An antibody that specifically binds to α-synuclein, and related products and uses thereof

Abstract

The present invention discloses an antibody specifically binding to α-synuclein, and related products and uses thereof. The present invention provides a novel monoclonal antibody targeting α-synuclein for the field. The monoclonal antibody can specifically bind to α-synuclein, and has good specificity and affinity. The present invention lays a foundation for the development of α-synuclein-related detection and / or auxiliary detection products in the field. At the same time, the present invention is expected to provide more diagnostic tools and treatment strategies for the diagnosis and / or treatment of α-synucleinopathies, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antibody that specifically binds to α-synuclein, and related products and uses thereof. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease. Its main pathological feature is the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta and the formation of Lewy bodies in the remaining neurons. In addition to motor symptoms such as muscle rigidity, bradykinesia, resting tremor, and postural gait abnormalities, PD also presents non-motor symptoms such as olfactory impairment, constipation, and rapid eye movement sleep disorder. Moreover, non-motor symptoms appear 10 - 20 years earlier than motor symptoms. By the time patients present with motor symptoms, the disease has reached an irreversible stage. Therefore, early diagnosis and treatment of PD are of particular importance.

[0003] α-Synuclein (SNCA) is the main structural component of Lewy bodies, which is one of the pathological features of PD and is formed by the aggregation of α-synuclein. In the brain tissue of PD patients, α-synuclein is abnormally expressed and aggregated, leading to biochemical reactions such as oxidative stress and the formation of oligomeric intermediate conformations, which play important roles in the pathogenesis of PD. α-Synuclein is a soluble protein expressed in the presynaptic and perinuclear regions of the central nervous system, consisting of 140 amino acids with a molecular weight of 14 kDa. α-Synuclein is the most important protein in the pathogenesis of PD.

[0004] Antibodies targeting α-synuclein can be used to detect and analyze the expression and aggregation of α-synuclein, and can also be used to further understand the pathogenesis of Parkinson's disease, providing clues for the development of new treatment methods. In addition, the antibody can also be used to detect the content of α-synuclein in cerebrospinal fluid, blood, or other biological samples, and can be used as a diagnostic reagent, providing an effective means for the early detection and diagnosis of Parkinson's disease. Therefore, the development and research of antibodies targeting α-synuclein have important clinical and scientific values. Currently, there is still an urgent need in this field to develop a monoclonal antibody product with good affinity and specificity for α-synuclein. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a novel antibody that specifically binds to α-synuclein, and related products and uses thereof for this field.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention purpose:

[0007] In the first aspect of the present invention, an antibody against α-synuclein is provided.

[0008] Further, HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 1;

[0009] LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence as shown in SEQ ID NO: 2.

[0010] Further, the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are respectively as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5;

[0011] The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are respectively as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0012] Further, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1 or has at least 75% homology with SEQ ID NO: 1;

[0013] Optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2 or has at least 75% homology with SEQ ID NO: 2.

[0014] In some embodiments, amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the present invention are also within the scope of protection of the present invention. The amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology include those obtained by one or more amino acid or nucleotide deletion, insertion, or substitution mutations of the parental sequence.

[0015] In some embodiments, the amino acid sequences corresponding to HCDR1, HCDR2, and HCDR3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and the amino acid sequences corresponding to LCDR1, LCDR2, and LCDR3 of the present invention are also not limited to the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Using any CDR numbering scheme (existing CDR numbering schemes or new CDR numbering schemes that may be developed in the future) to define CDR1, CDR2, and CDR3 in the heavy chain variable region shown in SEQ ID NO:1 and CDR1, CDR2, and CDR3 in the light chain variable region shown in SEQ ID NO:2 respectively, the amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (and the nucleotide sequences corresponding to the amino acid sequences) all fall within the protection scope of the present invention.

[0016] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can be defined by using any one or any combination (two or more) of the IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, Aho numbering scheme (or other existing CDR numbering schemes or new CDR numbering schemes that may be developed in the future) for the amino acid sequences corresponding to the heavy chain variable region and the light chain variable region of the antibody as described above in the present invention. The sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 defined by the above definition method also fall within the protection scope of the present invention.

[0017] In some embodiments, the functional variants of the antibody provided in the first aspect of the present invention are also included within the protection scope of the present invention.

