CN113912714B

CN113912714B - Antibody specifically binding to alpha-synuclein and application thereof

Info

Publication number: CN113912714B
Application number: CN202111526680.7A
Authority: CN
Inventors: 马咏翔; 庞晓静
Original assignee: Beijing Kaixiang Hongkang Biotechnology Co ltd
Current assignee: Beijing Kaixiang Hongkang Biotechnology Co ltd
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-02-22
Anticipated expiration: 2041-12-15
Also published as: CN113912714A

Abstract

The invention discloses an antibody specifically binding to alpha-synuclein and application thereof, wherein the monoclonal antibody is secreted by a hybridoma cell strain 25F5C5 and has CDR1-3 of a heavy chain shown as SEQ ID NO.1-3 and CDR1-3 of a light chain shown as SEQ ID NO. 11-13; meanwhile, the invention discloses a nucleic acid molecule for encoding the monoclonal antibody, a vector and a cell containing the nucleic acid molecule. The invention provides a new product for the detection or capture of alpha-synuclein.

Description

Antibody specifically binding to alpha-synuclein and application thereof

Technical Field

The invention belongs to the field of cell biotechnology and immunology, and relates to an antibody specifically binding to alpha-synuclein and application thereof.

Background

alpha-Synuclein (alpha-Syn) is a soluble protein widely existing in normal human brain tissue, is a main component of Lewy Body (LB), is related to nervous system degenerative diseases, and plays an important role in the pathogenesis of Parkinson disease. alpha-Syn is involved in synaptic function, and its physiological actions include regulation of synaptic plasticity, regulation of dopamine content in the synaptic site, regulation of lipid metabolism, and the development of random helices under normal physiological conditions, playing a key role in the regulation of Mitochondrial Homeostasis (Cali T, Ottolini D, Negro A, et al. alpha. -Synuclein Controls Mitochondrial Calcium by Enhancing endogenous bacterial metabolism-Mitochondria interaction, J Biol Chem, 2012, 287 (22):17914 and 17929.). However, the change of environment or gene is easy to cause the alpha-Syn to be misfolded and to generate oligomers, the fibrin is gradually increased, and then the lewy bodies are generated, and the biochemical reactions such as oxidative stress generated in the process, the intermediate conformation of the oligomers and the like play an important role in the pathogenesis of Parkinson. alpha-Syn and fragments thereof, such as Non-starch-like components (NAC), are considered important areas of alpha-Syn virulence and have been found to be involved in pathological damage to Parkinson's disease, such as The apoptotic mechanism, which is closely related to mitochondrial dysfunction (Wan OW, Chung KK. The role of alpha-synthesis oligomerization and aggregation in cellular and animal models of Parkinsons disease, Plos One,2012,7: 38545.), ultimately leading to programmed death of dopaminergic neurons. Typical pathological features are the formation of LB with alpha-Syn as the major component in Parkinson's disease patients, and degeneration and loss of dopaminergic neurons of the substantia nigra. α -Syn may affect the pathogenesis of Parkinson's disease in several ways. The detection of the concentration of the alpha-Syn in the peripheral blood can provide certain help for the diagnosis of the Parkinson's disease and the differential diagnosis of the misdiagnosed diseases which are easy to be diagnosed with the Parkinson's disease.

Pathological inclusion bodies composed of alpha-synuclein are commonly found in Parkinson's Disease (PD), Lewy body Dementia (DLB), Multiple System Atrophy (MSA), and some rare diseases, which are collectively referred to as alpha-synucleinopathies due to abnormal aggregation of synuclein in these diseases.

Alpha-synuclein is a target for the development of therapeutics for alpha-synucleinopathies (e.g., parkinson's disease). The main development strategies include inhibition of aggregate formation, gene silencing and aggregate removal. Currently antibodies against alpha-syn are antibodies against alpha-syn in different aggregation states (anti-monomeric, anti-oligomeric, anti-fibrotic antibodies); alpha-syn is divided into three domains, N-terminal, NAC region and C-terminal, so there are antibodies (anti-N-terminal, anti-C-terminal, anti-NAC region antibodies) directed against different regions of alpha-syn, but none are currently clinically applied.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide a hybridoma cell strain secreting anti-alpha-synuclein, an anti-alpha-synuclein monoclonal antibody and application thereof.

