1206 could bind to all the tested species of VMAT1 and VMAT2, JPF1039 only bound to hVMAT2, and JPF1254 only bound to different species of VMAT2.

[0319] Example 10 Cell transport inhibition experiment (experiment for verifying that the antibody has an inhibitory effect at the cell level)

[0320] The hVMAT2 sequence was cloned into the pCAG vector. 24-well plates were pre-coated with poly-D-lysine, and then HEK293 T cells were seeded into the plates at a density of 2 × 10⁻⁶ cells per well. 5 Cells were cultured at 37°C and 5% CO2 for 12 hours. hVMAT2 and ScFv plasmids (denoted as JPS) were co-transfected using Lipofectamine 3000. Forty-eight hours after transfection, cells were washed with PBS. Cells were then incubated for 15 minutes in a 37°C buffer (125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM NaH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES, pH 7.4), followed by incubation at 37°C in the dark with 2 μM FFN206 (Abcam) and 2 μM calci-AM (enzyme). After washing twice with frozen PBS, fluorescence was detected using EnSight (PerkinElmer).

[0321] The study was divided into an antibody experimental group containing antibodies JPS0514, JPS1026, JPS1039, JPS1206, JPS1212, JPS1217, JPS1258, JPS1254, JPS0503, and JPS0568; and a small molecule positive inhibitor control group containing TBZ tetrabenazine.

[0322] The results are as follows Figure 11 As shown in the results of the cell inhibition transport experiment, antibodies JPS0514, JPS1026, JPS1039, JPS1206, JPS1212, JPS1217, JPS1258, JPS1254, JPS0503 and JPS0568 all have the function of inhibiting VMAT2 transport.

[0323] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-VMAT2 antibody or antigen-binding fragment thereof, characterized in that, The anti-VMAT2 antibody or antigen-binding fragment thereof has three complementarity determining regions (CDRs) of a heavy chain variable region (HCDRs) and three complementarity determining regions (CDRs) of a light chain variable region (LCDRs) selected from the group consisting of: (a1) HCDR1 set forth in SEQ ID NO: 48, HCDR2 set forth in SEQ ID NO: 49, HCDR3 set forth in SEQ ID NO: 50, LCDR1 set forth in SEQ ID NO: 52, LCDR2 set forth in SEQ ID NO: 53, LCDR3 set forth in SEQ ID NO: 54; (a2) HCDR1 set forth in SEQ ID NO: 48, HCDR2 set forth in SEQ ID NO: 58, HCDR3 set forth in SEQ ID NO: 50, LCDR1 set forth in SEQ ID NO: 52, LCDR2 set forth in SEQ ID NO: 53, LCDR3 set forth in SEQ ID NO: 54; (a3) HCDR1 set forth in SEQ ID NO: 48, HCDR2 set forth in SEQ ID NO: 63, HCDR3 set forth in SEQ ID NO: 50, LCDR1 set forth in SEQ ID NO: 52, LCDR2 set forth in SEQ ID NO: 53, LCDR3 set forth in SEQ ID NO: 54; wherein the amino acid sequence set forth in SEQ ID NO: 53 is: RMS.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-VMAT2 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof against the cytosolic side of the VMAT2 protein.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein The antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain variable region and the light chain variable region of the anti-VMAT2 antibody or antigen-binding fragment thereof are selected from the group consisting of: (a) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 51; (b) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 57, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 59; (c) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 62, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:

51.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein, The heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region; and the light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.

6. A recombinant protein, characterized in that, The recombinant protein is comprised of: (i) the anti-VMAT2 antibody or antigen-binding fragment thereof of claim 1; and (ii) a tag sequence that optionally assists in expression and / or purification.

7. The recombinant protein of claim 6, wherein, The tag comprises a FLAG tag, a 6His tag, or a combination thereof.

8. A polynucleotide molecule, comprising, The polynucleotide molecule encodes the anti-VMAT2 antibody or antigen-binding fragment thereof of claim 1.

9. The polynucleotide molecule of claim 8, wherein, The polynucleotide molecule is DNA or RNA.

10. A vector, characterized in that, The vector contains the polynucleotide molecule of claim 8.

11. A host cell, characterized in that, The host cell contains the vector of claim 10, or the polynucleotide molecule of claim 8 integrated into its genome.

12. The host cell of claim 11, wherein The cell is a eukaryotic cell or a prokaryotic cell.

13. An antibody drug conjugate characterized in that, The antibody drug conjugate contains: (a) an antibody moiety comprising the anti-VMAT2 antibody or antigen-binding fragment thereof of claim 1; and (b) a conjugating moiety coupled to the antibody or antigen-binding fragment thereof, the conjugating moiety being a detectable label.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (i) the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 6, the polynucleotide molecule of claim 8, the vector of claim 10, or the host cell of claim 11, or the antibody drug conjugate of claim 13; and (ii) a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein, The pharmaceutical composition is in an injectable form.

16. The use of the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 6, or the antibody drug conjugate of claim 13, wherein, for preparing a reagent, an assay plate, or a kit; The reagent, assay plate, or kit is used for detecting a VMAT2 protein in a sample.

17. A method of making the anti-VMAT2 antibody or antigen binding fragment thereof of claim 1, wherein, The method comprises the following steps: (a) culturing the host cell of claim 11 under expression conditions, thereby expressing the anti-VMAT2 antibody or antigen-binding fragment thereof; (b) isolating and purifying the anti-VMAT2 antibody or antigen-binding fragment thereof of (a).

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