[0018] In some embodiments, the functional variant refers to a protein that has significant or remarkable sequence homology or similarity compared to the parental antibody (the antibody described in the first aspect of the present invention), and the functional variant retains the biological activity of the parental antibody. Functional variants encompass, for example, the following variants of the antibody (parental antibody) described herein, which retain the ability to recognize target cells to a similar extent, to the same extent, or to a higher extent compared to the parental antibody. With reference to the parental antibody, the functional variant may, for example, have at least about 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95% or higher homology with the parental antibody in the amino acid sequence.

[0019] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parental antibody having at least one conservative amino acid substitution. As an alternative or in addition, the functional variant may comprise the amino acid sequence of a parental antibody having at least one non-conservative amino acid substitution. In such cases, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parental antibody. Such functional variants obtained by conservative or non-conservative amino acid substitutions based on the parental antibody also fall within the scope of protection of the present invention.

[0020] In some embodiments, conservative amino acid substitutions are known in the art and include substituting one amino acid having specific physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be substituting one acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substituting an amino acid having a non-polar side chain with another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substituting one basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substituting an uncharged amino acid having a polar side chain with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substituting an amino acid having a β-branched side chain with another amino acid having a β-branched side chain (e.g., Ile, Thr, and Val), or substituting an amino acid having an aromatic side chain with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0021] The second aspect of the present invention provides a bispecific antibody.

[0022] Furthermore, the bispecific antibody comprises the antibody described in the first aspect of the present invention;

[0023] Optionally, the bispecific antibody further comprises a second antibody that specifically binds to another antigen.

[0024] In some embodiments, the second antibody that specifically binds to another antigen is not particularly limited and may be another antibody targeting α-synuclein or an antibody targeting any antigen other than α-synuclein, which can be conventionally selected by those skilled in the art according to actual needs.

[0025] In some embodiments, the bispecific antibody comprises binding specificities for two different antigens, one of which is α-synuclein and the other is any antigen other than α-synuclein. In other embodiments, the bispecific antibody comprises binding specificities for different epitopes or the same epitope of the same antigen (e.g., having different binding affinities and / or specific epitopes for the same antigen).

[0026] In some embodiments, the antigen other than α-synuclein includes, but is not limited to: LRRK2 (leucine-rich repeat kinase 2), dopamine receptors (D1 and D2 receptors), tau protein.

[0027] The third aspect of the present invention provides a nucleic acid molecule.

[0028] Furthermore, the nucleic acid molecule encodes the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention.

[0029] Those of ordinary skill in the art can easily mutate the nucleotide sequence corresponding to the antibody of the present invention by known methods, such as directed evolution and site-directed mutagenesis methods. As long as it encodes the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention, and is derived from the nucleotide sequence of the present invention and equivalent to the sequence of the present invention, it also falls within the protection scope of the present invention.

[0030] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained by conventional techniques or by using hybridoma techniques. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. In addition, the relevant sequence can also be synthesized by artificial synthesis methods, especially when the fragment length is short. Usually, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0031] The fourth aspect of the present invention provides a recombinant vector.

[0032] Furthermore, the recombinant vector comprises the nucleic acid molecule described in the third aspect of the present invention;

[0033] Optionally, the vector is a DNA vector, an RNA vector, a plasmid, a transposon vector, and / or a virus-derived vector;

[0034] Optionally, the virus-derived vector is an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a poxvirus vector, and / or a herpesvirus vector.

[0035] In some embodiments, the recombinant vector is an expression vector or a cloning vector.

[0036] In some embodiments, any vector capable of delivering nucleic acids is applicable to the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0037] The fifth aspect of the present invention provides a recombinant host cell.

[0038] Furthermore, the recombinant host cell comprises the recombinant vector described in the fourth aspect of the present invention;

[0039] Optionally, the host cell is a mammalian cell, an insect cell, and / or a yeast cell.

[0040] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cells include, but are not limited to: mammalian cells, insect cells, plant cells, yeast cells. In some embodiments, the host cells for expressing the antibody or bispecific antibody of the present invention include Escherichia coli, yeast cells, insect cells, COS cells, CHO cells, immune cells, etc. In some embodiments, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof.