In a first aspect, the present invention provides an anti- α -synuclein monoclonal antibody comprising three CDRs of a heavy chain variable region and three CDRs of a light chain variable region; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.11, 12 and 13.

Further, the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.4, 5, 6 and 7; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.14, 15, 16 and 17.

Further, the monoclonal antibody comprises:

(a) a heavy chain variable region sequence having at least 90%, preferably 95% sequence identity to the amino acid sequence of SEQ ID No. 9;

(b) a light chain variable region sequence having at least 90%, preferably 95% sequence identity to the amino acid sequence of SEQ ID No. 19; or

(c) A heavy chain variable region sequence as in (a) and a light chain variable region sequence as in (b).

Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19.

Further, the monoclonal antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the invention or a functional fragment thereof.

Further, the CDR1, CDR2, CDR3 nucleic acid molecule encoding the heavy chain variable region has a sequence having at least 90%, preferably 95% sequence identity to the nucleotide sequence set forth in SEQ ID No.21, 22, 23;

the CDR1, CDR2, CDR3 nucleic acid molecules encoding the variable region of the light chain have a sequence which has at least 90%, preferably 95% sequence identity to the nucleotide sequences shown in SEQ ID nos. 31, 32, 33.

Further, the nucleic acid molecules encoding framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region have a sequence which has at least 90%, preferably 95%, sequence identity to the nucleotide sequence set forth in SEQ ID nos. 24, 25, 26, 27; nucleic acid molecules encoding framework regions FR1, FR2, FR3 and FR4 of the light chain variable region have a sequence which has at least 90%, preferably 95%, sequence identity to the nucleotide sequences set forth in SEQ ID Nos. 34, 35, 36 and 37.

Further, the nucleic acid molecule encoding the heavy chain variable region has a sequence with at least 90%, preferably 95% sequence identity to the nucleotide sequence shown in SEQ ID No. 29; the nucleic acid molecule encoding the variable region of the light chain has a sequence which has at least 90%, preferably 95% sequence identity with the nucleotide sequence shown in SEQ ID NO. 39.

Preferably, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.29 and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 39.

In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.

Further, the vector comprises a first signal peptide operably linked to the heavy chain of the monoclonal antibody, and/or a second signal peptide operably linked to the monoclonal antibody.

Further, the amino acid sequence of the first signal peptide is shown as SEQ ID NO. 8; the amino acid sequence of the second signal peptide is shown as SEQ ID NO. 18.

Further, the nucleotide sequence for coding the first signal peptide is shown as SEQ ID NO.28, and the nucleotide sequence for coding the second signal peptide is shown as SEQ ID NO. 38.

In a fourth aspect, the invention provides a cell comprising a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

In a fifth aspect, the present invention provides a method for detecting α -synuclein in a sample, comprising contacting the sample with a monoclonal antibody according to the first aspect of the invention, and detecting the complex formed for a time and under conditions sufficient for an α -synuclein-monoclonal antibody complex to form, thereby detecting α -synuclein in the sample.

Further, the monoclonal antibody may be detectably labeled.

A sixth aspect of the invention provides the use of any one of:

1) use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a biological material according to the third aspect of the invention, a cell according to the fourth aspect of the invention for detecting a-synuclein;

2) use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a biological material according to the third aspect of the invention, a cell according to the fourth aspect of the invention and a product of an alpha-synucleinopathy;

3) use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a biological material according to the third aspect of the invention, or a cell according to the fourth aspect of the invention, for the preparation of a medicament for the prevention and/or treatment of an α -synucleinopathy.

Further, the product comprises a kit.

Further, the kit comprises: colloidal gold immunoassay kit, chemiluminescence kit, radioimmunoassay kit, enzyme linked immunosorbent assay (ELISA), fluorescence immunoassay kit and microfluid chip.

