Anti-VMAT2 antibody or antigen binding fragment thereof as well as composition and application thereof

Abstract

The invention provides an anti-VMAT2 antibody or an antigen binding fragment thereof as well as a composition and application thereof. Specifically, provided is an anti-VMAT2 antibody or an antigen-binding fragment thereof, which can effectively bind to an intracellular segment of a human VMAT2 protein. The invention also provides an antibody drug conjugate containing the antibody or the antigen binding fragment, and the antibody drug conjugate can be applied to preparation of drugs for treating overexpression of VMAT2 diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to an anti-VMAT2 antibody or an antigen-binding fragment thereof, and a composition and application thereof. Background Art

[0002] Vesicular monoamine transporter 2 (VMAT2) is a multi-transmembrane protein located on the synaptic vesicle membrane of neurons. It can transport a variety of monoamine neurotransmitters such as dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) to synaptic vesicles for storage. Under external stimulation, these neurotransmitters are released from synaptic vesicles into the synaptic cleft and bind to the corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signal transduction. VMAT2 is involved in regulating many functions such as movement, emotion, sleep, reward, attention, addiction, etc.

[0003] Overexpression or overtransport of VMAT2 can lead to a disorder in the monoamine neurotransmitter system, thus inducing Huntington's disease, addictive diseases, tardive dyskinesia, hypertension, etc. So far, there is an extreme lack of available treatments for such diseases.

[0004] Currently, small molecule inhibitors targeting VMAT2, such as reserpine, tetrabenazine, deutetrabenazine, valbenazine, GZ-793A, etc., are mostly used to treat Huntington's disease, tardive dyskinesia and hypertension.

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

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

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

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

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

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

[0011] HCDR2 shown in SEQ ID NO: 49,

[0012] HCDR3 shown in SEQ ID NO: 50,

[0013] LCDR1 shown in SEQ ID NO: 52,

[0014] LCDR2 shown in SEQ ID NO: 53,

[0015] LCDR3 shown in SEQ ID NO: 54;

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

[0017] HCDR2 shown in SEQ ID NO: 58,

[0018] HCDR3 shown in SEQ ID NO: 50,

[0019] LCDR1 shown in SEQ ID NO: 52,

[0020] LCDR2 shown in SEQ ID NO: 53,

[0021] LCDR3 shown in SEQ ID NO: 54;

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

[0023] HCDR2 shown in SEQ ID NO: 63,

[0024] HCDR3 shown in SEQ ID NO: 50,

[0025] LCDR1 shown in SEQ ID NO: 52,

[0026] LCDR2 shown in SEQ ID NO: 53,

[0027] LCDR3 shown in SEQ ID NO: 54;

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

[0029] HCDR2 shown in SEQ ID NO: 68,

[0030] HCDR3 shown in SEQ ID NO: 69,

[0031] LCDR1 shown in SEQ ID NO: 71,

[0032] LCDR2 shown in SEQ ID NO: 72,

[0033] LCDR3 shown in SEQ ID NO: 73;

[0034] (b2) HCDR1 shown in SEQ ID NO: 67,

[0035] HCDR2 shown in SEQ ID NO: 68,

[0036] HCDR3 shown in SEQ ID NO: 77,

[0037] LCDR1 shown in SEQ ID NO: 71,

[0038] LCDR2 shown in SEQ ID NO: 72,

[0039] LCDR3 shown in SEQ ID NO: 73;

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

[0041] HCDR2 shown in SEQ ID NO: 83,

[0042] HCDR3 shown in SEQ ID NO: 84,

[0043] LCDR1 shown in SEQ ID NO: 86,

[0044] LCDR2 shown in SEQ ID NO: 87,

[0045] LCDR3 shown in SEQ ID NO: 88;

[0046] (c2) HCDR1 shown in SEQ ID NO: 82,

[0047] HCDR2 shown in SEQ ID NO:92,

[0048] HCDR3 shown in SEQ ID NO:93,

[0049] LCDR1 shown in SEQ ID NO: 95,

[0050] LCDR2 shown in SEQ ID NO: 96,

[0051] LCDR3 shown in SEQ ID NO: 97;

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

[0053] (d1) HCDR1 shown in SEQ ID NO: 101,

[0054] HCDR2 shown in SEQ ID NO: 102,

[0055] HCDR3 shown in SEQ ID NO: 103,

[0056] LCDR1 shown in SEQ ID NO: 71,

[0057] LCDR2 shown in SEQ ID NO: 72,

[0058] LCDR3 shown in SEQ ID NO: 105;