[0041] In some embodiments, the recombinant host cell is prepared by the following method: introducing the nucleic acid molecule or expression vector as described above in the present invention into a host cell, and the introducing methods include, but are not limited to: physical methods, chemical methods, biological methods. The physical methods include, but are not limited to: calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation; the chemical methods include, but are not limited to: colloidal dispersion systems, lipid-based systems; the colloidal dispersion systems include, but are not limited to: macromolecular complexes, nanocapsules, microspheres, beads; the lipid-based systems include, but are not limited to: water-in-oil emulsions, micelles, mixed micelles, liposomes; the biological methods include, but are not limited to: DNA vectors, lentiviral vectors, vaccinia virus vectors, herpes simplex virus vectors, adenovirus vectors, adeno-associated virus vectors.

[0042] In some embodiments, the nucleic acid molecules or expression vectors of the present invention as described above can be introduced into host cells by various suitable means, not limited to the methods listed in the present invention, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (such as liposomes) or viral vector-mediated transfection (such as lentiviral infection, retroviral infection, adenoviral infection), as well as other physical, chemical or biological means for transferring into cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0043] The sixth aspect of the present invention provides any one of the following products:

[0044] (1) An antibody conjugate, which is a complex formed by directly or indirectly conjugating the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention to a detectable label;

[0045] (2) A detection reagent, which contains the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention or the antibody conjugate;

[0046] (3) A detection product, which contains the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate or the detection reagent;

[0047] (4) A pharmaceutical composition, which contains the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention;

[0048] (5) A biological preparation, which contains the pharmaceutical composition;

[0049] Optionally, the detectable label is a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitizing diagnostic agent, a radionuclide or a chemiluminescent agent;

[0050] Optionally, the detection product is a detection kit, a test strip or a detection chip.

[0051] In some embodiments, the bioluminescent agents include, but are not limited to: luciferin, luciferase, aequorin; the paramagnetic ions include, but are not limited to: chromium(III), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), manganese(II), iron(III), iron(II), cobalt(II), gadolinium(III), vanadium(II); the enzymes include, but are not limited to: horseradish peroxidase, catalase, alkaline phosphatase, urease, glucose oxidase, β-D-galactosidase, or glucoamylase; the photosensitizing diagnostic agents include, but are not limited to: dihydroxysilicon phthalocyanine, protoporphyrin, hematoporphyrin, methylene blue, photoporphyrin; the radionuclides include, but are not limited to: 68 Ga, 86 Y, 90 Y, 89 Zr, 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 31 I, 154-158 Gd, 32 F, 11 C, 67 Cu, 67 Ga, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 113 N, 15 O, 186 Re, 188 Re; the chemiluminescent agents include, but are not limited to: aromatic acridinium esters, imidazoles, luminol, isoluminol, acridinium salts, oxalates.

[0052] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient, which are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995).

[0053] In some embodiments, the dosage forms of the biological agents include, but are not limited to: solutions, emulsions, tablets, pills, powders, granules, capsules, suspensions, syrups, sterile aqueous solutions, non-aqueous solutions, suspending agents, emulsions, freeze-dried preparations or suppositories.

[0054] In some embodiments, the administration routes of the pharmaceutical composition or biologic agent include, but are not limited to: intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, oral, topical, intranasal, intrapulmonary, rectal, etc. When administered orally, it can be formulated with a coating that protects the active ingredient in the pharmaceutical composition or biologic agent from degradation in the stomach. In addition, the active ingredient can be administered by any device capable of delivering it to the target tissue.

[0055] In specific embodiments, the pharmaceutical composition or biologic agent provided by the present invention can be made into various dosage forms according to actual needs, and a clinician can determine a beneficial dose for the patient according to factors such as the type, age, weight, and general disease condition of the subject, and the administration route, etc. The administration route can be, for example, injection or any other suitable administration route known to those skilled in the art.

[0056] The seventh aspect of the present invention provides any one of the following methods:

[0057] (1) A method for preparing the recombinant host cell described in the fifth aspect of the present invention, the method comprising: introducing the recombinant vector described in the fourth aspect of the present invention into a host cell to obtain the recombinant host cell described in the fifth aspect of the present invention;

[0058] (2) A method for producing the antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention, the method comprising: culturing the recombinant host cell described in the fifth aspect of the present invention and isolating the antibody or bispecific antibody from the culture product of the recombinant host cell;

[0059] (3) A method for non-diagnostic and non-therapeutic detection of α-synuclein in a test sample, the method comprising: contacting the test sample with the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, a detection reagent or a detection product, and detecting the formation of an immune complex of α-synuclein with the antibody;

[0060] (4) A method for in vitro inhibiting the activity of α-synuclein in a sample, the method comprising: contacting the sample with the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, a detection reagent or a detection product.