Further, the alpha-synucleinopathy is a neurodegenerative disease.

Further, the neurodegenerative diseases include parkinson's disease, lewy body dementia, diffuse lewy body disease, lewy body variants of alzheimer's disease, combined alzheimer's and parkinson's disease, multiple system atrophy, neurodegeneration with brain iron accumulation type I.

Further, the neurodegenerative disease is parkinson's disease.

Drawings

FIG. 1 is a diagram of the recognition ability of western test immune mouse antisera against recombinant antigens;

FIG. 2 is a diagram showing the recognition ability of the culture supernatant of the 25F5C5 monoclonal antibody against recombinant antigens by western test;

FIG. 3 is a graph of the specificity of the combined detection of antigen binding by purified antibodies 53A2G6 and 25F5C 5.

Detailed Description

In order to prepare the antibody of alpha-synuclein with strong specificity and high affinity, the invention immunizes animals by preparing alpha-synuclein recombinant protein, thereby obtaining hybridoma cell strains secreting positive monoclonal antibodies, and further purifying to obtain the monoclonal antibodies with high specificity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, a monoclonal antibody encompasses a sequence having a certain degree of sequence identity or sequence homology with the amino acid sequence of the antibody or any nucleotide sequence encoding the antibody, and in the present invention, "homology" may be equivalent to "identity".

One skilled in the art will also appreciate that antibodies may be subjected to various post-translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are the deletion of a basic residue at the carboxy terminus (such as lysine or arginine) due to the action of carboxypeptidase.

As used herein, "identity" indicates that at any particular position in the aligned sequences, the amino acid residues between the sequences are identical. As used herein, "similarity" indicates that at any particular position of the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine may be substituted with isoleucine or valine. Other amino acids that may be substituted for one another in general include (but are not limited to): phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains), lysine, arginine and histidine (amino acids having basic side chains), aspartic acid and glutamic acid (amino acids having acidic side chains), asparagine and glutamine (amino acids having amide side chains), and cysteine and methionine (amino acids having sulfur-containing side chains).

Generally, modification of one or more amino acids in a protein does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative or variant. Typically, these changes are made over several amino acids to minimize changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, greater variations may be tolerated in some cases. Amino acid substitutions are typically of a single base; insertions will typically be on the order of about one to about twenty amino acid residues, although significantly larger insertions may be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases, deletions can be much larger.

Further, the amino acid sequence of the variable region including, but not limited to, the framework region, the hypervariable region, and particularly the CDR3 region, is modified. Typically, the light or heavy chain region includes three hypervariable regions (including three CDRs) and more conserved regions (so-called Framework Regions (FRs)). The hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops. Computer algorithms known to those skilled in the art, such as Gap or Bestfit, can be used to optimally align the amino acid sequences for comparison, and to define similar or identical amino acid residues. The parental monoclonal antibody or a part thereof can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis (oligonucleotide-directed mutagenesis) and site-directed mutagenesis (site-directed mutagenesis), or functional variants can be obtained by organic synthesis methods.

The antibodies and fragments disclosed herein are expressed at good levels from host cells. Thus, the properties of the antibody and/or binding fragment are suitable for expression on a commercial scale.

Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. "Fab" refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded; "Fab'" refers to a Fab fragment comprising part of the hinge region; "F (ab ') 2" refers to a dimer of Fab'; "Fv" refers to the smallest antibody fragment containing the heavy chain variable region, the light chain variable region of an antibody and having all antigen binding sites, and can be derived from any mammal, including but not limited to, human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more CDRs described herein, can be covalently linked to a secondary protein or small molecule compound and used as a target therapeutic agent for a particular target.

The expression vector of the present invention is not particularly limited, and its selection depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, bacteriophages and other vectors routinely used in, for example, genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors.

In one embodiment, the vector is an expression vector. The expression vector according to the invention is capable of directing the replication and expression of the nucleic acid molecule of the invention in a host and thus ensuring the expression of the variable chain domain of the anti-IgG antibody of the invention encoded thereby in the selected host. In a further embodiment, the one or more vectors comprise further sequences to ensure that not only said variable chain domain of the invention is expressed, but also a full length IgG antibody comprising said variable chain domain of the invention is expressed.