[0059] (d2) HCDR1 shown in SEQ ID NO: 109,

[0060] HCDR2 shown in SEQ ID NO: 110,

[0061] HCDR3 shown in SEQ ID NO: 111,

[0062] LCDR1 shown in SEQ ID NO: 71,

[0063] LCDR2 shown in SEQ ID NO: 72,

[0064] LCDR3 shown in SEQ ID NO: 105;

[0065] (d3) HCDR1 shown in SEQ ID NO: 116,

[0066] HCDR2 shown in SEQ ID NO: 117,

[0067] HCDR3 shown in SEQ ID NO: 118,

[0068] LCDR1 shown in SEQ ID NO: 120,

[0069] LCDR2 shown in SEQ ID NO: 72,

[0070] LCDR3 shown 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 cytoplasmic VMAT2 protein.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0086] In another preferred example, the heavy chain of the antibody or antigen-binding fragment thereof further includes a heavy chain constant region; the light chain of the antibody or antigen-binding fragment thereof further includes 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 mouse antibody, or a chimeric antibody.

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

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

[0091] In another preferred embodiment, the antibody is a full-length antibody 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 example, the antibody further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.

[0095] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

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

[0097] (ii) optionally a tag sequence to facilitate expression and / or purification.

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

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

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

[0101] In the third aspect of the present invention, a nucleotide molecule is provided, wherein the nucleotide molecule encodes the anti-VMAT2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

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

[0103] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the nucleotide molecule described in the third aspect of the present invention.

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

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

[0106] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the fourth aspect of the present invention, or the nucleotide molecule described in the third aspect of the present invention is integrated into its genome.

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

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

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

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

[0111] In another preferred embodiment, the cell is an immune cell, and a chimeric antigen receptor is simultaneously expressed on its surface.

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

[0113] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

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

[0115] In a sixth aspect of the present invention, an antibody-drug conjugate is provided, wherein the antibody-drug conjugate comprises:

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

[0117] (b) a conjugated moiety conjugated to the antibody or antigen-binding fragment thereof, wherein the conjugated moiety is 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 excessive 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-(XY)n;

[0121] in,

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

[0123] X is a linker;

[0124] Y is a coupling moiety, and the coupling moiety is a drug;

[0125] n is a positive integer ≤8;

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

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

[0128] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the nucleotide molecule according to the third aspect of the present invention, the vector according to the fourth aspect of the present invention, or the host cell according to the fifth aspect of the present invention, or the antibody-drug conjugate according to the sixth aspect of the present invention; 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 used to prepare a drug for treating a disease caused by overexpression or overtransport 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 the eighth aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, or an antibody-drug conjugate as described in the sixth aspect of the present invention, for use in preparing a medicament, a reagent, a test plate or a kit;

[0134] The reagent, detection plate or kit is used for: detecting VMAT2 protein in a sample;

[0135] The medicament is used for treating or preventing diseases caused by overexpression of 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 a VMAT2 cytoplasmic protein.

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

[0139] (a) culturing the host cell according to the fifth aspect of the present invention under expression conditions, thereby expressing the anti-VMAT2 antibody or antigen-binding fragment thereof;

[0140] (b) isolating and purifying the anti-VMAT2 antibody or antigen-binding fragment thereof described in (a).

[0141] In the 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) contacting a sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

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

[0144] In another preferred embodiment, the sample includes: human or animal tissue samples, 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 comprises step (3) analyzing the affinity between the antibody and the antigen.

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

[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 overlapped in sequence.

[0151] In the twelfth aspect of the present invention, a kit is provided, comprising:

[0152] (1) a first container, wherein the first container contains the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0153] (2) a second container, wherein the second container contains a second antibody against the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0154] (3) a third container, wherein the third container contains a cell lysis reagent;

[0155] or,

[0156] The kit contains the detection plate as described in the eleventh aspect of the present invention.

[0157] In another preferred embodiment, the antibody in the first container is detectably labeled.

[0158] In another preferred embodiment, the antibody in the second container is detectably labeled.

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

[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 within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0163] Figure 2 The results of the gradient ELISA antigen-antibody binding experiment of the culture supernatant of the positive wells of the ELISA experiment for screening antibodies targeting VMAT2 in Example 3 are shown.

