CN119735674A - Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof - Google Patents
Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof Download PDFInfo
- Publication number
- CN119735674A CN119735674A CN202411791297.8A CN202411791297A CN119735674A CN 119735674 A CN119735674 A CN 119735674A CN 202411791297 A CN202411791297 A CN 202411791297A CN 119735674 A CN119735674 A CN 119735674A
- Authority
- CN
- China
- Prior art keywords
- seq
- antibody
- variable region
- chain variable
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明提供了抗VMAT2的抗体或其抗原结合片段及其组合物和应用。具体地，提供了一种抗VMAT2抗体或其抗原结合片段，其能够有效结合人VMAT2蛋白的胞内区段。本发明还提供了包含该抗体或抗原结合片段的抗体药物偶联物等，可应用于制备治疗过表达VMAT2疾病的药物，具有良好的应用前景。The present invention provides an anti-VMAT2 antibody or an antigen-binding fragment thereof, and a composition and application thereof. Specifically, an anti-VMAT2 antibody or an antigen-binding fragment thereof is provided, which can effectively bind to the intracellular segment of human VMAT2 protein. The present invention also provides an antibody-drug conjugate containing the antibody or antigen-binding fragment, which can be used to prepare a drug for treating a disease that overexpresses VMAT2, and has good application prospects.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-VMAT 2 antibody or an antigen binding fragment thereof, and a composition and application thereof.
Background
Vesicle monoamine transporter 2 (VMAT 2) is a multiple transmembrane protein that localizes to neuronal synaptic vesicle membranes. Various monoamine neurotransmitters such as dopamine (dopamine, DA), serotonin (serotonin, 5-HT), norepinephrine (NE), and epinephrine (E) can be transported to synaptic vesicle stores. Under external stimuli, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, binding to the corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signaling. VMAT2 participates in regulating a number of functions such as exercise, mood, sleep, rewarding, attention, addiction, etc.
Overexpression or excessive transport of VMAT2 can lead to disturbance of monoamine neurotransmitter systems, thereby inducing Huntington's chorea, addictive disorders, tardive dyskinesia, hypertension, etc. To date, the available treatments for such diseases are extremely lacking.
Currently, small molecule inhibitors for VMAT2 as drug targets, such as reserpine (respine), tetrabenazine (tetrabenazine), deutetrabenazine (deutetrabenazine), valbenazine (valbenazine), GZ-793A, and the like, are used for treating Huntington's chorea, tardive dyskinesia, and hypertension.
However, these inhibitors have the disadvantages of short half-life, low target specificity, susceptibility to adverse reactions, and the like.
Therefore, there is a need in the art to develop new VMAT2 drugs with long half-life and high specificity, which make up for the defects of small molecule drugs.
Disclosure of Invention
The invention provides a novel VMAT2 drug with long half-life and high specificity.
In a first aspect of the invention, there is provided an anti-VMAT 2 antibody or antigen-binding fragment thereof, having three complementarity determining CDRs (HCDR) of a heavy chain variable region and three complementarity determining region CDRs (LCDR) of a 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 as shown in SEQ ID NO. 53,
LCDR3 as 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 as shown in SEQ ID NO. 53,
LCDR3 as 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 as shown in SEQ ID NO. 53,
LCDR3 as 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 as shown in SEQ ID NO. 72,
LCDR3 as 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 as shown in SEQ ID NO. 72,
LCDR3 as 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 as shown in SEQ ID NO. 86,
LCDR2 as shown in SEQ ID NO. 87,
LCDR3 as 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 as shown in SEQ ID NO. 96,
LCDR3 as 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 as shown in SEQ ID NO. 72,
LCDR3 as 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 as shown in SEQ ID NO. 72,
LCDR3 as 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 as shown in SEQ ID NO. 72,
LCDR3 as shown in SEQ ID NO. 105.
In another preferred embodiment, the anti-VMAT 2 antibody or antigen-binding fragment thereof is an anti-VMAT 2 protein cytoplasmic side antibody or antigen-binding fragment thereof.
In another preferred embodiment, the anti-VMAT 2 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof against a cytoplasmic VMAT2 protein.
In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.
In another preferred embodiment, the antigen binding fragment comprises a Fab fragment, a F (ab') 2 fragment, or an Fv fragment.
In another preferred embodiment, the heavy chain variable region and the light chain variable region of the anti-VMAT 2 antibody or antigen-binding fragment thereof are selected from the group consisting of:
(a) A heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 47, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 51;
(b) A heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 57, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 59;
(c) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 62, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 51;
(d) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 66, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 70;
(e) A heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 76, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 78;
(f) A heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 81, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 85;
(g) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 91, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 94;
(h) A heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 100, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 104;
(i) A heavy chain variable region having an amino acid sequence shown as SEQ ID NO. 108, and a light chain variable region having an amino acid sequence shown as SEQ ID NO. 112;
(j) A heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 115, and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 119.
In another preferred embodiment, the heavy chain of the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, and the light chain of the antibody or antigen-binding fragment thereof further comprises a light chain constant region.
In another preferred embodiment, the antibody is a single chain antibody, a diabody, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.
In another preferred embodiment, the heavy chain constant region is of human or murine origin.
In another preferred embodiment, the light chain constant region is of human or murine origin.
In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the antibody further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.
In a second aspect of the present invention, there is provided a recombinant protein having:
(i) An anti-VMAT 2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and
(Ii) Optionally a tag sequence that facilitates expression and/or purification.