The expression vector may be, for example, a cloning vector, a binary vector or an integrative vector. Expression includes transcription of the nucleic acid molecule, e.g., into translatable mRNA.

Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13, CAT-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitron), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edgesys) pTriEx-hygro (Novagen) and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris (Pichia pastoris) include, for example, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus (Xenopus) embryos, zebrafish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2 +.

In general, a vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes. In addition, the coding sequences contained in the vector may be linked to transcriptional regulatory elements and/or to other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure initiation of transcription, an Internal Ribosome Entry Site (IRES), and optionally regulatory elements that ensure termination of transcription and stabilization of the transcript. Non-limiting examples of such regulatory elements that ensure initiation of transcription include promoters, translation initiation codons, enhancers, insulators, and/or regulatory elements that ensure termination of transcription, which are included downstream of the nucleic acid molecules of the invention. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences which, depending on the expression system used, are capable of directing the expressed protein to a cell compartment or culture medium. The vector may also contain additional expressible polynucleotides encoding one or more chaperones to facilitate proper protein folding.

Additional examples of suitable origins of replication include, for example, full-length ColE1, truncated ColEI, SV40 virus, and M13 origins of replication, yet additional examples of suitable promoters include, but are not limited to, the Cytomegalovirus (CMV) promoter, SV 40-promoter, RSV-promoter (rous sarcoma virus), lacZ promoter, tetracycline promoter/operator (tetp/o), chicken β -actin promoter, CAG-promoter (a combination of chicken β -actin promoter and cytomegalovirus immediate early enhancer), gai10 promoter, human elongation factor 1 α -promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, lac, trp or tac promoter, T7 or T5 promoter, lacUV5 promoter, Autographa californica (Autographa) polynedron promoter or globin intron in mammalian and other animal cells. An example of an enhancer is, for example, the SV 40-enhancer. Additional non-limiting examples of regulatory elements that ensure transcription termination include the SV 40-polyadenylation site, tk-polyadenylation site, rho factor-independent lpp terminator or AcMNPV polyhedric polyadenylation signal. Further non-limiting examples of selectable markers include dhfr, which confers resistance to methotrexate, npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromomycin (paromycin), and hygro, which confers resistance to hygromycin. An additional selection gene has been described, trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to utilize histidinol instead of histidine; mannose 6-phosphate isomerase, which allows the cell to utilise mannose and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine DFMO or confers resistance to blasticidin S a deaminase from Aspergillus terreus (Aspergillus terreus).

In a further embodiment, the vector is a eukaryotic expression plasmid containing an expression cassette consisting of a5 'CMV promoter including intron a and a 3' BGH polyadenylation sequence.

Suitable bacterial expression hosts include, for example, strains derived from JM83, W3110, KS272, TG1, K12, BL21 (e.g.BL 21(DE3), BL21(DE3) PlysS, BL21(DE3) RIL, BL21(DE3) PRARE) or Rosetta â.

The nucleic acid molecules and/or vectors of the invention can be designed to be introduced into cells by, for example, chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection, non-chemical methods (electroporation, sonoporation, light transfection, gene electrotransfer, fluid delivery, or transformation that occurs naturally when cells are contacted with the nucleic acid molecules of the invention), particle-based methods (gene gun, magnetic transfection, transfections by puncture), phage-based vector methods, and viral methods.

To facilitate purification of the nucleic acid molecules of the invention, a tag (tag) sequence may be inserted into the expression vector. Examples of tags include, but are not limited to, a hexa-histidine tag, a myc tag, or a FLAG tag. Any tag known to those skilled in the art to facilitate purification may be used in the present invention.

In the present invention, any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., E.coli) and other microbial systems may be used, or eukaryotic (e.g., mammalian) host cell expression systems may also be used. Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin. As the mammalian cell, one selected from the group consisting of, but not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, a549 cell, HEK293 cell, and HEK293T cell can be preferably used as the host cell. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.