[0061] In some embodiments, the method for introducing the nucleic acid molecule into the host cell is well-known to those skilled in the art and includes, but is not limited to: physical methods, chemical methods, or biological methods.

[0062] In some embodiments, the sample or sample to be tested can be selected from blood, serum, plasma, brain tissue, cerebrospinal fluid, urine, saliva, ascites, non-tissue-associated cells, tissues, histological preparations, etc. derived from the subject to be tested. The present invention does not particularly limit the specific type of the sample or sample to be tested, and any sample that may contain alpha-synuclein can be used as the sample or sample to be tested.

[0063] The eighth aspect of the present invention provides any one of the following applications:

[0064] (1) The application of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention in the preparation of an antibody conjugate for detecting alpha-synuclein;

[0065] (2) The application of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, or the antibody conjugate described in the sixth aspect of the present invention in the preparation of a detection reagent for detecting alpha-synuclein;

[0066] (3) The application of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, or the detection reagent in the preparation of a detection product for detecting alpha-synuclein;

[0067] (4) The application of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent, or the detection product in the detection of alpha-synuclein for non-diagnostic and non-therapeutic purposes;

[0068] (5) The application of the antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant vector described in the fourth aspect of the present invention, the recombinant host cell described in the fifth aspect of the present invention, the antibody conjugate described in the sixth aspect of the present invention, the detection reagent, or the detection product in the preparation of a diagnostic product for diagnosing or assisting in diagnosing alpha-synucleinopathy;

[0069] (6) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the recombinant vector according to the fourth aspect of the present invention, or the recombinant host cell according to the fifth aspect of the present invention in the preparation of a medicament for treating and / or preventing α-synucleinopathy;

[0070] (7) Use of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the recombinant vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of a biological agent for treating and / or preventing α-synucleinopathy;

[0071] Optionally, the α-synucleinopathy is a neurodegenerative disease;

[0072] Optionally, the neurodegenerative disease is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Lewy body variant of Alzheimer's disease, diffuse Lewy body disease, combined Alzheimer's disease and Parkinson's disease, or neurodegeneration with brain iron accumulation type I;

[0073] Optionally, the neurodegenerative disease is Parkinson's disease.

[0074] In some embodiments, the α-synucleinopathy is a general term for a class of neurodegenerative diseases, and any disease caused by abnormal aggregation and deposition of α-synuclein is within the protection scope of the present invention, not limited to the specific disease types listed above.

[0075] The present invention provides a method for diagnosing and / or assisting in the diagnosis of α-synucleinopathy, the method comprising: detecting a test sample derived from a subject using the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the recombinant vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, the antibody conjugate, detection reagent or detection product according to the sixth aspect of the present invention, and detecting the presence or content of α-synuclein in the test sample derived from the subject through an antigen-antibody reaction to diagnose and / or assist in diagnosing whether the subject has α-synucleinopathy or the risk of having α-synucleinopathy.

[0076] The present invention provides a method for treating and / or preventing α-synucleinopathy, the method comprising: administering to a subject in need a therapeutically and / or prophylactically effective amount of the antibody according to the first aspect of the present invention, the bispecific antibody according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the recombinant vector according to the fourth aspect of the present invention, the recombinant host cell according to the fifth aspect of the present invention, the pharmaceutical composition or biological agent according to the sixth aspect of the present invention.

[0077] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0078] The present invention provides a novel monoclonal antibody targeting α-synuclein for the field. The monoclonal antibody can specifically bind to α-synuclein and has good specificity and affinity. The present invention lays a foundation for the development of α-synuclein-related detection and / or auxiliary detection products in the field. At the same time, the present invention is expected to provide more diagnostic tools and treatment strategies for the diagnosis and / or treatment of α-synucleinopathy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 : The affinity detection result diagram of the antibody targeting α-synuclein prepared by the present invention;

[0080] Figure 2 : The specificity detection result diagram of the antibody targeting α-synuclein prepared by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0081] The inventors of the present invention have conducted extensive and in-depth research, and screened a novel monoclonal antibody targeting α-synuclein by using monoclonal antibody technology. The monoclonal antibody can specifically bind to α-synuclein, has good specificity and affinity, and can be applied to the development of α-synuclein-related detection and / or auxiliary detection products.