In another preferred embodiment, the tag comprises an Fc tag, a FLAG tag, a 6His tag, or a combination thereof.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a third aspect of the invention, there is provided a nucleotide molecule encoding an anti-VMAT 2 antibody or antigen-binding fragment thereof according to the first aspect of the invention.
In another preferred embodiment, the nucleotide molecule is DNA, RNA or cDNA.
In a fourth aspect of the invention there is provided a vector comprising a nucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the vector comprises a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.
In another preferred embodiment, the vector is a eukaryotic expression vector.
In a fifth aspect of the invention there is provided a host cell comprising a vector according to the fourth aspect of the invention, or having integrated into its genome a nucleotide molecule according to the third aspect of the invention.
In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of E.coli, yeast cells, mammalian cells.
In another preferred embodiment, the prokaryotic cell is E.coli.
In another preferred embodiment, the cells are immune cells and have a chimeric antigen receptor expressed on their surface.
In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.
In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell).
In another preferred embodiment, the chimeric antigen receptor is an anti-VMAT 2 antibody or antigen-binding fragment thereof.
In a sixth aspect of the invention, there is provided an antibody drug conjugate comprising:
(a) An antibody moiety comprising an anti-VMAT 2 antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and
(B) A coupling moiety coupled to the antibody or antigen binding fragment thereof, the coupling moiety selected from the group consisting of a detectable label, a drug, or a combination thereof.
In another preferred embodiment, the agent is an agent that inhibits monoamine neurotransmitter over-transport.
In another preferred example, the monoamine neurotransmitters include dopamine (dopamine, DA), serotonin (serotonin, 5-HT), norepinephrine (NE) and epinephrine (E).
In another preferred embodiment, the antibody drug conjugate has the expression mAb- (X-Y) n;
Wherein,
The mAb is the anti-VMAT 2 antibody or antigen-binding fragment thereof;
x is a linker;
Y is a coupling moiety, which is a drug;
n is a positive integer less than or equal to 8;
the coupling moiety is coupled to the anti-VMAT 2 antibody or antigen-binding fragment thereof via a linker.
In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, a nucleotide molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a host cell according to the fifth aspect of the invention, or an antibody drug conjugate according to the sixth aspect of the invention, and
(Ii) A pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the pharmaceutical composition is in the form of an injection.
In another preferred embodiment, the pharmaceutical composition is used for the preparation of a medicament for treating a disease caused by overexpression or excessive transport of VMAT 2.
In another preferred embodiment, the disorder is selected from the group consisting of Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.
In an eighth aspect of the invention there is provided an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an antibody drug conjugate according to the sixth aspect of the invention for use in the preparation of a medicament, reagent, assay plate or kit;
The reagent, the detection plate or the kit is used for detecting VMAT2 protein in a sample;
the agent is useful for treating or preventing a disease that overexpresses the VMAT2 protein.
In another preferred embodiment, the VMAT2 protein is an intracellular segment of VMAT2 protein.
In another preferred embodiment, the VMAT2 protein is VMAT2 cytoplasmic side protein.
In a ninth aspect of the present invention, there is provided a method for preparing the anti-VMAT 2 antibody or antigen-binding fragment thereof of the first aspect of the invention, the method comprising the steps of:
(a) Culturing a host cell according to the fifth aspect of the invention under expression conditions, thereby expressing the anti-VMAT 2 antibody or antigen-binding fragment thereof;
(b) Isolating and purifying the anti-VMAT 2 antibody or antigen binding fragment thereof of (a).
In a tenth aspect of the present invention, there is provided a method of detecting VMAT2 protein in a sample, the method comprising the steps of:
(1) Contacting a sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of VMAT2 protein in the sample.
In another preferred embodiment, the sample comprises a human or animal tissue sample, an exfoliated cell sample.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method further comprises step (3) of analyzing the affinity of the antibody and the antigen.
In an eleventh aspect of the invention there is provided a test plate comprising a substrate (support) and a test strip comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or an antibody drug conjugate according to the sixth aspect of the invention.
In another preferred embodiment, the test strip further comprises an antigen spotting region.
In another preferred embodiment, the test strip is formed by sequentially overlapping a sample filtering paper, a chromatographic material, a nitrocellulose membrane and a water absorbing paper.
In a twelfth aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, and/or
(2) A second container comprising a second antibody against the antibody or antigen-binding fragment thereof according to the first aspect of the invention, and/or
(3) A third container containing a cell lysis reagent therein;
Or alternatively
The kit contains a detection plate according to the eleventh aspect of the invention.
In another preferred embodiment, the antibodies in the first container are detectably labeled.
In another preferred embodiment, the antibodies in the second container are detectably labeled.
In a thirteenth aspect of the invention, there is provided a method of treating a disease associated with overexpression or oversransportion of VMAT2, comprising the step of administering to a subject in need thereof a therapeutically effective amount of an anti-VMAT 2 antibody or antigen-binding fragment thereof according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, a nucleotide molecule according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, an antibody drug conjugate according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.
In another preferred embodiment, the disease associated with overexpression or overdransportion of VMAT2 is selected from the group consisting of Huntington's chorea, tardive dyskinesia, hypertension, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the results of sorting mouse IgG1 positive memory B cells by flow cytometry and sorting mouse IgG1 positive memory B cells by control after immunization.
FIG. 2 shows the results of a gradient ELISA antigen-antibody binding assay of ELISA assay positive well culture supernatants of the VMAT 2-targeted antibody screen of example 3.