The recombinant cells of the invention can then be used for expression as well as culture purposes for antibody expression for large scale drug production. Can also be used as active ingredient of pharmaceutical composition. Any suitable culture technique may be used, including but not limited to static culture, spinner flask culture, ascites fluid, hollow fiber-type bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.

As an alternative embodiment, the product of the invention comprises the antibody or functional fragment thereof prepared according to the invention. As another alternative embodiment, the article of manufacture of the invention comprises a diagnostic composition comprising at least one detectable label, such as a detectable moiety/agent. The tag may be non-covalently conjugated to a monoclonal antibody of the invention. The tag may also be conjugated directly to the monoclonal antibody by a covalent bond. Alternatively, the tag may be conjugated to the monoclonal antibody using one or more linking compounds. Techniques for conjugating tags to monoclonal antibodies are well known to those skilled in the art. The detectable moiety/agent as a label is preferably one selected from the group consisting of, but not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials and non-radioactive paramagnetic metal ions. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent substances include, but are not limited to, FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine-type labels, including TAMRA; dansyl; lissamine; cyanine; phycoerythrin; texas Red; and the like. Fluorescent labels can be conjugated to aldehyde groups contained in the target molecule using the techniques disclosed herein. Suitable luminescent substances include luminol, acridine compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and derivatives thereof; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125I, 131I, 111In, and 99 Tc.

The pharmaceutical composition of the present invention includes the monoclonal antibody of the present invention, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may additionally contain liquids such as water, physiological saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in the composition. These carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

Suitable administration forms include forms suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof according to the invention may be in dry form for reconstitution with a suitable sterile liquid prior to use. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection can also be prepared.

Once formulated, the compositions of the present invention can be administered directly to a subject. Accordingly, provided herein is the use of an antibody or antigen-binding fragment thereof according to the invention for the manufacture of a medicament.

The subject to be treated may be an animal. Preferably, the pharmaceutical composition according to the invention is adapted for administration to a human subject.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 preparation of anti-alpha-synuclein antibody

1. Immunogen treatment: the immunogen is recombinant alpha-synuclein, the purity and molecular weight of the protein are identified by SDS-PAGE, and the immunogenicity is enhanced by Immunoplus technology treatment.

2. Animal immunization: BALB/c mice were selected and immunized by the conventional method. After three times of immunization, the titer of the antiserum is tested by an indirect ELISA method, a mouse with high titer is selected for subsequent experiments, and the identification of the antiserum to the recombinant antigen is tested by using a western method.

3. Preparation of splenocytes: the mice were sacrificed by cervical drainage, the spleen was taken out under aseptic conditions and placed in a sterilized stainless steel mesh of 90-100 mesh. Injecting 3 ml of serum-free culture solution into the spleen by using a syringe, repeatedly pumping for several times to obtain cells, and then preparing a cell suspension. And injecting the cell suspension into a 50ml centrifuge tube, adding 10-20 ml of culture solution, gently blowing and beating for a plurality of times, and standing for 5 minutes at room temperature. And (4) centrifuging (800-1000 rpm) and counting for later use.

4. Cell fusion: mouse myeloma cells and mouse splenocytes were mixed as 1: 5, mixing the raw materials in a ratio of 5,the supernatant was discarded by centrifugation and excess supernatant was blotted with sterile filter paper. 1ml of 40% PEG solution was added dropwise to the cell pellet over 60 seconds while gently rotating the centrifuge tube. 1ml of serum-free culture medium was added dropwise over 60 seconds in a centrifuge tube which was continuously rotated. Then 20ml of serum-free medium was slowly added over 5 minutes. Centrifugation (800 rpm, 8 min), removal of supernatant, suspension in 10ml of complete medium and gentle mixing. The cell suspension was added to a 96-well plate at 50. mu.l per well. CO at 37 deg.C₂After 24 hours of culture in the incubator, HAT selective culture solution is replaced.