[0082] The amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody targeting α-synuclein is shown as SEQ ID NO:1, the amino acid sequence of the light chain variable region (VL) of the monoclonal antibody targeting α-synuclein is shown as SEQ ID NO:2, the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are shown as SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown as SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 respectively.

[0083] To better understand the technical solutions related to the present application, the following terms involved in the present application are explained as follows. Unless otherwise specified, the following terms involved in the present application refer to the following content.

[0084] As used herein, the term "comprising" or "including" refers to the compositions, methods, and their corresponding components present in a given embodiment, but is also open-ended, including unspecified elements.

[0085] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain CL.

[0086] Among them, the VH region and the VL region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), and the hypervariable regions are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Among them, the three CDR regions of the heavy chain are represented by HCDR1, HCDR2, and HCDR3 respectively, and the three CDR regions of the light chain are represented by LCDR1, LCDR2, and LCDR3 respectively. The variable regions of the heavy chain and the light chain contain binding domains that interact with the antigen.

[0087] As used herein, the term "homology" refers to the sequence similarity to a target amino acid sequence or nucleotide sequence. "Homology" includes amino acid sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology with the amino acid sequence of the antibody provided by the present invention; nucleotide sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology with the nucleotide sequence of the antibody provided by the present invention. Homology can be evaluated by the naked eye or computer software. When using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. The homology of 75% or more than 75% can be 75%, 80%, 85%, 90%, or 95% or more homology.

[0088] In some embodiments, modification of one or more amino acids in a protein generally does not affect the function of the protein. Those skilled in the art will recognize that changing a single amino acid or a small percentage of amino acids or individual additions, deletions, insertions, or substitutions to the amino acid sequence are conservative modifications, where the change to the protein results in a protein with similar function. Tables of conservative substitutions providing amino acids with similar function are well known in the art. Amino acid sequences obtained by such conservative substitutions are also included within the scope of the present invention.

[0089] Final derivatives or variants can be achieved using substitutions, deletions, insertions, or any combination thereof. Generally, these changes are made to a few amino acids to minimize changes to the molecule, particularly the immunogenicity and specificity of an antigen-binding protein. However, in certain cases, larger changes can be tolerated. Amino acid substitutions are typically single base; insertions will generally 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.

[0090] As used herein, the term "nucleic acid molecule" can include natural, non-natural, or altered nucleotides; and it can include natural, non-natural, or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester present between nucleotides in an unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions, and thus, nucleic acids formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of the present invention.

[0091] As used herein, the term "expression vector" can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. Vectors can be selected from the pUC series (Fermentas Life Sciences, GlenBumie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA).

[0092] In some embodiments, the expression vector further comprises a vector with a suitable promoter or control sequence. These expression vectors can be used to transform suitable host cells to enable them to express proteins. The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: bacterial cells such as Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, NSO or Bowes melanoma cells, etc. Host cells particularly suitable for the present invention are eukaryotic host cells, especially mammalian cells, such as CHO cells, 293 cells, etc.

[0093] As used herein, the term "cloning vector" refers to a DNA molecule having the ability to replicate autonomously in a host cell, such as a plasmid, cosmid or phage. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites and a marker gene, wherein the foreign DNA sequence is inserted in a defined manner at the restriction endonuclease recognition site without losing the essential biological functions of the vector, and the marker gene is suitable for the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes providing tetracycline resistance or ampicillin resistance.

[0094] In some embodiments, the present invention does not particularly limit the specific type of the vector, and its selection depends on the desired function. Non-limiting examples of vectors include, but are not limited to: plasmid vectors, virus-derived vectors (such as lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, herpesviral vectors, baculoviral vectors, papillomaviral vectors, polyomaviral vectors), phage vectors and other vectors commonly used in, for example, genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art.

[0095] In some embodiments, the vectors of the present invention 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. Additionally, established methods can be used to ligate the coding sequences contained in the vector to transcriptional regulatory elements and / or to other amino acid coding sequences. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure transcriptional initiation, internal ribosome entry sites (IRES), and optionally regulatory elements that ensure transcriptional termination and transcript stability. Non-limiting examples of such regulatory elements that ensure transcriptional initiation include promoters, translation initiation codons, enhancers, insulators, and / or regulatory elements that ensure transcriptional termination, which are included downstream of the nucleic acid molecules of the present 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 depending on the expression system used, which are capable of directing the expressed protein to a cellular compartment or the culture medium. The vector may also contain additional expressible polynucleotides encoding one or more protein chaperones to facilitate proper protein folding.