FIG. 3 shows the results of a gradient ELISA antigen-antibody binding assay of ELISA assay positive well culture supernatants of the VMAT 2-targeted antibody screen of example 3.
FIG. 4 shows schematic representation of VMAT2 protein expression and trafficking in cells.
FIG. 5 shows the supernatant results of single B cell cultures that bind to cell surface hVMAT.
FIG. 6 shows the supernatant results of single B cell cultures that did not bind to cell surface hVMAT 2.
FIGS. 7A-B show the heavy chain vector of human IgG1 CH1 used to construct an antibody, the light chain vector of human IgK used to construct an antibody, the heavy chain vector of human IgG1 constant region used to construct an antibody, and the ScFv vector.
FIG. 8 shows the results of a cell-stream antigen-antibody binding assay in example 6 of the present invention.
FIG. 9 shows the results of SPR antigen-antibody binding affinity assay in example 7 of the present invention.
FIG. 10 shows the results of verification of binding of Fab antibodies to VMAT2 and VMAT1 of different species in example 8 of the present invention.
FIG. 11 shows the experimental results of the inhibition of transport of cells by the antibody of example 9 of the present invention.
Detailed Description
The present inventors have made extensive and intensive studies to unexpectedly obtain a class of anti-VMAT 2 antibodies by mass screening. Experimental results show that the anti-VMAT 2 antibody has high affinity and good biological activity. And the antibodies of the invention can have cross-binding reactions with human VMAT2, murine VMAT2, and porcine VMAT2, and can also bind to VMAT 1. The antibody of the invention can obviously inhibit VMAT2 transport function. On this basis, the present invention has been completed.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the terms include "consisting of" and "consisting essentially of.
As used herein, the term "pharmaceutically acceptable carrier" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in and is acceptable to a human and/or animal. It will be appreciated by those of ordinary skill in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug being used, the severity of the disease, and the combination with other drugs.
VMAT2
Vesicle monoamine transporter 2 (VMAT 2) is a multiple transmembrane protein that localizes to neuronal synaptic vesicle membranes. Various monoamine neurotransmitters such as dopamine (dopamine, DA), serotonin (serotonin, 5-HT), norepinephrine (NE), and epinephrine (E) can be transported to synaptic vesicle stores. Under external stimuli, these neurotransmitters are released from synaptic vesicles into the synaptic cleft, binding to the corresponding target receptors on the postsynaptic membrane, thereby mediating downstream signaling. VMAT2 participates in regulating a number of functions such as exercise, mood, sleep, rewarding, attention, addiction, etc.
The prior art antibodies against the intracellular region of VMAT2 only show binding activity to the intracellular region, and no transport inhibitory activity. The antibody aiming at the VMAT2 intracellular region has better transportation inhibition activity besides the binding activity with the VMAT2 intracellular region.
Antibodies to
In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are isotetralin proteins having the same structural characteristics, which are composed 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, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes (isotype). 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, the heavy chain constant region consisting of three domains CH1, CH2, and CH 3. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region, the constant region of the light chain comprises a structural domain CL, the constant region of the light chain is paired with a CH1 structural domain of a constant region of the heavy chain, 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 binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-DEPENDENT CELL-mediated cytotoxicity), and the like. The heavy chain constant region comprises the IgG1, igG2, igG3, igG4 subtype, and the light chain constant region comprises Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an 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 an antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.
In the present invention, the terms "Fab" and "Fc" refer to papain that cleaves antibodies into two identical Fab fragments and one Fc fragment. The Fab fragment consists of VH and CH1 of the heavy chain and VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., the crystallisable fragment (fragment crystallizable, fc), consists of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where an antibody interacts with an effector molecule or cell.
In the present invention, the term "scFv" is a single chain antibody (SINGLE CHAIN antibody fragment, scFv) formed by the joining of the heavy and light chain variable regions of an antibody, typically via a 15-25 amino acid linker.
In the present invention, the term "variable" means that some portion of the variable region in an antibody differs in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the heavy and light chain variable regions, known as complementarity-DETERMINING REGION (CDR) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by 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 publication No.91-3242, vol. I, pp. 647-669 (1991)).
As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3 and H-FR4, respectively. Accordingly, the light chain variable domain may thus be referred to as (L-FR 1) - (LCDR 1) - (L-FR 2) - (LCDR 2) - (L-FR 3) - (LCDR 3) - (L-FR 4) and the heavy chain variable domain may thus be denoted as (H-FR 1) - (HCDR 1) - (H-FR 2) - (HCDR 2) - (H-FR 3) - (HCDR 3) - (H-FR 4). Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence identity of up to 85%, 90%, 95%, 96%, 97%, 98% or 99%.
Knowing the amino acid sequence of the CDRs, one skilled in the art can readily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and/or H-FR1, H-FR2, H-FR3, H-FR4.
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.
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 and heavy chains to fold into an exchanged double variable region immunoglobulin. In the present invention, preferred linkers refer to linkers Linker1 and Linker2, wherein Linker1 links VH and VL of a single chain antibody (scFv), and Linker2 is used to link scFv to the heavy chain of another antibody.
Examples of suitable linkers include mono glycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural element that needs to be achieved in the linker.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the specific antibody of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
| Initial residues | Representative substitution | Preferred substitution |
| 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 |
In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, e.g., less than about 10 -8M、10-9M、10-10M、10-11 M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) or the relative affinity of an antibody to antigen binding is determined using ELISA.