5. Cell culture after fusion: half the HAT culture solution is used for replacing the solution 7-10 days after fusion, and half the solution is replaced every 2-3 days later. Hybrid cell colonies appeared after 2-3 weeks. When the colonies proliferated and grown to 1/3 wells, the culture supernatants were subjected to antibody detection by ELISA. Recombinant protein is used as an antigen coating enzyme label plate, the concentration of the coating antigen is 1 mu g/ml, and 100 mu l/hole. Coating buffer was PBS (PH = 7.4). Left overnight at 4 ℃. The next day PBS was washed 3 times for 5 minutes each. Blocking with 1% BSA was performed by adding 200. mu.l per well. Incubate at 37 ℃ for 2 hours in an incubator. BSA was discarded and cell culture supernatant containing monoclonal antibody was added in an amount of 100. mu.l per well. The positive control was positive antiserum from mice and the negative control was blank culture supernatant. Incubate at 37 ℃ for 2 hours in an incubator. The primary antibody was discarded, washed 5 times with the washing solution, added with Peroxidase-affinity Goat Anti-Mouse IgG, and incubated at 37 ℃ for 1 hour in a thermostat. Adding substrate for color development, and measuring the light absorption value by an enzyme-labeling instrument.

And (3) performing another round of cloning culture on the detected positive cells, determining positive cell strains after verification, performing cryopreservation and in-vitro culture after proliferation.

6. Results

After three times of immunization, the results of titer detection for recognizing recombinant antigens by antiserum of six immunized mice (# 4142- # 4147) by using an indirect ELISA method are shown in Table 1, and the results show that the recombinant alpha-synuclein has better immunogenicity and can generate stronger immune response reaction after the mice are immunized.

TABLE 1 mouse serum potency assay

The western is used for detecting the recognition of the immune mouse antiserum to the recombinant antigen, the result is shown in figure 1, and the result shows that the immune mouse antiserum has better recognition capability to the recombinant antigen.

After the positive cell strain is subcloned, a plurality of positive cell strains which stably secrete monoclonal antibodies are obtained: 53A2G6, 22B5G6, 25F5C5, 34E10D8, 3D4H 7. The results of ELISA detection of the culture supernatant of hybridoma cell line 25F5C5 are shown in Table 2, which indicates that hybridoma cell line 25F5C5 produces an antibody against recombinant α -synuclein.

TABLE 2 ELISA assay for hybridoma cell lines

Example 2 purification and sequencing of monoclonal antibodies

Firstly, a culture solution for generating a monoclonal antibody is precipitated by using semi-saturated and saturated ammonium sulfate, and is primarily concentrated and purified; further purified by affinity chromatography.

1. Preliminary precipitation of ammonium sulfate solution

Salting out is carried out with a saturated ammonium sulfate solution. The required amount is taken before use, and the PH value is adjusted to 7.8 by 2mol/L NaOH; transferring the culture solution for generating the antibody into a beaker, dropwise adding 5ml of saturated ammonium sulfate solution while stirring, and continuously and slowly stirring for 30 minutes; centrifuging at 10000rpm/min for 15 minutes; discarding supernatant, suspending the precipitate with 1/3 saturated ammonium sulfate, stirring for 30 min, and centrifuging by the same method; repeating the previous step for 1-2 times; the precipitate was dissolved in PBS (0.01 mol/L pH 7.2) buffer.

Desalting the salted-out sample by dialysis. The dialysis bag was filled with 2% NaHCO₃Boiling in 1mmol/L EDTA solution for 10 min, washing the inner and outer surfaces of the dialysis bag with distilled water, boiling the dialysis bag with distilled water for 10 min, and cooling to room temperature. The salted-out sample was put into a dialysis bag and dialyzed against 50 to 100-fold volume of PBS buffer (4 ℃ C.) for 12 hoursAfter 24 hours, the dialysis solution was changed 5 times and measured with naphthalene reagent (11.5 g of mercuric iodide, 8g of potassium iodide, 50ml of distilled water, and 50ml of 20% NaOH after dissolution) until no yellow substance was formed in the dialysis solution.