[0096] In some embodiments, additional examples of suitable origins of replication include, but are not limited to: full-length ColE1, truncated ColEI, SV40 virus, and M13 origins of replication, while additional examples of suitable promoters include, but are not limited to: cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter, lacZ promoter, tetracycline promoter / operator, chicken β-actin promoter, CAG-promoter, gai10 promoter, human elongation factor 1α-promoter, AOX1 promoter, GAL1 promoter, etc. An example of an enhancer is, for example, the SV40-enhancer. Non-limiting additional examples of regulatory elements that ensure transcriptional termination include the SV40-polyadenylation site, the tk-polyadenylation site, the ρ-independent lpp terminator, or the AcMNPV polyhedron polyadenylation signal. Further non-limiting examples of selection markers include dhfr, which confers resistance to methotrexate, npt, which confers resistance to the aminoglycosides neomycin, kanamycin, and paromycin, and hygro, which confers resistance to hygromycin. Additional selectable genes have been described, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to utilize histinol instead of histidine; mannose 6-phosphate isomerase, which allows cells to utilize mannose, and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine (DFMO) or a deaminase from Aspergillus terreus that confers resistance to blasticidin S.

[0097] To facilitate the purification of the nucleic acid molecules of the present invention, a tag sequence can be inserted into the expression vector. Examples of tags include, but are not limited to: a hexahistidine tag, a myc tag, or a FLAG tag. Any tag known to those skilled in the art for facilitating purification can be used in the present invention.

[0098] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, mice, amphibians, and reptiles. In a specific embodiment of the present invention, the subject is preferably human.

[0099] As used herein, the term "therapeutically and / or prophylactically effective amount" refers to the amount of the antibodies, bispecific antibodies, pharmaceutical compositions, and / or biological agents described herein that can effectively inhibit, prevent, arrest, delay, or treat the symptoms of a particular disease, disorder, or side effect. Such diseases, disorders, and side effects include, but are not limited to, those pathological conditions associated with the abnormal aggregation and deposition of α-synuclein, wherein treatment or prevention includes, for example, inhibiting its activity by contacting cells, tissues, or receptors with the antibodies, bispecific antibodies, pharmaceutical compositions, and / or biological agents described in the present invention. The therapeutically and / or prophylactically effective amount can vary depending on the route of administration and dosage form, the age and weight of the subject being treated, and / or the disease or condition being treated.

[0100] The present invention will be further illustrated below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and spirit of the present invention, various changes, modifications, substitutions, and variations can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents.

[0101] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained from commercial sources. The experimental methods without specific conditions described in the present invention are usually carried out under the conventional conditions in the art or according to the conditions recommended by the manufacturer. In particular, the following embodiments are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and their results described in the following embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0102] Example 1 Preparation of an Antibody Targeting α-Synuclein

[0103] 1. Preparation and Treatment of Immunogen

[0104] The immunogen is recombinant α-synuclein. First, its purity and molecular weight are identified by SDS-PAGE technology. To ensure enhanced immunogenicity, we used ImmunoPlus technology to process the protein.

[0105] 2. Mouse immunization protocol

[0106] BALB / c strain mice are selected for conventional immunization. After three immunization cycles, the titer of the antiserum is detected by indirect ELISA technology, and the mouse with the highest titer is selected for subsequent experiments. Meanwhile, the specific recognition ability of the antiserum to the recombinant antigen is verified by Western blotting technology.

[0107] 3. Isolation and preparation of splenocytes

[0108] After the selected mice are sacrificed by cervical dislocation, the spleens are quickly removed under sterile conditions. The spleens are placed in a sterilized 90 - 100 mesh stainless steel mesh, and 3 mL of serum-free culture medium is injected into the spleens using a syringe, and cells are obtained by repeated aspiration. Subsequently, the cells are made into a suspension and injected into a 50 mL centrifuge tube, and an appropriate amount of culture medium is added for pipetting and static treatment. After centrifugation and counting, the splenocytes are reserved for use.