In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. An epitope of the invention is a region of an antigen to which an antibody binds.
Polynucleotides, vectors and host cells
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences and various additional coding sequences for the mature polypeptide, coding sequences (and optionally additional coding sequences) for the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means (1) hybridization and elution at a relatively low ionic strength and a relatively high temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc., or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Representative examples are E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, drosophila S2 or Sf9 insect cells, CHO, COS7, 293 cell animal cells, and the like.
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, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl 2 method using procedures well known in the art. Another approach is to use MgCl 2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to the antibodies of the invention include, but are not limited to, 1. Radionuclides, 2. Biotoxics, 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 proteins (BPHL)), 10. Chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
Pharmaceutical composition and application
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection. In the present invention, the term "pharmaceutical composition" means that the specific antibodies of the present invention can be combined with pharmaceutically acceptable carriers to form pharmaceutical formulation compositions for more stable therapeutic effects, which formulations can ensure the conformational integrity of the amino acid core sequences of the specific antibodies disclosed herein, while also protecting the multifunctional groups of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation). The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the specific antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the specific antibodies of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the specific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.
Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically 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 glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide, 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 a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodine, bromine, or chlorinated), haloesters (e.g., iodine, bromine, or chlorinated), halomethyl ketones (e.g., iodine, bromine, or chlorinated), benzyl halides (e.g., iodine, bromine, or chlorinated), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3, 6-bis- (mercury methyl) dioxane, with the counter ion being acetate, chloride, or nitrate, and polymethylene dimethyl sulfide thiosulfate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.
The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.
Useful classes of drugs 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, and the like. In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
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, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne 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 attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a 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.
The invention also provides methods of making ADCs, which may further comprise combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
In some embodiments, the antibody drug conjugate ADC is of the formula:
Wherein:
Ab is an antibody that is conjugated to a polypeptide,
LU is the linker;
d is a drug;
And subscript p is a value selected from 1 to 8.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Application of
The invention provides the use of an antibody of the invention, for example, in the preparation of a diagnostic formulation, or in the preparation of a medicament for the prevention and/or treatment of WAMT-related diseases.
In a preferred embodiment, the WAMT-related disorder is a disorder associated with overexpression or excessive transport of WAMT 2. In a preferred embodiment, the disorder comprises Huntington's disease, tardive dyskinesia, hypertension, or a combination thereof.
The main advantages of the invention include:
(a) The anti-VMAT 2 antibody has better specificity and binding activity with human VMAT2, and can inhibit the over-expression or over-transportation of the VMAT2 to the synaptic vesicle storage of various monoamine neurotransmitters such as Dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E), thereby improving the disturbance of the monoamine neurotransmitter system.
(B) The VMAT2 antibody of the present invention has human, rat, mouse and pig cross-binding activity.
(C) Compared with the VMAT2 small molecule inhibitor, the antibody has long half-life and high specificity.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (NewYork: coldSpringHarborLaboratoryPress, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 acquisition of human VMAT2 protein mimicking the native conformation
In this example, the full-length human VMAT2 gene sequence (EBI: ENST 00000644641.2) is constructed into a mammalian cell expression vector, such as a pCAG vector, and exogenous expression is performed using HEK293 cells to obtain a large number of VMAT2 protein samples.
The expressed protein will be purified using methods conventional in the art of biochemistry, and the purification conditions are optimized to preserve as much as possible the native conformation of the protein, followed by packaging of the purified VMAT2 protein using Amphipol reagents to maximize the native conformation of the simulated VMAT2 protein on the cell membrane surface.
EXAMPLE 2 mouse immunization, mouse B cell sorting and culture
The VMAT2 protein obtained in example 1 was immunized according to a method conventionally known in the art, wherein the immunization strategy was to select 10-12 weeks C57BL/6 mice, respectively, and to set an immunization group and a control group, each of which was immunized 1 time every 3-4 days with 30. Mu.g and CpGOND2395 20. Mu.g (5'-tcgtcgttttcggcgcgcgccg-3') (SEQ ID NO: 123) of the VMAT2 protein (antigen) prepared in example 1 described above and an equal volume of AddaVax TM (InvivoGen) for intraperitoneal and tarsal joint injection, respectively, and the control group was injected with 20. Mu.g of CpGOND2395 and an equal volume of AddaVax TM, and immunized 7 times every 3-4 days.
Sorting of mouse B cells the experimental and control mice were sacrificed 3-7 days after the last needle immunization, spleen and lymph node tissues were obtained, single cell suspensions were prepared from spleen and lymph nodes (popliteal fossa and inguinal canal), and then erythrocytes were removed with erythrocyte lysis buffer (SangonBiotechcat.b 541001-0100). After washing twice with 2% fbs in PBS (FACS buffer), cells were ready for staining, and then stained round-door strategy for obtaining target cells was selected using CytoflexSRT (BeckmanCoulter) cell sorter:
DAPI-CD19+CD38+GL7-IgG1+;
The reagents used were as follows:
DAPI(live/dead;Invitrogen Cat.D1306),
anti-mouseCD19(APC/Cyanine7;Biolegend Cat.115530),
anti-mouseCD38(PE/Cyanine7;Biolegend Cat.102718),
anti-mouse/humanGL7Antigen(TandBcellActivationMarker),488;BiolegendCat.144612);
anti-mouseIgG1(APC;BiolegendCat.406610)。
Mouse IgG1 positive memory B cells were sorted by flow cytometry after immunization.