2. Affinity chromatography for antibody purification

And filtering the primarily purified antibody solution through a protein A/G affinity chromatography column, and obtaining the high-purity antibody through combination, elution and collection. Antibody concentration was determined using a spectrophotometer. The purified antibody was aliquoted and stored at-80 ℃.

3. Determination of monoclonal antibody sequences

Hybridoma 25F5C5 in the logarithmic growth phase was taken, total RNA was extracted by Trizol from Invitrogen, and cDNA was generated by reverse transcription. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And (3) after the PCR product is purified by electrophoresis, inserting the PCR product into a vector by TA cloning, sequencing and carrying out sequence analysis.

4. Results

The sequence detection results are shown in Table 3, and the amino acid sequences of the CDR1-3 of the heavy chain and the CDR1-3 of the light chain are respectively shown in SEQ ID NO.1-3 and SEQ ID NO. 11-13; the amino acid sequences of the heavy chain variable region and the light chain variable region are shown as SEQ ID NO.9 and SEQ ID NO.19, respectively.

TABLE 3 monoclonal antibody sequences

EXAMPLE 3 identification of monoclonal antibodies

1. The detection was performed using alpha-synuclein in clinical samples as an antigen, monoclonal culture supernatants of mouse hybridoma 25F5C5, and the ability of the antibody to recognize the antigen was determined using an indirect ELISA method.

2. The alpha-synuclein in clinical samples is used as an antigen, the monoclonal antibody-containing mouse hybridoma 25F5C5 culture supernatant is used for detection, and the binding capacity of the antibody and the antigen is detected by using western blot.

3. A double-antibody sandwich ELISA method is applied, 53A2G6 is used as a coating antibody, and the following antibodies are used as detection antibodies: 3D4H7-biotin, 22B5G6-biotin, 25F5C5-biotin, 34E10D8-biotin, 49B2G9-biotin, 53A2G6-biotin, 63E12C2-biotin, 68E6D6-biotin, 71G2F7-biotin, 75G12C8-biotin, 77G9C11-biotin, and detecting the recognition and capture effects of the antibody on the antigen.

The plate was coated with 2.5. mu.g/ml of the purified antibody in PBS (pH = 7.4) and left overnight at 4 ℃. Washing with washing solution for 3 times, adding recombinant protein as antigen at concentrations of 0, 1, 10, and 100ng/ml, respectively. Incubate at 37 ℃ for 1 hour. The washing was carried out 3 times, and a biotin-labeled detection antibody was added at a concentration of 1. mu.g/ml. Incubate at 37 ℃ for 1 hour. Washing 3 times, adding HRP labeled streptavidin, combining with detection antibody, the concentration is 1mg/ml, diluting 1:10,000, adding 100 μ l per hole. Incubate at 37 ℃ for 30 minutes. Adding substrate for color development, and measuring light absorption value by an enzyme-labeling instrument.

4. Monoclonal antibody 53A2G6 was paired with antibody 25F5C5-biotin, the specificity of binding of the purified antibody to the antigen was examined, the antigen was diluted in multiples and plotted against antigen concentration and absorbance.

5. Results

The detection result of the indirect ELISA method is shown in Table 4, the absorbance value of the antibody is more than 3 and is far greater than the negative control value of 0.154, which indicates that the antibody has good affinity to alpha-synuclein.

TABLE 4 ELISA detection of monoclonal antibodies

The Western detection result is shown in FIG. 2, and a strong positive band appears at a position of about 14KD, which indicates that the antibody has strong binding force to the antigen in the clinical sample.

The results of the double antibody sandwich ELISA are shown in Table 5, the antibody 53A2G6 successfully pairs with 25F5C5-biotin, and the absorbance value rises with the increase of the antibody content. When the antigen concentration is 100ng/ml, the absorbance value is more than 3, which is obviously higher than that of the negative control. Indicating that the antibody has strong recognition and capture effects on the antigen.

TABLE 5 recognition of antigen by purified antibody

The combination of monoclonal antibody 53A2G6 and antibody 25F5C5-biotin pairing to detect specific binding of antigen and antibody is shown in FIG. 3, and the absorbance increases with the antigen concentration, indicating that the antibody has specificity for antigen binding.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Beijing Kaixiang Hongkang Biotechnology Co Ltd

<120> antibody specifically binding to alpha-synuclein and use thereof

<141> 2021-11-22

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aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgaaagtgt tgagtctgtt gtacctgttg acagccattc ctggtatcct gtctgatgta 60

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tctgtcactg gctactccat caccagtggt ttttactgga attggatccg gcagtttccg 180

ggaaacaaac tggaatggat gggctacata aacagcgatg gtagcaataa ctacaaccca 240

tctctcaaaa atcgaatctc catcactcgt gacacatcta agaaccagtt tttcctgaag 300

ttgaattctg tgactactga ggacatagcc acatattact gtgtaagagg ggactgggac 360

tggggccaag ggactctggt cactgtctct gca 393

<210> 31

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aagtcaagtc agagcctctt agatgttgat ggagagacat atttgaat 48

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ctggtgtcta aactggactc t 21

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tggcaaggta cacattttcc tcagacg 27

<210> 34

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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<210> 37

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ttcggtggag gcaccaagct ggaaatcaaa 30

<210> 38

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

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<210> 39

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gatattgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60

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ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgataggtt cactggcagt ggatcaggga cagatttcac gctgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300

cagacgttcg gtggaggcac caagctggaa atcaaa 336

<210> 40

<211> 393

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atgagtcctg cccagttcct gtttctgtta gtgctctgga ttcgggaaac caacggtgat 60

attgtgatga cccagactcc actcactttg tcggttacca ttggacaacc agcctccatc 120

tcttgcaagt caagtcagag cctcttagat gttgatggag agacatattt gaattggttg 180

ttacagaggc caggccagtc tccaaagcgc ctaatctatc tggtgtctaa actggactct 240

ggagtccctg ataggttcac tggcagtgga tcagggacag atttcacgct gaaaatcagc 300

agagtggagg ctgaggattt gggagtttat tattgctggc aaggtacaca ttttcctcag 360

acgttcggtg gaggcaccaa gctggaaatc aaa 393

Claims

1. An anti- α -synuclein monoclonal antibody, wherein the monoclonal antibody comprises three CDRs from the heavy chain variable region and three CDRs from the light chain variable region; wherein, the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.1, 2 and 3, and the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO.11, 12 and 13.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises heavy chain variable region framework regions FR1, FR2, FR3 and FR 4; the light chain variable region further comprises light chain variable region framework regions FR1, FR2, FR3 and FR4, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO.4, 5, 6 and 7; the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO.14, 15, 16 and 17.

3. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody comprises:

(a) a heavy chain variable region sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID No. 9;

(b) a light chain variable region sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID No. 19; or

4. A nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1-3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. The vector of claim 5, further comprising a first signal peptide operably linked to the heavy chain of the monoclonal antibody, and/or a second signal peptide operably linked to the monoclonal antibody.

7. The vector of claim 6, wherein the amino acid sequence of the first signal peptide is as set forth in SEQ ID No. 8; the amino acid sequence of the second signal peptide is shown as SEQ ID NO. 18.

8. A cell comprising the nucleic acid molecule of claim 4 or the vector of any one of claims 5 to 7.

9. A method for the detection of alpha-synuclein in a sample for non-diagnostic purposes,

comprising contacting the sample with the monoclonal antibody of any one of claims 1-3, and detecting the complex formed for a time and under conditions sufficient for formation of an alpha-synuclein-monoclonal antibody complex, thereby detecting alpha-synuclein in the sample.

10. Use according to any one of the following:

1) use of the monoclonal antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5-7, the cell according to claim 8 for the detection of α -synuclein for non-diagnostic purposes;

2) use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5 to 7, the cell according to claim 8 for the preparation of a product for the diagnosis of an α -synucleinopathy;

3) use of the monoclonal antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the vector according to any one of claims 5-7, the cell according to claim 8 for the preparation of a medicament for the prevention and/or treatment of an α -synuclein-associated disease.