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

[0001] This case is a divisional application of Chinese invention patent application filed on December 6, 2024, with application number CN 202411791297.8 and invention title "Antibody to VMAT2 or antigen-binding fragment thereof and composition thereof and application thereof". Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to antibodies against VMAT2 or their antigen-binding fragments, compositions thereof, and applications. Background Technology

[0003] Vesicular monoamine transporter 2 (VMAT2) is a multi-transmembrane protein located on the synaptic vesicle membrane of neurons. It transports various monoamine neurotransmitters, including dopamine (DA), serotonin (5-HT), norepinephrine (NE), and epinephrine (E), to synaptic vesicles for storage. Upon external stimulation, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, where they bind to their corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signal transduction. VMAT2 participates in the regulation of numerous functions, including movement, mood, sleep, reward, attention, and addiction.

[0004] Overexpression or overtransportation of VMAT2 can lead to disorders of the monoamine neurotransmitter system, thereby inducing Huntington's disease, addictive disorders, tardive dyskinesia, hypertension, and other conditions. To date, there is a severe lack of available treatments for these diseases.

[0005] Currently, small molecule inhibitors targeting VMAT2, such as reserpine, tetrabenazine, deutetrabenazine, valbenazine, and GZ-793A, are mostly used to treat Huntington's disease, tardive dyskinesia, and hypertension.

[0006] However, these inhibitors have drawbacks such as short half-life, low target specificity, and a tendency to cause adverse reactions.

[0007] Therefore, there is an urgent need in this field to develop new VMAT2 drugs with long half-lives and high specificity to make up for the shortcomings of small molecule drugs. Summary of the Invention

[0008] This invention provides a novel VMAT2 drug with a long half-life and high specificity.

[0009] In a first aspect of the invention, an anti-VMAT2 antibody or an antigen-binding fragment thereof is provided, said anti-VMAT2 antibody or antigen-binding fragment having three complementarity-determining CDRs (HCDRs) of the heavy chain variable region and three complementarity-determining CDRs (LCDRs) of the light chain variable region selected from the group consisting of:

[0010] (A) Selected from three HCDRs and three LCDRs of group A:

[0011] (a1)HCDR1 shown in SEQ ID NO: 48,

[0012] HCDR2, as shown in SEQ ID NO: 49,

[0013] HCDR3, as shown in SEQ ID NO: 50,

[0014] LCDR1 shown in SEQ ID NO: 52,

[0015] The LCDR2 shown in SEQ ID NO: 53,

[0016] LCDR3 as shown in SEQ ID NO: 54;

[0017] (a2)HCDR1 shown in SEQ ID NO: 48,

[0018] HCDR2, as shown in SEQ ID NO: 58,

[0019] HCDR3, as shown in SEQ ID NO: 50,

[0020] LCDR1 shown in SEQ ID NO: 52

[0021] The LCDR2 shown in SEQ ID NO: 53,

[0022] LCDR3 as shown in SEQ ID NO: 54;

[0023] (a3)HCDR1 shown in SEQ ID NO: 48,

[0024] HCDR2, as shown in SEQ ID NO: 63,

[0025] HCDR3, as shown in SEQ ID NO: 50,

[0026] LCDR1 shown in SEQ ID NO: 52

[0027] The LCDR2 shown in SEQ ID NO: 53,

[0028] LCDR3 as shown in SEQ ID NO: 54;

[0029] (B) Selected from three HCDRs and three LCDRs of group B:

[0030] (b1)HCDR1 shown in SEQ ID NO: 67,

[0031] HCDR2, as shown in SEQ ID NO: 68,

[0032] HCDR3, as shown in SEQ ID NO: 69,

[0033] LCDR1 shown in SEQ ID NO: 71

[0034] LCDR2 shown in SEQ ID NO: 72

[0035] LCDR3 as shown in SEQ ID NO: 73;

[0036] (b2)HCDR1 shown in SEQ ID NO: 67,

[0037] HCDR2, as shown in SEQ ID NO: 68,

[0038] HCDR3, as shown in SEQ ID NO: 77,

[0039] LCDR1 shown in SEQ ID NO: 71

[0040] LCDR2 shown in SEQ ID NO: 72

[0041] LCDR3 as shown in SEQ ID NO: 73;

[0042] (C) Selected from three HCDRs and three LCDRs of group C:

[0043] (c1)HCDR1 shown in SEQ ID NO: 82,

[0044] HCDR2, as shown in SEQ ID NO: 83,

[0045] HCDR3, as shown in SEQ ID NO: 84,

[0046] LCDR1 shown in SEQ ID NO: 86

[0047] LCDR2 shown in SEQ ID NO: 87

[0048] LCDR3 as shown in SEQ ID NO: 88;

[0049] (c2)HCDR1 shown in SEQ ID NO: 82,

[0050] HCDR2, as shown in SEQ ID NO: 92,

[0051] HCDR3, as shown in SEQ ID NO: 93,

[0052] LCDR1 shown in SEQ ID NO: 95

[0053] LCDR2 shown in SEQ ID NO: 96

[0054] LCDR3 as shown in SEQ ID NO: 97;

[0055] (D) Selected from three HCDRs and three LCDRs of group D:

[0056] (d1)HCDR1 shown in SEQ ID NO: 101,

[0057] HCDR2, as shown in SEQ ID NO: 102,

[0058] HCDR3, as shown in SEQ ID NO: 103,

[0059] LCDR1 shown in SEQ ID NO: 71

[0060] LCDR2 shown in SEQ ID NO: 72

[0061] LCDR3 as shown in SEQ ID NO: 105;

[0062] (d2)HCDR1 shown in SEQ ID NO: 109,

[0063] HCDR2, as shown in SEQ ID NO: 110,

[0064] HCDR3, as shown in SEQ ID NO: 111,

[0065] LCDR1 shown in SEQ ID NO: 71

[0066] LCDR2 shown in SEQ ID NO: 72

[0067] LCDR3 as shown in SEQ ID NO: 105;

[0068] (d3)HCDR1 shown in SEQ ID NO: 116

[0069] HCDR2, as shown in SEQ ID NO: 117,

[0070] HCDR3, as shown in SEQ ID NO: 118,

[0071] LCDR1 shown in SEQ ID NO: 120

[0072] LCDR2 shown in SEQ ID NO: 72

[0073] LCDR3 as shown in SEQ ID NO: 105.

[0074] In another preferred embodiment, the anti-VMAT2 antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment on the cytoplasmic side of the anti-VMAT2 protein.

[0075] In another preferred embodiment, the anti-VMAT2 antibody or its antigen-binding fragment is an antibody against the cytoplasmic VMAT2 protein or its antigen-binding fragment.

[0076] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0077] In another preferred embodiment, the antigen-binding fragment includes the Fab fragment, the F(ab')2 fragment, and the Fv fragment.

[0078] In another preferred embodiment, the heavy chain variable region and light chain variable region of the anti-VMAT2 antibody or its antigen-binding fragment are selected from the group consisting of:

[0079] (a) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 47, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 51;

[0080] (b) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 57, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 59;

[0081] (c) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 62, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 51;

[0082] (d) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 66, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 70;

[0083] (e) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 76, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 78;

[0084] (f) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 81, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 85;

[0085] (g) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 94;

[0086] (h) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 100, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 104;

[0087] (i) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 108, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 112;

[0088] (j) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO: 115, and the light chain variable region with amino acid sequence as shown in SEQ ID NO: 119.

[0089] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0090] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0091] In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

[0092] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0093] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0094] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0095] In another preferred embodiment, the antibody is a monoclonal antibody.

[0096] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0097] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0098] In a second aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0099] (i) the anti-VMAT2 antibody or its antigen-binding fragment as described in the first aspect of the present invention; and

[0100] (ii) Tag sequences that optionally assist in expression and / or purification.

[0101] In another preferred embodiment, the label includes an Fc label, a FLAG label, a 6His label, or a combination thereof.

[0102] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0103] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0104] In a third aspect of the invention, a nucleotide molecule is provided that encodes the anti-VMAT2 antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0105] In another preferred embodiment, the nucleotide molecule is DNA, RNA, or cDNA.

[0106] In a fourth aspect of the invention, a carrier is provided, the carrier containing the nucleotide molecules described in the third aspect of the invention.

[0107] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0108] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0109] In a fifth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fourth aspect of the invention, or having nucleotide molecules described in the third aspect of the invention integrated into its genome.

[0110] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0111] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0112] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0113] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0114] In another preferred embodiment, the cell is an immune cell, and its surface simultaneously expresses a chimeric antigen receptor.

[0115] In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.

[0116] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0117] In another preferred embodiment, the chimeric antigen receptor is an anti-VMAT2 antibody or its antigen-binding fragment.

[0118] In a sixth aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising:

[0119] (a) An antibody portion comprising the anti-VMAT2 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0120] (b) A coupling portion conjugated to the antibody or its antigen-binding fragment, the coupling portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0121] In another preferred embodiment, the drug is a drug that inhibits the excessive transport of monoamine neurotransmitters.

[0122] In another preferred embodiment, the monoamine neurotransmitters include: dopamine (DA), serotonin (5-HT), norepinephrine (NE), and epinephrine (E).

[0123] In another preferred embodiment, the expression for the antibody-drug conjugate is: mAb-(XY)n;

[0124] in,

[0125] mAb is the anti-VMAT2 antibody or its antigen-binding fragment;

[0126] X is a connector;

[0127] Y represents the coupling portion, which is a drug;

[0128] n is a positive integer ≤ 8;

[0129] The conjugation portion is conjugated to the anti-VMAT2 antibody or its antigen-binding fragment via a linker.

[0130] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0131] (i) 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, a nucleotide molecule as described in the third aspect of the present invention, a vector as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, or an antibody-drug conjugate as described in the sixth aspect of the present invention; and

[0132] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0133] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0134] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating diseases caused by VMAT2 overexpression or overtransportation.

[0135] In another preferred embodiment, the disease is selected from the group consisting of Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0136] 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.

[0137] The reagents, detection plates, or kits are used to detect VMAT2 protein in samples.

[0138] The agent is used to treat or prevent diseases that overexpress the VMAT2 protein.

[0139] In another preferred embodiment, the VMAT2 protein is the intracellular segment of the VMAT2 protein.

[0140] In another preferred embodiment, the VMAT2 protein is the VMAT2 cytoplasmic side protein.

[0141] 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:

[0142] (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;

[0143] (b) Isolate and purify the anti-VMAT2 antibody or its antigen-binding fragment described in (a).

[0144] 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:

[0145] (S1) Contact the sample with an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0146] (S2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of VMAT2 protein in the sample.

[0147] In another preferred embodiment, the sample includes: human or animal tissue samples, or exfoliated cell samples.

[0148] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0149] In another preferred embodiment, the method is an in vitro method.

[0150] In another preferred embodiment, the method further includes step (3) analyzing the affinity between the antibody and the antigen.

[0151] In an eleventh aspect of the present invention, a detection plate is provided, the detection plate comprising a substrate (support plate) and a test strip, the test strip containing 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.

[0152] In another preferred embodiment, the test strip also contains an antigen spotting area.

[0153] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper stacked in sequence.

[0154] In a twelfth aspect of the present invention, a kit is provided, the kit comprising:

[0155] (1) A first container containing an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and / or

[0156] (2) A second container containing a secondary antibody against the antibody or antigen-binding fragment thereof as described in the first aspect of the invention; and / or

[0157] (3) A third container containing a cell lysis reagent;

[0158] or,

[0159] The kit contains a detection plate as described in the eleventh aspect of the present invention.

[0160] In another preferred embodiment, the antibody in the first container is labeled with a detectable tag.

[0161] In another preferred embodiment, the antibody in the second container is labeled with a detectable tag.

[0162] In a thirteenth aspect of the invention, a method for treating a disease associated with VMAT2 overexpression or overtransportation is provided, comprising the steps of administering to a subject requiring treatment a therapeutically effective amount of the anti-VMAT2 antibody or its antigen-binding fragment as described in the first aspect of the invention, the recombinant protein as described in the second aspect of the invention, the nucleotide molecule as described in the third aspect of the invention, the expression vector as described in the fourth aspect of the invention, the antibody-drug conjugate as described in the sixth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0163] In another preferred example, diseases associated with VMAT2 overexpression or overtransport are selected from the group consisting of Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0164] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0165] Figure 1 The results show the sorting of mouse IgG1-positive memory B cells by flow cytometry after immunization and the sorting of mouse IgG1-positive memory B cells by control group.

[0166] Figure 2 The results of gradient ELISA antigen-antibody binding assays using the culture supernatant of the positive wells from the ELISA screening of antibodies targeting VMAT2 in Example 3 are shown.

[0167] Figure 3 The results of gradient ELISA antigen-antibody binding assays using the culture supernatant of the positive wells from the ELISA screening of antibodies targeting VMAT2 in Example 3 are shown.

[0168] Figure 4 This diagram illustrates the expression and transport of the VMAT2 protein within cells.

[0169] Figure 5 The results of supernatant culture of a single B cell containing hVMAT2 on the cell surface are shown.

[0170] Figure 6 The results of the supernatant from a single B cell culture without hVMAT2 binding to the cell surface are shown.

[0171] Figure 7A -B shows the heavy chain vector for human IgG1 CH1 used to construct the antibody; the light chain vector for human IgK used to construct the antibody; the heavy chain vector for the human IgG1 constant region used to construct the antibody; and the ScFv vector.

[0172] Figure 8 The results of the cell flow cytometry antigen-antibody binding experiment in Example 7 of the present invention are shown.

[0173] Figure 9 The results of SPR antigen-antibody binding affinity detection in Example 8 of the present invention are shown.

[0174] Figure 10 The results of the binding verification of Fab antibody with VMAT2 and VMAT1 of different species in Example 9 of the present invention are shown.

[0175] Figure 11 The experimental results of antibody inhibition of cell transport in Example 10 of the present invention are shown. Detailed Implementation

[0176] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly obtained a class of anti-VMAT2 antibodies. Experimental results show that the anti-VMAT2 antibodies of this invention possess high affinity and good biological activity. Furthermore, the antibodies of this invention exhibit cross-binding reactions with human VMAT2, mouse VMAT2, and porcine VMAT2, and can also bind to VMAT1. The antibodies of this invention can significantly inhibit VMAT2 transport function. Based on these findings, this invention was completed.

[0177] the term

[0178] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention 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 terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0179] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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.).

[0180] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0181] 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 reactions), i.e., a reasonable benefit / risk ratio.

[0182] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic 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 whether it is used in combination with other drugs.

[0183] VMAT2

[0184] Vesicular monoamine transporter 2 (VMAT2) is a multi-transmembrane protein located on the synaptic vesicle membrane of neurons. It transports various monoamine neurotransmitters, including dopamine (DA), serotonin (5-HT), norepinephrine (NE), and epinephrine (E), to synaptic vesicles for storage. Upon external stimulation, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, where they bind to their corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signal transduction. VMAT2 participates in the regulation of numerous functions, including movement, mood, sleep, reward, attention, and addiction.

[0185] Existing antibodies targeting the intracellular region of VMAT2 only exhibit binding activity to the intracellular region, without any transport inhibitory activity. However, the antibody targeting the intracellular region of VMAT2 obtained in this invention not only has binding activity to the VMAT2 intracellular region but also exhibits good transport inhibitory activity.

[0186] Antibody

[0187] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0188] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0189] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain, typically linked by a short peptide (linker) of 15 to 25 amino acids.

[0190] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0191] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has 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 be represented as (L-FR1)-(LCDR1)-(L-FR2)-(LCDR2)-(L-FR3)-(LCDR3)-(L-FR4), and the heavy chain variable domain 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, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0192] Knowing the amino acid sequence of the CDR, those 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.

[0193] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0194] As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the light and heavy chains to fold into one or more amino acid residues of an exchangeable dual variable region immunoglobulin. In this invention, preferred linkers are Linker1 and Linker2, wherein Linker1 links the VH and VL of a single-chain antibody (scFv), while Linker2 is used to link the scFv to the heavy chain of another antibody.

[0195] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0196] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the specific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0197] Table A

[0198]

[0199]

[0200] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0201] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0202] Polynucleotides, vectors and host cells

[0203] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0204] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0205] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0206] The present invention also relates to polynucleotides that hybridize with the above-described 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 hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0207] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0208] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0209] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0210] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0211] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0212] 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*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0213] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0214] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0215] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0216] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0217] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0218] Pharmaceutical Compositions and Applications

[0219] This invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In this invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition in which the specific antibody of this invention can be combined with a pharmaceutically acceptable carrier to exert its therapeutic effect more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the specific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described specific antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. 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. Furthermore, the specific antibody of the present invention can also be used with other therapeutic agents.

[0220] When using a pharmaceutical composition, a safe and effective amount of the specific antibody or its immunoconjugate is administered to a mammal. This safe and effective amount is typically 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 between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0221] Antibody-drug conjugates (ADCs)

[0222] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0223] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0224] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0225] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0226] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., 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 (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0227] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl 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, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0228] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0229] 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 this invention, the drug-linker can be used to form an ADC in a single, simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in two or more steps. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in subsequent steps, functional groups on the linker react with the drug to form an ADC.

[0230] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable 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 via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (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 linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0231] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0232] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0233] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0234]

[0235] in:

[0236] Ab is an antibody.

[0237] LU stands for connector;

[0238] D is a drug;

[0239] Furthermore, the subscript p is a value selected from 1 to 8.

[0240] Detection uses and kits

[0241] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0242] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0243] The samples used in this invention include fixed or preserved cell or tissue samples.

[0244] 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 includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0245] application

[0246] This invention provides the use of the antibody of this invention, for example, for the preparation of diagnostic agents, or for the preparation of drugs for the prevention and / or treatment of WAMT2-related diseases.

[0247] In a preferred embodiment, the WAMT2-related disease is a disease related to WAMT2 overexpression or overtransportation. In a preferred embodiment, the disease includes: Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.

[0248] The main advantages of this invention include:

[0249] (a) The anti-VMAT2 antibody of the present invention has good specificity and binding activity with human VMAT2, and can inhibit the overexpression or overtransport of various 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.

[0250] (b) The VMAT2 antibody of the present invention has cross-binding activity in humans, rats, mice and pigs.

[0251] (c) Compared with VMAT2 small molecule inhibitors, the antibody in this application has a longer half-life and higher specificity.

[0252] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0253] Example 1: Obtaining human VMAT2 protein with a natural conformation

[0254] In this example, conventional techniques in the field were used to construct the full-length human VMAT2 gene sequence (EBI: ENST00000644641.2) into a mammalian cell expression vector, such as the pCAG vector, and HEK293 cells were used for exogenous expression in order to obtain a large number of VMAT2 protein samples.

[0255] The expressed protein will be purified using conventional biochemical techniques in the field, and the purification conditions will be optimized to preserve the native conformation of the protein as much as possible. The purified VMAT2 protein will then be packaged using Amphipol reagent to maximize the mimicry of the native conformation of the VMAT2 protein on the cell membrane surface.

[0256] Example 2: Mouse immunization, mouse B cell sorting and culture

[0257] The VMAT2 protein obtained in Example 1 was used to immunize mice using conventional and known techniques. The immunization strategy was as follows: 10-12 week old C57BL / 6 mice were selected and divided into an immunization group and a control group. Each mouse in the immunization group was given 30 μg of 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) Intraperitoneal and tarsal joint injections were administered, while the control group received 20 μg of CpGOND2395 and an equal volume of AddaVax. TM Vaccinate once every 3-4 days, for a total of 7 times.

[0258] Sorting of mouse B cells: 3-7 days after the last immunization, mice in both the experimental and control groups were sacrificed, and spleen and lymph node tissues were obtained. Single-cell suspensions were prepared from the spleen and lymph nodes (popliteal fossa and groin), and then red blood cells were removed using erythrocyte lysis buffer (Sangon Biotech Cat.b541001-0100). After washing twice with 2% FBS in PBS (FACS buffer), the cells were prepared for staining. A Cytoflex SRT (Beckman-Coulter) cell sorting system was then used to sort the cells to obtain the target cells. The staining gating strategy was as follows:

[0259] DAPI-CD19+CD38+GL7-IgG1+;

[0260] The following reagents were used:

[0261] DAPI (live / dead; Invitrogen Cat.D1306),

[0262] anti-mouseCD19(APC / Cyanine7;Biolegend Cat.115530),

[0263] anti-mouseCD38(PE / Cyanine7; Biolegend Cat.102718),

[0264] anti-mouse / humanGL7Antigen(TandBcellActivationMarker), 488; Biolegend (Cat. 144612);

[0265] anti-mouseIgG1 (APC; BiolegendCat. 406610).

[0266] Following immunization, mouse IgG1-positive memory B cells were sorted by flow cytometry.

[0267] Example results of flow sorting are as follows Figure 1 As shown. By Figure 1 It can be seen that after using the immunization methods in the protocol, a large number of IgG1 positive memory B cells can be isolated from the experimental group mice, and the proportion of IgG1 positive memory B cells to total B cells is higher. The proportion of IgG1 positive memory B cells in the control group is much lower than that in the experimental group.

[0268] Culture of mouse B cells: The IgG1-positive memory B cells obtained above were cultured. The culture method can be referred to Hanida and Kitamura, (2019). Induced Germinal Center B Cell Culture System, Bio-protocol 9(4): e3163. DOI: 10.21769 / BioProtoc.3163. The feeding medium was: RPMI-1640 (Gibco, Cat. 11875-093), 10% Fetal Bovine Serum (FBS, Viva Cell, Cat. C04002-500), 55 μM 2-mercaptoethanol (Thermo Fisher, Cat. 21985), 100 units / ml Penicillin (Biosharp), 100 μg / 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) were used. One day before sorting, feeder cells expressing CD40L and BAFF were seeded into 96-well plates at 1000 cells per well. Single IgG1-positive B cells were sorted into each well, and 2-10 ng / ml IL-4 was added to the feeder medium and cultured for two days. Then, 4-10 ng / ml IL-21 was added and cultured for another 6-8 days, changing the medium daily. On day 10, the supernatant was collected and stored at 4°C. The 96-well plates were stored at -80°C for subsequent lysis to obtain RNA from single B cells.

[0269] Example 3: ELISA experiment and results analysis for screening antibodies targeting VMAT2

[0270] The VMAT2 protein obtained in Example 1 was coated onto a 96-well ELISA plate and incubated overnight at 4°C under humid conditions. After discarding the coating buffer, 100-120 μl of blocking buffer (4% BSA in 1×PBS) was added to the plate and incubated at room temperature for 2 hours. After removing the blocking buffer, 20-60 μl of single-cell culture supernatant collected in Example 2 was added to each well and incubated overnight at 4°C under humid conditions. After washing three times, 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat. 1030-04) was added to each well and incubated at room temperature for 2 hours. After washing four times, 20-30 μl of chromogenic solution containing disodium 4-nitrophenyl phosphate hexahydrate (CSNpharm, Cat. CSN66207) was added to each well. OD405 was measured using an MDSpectraMaxiD3.

[0271] The results are shown in Tables 1-4 below: Partial detection data of ELISA assay using 96-well plates.

[0272] Tables 1-4 show the detection of plate 1 antigen and plate 1 secreted IgG1 antibody, respectively; and the detection of plate 2 antigen and plate 2 secreted IgG1 antibody.

[0273] Table 1. Antigen Specificity Detection (Panel 1)

[0274] 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

[0275] Table 2. Detection of IgG1 antibodies secreted by Plate 1 cells.

[0276] 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

[0277] Table 3. Antigen Specificity Detection (Patent 2)

[0278] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.10 0.11 0.09 0.10 0.10 0.11 0.10 0.10 0.11 0.10 0.10 0.09 B 0.10 0.12 0.10 0.10 0.10 0.11 0.11 0.10 0.11 0.11 0.10 0.09 C 0.17 1.18 0.11 0.10 0.11 0.11 0.27 0.10 0.10 0.11 0.11 0.11 D 0.10 0.12 0.10 0.10 0.11 0.11 0.12 0.11 0.11 0.11 0.12 0.09 E 0.09 0.10 0.10 0.10 0.10 0.10 0.11 0.12 0.10 0.10 0.10 2.30 F 0.10 0.11 0.10 0.10 0.10 0.11 0.10 0.10 0.10 0.11 0.10 0.08 G 0.09 0.10 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.08 H 0.08 0.09 0.09 0.09 0.09 0.09 0.09 0.11 0.09 1.49 0.09 0.07

[0279] Table 4. Detection of IgG1 antibodies secreted by platelet-2 cells.

[0280] 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

[0281] ELISA results showed that the supernatant of cultured IgG1+ positive B cells contained antibodies that specifically bind to the VMAT2 protein, and the color development varied depending on the antibody binding affinity or concentration in the culture supernatant. Further ELISA concentration gradient assays showed that all of these antibodies possessed antigen-antibody binding activity.

[0282] Example 4: Detection of antibody binding to VMAT2 antigen on cell surface (antibody detection from single B cell culture supernatant), screening for antibodies binding to the cytoplasmic side of hVMAT2.

[0283] HEK293 T cells were used at a rate of 1×10 7 Cells were seeded at a density of 10 cm in 10 cm cell culture dishes and cultured in a CO2 incubator (37°C, 5% CO2) for 12 hours. Cells were then co-transfected with Lipofectamine 3000, a full-length human VAMT2 plasmid (constructed using standard techniques in the art), and a pMax GFP plasmid. After 24 hours, the culture medium was replaced with fresh medium, and the cells were cultured again. Forty-eight hours after transfection, cells were treated with 2 mM EDTA-PBS buffer, followed by washing with 2% FBS-containing PBS buffer. The single-cell suspension was filtered and placed in 96-well plates. The transfected cells were incubated with the supernatant from a single B cell culture for 1 hour, followed by washing twice with FACS. Cells were then co-incubated with DAPi (live / dead; Invitrogen Cat. D1306) and anti-mouse IgG1 (APC; Biolegend Cat. 406610) for 15 minutes. All staining procedures were performed on ice. DAPI-GFP+IgG1+ cells were analyzed using a CytoFLEX LX (Beckman Coulter) flow cytometer.

[0284] like Figure 4 As shown, after ribosomes synthesize the VMAT2 polypeptide chain, they transfer it to the endoplasmic reticulum and Golgi apparatus for further synthesis, processing, and modification. Ultimately, in vivo, VMAT2 is located on the synaptic vesicle membrane of cells. In antibody screening experiments, human VMAT2 (hVMAT2) is overexpressed on the surface of HEK293T cells. During this process, VMAT2 flips from the lumen-side region of the vesicle membrane to the extracellular region (i.e., the cell membrane surface region), while the cytoplasmic side of the vesicle membrane remains the plasma membrane cytoplasmic side after the flipping. Therefore, in the initial antibody screening, this invention excludes antibodies that bind to the cell surface VMAT2. The remaining antibodies that are positive by ELISA screening (i.e., anti-VMAT2 cytoplasmic side antibodies) will be cloned for further verification.

[0285] In ELISA assays, VMAT2 protein is an intact protein, including both its luminal and cytoplasmic sides. However, in ELISA, VMAT2 protein is not located on the cell membrane; both its luminal and cytoplasmic sides are exposed. When VMAT2 protein encounters antibodies, antibodies bound to both sides can be detected. In contrast, in flow cytometry, VMAT2 is expressed on the cell membrane, thus there are luminal and cytoplasmic sides. In this case, only antibodies bound to the cell surface can be detected; antibodies bound to the cytoplasmic side cannot be detected. Therefore, it is considered that ELISA-positive samples, excluding those bound to the overexpressing cell surface, represent samples with antibodies bound to the cytoplasmic side.

[0286] Specifically, the streaming results are as follows: Figure 5-6 As shown in the figure, cell surface binding assays revealed that murine monoclonal antibodies bind to hVMAT2 overexpressed on the cell surface, corresponding to hVMAT2 in the protruding vesicles, specifically binding to one side of the vesicle cavity. Such antibodies will be excluded during screening.

[0287] Further screening revealed antibodies binding to the cytoplasmic side of hVMAT2. Therefore, in this embodiment, the final antigen-antibody binding positive antibody was the one bound to the cytoplasmic side of hVMAT2. This cytoplasmic antibody of hVMAT2 can serve as both a marker antibody for the VMAT2 antigen and a functional antibody to inhibit its transport.

[0288] Example 5: ELISA gradient detection of single B cell culture supernatant

[0289] The VMAT2 protein obtained in Example 1 was coated onto a 96-well ELISA plate and incubated overnight at 4°C under humid conditions. After discarding the coating buffer, 100-120 μl of blocking buffer (4% BSA in 1×PBS) was added to the plate and incubated at room temperature for 2 hours. After removing the blocking buffer, single-cell culture supernatant, which showed antigen-antibody binding in both Examples 3 and 4, was added to each well. The single-cell culture supernatant was serially diluted (4-fold, 16-fold, 64-fold, and 256-fold) and added to each well. Add 20-60 μl of the diluted single-cell culture supernatant collected on day 10 in Example 2, incubate overnight at 4°C under humid conditions, wash 3 times, add 20-30 μl of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat. 1030-04), incubate at room temperature for 2 hours, wash 4 times, and add 20-30 μl of colorimetric solution containing 4-nitrophenyl phosphate disodium hexahydrate (CSNpharm, Cat. CSN66207) to each well.

[0290] OD405 was measured using MDSpectraMaxiD3, and the results are as follows: Figure 2-3 As shown in the figure, all samples exhibit strong antigen-antibody binding activity.

[0291] Example 6: Molecular cloning method and obtaining the monoclonal antibody VDJ / VJ sequence

[0292] Based on the analysis of the ELISA results from Examples 3 and 5 and the antigen-antibody cell surface binding detection results from Example 4, cells corresponding to the positive wells (antigen-antibody binding) of 96-well cell culture plates that were pre-frozen at -80°C were selected for RNA extraction, which was used for subsequent molecular cloning to obtain the VDJ / VJ sequence.

[0293] Total RNA was extracted from B cells in 96-well cell culture plates that tested positive (antigen-antibody binding) using TRIzolReagent (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). In short, cDNA synthesis was performed using Maxima H Minus reverse transcriptase (Thermo Fisher) at the following temperatures: 42°C, 5 min, 25°C, 10 min, 50°C, 60 min, and 94°C. Two rounds of semi-nested PCR were then performed using HotStar DNA polymerase (Qiagen) to enrich the heavy and light chains. The PCR products were purified and sequenced. Sequencing results were analyzed using IgBlast and the IMGT database. The VDJ / VJ fragment was amplified using gene-specific primers (Table 5), and the VDJ heavy chain and VJ light chain vectors were cloned using homologous recombination or T4 ligase ligation methods, respectively.

[0294] Table 5 Gene-specific primers for amplifying VDJ / VJ

[0295]

[0296]

[0297] The primers used for the two-round semi-nested PCR are shown in Table 6. The PCR program is as follows: 95℃ for 15 minutes, 95℃ for 30 seconds, 50-65℃ for 30 seconds, and 72℃ for 5 minutes.

[0298] Table 6. Primers for semi-nested PCR

[0299]

[0300] The VDJ / VJ-specific primers in this embodiment include homologous arms of the heavy / light chain vectors, as well as specific portions of the VDJ / VJ fragments. Generally, the specific primers will use different homologous arms depending on the cloning vector used. This application is not limited to the specific primers used in the embodiments.

[0301] The nucleotide and amino acid sequences of the VDJ / VJ variable region of the antibody were obtained after reverse transcription and two rounds of semi-nested PCR, as shown in Table 7.

[0302] Table 7

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Human heavy chain constant region CH1 vector and light chain vector (expressing Fab) with strep II and His tags, as well as human full-length constant region heavy and light chain vector (expressing human IgG1) and SCFV vector, were constructed. Antibody plasmids were extracted and expressed in Expi293F suspension cells. The full-length antibody was isolated and purified using rProtein A Beads. Fab was obtained after strep II-tagged agarose resin purification. In this invention, Fab vector, full-length human IgG1 heavy and light chain vector, and scFV vector were used to prepare the VMAT2 antibody. The vector maps are shown below. Figure 7A -B is shown.

[0310] Example 7 Antigen-antibody binding experiment

[0311] 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.

[0312] 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.

[0313] Example 8: SPR Experiment, Results, and Analysis

[0314] 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.

[0315] 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.

[0316] Table 8

[0317]

[0318]

[0319] Example 9: Verification of the binding of Fab antibody to VMAT2 and VMAT1 from different species

[0320] hVMAT2 (human VMAT2, already constructed in Example 1), hVMAT1 (human VMAT1, Ensembl: ENSG00000036565), and rVMAT2 (rat VMAT2, Ensembl: ENSG00000036565) were constructed respectively.

[0321] ENSRNOG00000008890), rVMAT1 (rat VMAT1, Ensembl: ENSRNOG00000011992), mVMAT2 (mouse VMAT2, Ensembl: ENSMUSG00000025094), mVMAT1 (mouse VMAT1, Ensembl: ENSMUSG00000036330), pVMAT2 (pig VMAT2, Ensembl:

[0322] Ensembl: ENSSCG00000020671), pVMAT1 (pig VMAT1, Ensembl: ENSSCG00000009601), plasmids (human, rat, mouse, and pig VAMT2 or VAMT1 plasmids constructed using conventional techniques in the field), HEK293 T cells were injected at 1×10 7Cells were seeded at a density of 10 cm in 10 cm cell culture dishes and cultured in a CO2 incubator (37°C, 5% CO2) for 12 hours. Following standard techniques, cells were co-transfected with Lipofectamine 3000 and plasmids expressing hVMAT2 (human VMAT2), rVMAT2 (rat VMAT2), rVMAT1 (rat VMAT1), mVMAT1 (mouse VMAT1), pVMAT2 (porcine VMAT2), and pVMAT1 (porcine VMAT1), respectively, along with pMax GFP plasmid. After 24 hours, the culture medium was replaced with fresh medium, and cells were cultured for another 48 hours. Cells were then isolated in PBS with 2 mM EDTA, washed with PBS, and the single-cell suspension was filtered and placed into 96-well plates. 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 and removal of the supernatant, osmosis buffer (Thermo Fisher Scientific; cat.no. 00-8333-56) was added, followed by centrifugation and removal of the supernatant. Fab antibody was added and incubated for 1 hour, followed by incubation with anti-human Ig light chain κ antibody (Invitrogen, Cat. 2806744) for 15 minutes. All staining procedures were performed on ice. DAPI-GFP+Igκ+ cells were analyzed using a CytoFLEX LX (Beckman Coulter) flow cytometer. The detection principle was the same as in Example 4.

[0323] The results are as follows Figure 10 As shown, after validation of the binding of different antibodies and VMAT2 and VMAT1 of different species, it was found that at 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 VMAT1 and VMAT2 of all detected species, JPF1039 only bound to hVMAT2, and JPF1254 only bound to VMAT2 of different species.

[0324] Example 10 Cell Transport Inhibition Assay (An experiment to verify the inhibitory effect of antibodies at the cellular level)

[0325] 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).

[0326] 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.

[0327] 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.

[0328] 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 its antigen-binding fragment, characterized in that, The anti-VMAT2 antibody or its antigen-binding fragment has three complementarity-determining CDRs (HCDRs) selected from the group consisting of three complementarity-determining CDRs (LCDRs) of the heavy chain variable region and three complementarity-determining CDRs (LCDRs) of the light chain variable region: (1) HCDR1 shown in SEQ ID NO: 82, HCDR2, as shown in SEQ ID NO: 83, HCDR3, as shown in SEQ ID NO: 84, LCDR1 shown in SEQ ID NO: 86 LCDR2 shown in SEQ ID NO: 87 LCDR3 as shown in SEQ ID NO: 88; (2) HCDR1 shown in SEQ ID NO: 82, HCDR2, as shown in SEQ ID NO: 92, HCDR3, as shown in SEQ ID NO: 93, LCDR1 shown in SEQ ID NO: 95 LCDR2 shown in SEQ ID NO: 96 LCDR3 as shown in SEQ ID NO: 97; 2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-VMAT2 antibody or its antigen-binding fragment is an anti-VMAT2 protein cytoplasmic side antibody or its antigen-binding fragment.

3. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the anti-VMAT2 antibody or its antigen-binding fragment are selected from the following group: (a) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 81, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 85; (b) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 91, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

94.

4. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antiVMAT2 antibody or its antigen-binding fragment as claimed in claim 1; and (ii) Tag sequences that optionally assist in expression and / or purification.

5. A polynucleotide molecule, characterized in that, The nucleotide molecule encodes the anti-VMAT2 antibody of claim 1 or its antigen-binding fragment.

6. A carrier, characterized in that, The carrier contains the polynucleotide molecule as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector of claim 6, or has the polynucleotide molecule of claim 5 integrated into its genome.

8. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) an antibody portion comprising the anti-VMAT2 antibody of claim 1 or an antigen-binding fragment thereof; and (b) A coupling portion conjugated to the antibody or its antigen-binding fragment, the coupling portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the antibody or its antigen-binding fragment as claimed in claim 1, the recombinant protein as claimed in claim 4, the polynucleotide molecule as claimed in claim 5, the vector as claimed in claim 6, or the host cell as claimed in claim 7, or the antibody-drug conjugate as claimed in claim 8; and (ii) Pharmaceutically acceptable carriers, diluents or excipients.

10. The use of the antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 4, and the antibody-drug conjugate as described in claim 8, characterized in that, Used in the preparation of pharmaceuticals, reagents, test plates, or kits; The reagents, detection plates, or kits are used to detect VMAT2 protein in samples. The agent is used to treat or prevent diseases that overexpress the VMAT2 protein.

Images