An example result of the flow sort is shown in fig. 1. As can be seen from FIG. 1, after the immunization method in the scheme is adopted, the mice in the experimental group can be sorted into a large number of IgG1 positive memory B cells, the proportion of the IgG1 positive memory B cells in the total B cells is higher, and the proportion of the IgG1 positive memory B cells in the control group is far less than that of the IgG1 positive memory B cells in the experimental group.
Culturing the mouse B cells, namely culturing the IgG1 positive memory B cells obtained by sorting, wherein the culturing method can refer to Haniuda and Kitamura,(2019).Induced Germinal Center B Cell Culture System,Bio-protocol9(4)：e3163.DOI：10.21769/BioProtoc.3163,, wherein the culture medium is ：RPMI-1640,(Gibco,Cat.11875-093),10％ Fetal Bovine Serum(FBS,VivaCell,Cat.C04002-500),55μM 2-mercaptoethanol(ThermoFisher,Cat.21985),100units/ml Penicillin(Biosharp),100μg/ml Streptomycin(Biosharp),10mM HEPES(ThermoFisher,Cat.15630-080),1mM Sodium Pyruvate(ThermoFisher,Cat.11360-070)and 0.1mM MEM nonessential amino acid(ThermoFisher,Cat.11140-050),, 1 day before sorting, inoculating feeder layer cells expressing CD40L and BAFF onto a 96-well plate, 1000 cells per well, sorting single IgG1 positive B cells into each well, culturing for two days by adding 2-10ng/ml IL-4 into the culture medium, adding 4-10ng/ml IL-21, continuously culturing for 6-8 days, changing liquid every day, collecting supernatant on the 10 th day, storing at 4 ℃, and storing at 96-80 ℃ for subsequent cracking to obtain RNA of the single B cells.
Example 3 ELISA experiments, results and analysis of VMAT 2-targeting antibody screening
The VMAT2 protein obtained in example 1 was coated in 96-well ELISA plates, incubated overnight at 4℃in wet condition, 100-120. Mu.l of blocking buffer (4% BSA in1 XPBS) was added to the plates after discarding the coating buffer, incubated at room temperature for 2 hours, after removing the blocking buffer, 20-60. Mu.l of single cell culture supernatant collected in example 2 was added per well, incubated overnight at 4℃in wet condition, washed 3 times, 20-30. Mu.l 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 20-30ul of developing solution containing 4-nitrophenyl disodium phosphate hexahydrate (CSNpharm, cat.CSN66207) was added per well. OD405 was measured using MDSpectraMaxiD.
The results are shown in the following Table 1-Table 4: partial assay data for ELISA assays performed in 96-well plates.
Tables 1-4 show the plate 1 antigen assay and plate 1 IgG1 antibody secretion assay, respectively, and the plate 2 antigen assay and plate 2 IgG1 antibody secretion assay, respectively.
TABLE 1 Board 1 antigen specificity assay
| 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 |
TABLE 2 detection of IgG1 antibodies secreted by plate 1 cells
| 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 |
TABLE 3 Board 2 antigen-specific assay
TABLE 4 detection of IgG1 antibodies secreted by plate 2 cells
| 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 |
ELISA detection results show that the supernatant of the screened IgG1+ positive B cell culture contains the antibody specifically bound by the VMAT2 protein, and the color development is different due to different antibody binding force or antibody concentration in the culture supernatant. ELISA concentration gradient detection is further carried out, and the antibodies have antigen-antibody binding activity.
EXAMPLE 4 detection of antibody binding to cell surface VMAT2 antigen (detection of antibody from Single B cell culture supernatant), screening for antibodies that bind to the hVMAT2 cytoplasmic side
HEK 293T cells were seeded at a density of 1×10 7 cells in 10cm cell culture dishes and cultured in a carbon dioxide incubator (37 ℃ 5% CO 2) for 12 hours, cells were CO-transfected with Lipofectamine 3000 with plasmid expressing full length human VAMT2 (VAMT plasmid constructed by means of conventional techniques in the art) and pMax GFP plasmid, and after 24 hours, fresh medium was changed to continue culturing cells. 48 hours after transfection, cells were treated with 2mM EDTA-PBS buffer, then washed with 2% FBS-containing PBS buffer, and single cell suspensions were filtered and placed in 96-well plates. The transfected cells were incubated with the supernatant of single B cell culture for 1 hour, then washed 2 times with FACS, cells were incubated with DAPi (live/dead; invitrogen Cat.D 1306) and anti-mouse IgG1 (APC; bioleged Cat.406610) for 15 minutes, and all staining procedures were completed on ice. DAPI-GFP+IgG1+ cells were analyzed using CytoFLEX LX (Beckman Coulter) flow cytometer.
As shown in FIG. 4, the ribosome is transferred to the endoplasmic reticulum and Golgi apparatus for further synthesis, processing and modification after synthesizing the VMAT2 polypeptide chain. Eventually, in vivo, VMAT2 localizes to synaptic vesicle membranes in cells. In the antibody screening experiment, the human VMAT2 (hVMAT 2) is over-expressed on the surface of HEK293T cell membrane, in the process, VMAT2 is turned into extracellular region (namely cell membrane surface region) in the vesicle membrane cavity side region, and the vesicle membrane cytoplasmic side is still the plasma membrane cytoplasmic side after turning. Thus, in the initial screening of antibodies, the invention excludes binding to cell surface VMAT2 antibodies and the remaining antibodies positive for ELISA screening (i.e., anti-VMAT 2 cytoplasmic side antibodies) will be cloned for further validation.