[0164] Figure 3 The results of the gradient ELISA antigen-antibody binding experiment of the culture supernatant of the positive wells of the ELISA experiment for screening antibodies targeting VMAT2 in Example 3 are shown.

[0165] Figure 4 A schematic diagram of VMAT2 protein expression and trafficking in cells is shown.

[0166] Figure 5 Results are shown for supernatants from single B cell cultures that bind cell surface hVMAT2.

[0167] Figure 6 Results are shown for supernatants from single B cell cultures that did not bind cell surface hVMAT2.

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

[0169] Figure 8 The results of the cell flow cytometry antigen-antibody binding experiment in Example 6 of the present invention are shown.

[0170] Fig. 9 The results of the SPR antigen-antibody binding affinity test in Example 7 of the present invention are shown.

[0171] Fig.10 The results of binding verification of the Fab antibody in Example 8 of the present invention with VMAT2 and VMAT1 of different species are shown.

[0172] Fig.11 The experimental results of the antibody in Example 9 of the present invention inhibiting cell transport are shown. DETAILED DESCRIPTION

[0173] The inventors have obtained a class of anti-VMAT2 antibodies unexpectedly through extensive and in-depth research and a large number of screenings. The experimental results show that the anti-VMAT2 antibodies of the present invention have high affinity and good biological activity. In addition, the antibodies of the present invention can have cross-binding reactions with human VMAT2, mouse VMAT2 and pig VMAT2, and can also bind to VMAT1. The antibodies of the present invention can significantly inhibit the transport function of VMAT2. On this basis, the present invention is completed.

[0174] the term

[0175] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0176] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. 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 "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

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

[0179] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0180] VMAT2

[0181] Vesicular monoamine transporter 2 (VMAT2) is a multi-transmembrane protein located on the synaptic vesicle membrane of neurons. It can transport a variety of monoamine neurotransmitters such as dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) to synaptic vesicles for storage. Under external stimulation, these neurotransmitters are released from synaptic vesicles into the synaptic cleft and bind to the corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signal transduction. VMAT2 is involved in regulating many functions such as movement, emotion, sleep, reward, attention, addiction, etc.

[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 invention not only have binding activity to the intracellular region of VMAT2 but also have good transport inhibition activity.

[0183] Antibody

[0184] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" 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 the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between 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 a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by disulfide bonds 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 inter-polypeptide disulfide bonds formed between the hinge regions.

[0185] In the present invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave antibodies into two identical Fab segments and one Fc segment. The Fab segment is composed 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 segment is the crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

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

[0187] In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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, Volume I, pp. 647-669 (1991)).

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

[0189] Knowing the amino acid sequences of the CDRs, one skilled in the art can easily 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 the framework region of a naturally occurring human antibody.

[0191] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light chain and the heavy chain to fold into an exchange dual variable region immunoglobulin. In the present invention, preferred linkers refer to linkers Linker1 and Linker2, wherein Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect scFv to the heavy chain of another antibody.

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

[0193] In the present invention, the antibody of the present invention also includes its conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared with the amino acid sequence of the specific antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0194] Table A

[0195] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; 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) Ile; Leu; Met; Phe; Ala Leu

[0196] In the present invention, the terms "anti", "binding", and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The equilibrium dissociation constant (KD) of M or less binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium 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 the antibody and the antigen is measured in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviation SPR) or the relative affinity of the antibody and antigen binding is measured using ELISA.

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

[0198] Polynucleotides, vectors and host cells

[0199] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention 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 a coding strand or a non-coding strand.

[0200] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0201] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0202] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. 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) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0203] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

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

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

[0206] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0207] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0208] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0209] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[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 separated 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, 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 invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, 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 a detectable product.

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

[0214] Pharmaceutical compositions and applications

[0215] 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 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 may vary with the properties of the formulated substance and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection. In the present invention, the term "pharmaceutical composition" means that the specific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert the therapeutic effect more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the specific antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation). 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 specific antibody of the present invention (or its conjugate) 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 match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the specific antibodies of the present invention can also be used with other therapeutic agents.

[0216] When using a pharmaceutical composition, a safe and effective amount of a specific antibody or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0217] Antibody-drug conjugates (ADCs)

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

[0219] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

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

[0221] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0222] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0223] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0224] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0225] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, drug-linkers can be used to form ADCs in a simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step method. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0226] Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other connection strategies, such as those described in Bioconjugation Technology, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.