[0109] 4. Cell fusion operation

[0110] Mouse myeloma cells and splenocytes are mixed at a ratio of 1:5, and the supernatant is removed by centrifugation. Then, 1 mL of 40% PEG solution is added dropwise within 1 minute, and the centrifuge tube is continuously rotated slightly to promote cell fusion. Subsequently, while continuously rotating, 1 mL of serum-free culture medium is added dropwise within 60 seconds, and then 20 mL of serum-free medium is slowly added within 5 minutes. After centrifugation, the supernatant is removed, the cells are suspended with complete culture medium, and gently mixed. Finally, the cell suspension is evenly distributed into 96-well plates and cultured in a 37°C, CO2 incubator for 24 hours, and then replaced with HAT selective culture medium.

[0111] 5. Culture of fused cells and antibody detection

[0112] The fused cells are subjected to half-volume medium change within 7 - 10 days, and then half-volume medium change is performed every 2 - 3 days. After 2 - 3 weeks of culture, hybrid cell colonies gradually form. When the colonies proliferate to 1 / 3 of the well area, the culture supernatant is taken for antibody detection. The detection uses the ELISA method. The recombinant protein is used as the antigen to coat the enzyme-linked immunosorbent assay (ELISA) plate, and the coated antigen concentration is set at 1 μg / mL, with 100 μL added to each well. The coating buffer used is PBS (pH = 7.4), and it is placed overnight at 4°C. The next day, after washing 3 times with PBS, it is blocked with 1% BSA. Subsequently, the cell culture supernatant containing the monoclonal antibody is added for incubation, and the positive antiserum of mice and the blank culture supernatant are used as positive and negative controls respectively. After incubation, washing, and adding the secondary antibody treatment, the substrate is added for color development, and the absorbance value is measured using an ELISA reader to determine the antibody positive situation. Finally, the cells detected as positive are further subjected to cloning culture. After verification, the positive cell line is determined, and operations such as proliferation, cryopreservation, and in vitro culture are carried out.

[0113] 6. Purification and Sequencing of Cloned Antibodies

[0114] The monoclonal antibody in the culture solution of mouse hybridoma cells is purified using conventional purification methods (first, the culture solution producing the monoclonal antibody is precipitated with semi-saturated and saturated ammonium sulfate for preliminary concentration and purification; then further purified by affinity chromatography), and the purified monoclonal antibody is sequenced (using Trizol from Invitrogen to extract total RNA, reverse transcribed to generate cDNA, and then the variable region genes of its heavy and light chains are amplified separately by PCR using specific primers. After the PCR products are purified by electrophoresis, they are inserted into the vector through TA cloning, sequenced, and sequence analysis is carried out).

[0115] 7. Experimental Results

[0116] The sequence information corresponding to the screened antibody targeting α-synuclein is shown in Table 1. The amino acid sequences of its corresponding heavy chain variable region HCDR1 - 3 are shown as SEQ ID NO:3 - 5 respectively, the amino acid sequences of its corresponding light chain variable region LCDR1 - 3 are shown as SEQ ID NO:6 - 8 respectively, the amino acid sequence of its corresponding heavy chain variable region VH is shown as SEQ ID NO:1, and the amino acid sequence of its corresponding light chain variable region VL is shown as SEQ ID NO:2.

[0117] Table 1 Sequence Information of Monoclonal Antibodies Targeting α-Synuclein

[0118]

[0119] Example 2 Functional Study of Antibodies Targeting α-Synuclein

[0120] 1. Affinity Detection of Anti-α-Synuclein Antibody

[0121] (1) Experimental Method

[0122] a. Coating Antigen

[0123] Dilute the α-synuclein antigen with phosphate-buffered saline (PBS, pH = 7.4) to a concentration of 1 μg / mL. Then add it to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well. Place the ELISA plate in a refrigerator at 4°C overnight.

[0124] b. Washing

[0125] Take out the ELISA plate and discard the antigen solution in the wells. Use PBS buffer containing Tween-20 as the washing solution, fill each well, gently shake the ELISA plate to allow the washing solution to fully contact the well walls, and then discard the washing solution. Repeat the above washing process 3 times. After each washing, fully discard the washing solution and pat the ELISA plate dry to ensure that there is no residual washing solution in the wells.

[0126] c. Adding the Primary Antibody

[0127] Dilute the purified antibody with PBS buffer in a serial dilution (1:1000 to 1:512000). Then, add the diluted primary antibody solution to each well of the ELISA plate, 100 μL per well. Place the ELISA plate in an incubator at 37°C for 1 hour to allow the primary antibody to fully bind to the antigen.

[0128] d. Adding the Secondary Antibody

[0129] Wash the ELISA plate 5 times and pat it dry. Add the secondary antibody solution diluted 1:10,000, 100 μL per well. Place the ELISA plate in an incubator at 37°C for 30 minutes to allow the secondary antibody to fully bind to the primary antibody.

[0130] e. Adding Substrate for Color Development

[0131] Wash the ELISA plate 5 times and pat it dry. Add 50 μL of each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of the stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0132] Evaluate the affinity of the anti-α-synuclein antibody by comparing the absorbance values corresponding to different concentrations of the antigen.

[0133] (2) Experimental Results

[0134] The results are as Figure 1As shown, the absorbance value of the high-concentration antibody was 2.652, which was much greater than the negative control value of 0.084, indicating that the antibody targeting α-synuclein prepared in Example 1 of the present invention had good affinity for the recombinant protein antigen (α-synuclein).

[0135] 2. Specificity Detection of the Antibody Targeting α-Synuclein

[0136] (1) Experimental Method

[0137] The protein immunoblotting method was used to detect the recognition specificity of the antibody for α-synuclein in plasma samples. A 10% SDS-PAGE gel was prepared, 10 μL of sample was loaded into each well, and electrophoresis was performed using a PowerPac™ HC high-current electrophoresis apparatus. Transfer was carried out to a PVDF membrane using a multi-channel semi-dry transfer apparatus. The PVDF membrane was blocked with 5% skim milk powder for 30 min, and the purified α-synuclein antibody was added, and incubated with shaking at room temperature for 2 h. After washing the membrane with TBST, an HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. Color development was carried out using an Omni-ECLTM ultrasensitive chemiluminescence detection kit, and the image was taken on an electronic film imager.

[0138] (2) Experimental Results

[0139] The results were as Figure 2 shown. When the plasma sample was detected with this antibody, a target band could be shown at around 15 KD, and the band was single, indicating that the antibody targeting α-synuclein prepared in Example 1 of the present invention could specifically detect α-synuclein in the sample.

Claims

1. An anti-α-synuclein antibody, characterized in that, The HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are the HCDR1, HCDR2, and HCDR3 in the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:1; The LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are the LCDR1, LCDR2, and LCDR3 in the light chain variable region with the amino acid sequence shown in SEQ ID NO:

2.

2. The antibody according to claim 1, wherein The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 respectively; The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 respectively.

3. The antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule according to claim 4.

6. A recombinant host cell, characterized in that, The recombinant host cell contains the recombinant vector according to claim 5.

7. An antibody conjugate, characterized in that, The antibody conjugate is a complex formed by directly or indirectly conjugating the antibody according to any one of claims 1-3 to a detectable label.

8. A detection reagent, characterized in that, The detection reagent contains the antibody according to any one of claims 1-3 or the antibody conjugate according to claim 7.

9. A detection product, characterized in that, The detection product contains the antibody according to any one of claims 1-3, the antibody conjugate according to claim 7, or the detection reagent according to claim 8.

10. A method for preparing the recombinant host cell according to claim 6, characterized in that, The method includes: introducing the recombinant vector according to claim 5 into a host cell to obtain the recombinant host cell according to claim 6.

11. A method for producing an antibody according to any one of claims 1-3, characterized in that, The method includes: culturing the recombinant host cell according to claim 6 and isolating the antibody from the culture product of the recombinant host cell.

12. A method for detecting α-synuclein in a sample to be tested for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes: contacting the test sample with the antibody according to any one of claims 1-3, the antibody conjugate according to claim 7, the detection reagent according to claim 8, or the detection product according to claim 9, and detecting the formation of an immune complex of α-synuclein and the antibody.

13. Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, or the recombinant host cell according to claim 6 in the preparation of an antibody conjugate for detecting α-synuclein.

14. Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, or the antibody conjugate according to claim 7 in the preparation of a detection reagent for detecting α-synuclein.

15. Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the antibody conjugate according to claim 7, or the detection reagent according to claim 8 in the preparation of a detection product for detecting α-synuclein.

16. Use of the antibody according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the antibody conjugate according to claim 7, the detection reagent according to claim 8, or the detection product according to claim 9 in detecting α-synuclein for non-diagnostic and non-therapeutic purposes.

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