In ELISA experiments the VMAT2 protein was intact, including the luminal and cytoplasmic sides, but in ELISA experiments the VMAT2 protein was not on the cell membrane, both the luminal and cytoplasmic sides of the protein were exposed. When VMAT2 protein encounters an antibody, both sides of the bound antibody can be detected. In the case of cell flow experiments, VAMT2 is expressed on the cell membrane, so VMAT2 has a cavity side and a cytoplasmic side, and only antibodies bound to the cell surface can be detected, but antibodies bound to the cytoplasmic side cannot be detected. Therefore, ELISA positive samples were considered to be removed from the sample bound to the surface of the overexpressed cells, and the remaining ELISA positive samples were samples of the cytoplasmic bound antibodies.
In particular, the streaming results are shown in fig. 5-6. By cell surface binding assays, it was shown that murine monoclonal antibodies bind to cell surface overexpressed hVMAT, corresponding to hVMAT in the protruding vesicles, i.e., on the luminal side, and that such antibodies would be excluded from the screen.
Antibodies binding to the hVMAT cytoplasmic side were further screened, so that the antibodies that bound positively to the antigen antibodies screened last in this example were antibodies binding to the hVMAT cytoplasmic side. The hVMAT cytoplasmic antibody can be used as a labeled antibody for VMAT2 antigen or as a functional antibody for inhibiting its transport.
EXAMPLE 5 Single B cell culture supernatant ELISA gradient assay
The VMAT2 protein obtained in example 1 was coated in 96-well ELISA plates, incubated overnight at 4℃in wet conditions, 100-120. Mu.l of blocking buffer (4% BSA in1 XPBS) was added to the plates after discarding the coating buffer, incubated at room temperature for 2 hours, after removal of the blocking buffer, single cell culture supernatants showing antigen-antibody binding in both example 3 and example 4 were added per well, the single cell culture supernatants were subjected to gradient dilutions, i.e.4-fold, 16-fold, 64-fold, 256-fold, 20-60. Mu.l of single cell culture supernatant dilutions collected on day 10 in example 2 were added per well, incubated overnight at 4℃in wet conditions, 20-30. Mu.l of secondary antibody (AKP goat anti-mouse IgG1, southernBiotech, cat.1030-04) were added after washing for 3 times, incubated at room temperature for 2 hours, and 20-30. Mu.l of color development solution containing 4-nitrophenyl disodium phosphate (CSNpharm, cat.CSN66207) was added per well.
OD405 results were measured using MDSpectraMaxiD and are shown in fig. 2-3. As can be seen from the figure, each sample in the figure shows a strong antigen-antibody binding activity.
Example 6 molecular cloning method and obtaining monoclonal antibody VDJ/VJ sequences
Through the ELISA of example 3 and example 5 and the analysis of the detection results of the surface binding of antigen-antibody cells of example 4, cells corresponding to positive wells (antigen-antibody binding) of 96-well cell culture plates pre-frozen in a-80 ℃ refrigerator were selected for RNA extraction for subsequent molecular cloning to obtain VDJ/VJ sequences.
Total RNA of B cells in 96-well cell culture plates that were detected positive (antigen-antibody binding) was extracted with TRIzol Reagent (Thermo Fisher). Reverse transcription and PCR were performed according to 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 42 ℃, 5min, 25 ℃,10 min, 50 ℃,60 min and 94 ℃, followed by two rounds of semi-nested PCR with HotStar DNA polymerase (Qiagen) to enrich the heavy and light chains, and the PCR products were purified and sequenced. Sequencing results were analyzed using IgBlast tools and IMGT database. The VDJ/VJ fragment (Table 5) was amplified using gene specific primers and the VDJ fragment heavy chain and VJ fragment light chain vectors were cloned by homologous recombination or T4 ligase ligation, respectively.
TABLE 5 Gene-specific primers for amplification of VDJ/VJ
The two rounds of semi-nested PCR used primers as in Table 6 PCR procedure were 95℃for 15 minutes, 95℃for 30 seconds, 50-65℃for 30 seconds, 72℃for 5 minutes.
TABLE 6 semi-nested PCR primers
The VDJ/VJ specific primers in this example comprise the homology arm portions of the heavy/light chain vector, while comprising the specific portions of the VDJ/VJ fragment, and generally the specific primers will use different homology arms depending on the cloning vector used. The present application is not limited to the specific primers used in the examples.
The nucleotide and amino acid sequences of the VDJ/VJ variable regions of the antibodies were sequenced after reverse transcription and 2 rounds of semi-nested PCR are shown in Table 7.
TABLE 7
Constructed to a human heavy chain constant region CH1 vector and a light chain vector (expressing Fab) with strep II tag and His tag, and a human full-length constant region heavy and light chain vector (expressing human IgG 1) and SCFV vector respectively. And finally, extracting antibody plasmids, carrying out antibody expression in the Expi293F suspension cells, and separating and purifying the full-length antibody through rProtein A Beads. And (3) separating and purifying by strep II tag agarose resin to obtain Fab. In the present invention, fab vectors, full-length human IgG1 heavy and light chain vectors, scFV vectors were used to produce antibodies to VMAT2, and the respective vector profiles are shown in fig. 7A-B.