[0227] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0228] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the 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 has the following molecular formula:

[0230]

[0231] in:

[0232] Ab is antibody,

[0233] LU is the connector;

[0234] D is for medicine;

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

[0236] Test Uses and Kits

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

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

[0239] Samples used in the present invention include fixed or preserved cell or tissue samples.

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

[0241] application

[0242] The present invention provides uses of the antibodies of the present invention, for example, for preparing diagnostic preparations, or preparing drugs for preventing and / or treating WAMT2-related diseases.

[0243] In a preferred embodiment, the WAMT2-related disease is a disease associated with WAMT2 overexpression or over-transport. In a preferred embodiment, the disease includes Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

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

[0245] (a) The anti-VMAT2 antibody of the present invention has good specificity and binding activity to human VMAT2, and has the effect of inhibiting the overexpression of VMAT2 or the overtransport of multiple monoamine neurotransmitters such as dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E) to synaptic vesicles for storage, thereby improving the disorder of the monoamine neurotransmitter system.

[0246] (b) The VMAT2 antibody of the present invention has cross-binding activity to human, rat, mouse and pig.

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

[0248] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0249] Example 1 Obtaining human VMAT2 protein simulating the native conformation

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

[0251] The expressed protein will be purified using 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. The purified VMAT2 protein will then be packaged using Amphipol reagent to maximize the simulation of the native conformation of the 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 was used to immunize mice according to conventional known methods, wherein the immunization strategy was as follows: 10-12 week old C57BL / 6 mice were selected to set up an immunization group and a control group, respectively. Each mouse in the immunization group was immunized with 30 μg of the VMAT2 protein (antigen) prepared in Example 1 and 20 μg of CpGOND2395 (5'-tcgtcgttttcggcgcgcgccg-3') (SEQ ID NO: 123) and an equal volume of AddaVax TM (InvivoGen) were injected intraperitoneally and in the tarsal joint. The control group was injected with 20 μg of CpGOND2395 and an equal volume of AddaVax TM , immunization once every 3-4 days, 7 times in total.

[0254] Mouse B cell sorting: The experimental and control mice were killed 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 fossa and inguinal), and then red blood cells were removed with red blood cell lysis buffer (Sangon Biotech Cat. b541001-0100). After washing twice with 2% FBS in PBS (FACS buffer), the cells were ready for staining, and then the Cytoflex SRT (Beckman Coulter) cell sorter was used to sort the target cells. The staining gate strategy was as follows:

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

[0256] The reagents used are as follows:

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

[0258] anti-mouseCD19(APC / Cyanine7;Biolegend Cat.115530),

[0259] anti-mouseCD38(PE / Cyanine7; Biolegend Cat.102718),

[0260] anti-mouse / humanGL7Antigen(TandBcellActivationMarker), 488; BiolegendCat.144612);

[0261] anti-mouseIgG1 (APC; BiolegendCat. 406610).

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

[0263] The flow cytometry results are shown in the following figure. Figure 1 As shown. Figure 1 It can be seen that after adopting the immunization methods in the scheme, a large number of IgG1-positive memory B cells can be sorted out from the experimental group mice, 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 IgG1-positive memory B cells obtained by the above sorting were cultured. The culture method can be referred to Haniuda and Kitamura, (2019). Induced Germinal Center B Cell Culture System, Bio-protocol 9(4): e3163. DOI: 10.21769 / BioProtoc.3163, wherein the feeding medium is: RPMI-1640, (Gibco, Cat. 11875-093), 10% Fetal Bovine Serum (FBS, VivaCell, Cat. C04002-500), 55 μM 2-mercaptoethanol (ThermoFisher, Cat. 21985), 100 units / ml Penicillin (Biosharp), 100 μg / ml Streptomycin (Biosharp), 10 mM HEPES (ThermoFisher, Cat.15630-080), 1mM Sodium Pyruvate (ThermoFisher, Cat.11360-070) and 0.1mM MEMnonessential amino acid (ThermoFisher, Cat.11140-050). One day before sorting, feeder cells expressing CD40L and BAFF were seeded into 96-well plates, with 1000 cells per well. Single IgG1-positive B cells were sorted into each well, and 2-10ng / ml IL-4 was added to the feeder medium and cultured for two days. 4-10ng / ml IL-21 was added and cultured for 6-8 days. The medium was changed every day. The supernatant was collected on the 10th day and stored at 4°C. The 96-well plate cells were stored at -80°C for subsequent lysis to obtain RNA of single B cells.

[0265] Example 3 ELISA experiment and 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 overnight at 4°C humid conditions, discarded the coating buffer, added 100-120 μl of blocking buffer (4% BSA in 1×PBS) to the plate, incubated at room temperature for 2 hours, removed the blocking buffer, added 20-60 μl of the single cell culture supernatant collected in Example 2 to each well, incubated overnight at 4°C humid conditions, washed 3 times, added 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, SouthernBiotech, cat.1030-04), incubated at room temperature for 2 hours, washed 4 times, and added 20-30 ul of a colorimetric solution containing 4-nitrophenyl phosphate disodium hexahydrate (CSNpharm, Cat.CSN66207) to each well. OD405 was measured using MDSpectraMaxiD3.

[0267] The results are shown in Tables 1 to 4 below: Partial test data of ELISA test using 96-well plate.

[0268] Tables 1-4 show the 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-specific 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 Detection of IgG1 antibodies secreted by cells in plate 1

[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 Detection of IgG1 antibodies secreted by cells in plate 2

[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 test results showed that the supernatant of the screened IgG1+ positive B cell culture contained antibodies that specifically bound to the VMAT2 protein, and the color was different due to different antibody binding strengths or antibody concentrations in the culture supernatant. Further ELISA concentration gradient detection showed that the above antibodies all had antigen-antibody binding activity.

[0279] Example 4 Antibody Binding Detection to VMAT2 Antigen on Cell Surface (Antibody Detection in Single B Cell Culture Supernatant), Screening for Antibodies Binding to the Cytoplasmic Side of hVMAT2

[0280] HEK293 T cells were cultured at 1×10 7 The cells were inoculated in a 10 cm cell culture dish at a density of 10 cells and cultured in a carbon dioxide incubator (37°C, 5% CO2) for 12 hours. The cells were co-transfected with a plasmid expressing full-length human VAMT2 (the VAMT2 plasmid was constructed using conventional techniques in the art) and a pMax GFP plasmid using Lipofectamine 3000. After 24 hours, the cells were replaced with fresh culture medium and continued to be cultured. After 48 hours of transfection, the cells were treated with 2mM EDTA-PBS buffer, then washed with 2% PBS buffer containing FBS, and the single cell suspension was filtered and placed in a 96-well plate. The transfected cells were incubated with the supernatant of a single B cell culture for 1 hour, then washed twice with FACS, and the cells were co-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. DAPI-GFP+IgG1+ cells were analyzed using a CytoFLEX LX (Beckman Coulter) flow cytometer.

[0281] like Figure 4 As shown, after the ribosome synthesizes the VMAT2 polypeptide chain, it will be transferred to the endoplasmic reticulum and the Golgi apparatus for further synthesis, processing, and modification. Ultimately, in vivo, VMAT2 is localized on the synaptic vesicle membrane in the cell. In the antibody screening experiment, human VMAT2 (hVMAT2) was overexpressed on the surface of the HEK293T cell membrane. In this process, VMAT2 flipped to the extracellular region (i.e., the cell membrane surface region) in the vesicle membrane cavity side region, and the vesicle membrane cytoplasmic side after flipping was still the plasma membrane cytoplasmic side. Therefore, when initially screening antibodies, the present invention excludes antibodies that bind to cell surface VMAT2, and the remaining antibodies (i.e., anti-VMAT2 cytoplasmic side antibodies) that are positive for ELISA screening will be cloned and further verified.

[0282] In the ELISA experiment, VMAT2 protein is a complete protein, including the luminal side and the cytoplasmic side. However, in the ELISA experiment, VMAT2 protein is not on the cell membrane, and the luminal side and the cytoplasmic side of the protein are exposed. When the VMAT2 protein encounters antibodies, both the antibodies bound to the two sides can be detected. In the cell flow cytometry experiment, VAMT2 is expressed on the cell membrane, so VMAT2 has a luminal side and a cytoplasmic side. At this time, the test can only detect antibodies bound to the cell surface, and antibodies bound to the cytoplasmic side cannot be detected. Therefore, it is believed that the ELISA-positive samples are ELISA-positive samples except for the samples bound to the surface of overexpressed cells, and the remaining ELISA-positive samples are the samples of antibodies bound to the cytoplasmic side.

[0283] Specifically, the flow results are as follows Figure 5-6 As shown. Through the cell surface binding assay, it is shown that the mouse monoclonal antibody binds to the hVMAT2 overexpressed on the cell surface, which corresponds to the hVMAT2 in the protruding vesicles, that is, it binds to the vesicle cavity side. This type of antibody will be excluded in the screening.

[0284] Further screening was performed for antibodies that bind to the cytoplasm of hVMAT2. Therefore, the antibodies that were finally screened for antigen-antibody binding positive in this embodiment were antibodies that bind to the cytoplasm of hVMAT2. The antibodies to the cytoplasm of hVMAT2 can be used as both marker antibodies for VMAT2 antigen and functional antibodies that inhibit 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 and incubated overnight at 4°C in a humidified environment. After discarding the coating buffer, 100-120 μl of blocking buffer (4% BSA in1×PBS), incubated at room temperature for 2 hours, after removing the blocking buffer, the single cell culture supernatant showing antigen-antibody binding in both Example 3 and Example 4 was added to each well, the single cell culture supernatant was gradiently diluted, i.e. 4 times, 16 times, 64 times, and 256 times, and 20-60 μl of the single cell culture supernatant dilution collected on the 10th day in Example 2 was added to each well, incubated overnight under humid conditions at 4°C, washed 3 times, and then 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, SouthernBiotech, cat.1030-04) was added, incubated at room temperature for 2 hours, washed 4 times, and then 20-30 μl of a color developing solution containing 4-nitrophenyl phosphate disodium hexahydrate (CSNpharm, Cat.CSN66207) was added to each well.

[0287] The OD405 results were measured using MDSpectraMaxiD3. Figure 2-3As shown in the figure, each sample in the figure shows strong antigen-antibody binding activity.

[0288] Example 6 Molecular cloning method and obtaining monoclonal antibody VDJ / VJ sequence

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

[0290] Total RNA of B cells in 96-well cell culture plates that were tested positive (antigen-antibody binding) was extracted using 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) at the following program: 42°C, 5 minutes, 25°C, 10 minutes, 50°C, 60 minutes and 94°C, and then two rounds of semi-nested PCR were performed using HotStar DNA polymerase (Qiagen) to enrich heavy and light chains, and the PCR products were purified and sequenced. The sequencing results were analyzed using the IgBlast tool and the IMGT database. The VDJ / VJ segments were amplified using gene-specific primers (Table 5), and the VDJ segment heavy chain and VJ segment light chain vectors were cloned separately 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 was: 95°C for 15 min, 95°C for 30 sec, 50-65°C for 30 sec, and 72°C for 5 min.

[0294] Table 6 Semi-nested PCR primers

[0295]

[0296] The VDJ / VJ specific primers in this embodiment include the homology arm portion of the heavy / light chain vector and the specific portion of the VDJ / VJ fragment. The specific primers will use different homology arms depending on the cloning vector used. This application is not limited to the specific primers used in the embodiment.

[0297] The nucleotide and amino acid sequences of the VDJ / VJ variable regions of the antibodies were sequenced after reverse transcription and two rounds of semi-nested PCR and 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 (expressing Fab) with strep II tags and His tags, as well as the human full-length constant region heavy and light chain vectors (expressing human IgG1) and scFV vectors were constructed respectively. Finally, the antibody plasmid was extracted, the antibody was expressed in Expi293F suspension cells, and the full-length antibody was separated and purified by rProtein A Beads. Fab was obtained by separation and purification with strep II tag agarose resin. In the present invention, Fab vectors, full-length human IgG1 heavy and light chain vectors, and scFV vectors were used to prepare VMAT2 antibodies. The maps of each vector are shown in the figure. Fig. 7A -B.

[0306] Example 7 Antigen-antibody binding experiment

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

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

[0309] Example 8 SPR experiment and results and analysis

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

[0311] The results are as follows Fig. 9 As shown, through the above SPR antigen-antibody binding affinity test, the antibody binding and dissociation constants are shown in Table 8, and the screened antibodies all have 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 Validation of binding of Fab antibodies to VMAT2 and VMAT1 of 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] HEK293 T cells were cultured at 1×10 7The cells were inoculated in a 10 cm cell culture dish at a density of 10 cells, cultured in a carbon dioxide incubator (37°C, 5% CO2) for 12 hours, and co-transfected with Lipofectamine3000 and plasmids expressing hVMAT2 (human VMAT2), rVMAT2 (rat VMAT2), rVMAT1 (rat VMAT1), mVMAT1 (mouse VMAT1), pVMAT2 (pig VMAT2), pVMAT1 (pig VMAT1) and pMax GFP plasmid, respectively, according to conventional techniques in the art. After 24 hours, fresh culture medium was replaced to continue culturing the cells. After 48 hours of transfection, the cells were separated in PBS with 2 mM EDTA, then washed with PBS, and the single cell suspension was filtered and placed in a 96-well plate. The transfected cells were incubated with live / dead eflour450 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 then the permeation buffer (Thermo Fisher scientific; cat. no.; 00-8333-56) was added. After centrifugation and the supernatant was removed, Fab antibody was added and incubated for 1 hour, and then anti-human Ig light chainκAntibody (Invitrogen, Cat. 2806744) was added and incubated for 15 minutes. All staining processes were completed on ice. DAPI-GFP+Igκ+ cells were analyzed using CytoFLEX LX (Beckman Coulter) flow cytometer. The detection principle is the same as in Example 4.

[0318] The results are as follows Fig.10 As shown, after the above-mentioned different antibodies and VMAT2 and VMAT1 of different species were verified to bind, it was found that when the antibody concentration was 10 μg / ml, JPF1026, JPF1217, JPF1254, JPF1258, JPF0514, JPF0568, JPF0503, JPF1212, JPF1039, and JPF1206 could bind to hVMAT2. JPF1026, JPF1217, JPF1258, JPF0568, JPF1212, and JPF1206 could bind to VMAT1 and VMAT2 of all tested species, JPF1039 only bound to hVMAT2, and JPF1254 only bound to VMAT2 of different species.

[0319] Example 10 Cell transport inhibition experiment (experiment to verify the inhibitory effect of antibodies at the cell level)

[0320] The hVMAT2 sequence was cloned into the pCAG vector. A 24-well plate was coated with poly-D-lysine in advance, and HEK293 T cells were inoculated in the 24-well plate at a density of 2×10 5 Cells were cultured at 37°C, 5% CO2 for 12 hours. hVMAT2 and ScFv plasmids (expressed as JPS) were co-transfected with Lipofectamine 3000. After 48 hours of transfection, cells were washed with PBS. Then, cells were incubated in 37°C buffer (125mM sodium gluconate, 4.8mM potassium gluconate, 1.2mM NaH2PO4, 1.2mM MgSO4, 1.3mM CaCl2, 5.6mM glucose and 25mM HEPES pH 7.4) for 15 minutes, and then incubated with 2μM FFN206 (Abcam) and 2μM calcein-AM (enzyme) for 1 hour at 37°C in the dark. Fluorescence was detected using EnSight (PerkinElmer) after washing twice with chilled PBS.

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

[0322] The results are as follows Fig.11 As shown, the results of the cell transport inhibition experiment showed that 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 the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings 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 claims attached to this application.

Claims

1. An anti-VMAT2 antibody or an antigen-binding fragment thereof, characterized in that: The anti-VMAT2 antibody or antigen-binding fragment thereof has three complementarity determining CDRs (HCDRs) of the heavy chain variable region and three complementarity determining regions CDRs (LCDRs) of the light chain variable region selected from the group consisting of: (A) three HCDRs and three LCDRs selected from Group A: (a1) HCDR1 shown in SEQ ID NO: 48, HCDR2 shown in SEQ ID NO: 49, HCDR3 shown in SEQ ID NO: 50, LCDR1 shown in SEQ ID NO: 52, LCDR2 shown in SEQ ID NO: 53, LCDR3 shown in SEQ ID NO: 54; (a2) HCDR1 shown in SEQ ID NO: 48, HCDR2 shown in SEQ ID NO: 58, HCDR3 shown in SEQ ID NO: 50, LCDR1 shown in SEQ ID NO: 52, LCDR2 shown in SEQ ID NO: 53, LCDR3 shown in SEQ ID NO: 54; (a3) HCDR1 shown in SEQ ID NO: 48, HCDR2 shown in SEQ ID NO: 63, HCDR3 shown in SEQ ID NO: 50, LCDR1 shown in SEQ ID NO: 52, LCDR2 shown in SEQ ID NO: 53, LCDR3 shown in SEQ ID NO: 54; (B) three HCDRs and three LCDRs selected from Group B: (b1) HCDR1 shown in SEQ ID NO: 67, HCDR2 shown in SEQ ID NO: 68, HCDR3 shown in SEQ ID NO: 69, LCDR1 shown in SEQ ID NO: 71, LCDR2 shown in SEQ ID NO: 72, LCDR3 shown in SEQ ID NO: 73; (b2) HCDR1 shown in SEQ ID NO: 67, HCDR2 shown in SEQ ID NO: 68, HCDR3 shown in SEQ ID NO: 77, LCDR1 shown in SEQ ID NO: 71, LCDR2 shown in SEQ ID NO: 72, LCDR3 shown in SEQ ID NO: 73; (C) three HCDRs and three LCDRs selected from Group C: (c1) HCDR1 shown in SEQ ID NO: 82, HCDR2 shown in SEQ ID NO: 83, HCDR3 shown in SEQ ID NO: 84, LCDR1 shown in SEQ ID NO: 86, LCDR2 shown in SEQ ID NO: 87, LCDR3 shown in SEQ ID NO: 88; (c2) HCDR1 shown in SEQ ID NO: 82, HCDR2 shown in SEQ ID NO:92, HCDR3 shown in SEQ ID NO:93, LCDR1 shown in SEQ ID NO: 95, LCDR2 shown in SEQ ID NO: 96, LCDR3 shown in SEQ ID NO: 97; (D) three HCDRs and three LCDRs selected from Group D: (d1) HCDR1 shown in SEQ ID NO: 101, HCDR2 shown in SEQ ID NO: 102, HCDR3 shown in SEQ ID NO: 103, LCDR1 shown in SEQ ID NO: 71, LCDR2 shown in SEQ ID NO: 72, LCDR3 shown in SEQ ID NO: 105; (d2) HCDR1 shown in SEQ ID NO: 109, HCDR2 shown in SEQ ID NO: 110, HCDR3 shown in SEQ ID NO: 111, LCDR1 shown in SEQ ID NO: 71, LCDR2 shown in SEQ ID NO: 72, LCDR3 shown in SEQ ID NO: 105; (d3) HCDR1 shown in SEQ ID NO: 116, HCDR2 shown in SEQ ID NO: 117, HCDR3 shown in SEQ ID NO: 118, LCDR1 shown in SEQ ID NO: 120, LCDR2 shown in SEQ ID NO: 72, LCDR3 shown in SEQ ID NO:

105.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-VMAT2 antibody or antigen-binding fragment thereof is an anti-VMAT2 protein cytoplasmic antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region and light chain variable region of the anti-VMAT2 antibody or antigen-binding fragment thereof are selected from the following group: (a) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 47, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 51; (b) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 57, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 59; (c) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 62, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 51; (d) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 66, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 70; (e) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 76, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 78; (f) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 81, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 85; (g) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:91, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:94; (h) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 100, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 104; (i) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 108, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 112; (j) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 115, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

119.

4. A recombinant protein, characterized in that The recombinant protein has: (i) the anti-VMAT2 antibody or antigen-binding fragment thereof according to claim 1; and (ii) optionally a tag sequence to facilitate expression and / or purification.

5. A nucleotide molecule, characterized in that The nucleotide molecule encodes the anti-VMAT2 antibody or antigen-binding fragment thereof according to claim 1.

6. A carrier, characterized in that The vector contains the nucleotide molecule according to claim 5.

7. A host cell, characterized in that The host cell contains the vector according to claim 6, or the nucleotide molecule according to claim 5 is integrated into its genome.

8. An antibody-drug conjugate, characterized in that: The antibody drug conjugate contains: (a) an antibody portion, the antibody portion comprising the anti-VMAT2 antibody or antigen-binding fragment thereof of claim 1; and (b) a conjugated moiety conjugated to the antibody or antigen-binding fragment thereof, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 4, the nucleotide molecule according to claim 5, the vector according to claim 6, or the host cell according to claim 7, or the antibody-drug conjugate according to claim 8; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

10. The antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 4, or the antibody-drug conjugate according to claim 8, wherein: For preparing pharmaceutical agents, reagents, test plates or test kits; The reagent, detection plate or kit is used for: detecting VMAT2 protein in a sample; The medicament is used for treating or preventing diseases caused by overexpression of VMAT2 protein.

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