Example 7 antigen-antibody binding assay
Polystyrene Protein A (Spherech) microspheres were resuspended in 1 XPBS at room temperature, centrifuged, then resuspended in anti-flag (hIgG 1) antibody at a concentration of 10. Mu.g/ml, incubated for 15 minutes with stirring, washed twice with 1 XPBS, incubated for 1-2h with each 2. Mu.g/ml hVMAT-2% FACS solution, washed 2 times with 2% FACS solution, and then incubated for 1 hour with 50. Mu.l of 2% FACS solution diluted Fab antibody (expressed as JFF). After washing twice with 1 XPBS, 50. Mu.l 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. Flow analysis was performed using CytoFLEX LX (Beckman) and MFI was analyzed.
As a result, as shown in FIG. 8, it was found by the antigen-antibody binding experiments of polystyrene Protein A that antibodies JPF0514, JPF1217, JPF1254, JPF1258, JPF1026, JPF1206, JPF1212, JPF0568, JPF1039 and JP0503 had antigen binding activity.
Example 8SPR experiments and results and analysis
Surface Plasmon Resonance (SPR) binding experiments were performed using Biacore 8K+ (Cytiva) at 25 ℃,10 mM HEPES sodium salt (pH 7.4), 150mM NaCl, and 0.05% (V/V) Tween 20. Briefly, anti-flag antibody (hIgG 1) was immobilized on a Cytiva Protein A sensor chip, followed by sequential binding of hVMAT Protein and Fab fragments. KD values were calculated in Biacore Insight Evaluation software at Cytiva.
The results are shown in FIG. 9, and the binding and dissociation constants of the antibodies obtained by the above SPR antigen-antibody binding affinity assay are shown in Table 8, and the antibodies obtained by the screening have antigen-binding activity.
TABLE 8
| 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 |
EXAMPLE 9Fab antibody binding validation of different species VMAT2, VMAT1
HVMAT2 (humanized VMAT2, example 1 has been constructed), hVMAT1 (humanized VMAT1, ensembl: ENSG 00000036565), rVMAT2 (rat VMAT2, ensembl:
ENSRNOG 00000008890), rVMAT1 (rat VMAT1, ensembl: ENSRNOG 00000011992), mVMAT2 (mouse VMAT2, ensembl: ENSMUSG 00000025094), mVMAT1 (mouse VMAT1, ensembl: ENSMUSG 00000036330), pVMAT2 (pig VMAT2, ensembl:
Ensembl: ENSSSCG 00000020671), pVMAT1 (porcine VMAT1, ensembl: ENSSSCG 00000009601), plasmids (human, rat, mouse and porcine VAMT or VAMT plasmids constructed by conventional techniques in the art), HEK 293T cells were inoculated into 10cm cell culture dishes at a density of 1X 10 7 cells, cultured in a carbon dioxide incubator (37 ℃ C., 5% CO 2) for 12 hours, and the cells were CO-transfected with Lipofectamine 3000 and expression hVMAT (human VMAT 2), rVMAT2 (rat VMAT 2), rVMAT (rat VMAT 1), mVMAT1 (mouse VMAT 1), pVMAT2 (porcine VMAT 2), pVMAT1 (porcine VMAT 1) plasmids, respectively, and pMax GFP, and after 24 hours, the cells were continuously cultured by changing fresh medium. After 48 hours of transfection, cells were isolated in PBS with 2mM EDTA, then washed with PBS and single cell suspensions were filtered and placed in 96-well plates. The transfected cells were incubated with live/dead eflour450 dye (Thermo FISHER SCIENTIFIC; cat. No. 65-0863-14) for 15 min, then washed 1 time with PBS, then incubated with fixing buffer (Thermo FISHER SCIENTIFIC; cat. No. 00-8222-49) for 20 min, centrifuged to remove supernatant, then permeation buffer (Thermo FISHER SCIENTIFIC; cat. No.; 00-8333-56) was added, centrifuged to remove supernatant, then Fab Antibody was added for incubation for 1h, then anti-human IG LIGHT CHAIN kappa Antibody (Invitrogen, cat. 2806744) was added and incubated for 15 min, all staining procedures were completed on ice. DAPI-GFP+Igkappa+ cells were analyzed using CytoFLEX LX (Beckman Coulter) flow cytometer. The detection principle is the same as in example 4.
As a result, as shown in FIG. 10, it was found by the above-mentioned binding verification of VMAT2 and VMAT1 of different antibodies and different species that, at an antibody concentration of 10. Mu.g/ml, each of JPF1026, JPF1217, JPF1254, JPF1258, JPF0514, JPF0568, JPF0503, JPF1212, JPF1039, and JPF1206 could bind to hVMAT. JPF1026, JPF1217, JPF1258, JPF0568, JPF1212, JPF1206 can bind to VMAT1 and VMAT2 of all detected species, JPF1039 binds to hVMAT only, JPF1254 binds to VMAT2 of different species only.
Example 10 cell transport inhibition assay (assay for verifying at the cellular level that antibodies have inhibitory effects)
HVMAT2 sequences were cloned into the pCAG vector. The 24-well plate was coated with poly-D-lysine in advance, HEK 293T cells were then seeded in the 24-well plate at a density of 2X 10 5 cells per well and cultured at 37℃under 5% CO 2 for 12 hours. hVMAT2 and ScFv plasmids (expressed as JPS) were co-transfected with Lipofectamine 3000. After 48 hours of transfection, the cells were washed with PBS. Cells were then incubated in 37 ℃ buffer (125 mM 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 min followed by incubation with 2 μm FFN206 (Abcam) and 2 μm calcitonin-AM (ferment) for 1 hour at 37 ℃ in the dark. Fluorescence was detected with EnSight (PerkinElmer) after washing twice with frozen PBS.
The kit is divided into antibody experimental groups, namely antibodies JPS0514, JPS1026, JPS1039, JPS1206, JPS1212, JPS1217, JPS1258, JPS1254, JPS0503 and JPS0568, and a small molecule positive inhibitor control group, namely TBZ tetrabenazine (tetrabenazine).
As shown in FIG. 11, it was found from the results of the cytostatic transport experiments that antibodies JPS0514, JPS1026, JPS1039, JPS1206, JPS1212, JPS1217, JPS1258, JPS1254, JPS0503 and JPS0568 each have a function of inhibiting VMAT2 transport.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411791297.8A CN119735674A (en) | 2024-12-06 | 2024-12-06 | Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411791297.8A CN119735674A (en) | 2024-12-06 | 2024-12-06 | Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119735674A true CN119735674A (en) | 2025-04-01 |
Family
ID=95134858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411791297.8A Pending CN119735674A (en) | 2024-12-06 | 2024-12-06 | Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119735674A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1918180A (en) * | 2003-12-22 | 2007-02-21 | 葛兰素集团有限公司 | Nogo-a neutralising immunoglobulins for treatment of neurological diseases |
| CN101626783A (en) * | 2006-08-04 | 2010-01-13 | 诺华有限公司 | EPHB3-specific antibodies and applications thereof |
| US20220089704A1 (en) * | 2018-10-29 | 2022-03-24 | Biogen Ma Inc. | Humanized and stabilized fc5 variants for enhancement of blood brain barrier transport |
-
2024
- 2024-12-06 CN CN202411791297.8A patent/CN119735674A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1918180A (en) * | 2003-12-22 | 2007-02-21 | 葛兰素集团有限公司 | Nogo-a neutralising immunoglobulins for treatment of neurological diseases |
| CN101626783A (en) * | 2006-08-04 | 2010-01-13 | 诺华有限公司 | EPHB3-specific antibodies and applications thereof |
| US20220089704A1 (en) * | 2018-10-29 | 2022-03-24 | Biogen Ma Inc. | Humanized and stabilized fc5 variants for enhancement of blood brain barrier transport |
Non-Patent Citations (3)
| Title |
|---|
| JIPING YANG等: "An amphipol-stabilized multi-pass transmembrane protein as an immunogen to generate mouse memory B cells against native VMAT2", 《FEBS LETTERS》, 17 June 2025 (2025-06-17), pages 1 - 15 * |
| MATTHEW FREEBY等: "Vesicular monoamine transporter, type 2 (vmat2) expression as it compares to insulin and pancreatic polypeptide in the head, body and tail of the human pancreas", 《ISSN》, vol. 4, no. 6, 1 November 2012 (2012-11-01), pages 396 * |
| 满建梅等: "A53T α-synuclein 抑制SH-SY5Y 细胞2 型 囊泡单胺转运体的表达", 《中国比较医学杂志》, vol. 26, no. 8, 31 August 2016 (2016-08-31), pages 66 - 69 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US12018078B2 (en) | SEMA4D antibody, preparation method therefor and use thereof | |
| CN113166257B (en) | CD47 antibody and preparation method and application thereof | |
| EP4215549A1 (en) | Anti-4-1bb-anti-pd-l1 bispecific antibody, and pharmaceutical composition and use thereof | |
| CN113348180B (en) | OX40 antibodies, methods of making and uses thereof | |
| CN117024590A (en) | Monoclonal antibody against human B7-H3 and application thereof | |
| JP7568238B2 (en) | Anti-B7-H3 antibodies and their preparation and applications | |
| CN114106190A (en) | anti-VEGF/PD-L1 bispecific antibody and application thereof | |
| CN116731169B (en) | Nano antibody with sortilin 1 specificity and application thereof | |
| CN113166251B (en) | Anti-human TIM-3 monoclonal antibody and application thereof | |
| JP2022514786A (en) | MUC18-specific antibody | |
| US20230002503A1 (en) | Nano-antibody targeting caix antigen and application thereof | |
| CN114763383A (en) | Monoclonal antibody targeting human BCMA and application thereof | |
| JP2023501182A (en) | Anti-IL-4R single domain antibody and its application | |
| CN117624366A (en) | 5T4 nanobody and application thereof | |
| CN113166252B (en) | Fully human anti-GITR antibodies and methods of making same | |
| CN114763382A (en) | Monoclonal antibody targeting human CD276 and application thereof | |
| CN115304680B (en) | Preparation and application of bispecific cell adapter molecules constructed based on Pep42 | |
| CN114805581A (en) | Antibodies targeting IL13RA2, chimeric antigen receptors, and uses thereof | |
| CN119735674A (en) | Anti-VMAT2 antibodies or antigen-binding fragments thereof and compositions and uses thereof | |
| CN117402255A (en) | Bispecific antibodies against human PD-L1 and human OX40 and uses thereof | |
| WO2025098446A1 (en) | Anti-bcma single-domain antibody and use thereof | |
| TW202438528A (en) | Antibodies specifically binding to Claudin 18.2 and preparation and application thereof | |
| CN117285628A (en) | anti-VISTA antibodies and uses thereof | |
| CN119899264A (en) | Anti-p-Tau181 protein monoclonal antibody and preparation method and application thereof | |
| HK40043681A (en) | Sema4d antibody, preparation method therefor and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |