Anti-α-Synuclein antibodies and uses thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to α-synuclein, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, host cells containing the same, and related uses. Furthermore, the invention relates to the therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Description

Technical Field

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to α-synuclein, pharmaceutical compositions containing such antibodies or antigen-binding fragments, nucleic acid molecules encoding such antibodies or antigen-binding fragments, host cells containing such antibodies or antigen-binding fragments, and related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments. Background Art

[0002] Synuclein was discovered in 1991 and was named after its location in synapses and its subcellular localization on the nuclear membrane. Currently, there are three synuclein isoforms: α, β, and γ. α-synuclein can be structurally divided into three parts: the amino terminus (aa 1-60), which contains four lipid-binding motifs (KTKEGV) and readily forms an amphipathic α-helix, similar to the lipid-binding region of apolipoproteins. This region mediates the binding of α-synuclein to lipid membranes, and all five mutation sites in familial Parkinson's disease (PD) are located in this domain. The central, hydrophobic region of the NAC region (aa 61-95) readily forms a β-sheet structure, is highly susceptible to aggregation in vitro, and can promote the aggregation of full-length α-synuclein. The carboxyl terminus (aa96-140), rich in acidic amino acids and proline, carries a large negative charge and is highly hydrophilic. Three conserved tyrosine residues are considered to be hallmarks of α- and β-synuclein.

[0003] α-synuclein is the main component of Lewy bodies. Studies have found that α-synuclein in Lewy bodies has a variety of pathological changes, such as post-transcriptional modifications (such as phosphorylation, nitration and oxidation, C-terminal enzymatic truncation, etc.) and mutations (such as Ala53Thr, Ala30Pro, Glu46Lys, Gly51Asp, etc.). Since the discovery in 1997 that α-synuclein is associated with familial Parkinson's disease gene (SCNA) mutations and the main component of Lewy bodies, it has become a focus in the molecular pathogenesis of Parkinson's disease. The evidence for the pathological relationship between α-synuclein and PD is as follows: (1) α-synuclein gene mutations have been found in familial PD patients; (2) Increased α-synuclein gene copy number (2 / 3 copies) can lead to PD, and the severity is copy number dependent; (3) Overexpression of α-synuclein in cells and mice will cause the main symptoms of the disease.

[0004] Currently, Parkinson's disease is incurable, and existing treatment options aim to control symptoms while minimizing drug side effects. Existing PD treatments (e.g., levodopa, dopamine agonists, catechol-O-methyltransferase inhibitors, monoamine oxidase inhibitors, anticholinergics, amantadine, and rivastigmine) primarily improve symptoms by regulating dopamine synthesis and metabolism. Currently, there are no effective drugs that can slow the progression of the disease, and new mechanisms of action that can intervene in the disease process are needed to meet clinical needs.

[0005] Basic research suggests that oligomeric or profibrillar forms of α-synuclein play a primary pathological role, spreading between neurons in a "seed" manner, driving disease progression. In vitro studies have shown that antibodies targeting α-synuclein can reduce the ability of these "seed" forms to spread, potentially slowing disease progression.

[0006] Although there is already some theoretical knowledge in the field of treatment and prevention of Parkinson's disease, the development of antibody drugs targeting α-synuclein is in its infancy, and no related antibody drugs are currently on the market. There is an urgent need in this field to develop new antibodies targeting α-synuclein (especially fully human monoclonal antibodies) for the treatment of Parkinson's disease and other diseases. Summary of the Invention

[0007] After extensive research, the inventors of this application have screened and obtained fully human antibodies against α-Synuclein, which can effectively inhibit the polymerization of α-Synuclein, inhibit the spread of α-Synuclein, inhibit the phosphorylation of α-Synuclein, promote the phagocytosis of α-Synuclein by microglia, specifically recognize α-Synuclein deposits (such as Lewy bodies or Lewy neurofilaments), and / or, administering the antibodies of the present invention in PD mouse models can significantly improve the motor impairment caused by PFFs, and has the potential to improve Parkinson's disease motor ability. In addition, the antibodies of the present invention also have cross-reactivity with human, monkey, and mouse α-Synuclein. Based on this, the present application also provides a pharmaceutical composition containing the antibody or its antigen-binding fragment, a nucleic acid molecule encoding the antibody or its antigen-binding fragment, a host cell containing the same, and related uses.

[0008] Antibodies or antigen-binding fragments thereof

[0009] P21

[0010] Therefore, in a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to α-Synuclein, wherein the antibody or the antigen-binding fragment thereof comprises:

[0011] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 11; and / or VLCDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 15;

[0012] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0014] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 11; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 15.

[0015] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat numbering system.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0017] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 12, VH CDR2 of SEQ ID NO: 13, and VH CDR3 of SEQ ID NO: 14; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 16, VL CDR2 of SEQ ID NO: 17, and VL CDR3 of SEQ ID NO: 18;

[0018] Wherein, the CDRs are defined by the Kabat numbering system.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0020] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 36, VH CDR2 of SEQ ID NO: 37, and VH CDR3 of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 39, VL CDR2 of SEQ ID NO: 40, and VL CDR3 of SEQ ID NO: 41;

[0021] Wherein, the CDR is defined by the IMGT numbering system.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0023] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 48, VH CDR2 of SEQ ID NO: 49, and VH CDR3 of SEQ ID NO: 50; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 51, VL CDR2 of SEQ ID NO: 52, and VL CDR3 of SEQ ID NO: 53;

[0024] Wherein, the CDRs are defined by the Chothia numbering system.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0028] (i) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 11 or a variant thereof; and / or, (ii) a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 15 or a variant thereof;

[0029] Wherein, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:11 and a VL comprising the sequence shown in SEQ ID NO:15.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope contained within amino acid residues 126-140 of α-synuclein. In certain embodiments, the amino acid sequence of amino acid residues 126-140 of α-synuclein is as shown in SEQ ID NO:34.

[0032] P22

[0033] In a second aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to α-Synuclein, wherein the antibody or the antigen-binding fragment thereof comprises:

[0034] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as set forth in SEQ ID NO: 19; and / or VLCDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as set forth in SEQ ID NO: 23;

[0035] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0037] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 19; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 23.

[0038] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering systems. In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat numbering system.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0040] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 20, VH CDR2 of SEQ ID NO: 21, and VH CDR3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 24, VL CDR2 of SEQ ID NO: 25, and VL CDR3 of SEQ ID NO: 26;

[0041] Wherein, the CDRs are defined by the Kabat numbering system.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0043] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 42, VH CDR2 of SEQ ID NO: 43, and VH CDR3 of SEQ ID NO: 44; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 45, VL CDR2 of SEQ ID NO: 46, and VL CDR3 of SEQ ID NO: 47;

[0044] Wherein, the CDR is defined by the IMGT numbering system.

[0045] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0046] A heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 54, VH CDR2 of SEQ ID NO: 55, and VH CDR3 of SEQ ID NO: 56; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 57, VL CDR2 of SEQ ID NO: 58, and VL CDR3 of SEQ ID NO: 59;

[0047] Wherein, the CDRs are defined by the Chothia numbering system.

[0048] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene, and / or a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0051] (i) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 19 or a variant thereof; and / or, (ii) a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 23 or a variant thereof;

[0052] Wherein, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0053] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 19 and a VL comprising the sequence shown in SEQ ID NO: 23.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope contained within amino acid residues 126-140 of α-synuclein. In certain embodiments, the amino acid sequence of amino acid residues 126-140 of α-synuclein is as shown in SEQ ID NO:34.

[0055] In certain embodiments, the antibody or antigen-binding fragment thereof of the first or second aspect further comprises a constant region derived from a human immunoglobulin.

[0056] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; and / or,

[0057] The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.

[0058] In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative substitutions of amino acids compared to the sequence from which it is derived. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function compared to the wild-type sequence from which it is derived. In other embodiments, the variant of the heavy chain constant region (CH) may have altered (e.g., reduced or enhanced) effector function compared to the wild-type sequence from which it is derived.

[0059] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 27.

[0060] In certain exemplary embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO:27.

[0061] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ). In certain embodiments, the light chain constant region comprises the sequence shown in SEQ ID NO: 28 or 29.

[0062] In certain exemplary embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO: 28 or 29.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect comprises a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 31.

[0064] In certain embodiments, the antibody or antigen-binding fragment thereof of the second aspect comprises a heavy chain as shown in SEQ ID NO:32 and a light chain as shown in SEQ ID NO:33.

[0065] In certain embodiments, the antibody or antigen-binding fragment thereof of the first or second aspect further comprises a constant region derived from a murine immunoglobulin.

[0066] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0067] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of murine IgG1. In certain exemplary embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in NCBI Sequence ID: AAK53870.1.

[0068] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a murine immunoglobulin (eg, kappa or lambda).

[0069] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from murine immunoglobulin kappa (eg, see Uniprot ID: P01837).

[0070] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from murine immunoglobulin lambda (e.g., see Uniprot ID: A0A0G2JE99). In certain embodiments, X in the light chain constant region shown in Uniprot ID: A0A0G2JE99 is G.

[0071] In certain embodiments, in the first aspect or the second aspect, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single-domain antibody (sdAb); and / or, the antibody is a murinized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

[0072] In certain embodiments, in the first aspect or the second aspect, the antibody or antigen-binding fragment thereof is a fully human antibody.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect or the second aspect has one or more of the following characteristics:

[0074] (i) inhibiting the polymerization of α-synuclein in vitro or in vivo in a subject (e.g., a human, monkey, or mouse);

[0075] (ii) inhibiting the propagation of α-synuclein in vitro or in vivo in a subject (e.g., a human, monkey, or mouse);

[0076] (iii) inhibiting the phosphorylation of α-synuclein in vitro or in vivo in a subject (e.g., a human, monkey, or mouse);

[0077] (iv) promoting the phagocytosis of α-synuclein by microglia in vitro or in vivo in a subject (e.g., human, monkey, or mouse);

[0078] (v) ameliorating motor impairment caused by PFFs in vitro or in vivo in a subject (e.g., human, monkey, or mouse);

[0079] (vi) preventing and / or treating a disease associated with α-Synuclein or ameliorating at least one symptom or pathological manifestation of the disease in a subject (eg, a human, monkey, or mouse);

[0080] (vii) Specific recognition of α-synuclein deposits (e.g., Lewy bodies or Lewy neurofilaments).

[0081] In a third aspect, the present application also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain.

[0082] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0083] In a fourth aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0084] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0085] In a fifth aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0086] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a microorganism.

[0087] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0088] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0089] In a sixth aspect, the present application also provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first or second aspect, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0090] Therapeutic uses

[0091] In a seventh aspect, the present application also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0092] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0093] In an eighth aspect, the present application further provides the use of the antibody or antigen-binding fragment thereof as described in the first or second aspect, the isolated nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the host cell as described in the fifth aspect, or the pharmaceutical composition as described in the seventh aspect for preparing a drug for preventing and / or treating a disease associated with α-Synuclein in a subject.

[0094] In certain embodiments, the disease associated with α-Synuclein is characterized by the presence of Lewy bodies or pathological aggregates of α-Synuclein in the brain. In certain embodiments, the disease associated with α-Synuclein is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or a combination thereof.

[0095] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0096] In certain embodiments, the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent, eg, administered simultaneously or sequentially.

[0097] In a ninth aspect, the present application further provides a method for preventing and / or treating a disease associated with α-Synuclein in a subject, comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof as described in the first or second aspect, or the pharmaceutical composition as described in the seventh aspect.

[0098] In certain embodiments, the disease associated with α-Synuclein is characterized by the presence of Lewy bodies or pathological aggregates of α-Synuclein in the brain. In certain embodiments, the disease associated with α-Synuclein is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or a combination thereof.

[0099] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof of the first or second aspect or the pharmaceutical composition of the seventh aspect may be administered in combination with another pharmaceutically active agent, for example, simultaneously or sequentially.

[0101] The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0102] The antibody of the application or its Fab or pharmaceutical composition of the present invention can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technical personnel should understand that route of administration and / or mode will change according to intended purpose. In certain embodiments, antibody of the present invention or its Fab or pharmaceutical composition are given by intravenous injection or push injection.

[0103] Detection purpose

[0104] In the tenth aspect, the present application also provides a conjugate comprising the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0105] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0106] In the eleventh aspect, the present application also provides a kit comprising the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, or the conjugate as described in the tenth aspect.

[0107] In certain embodiments, the kit comprises a detection buffer.

[0108] In certain embodiments, the kit comprises the conjugate of the tenth aspect.

[0109] In certain embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0110] In a twelfth aspect, the present application also provides a method for detecting the presence or level of α-Synuclein in a sample, which comprises using the antibody or antigen-binding fragment thereof as described in the first or second aspect or the conjugate as described in the tenth aspect.

[0111] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0112] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0113] In certain embodiments, the method comprises using the conjugate of the tenth aspect.

[0114] In certain embodiments, the method comprises using the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).

[0115] In certain embodiments, the method comprises: (1) contacting the sample with an antibody, or antigen-binding fragment thereof, or conjugate thereof; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of α-synuclein or cells expressing α-synuclein.

[0116] The present application also provides a method for diagnosing a disease associated with α-Synuclein, comprising detecting the presence or level of α-Synuclein in a sample from a subject using the method described in aspect 12. In certain embodiments, when α-Synuclein or α-Synuclein deposits (e.g., Lewy bodies and Lewy neurofilaments) are present, an increase in the level of α-Synuclein or α-Synuclein deposits (e.g., Lewy bodies and Lewy neurofilaments) compared to a reference level (e.g., compared to a healthy control) indicates that the subject suffers from a disease associated with α-Synuclein.

[0117] In a thirteenth aspect, the present application further provides use of the antibody or antigen-binding fragment thereof as described in the first or second aspect, or the conjugate as described in the tenth aspect, in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of α-Synuclein in a sample and / or diagnose a disease associated with α-Synuclein.

[0118] In certain embodiments, the detection reagent detects the presence or level of α-Synuclein in a sample by the method of aspect 12.

[0119] In certain embodiments, the detection reagent detects the presence or level of α-synuclein in a sample by the method described in aspect 12 to diagnose a disease associated with α-synuclein. In certain embodiments, when α-synuclein or α-synuclein deposits (e.g., Lewy bodies and Lewy neurofilaments) are present, an increase in the level of α-synuclein or α-synuclein deposits (e.g., Lewy bodies and Lewy neurofilaments) compared to a reference level (e.g., compared to a healthy control) indicates that the subject suffers from a disease associated with α-synuclein.

[0120] In certain embodiments, the sample is a tissue sample (eg, central and peripheral nervous tissue) from a subject (eg, a mammal, preferably a human, monkey, or mouse).

[0121] Definition of terms

[0122] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0123] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0124] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0125] As used herein, unless otherwise indicated herein or clearly contradicted by context, the term "α-Synuclein" is intended to encompass α-Synuclein monomers and protofibrils (PFFs) thereof.

[0126] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domains are not directly involved in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0127] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0128] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0129] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0130] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0131] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0132] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as human; or can be chimeric or murinized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0133] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0134] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0135] As used herein, the term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0136] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0137] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0138] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

[0139] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity to at least two or more (e.g., three or four) different antigens (or epitopes). Bispecific antibodies or multispecific antibodies comprise multiple antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by a bispecific antibody or multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv fragment, Fab fragment, F(ab')2 fragment or scFv). In some cases, each antigen-binding domain is connected by a peptide linker.

[0140] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0141] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0142] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0143] As used herein, the term "murinized antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from fully human antibodies, while the heavy chain constant region and light chain constant region of the antibody are derived from murine constant regions. As used herein, the term "fully human antibody" refers to an antibody whose entire amino acid sequence is derived from humans, which can be obtained through phage and yeast display, transgenic animals, single B cells, and other technologies.

[0144] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0145] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0146] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0147] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0148] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D(See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0149] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0150] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0151] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0152] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0153] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0154] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0155] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0156] As used herein, the term "subject" refers to a mammal, such as a human, monkey, or mouse. In certain embodiments, the subject (e.g., human, monkey, or mouse) suffers from a disease associated with α-synuclein (e.g., Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or a combination thereof), or is at risk of suffering from such a disease.

[0157] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (Parkinson's disease, Lewy body dementia, multiple system atrophy, or a combination thereof) refers to an amount sufficient to prevent, stop, or delay the onset of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0158] Advantageous Effects of the Invention

[0159] The fully human anti-α-synuclein antibodies of the present invention can effectively inhibit the polymerization and propagation of α-synuclein, inhibit the phosphorylation of α-synuclein, promote the phagocytosis of α-synuclein by microglia, specifically recognize α-synuclein deposits (e.g., Lewy bodies or Lewy neurofilaments), and / or, in a PD mouse model, significantly improve motor impairment caused by PFFs, showing the potential to improve motor function in Parkinson's disease. Furthermore, the antibodies of the present invention have cross-reactivity with human, monkey, and mouse α-synuclein.

[0160] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1 The results of the detection of α-Synuclein monomer and PFF, control antibody P01-hIgG1, candidate molecules P21-hIgG1 and P22-hIgG1 are shown. The results of Native SDS-PAGE detection and ThT detection of α-Synuclein monomer and PFF are shown in Figure 2. Figure 1 A and Figure 1 As shown in C, the electron microscopy results of α-Synuclein PFF are as follows Figure 1 As shown in B, the SDS-PAGE detection results of the control antibody P01-hIgG1 are as follows Figure 1 As shown in D, the SDS-PAGE detection results of candidate molecules P21-hIgG1 and P22-hIgG1 are as follows Figure 1 As shown in E; Figure 1 In A, lane M: protein molecular weight marker; Figure 1 In B, the carrier used for electron microscopy was imported 200-mesh copper mesh ultra-thin film, and the microscope was examined at 100,000×; Figure 1 In C, the horizontal axis is the test sample, and the vertical axis is the ThT fluorescence intensity.

[0162] Figure 2 The results of the inhibition of α-Synuclein polymerization by fully human anti-α-Synuclein monoclonal antibodies P21-hIgG1, P22-hIgG1 and negative control antibodies are shown.

[0163] Figure 3 The results of the inhibition of α-Synuclein phosphorylation by the fully human anti-α-Synuclein monoclonal antibody P22-hIgG1 and the control antibody (P01-hIgG1, negative control antibody of the same subtype) are shown.

[0164] Figure 4A The results show that the fully human anti-α-Synuclein monoclonal antibody P22-hIgG1 inhibits the spread of α-Synuclein.

[0165] Figure 4BThe results of the inhibition of α-Synuclein propagation by fully human anti-α-Synuclein monoclonal antibodies P21-hIgG1 and P22-hIgG1 are shown; among them, "P21" indicates "P21-hIgG1" and "P22" indicates "P22-hIgG1".

[0166] Figure 5 shows the results of fully human anti-α-Synuclein monoclonal antibodies P21-hIgG1 and P22-hIgG1 promoting microglial phagocytosis of α-Synuclein and the detection of inflammatory factors during phagocytosis, wherein Figure 5A shows the results of high-content imaging detection of antibody-induced phagocytosis, and Figure 5B shows the results of ELISA detection of mTNF-α release; Figure 5C The results show that the fully human anti-α-Synuclein monoclonal antibodies P21-hIgG1 and P22-hIgG1 promote the phagocytosis of α-Synuclein by microglia, as detected by an optimized method; among them, "P21" indicates "P21-hIgG1" and "P22" indicates "P22-hIgG1".

[0167] Figure 6 The results of the efficacy test of P21-mIgG1, P22-mIgG1 murine antibodies and negative control antibodies in PD model mice are shown. Figure 6 A is the in vivo deposition of α-Synuclein detected by immunohistochemistry in mouse brain. Figure 6 B is a quantitative assessment of α-Synuclein deposition in the mouse brain.

[0168] Figure 7 The results showed that P21-mIgG1, P22-mIgG1 murine antibodies and negative control antibodies protected dopamine neurons in PD model mice. Figure 7 A is immunohistochemical staining of dopamine neurons. Figure 7 B is the quantitative evaluation of neuron number.

[0169] Figure 8 The results of neurotransmitter evaluation of P21-mIgG1, P22-mIgG1 murinized antibodies, and negative control antibodies in PD model mice are shown.

[0170] Figure 9 The results of the efficacy test of P21-mIgG1, P22-mIgG1 murine antibodies and negative control antibodies in PD model mice are shown; Figure 9 A is the result of Rotarod test. Figure 9 B is the result of the wire hang test. Figure 9 C is the result of the pole test. Figure 9 D is the result of the balance beam experiment.

[0171] Figure 10 The results of in vitro immunohistochemical detection of brain tissue of PD patients using murine antibodies P21-mIgG1, P22-mIgG1 and control antibody (P01-mIgG1) are shown. Figure 10 A, 10C, and 10E show the immunohistochemical results of brain tissues from PD patients using antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1, respectively; Figure 10 B, 10D, and 10F show the immunohistochemical results of hippocampal tissues from PD patients using antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1, respectively.

[0172] Sequence information

[0173] A description of the sequences involved in this application is provided in the table below.

[0174] Table 1: Sequence information

[0175]

[0176]

[0177] DETAILED DESCRIPTION

[0178] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0179] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0180] Unless otherwise specified and the percentage identity is not provided, "%" in the present invention represents mass volume percentage.

[0181] Example 1: Preparation of α-Synuclein Antigen and Positive Control Antibody

[0182] 1. Preparation of α-Synuclein Monomers, Protofibrils (PFFs), and Truncated Antigens

[0183] Preparation of α-Synuclein Monomers: The nucleotide sequence encoding human α-Synuclein (amino acid sequence shown in SEQ ID NO: 1) (SEQ ID NO: 2) was synthesized and inserted into the pET29a(+) prokaryotic expression vector. The product was transformed into BL21(DE3) cells and induced for expression. Anion exchange purification was performed to remove impurities, yielding α-Synuclein monomers with a purity >90%. Further purification was performed using cation exchange. Detection was performed by native electrophoresis.

[0184] Preparation of α-synuclein fibrils (PFFs): The purified α-synuclein monomers were buffer exchanged and concentrated (using DPBS at a concentration greater than 5 mg / ml). The polymers were then detoxified, sterilized by filtration, and tested for endotoxin (endotoxin <25 EU / mg). Polymerization was performed at a concentration of 5 mg / ml on a thermostat at 37°C, 1000 rpm, for 7 days. The polymerized samples were sonicated at 20% amplitude for 70 seconds (1 second on / off). The polymers were analyzed by native electrophoresis, ThT analysis, and electron microscopy.

[0185] Testing conditions:

[0186] 1) Native electrophoresis: 10 μg sample load, electrophoresis conditions: 4-20% Bio-Rad precast gel, 150 V constant voltage, 3.5 h;

[0187] 2) ThT detection: add 10 μl of sample to 100 μl of ThT (final concentration 5 μM) and detect at 450 nm and 485 nm;

[0188] 3) Electron microscopy: With a sample concentration of 5 μM and a sample volume of 20 μl on a copper grid (hanging drop method), incubate with 1% phosphotungstic acid hanging drops for 3 minutes, absorb with the edge of filter paper, and air dry for examination. Electron microscopy detection conditions are 200 kV.

[0189] The native electrophoresis results and ThT detection results of α-Synuclein monomer and PFF are as follows: Figure 1 A and Figure 1 As shown in C, the electron microscopy results of α-Synuclein PFF are as follows Figure 1 As shown in B, the results show PFF aggregation hallmarks: 1) Native electrophoresis shows polymerized macromolecules (at the gel pores); 2) ThT detection values ​​are around 3 to 5E7; 3) Electron microscopy shows fiber-like filaments.

[0190] Preparation of truncated α-Synuclein antigen:

[0191] The genes encoding α-Synuclein (aa 1-60, SEQ ID NO: 3), α-Synuclein (aa 1-95, SEQ ID NO: 5), α-Synuclein (aa 61-140, SEQ ID NO: 7), and α-Synuclein (aa 96-140, SEQ ID NO: 9) were respectively constructed into the pGS003-N-hIgG1Fc eukaryotic expression vector to obtain transient expression vectors of α-Synuclein truncated antigens with a human IgG1Fc tag (SEQ ID NO: 35) fused to the N-terminus.

[0192] Using FreeStyle TM 293E cells were transiently transfected in Freestyle medium. 24 hours before transfection, 0.5×10 6 30 ml of 293E cells at a concentration of 1 cell / ml were cultured in a 37°C, 5% CO2 incubator with a shaker at 130 rpm. For transfection, 60 μl of 293E Fectin was added to 1 ml of Opti-MEM, mixed thoroughly, and incubated at room temperature for 5 minutes. Simultaneously, 30 μg of the recombinant vector plasmid DNA was dissolved in 1 ml of Opti-MEM. The plasmid DNA and 293E Fectin were thoroughly mixed to a total volume of 2 ml and incubated at room temperature for 15 minutes. The entire mixture was then added to the cell culture wells, mixed thoroughly, and cultured in a 37°C, 5% CO2 incubator with a shaker at 130 rpm for 7 days. The culture medium was centrifuged at high speed, and the supernatant was vacuum filtered through a microporous filter.

[0193] The purified α-Synuclein truncated antigens were fused to the human IgG1 Fc tag at the N-terminus by purification using a MabSelect SuRe affinity chromatography column (GE), eluted with 20 mM citric acid-sodium citrate, pH 3.0, and adjusted to neutral pH with 1 M Tris base.

[0194] 2. Preparation of positive control antibodies

[0195] pGS003-hIgG1CH and pGS003-hIgLCL were selected as expression vectors for the heavy and light chains of the anti-human α-synuclein positive control antibody P01-hIgG1 (full-length antibody amino acid sequence reference KEGG Entry ID: D11467), respectively. After codon optimization of the nucleotide sequence encoding the variable region of the positive control antibody, the VH and VL genes of the positive control antibody were cloned into pGS003-hIgG1CH (which contains a nucleotide sequence encoding the antibody heavy chain constant region as shown in SEQ ID NO: 27) and pGS003-hIgLCL (which contains a nucleotide sequence encoding the antibody light chain constant region as shown in SEQ ID NO: 29, wherein the G at position 1 of SEQ ID NO: 29 is mutated to S) using a recombinant method, respectively, to obtain the heavy and light chain transient expression vectors pGS003-P01VH-hIgG1CH and pGS003-P01VL-hIgLCL of the positive control antibody.

[0196] The expression vectors pGS003-P01VH-hIgG1CH and pGS003-P01VL-hIgLCL were transformed into TM Transient expression was performed in 293E cells using Freestyle medium.

[0197] The purified positive antibody P01-hIgG1 was obtained by purification on a MabSelect SuRe affinity chromatography column (GE) using 20 mM citric acid-sodium citrate, pH 3.0, and adjusting the pH to neutral with 1 M Tris base. The SDS-PAGE detection results are shown in Figure 1 D.

[0198] Example 2: Construction of a natural fully human single-chain antibody phage display library

[0199] Healthy elderly people aged ≥80 years without neurological diseases were recruited to donate blood. Lymphocytes were isolated using lymphocyte separation fluid, total RNA was extracted, and first-strand cDNA was synthesized by reverse transcription. Based on the antibody gene sequence information published on the Kabat, V-base2, and IMGT websites, primers for amplifying human antibody heavy and light chains were designed (primer sequences are shown in CN108250296A). Using cDNA as a template, PCR amplification was performed to obtain the heavy and light chain variable regions of the antibody gene. VL and VH were sequentially constructed into phagemid vectors to construct human VH and VL single-chain antibody libraries with a storage capacity of ≥1E9.

[0200] Example 3: Phage display and screening of a fully human antibody library

[0201] 3.1 Phage display and panning of antibody libraries

[0202] Take 100 times the library volume of the above-mentioned fully human VH and VL single-chain antibody library bacterial solution to inoculate 880 ml of 2YT-AG medium (containing 100 μg / ml ampicillin and 2% glucose), culture at 37°C and 200 rpm to OD600 = 0.5-0.6, add helper phage with a cell density of 100 times, infect for 1.5 hours, collect the bacteria by centrifugation, resuspend the cells with 400 ml of 2YT-AK medium (containing 100 μg / ml ampicillin and 75 μg / ml kanamycin), and culture overnight at 30°C and 200 rpm.

[0203] Centrifuge the culture from the previous step at 4100 rpm and 4°C for 30 min, collect the supernatant, add 1 / 4 volume of PEG / NaCl, mix well, and let it stand on ice for 1 hour; centrifuge at 4100 rpm and 4°C for 30 min, discard the supernatant, and turn the centrifuge tube upside down on paper to remove all the liquid; resuspend the phage pellet in 2 ml of pre-cooled 1× PBS, centrifuge at 12000g at 4°C for 10 min; transfer the supernatant to a new 15 ml centrifuge tube to obtain the first round of starting phage. First, take an appropriate amount of PBS-diluted library phage solution 10 13 , placed in a pre-clear SA immunotube and incubated at room temperature for 1 hour; remove the pre-clear library phage, add 1ml of 5% MPBS, and divide equally into two sealed 2ml centrifuge tubes. Add an appropriate amount of antigen to the R tube and an equal volume of PBS to the CK tube, and incubate at room temperature for 1 hour; then wash away unbound phage with PBST and elute the phage with 1ml of Glycine-HCl (pH 2.2). The eluted phage is reinfected with TG1, and the eluted product is amplified. The phage is purified by PEG / NaCl precipitation and used for the next round of screening. A total of four rounds of phage library enrichment screening were performed, with decreasing antigen amount and increasing washing intensity. The elution product was titered in each round.

[0204] 3.2 Monoclonal expression and ELISA screening

[0205] The bacterial suspension after the 1st to 4th rounds of panning was spread on plates by limiting dilution and cultured overnight; single clones were picked and cultured overnight in 96-well deep-well plates containing 0.5 ml / well 2YT-AG medium; the overnight culture was then transferred to a 96-well deep-well plate containing 0.5 ml / well 2YT-AG medium at a ratio of 1:10 (v / v) and cultured to OD600 = 0.5-0.6. The cells were induced with 2YT-AG medium (containing 100 μg / ml ampicillin and 1 mM IPTG) at 30°C overnight, and the supernatant was transferred to a clean 96-well deep-well plate by centrifugation the next day. A 96-well ELISA plate was coated with α-Synuclein (SEQ ID NO: 1) as the antigen. After blocking, 50 μl of monoclonal supernatant sample was added to each well and incubated at 25°C for 1 h. Then, 250 μl of PBST was added to each well, the plate was shaken for 5-10 s, the solution was discarded, and this was repeated three times. Then, 50 μl of anti-His-HRP antibody (Sino Biological) diluted in PBS at 1:20,000 (v / v) was added to each well and incubated at 25°C for 1 h. Then, 250 μl of PBST was added to each well, the plate was shaken for 5-10 s, the solution was discarded, and this was repeated three times. 50 μl of TMB color development solution was added to each well and color was developed for 5 min. Then, 50 μl of 2 M H2SO4 was added to each well to stop color development. The OD450 value was measured using a microplate reader. A monoclonal clone specifically binding to α-Synuclein (SEQ ID NO: 1) was selected for sequencing; the overnight culture in 2YT-AG medium in a 96-well deep-well plate was taken for sequencing analysis and ELISA re-testing, and two scFv monoclonal antibodies (P20 and P21) were selected. P20 was randomly mutated to construct a random mutation library, and then screened by the same phage display method to obtain clones with improved affinity for testing. After sequencing analysis and ELISA re-testing, an affinity-matured scFv monoclonal antibody of P20 (P22) was selected. Finally, two scFv monoclonal antibodies (P21 and P22) were selected for the preparation of fully human full-length antibodies. The variable region and CDR sequence information of the monoclonal antibodies P21 and P22 are shown in the following table.

[0206]

[0207] Example 4: Preparation of fully human full-length antibodies and physical and chemical testing

[0208] 4.1 Construction of full-length antibody transient transfection expression vector

[0209] pGS003-hIgG1CH and pGS003-hIgKCL or pGS003-hIgLCL were selected as expression vectors for the heavy and light chains of the full-length anti-human α-Synuclein antibodies. Primers were designed based on the gene sequences of VH and VL and the multiple cloning sites in the vector. After PCR amplification, the two VH and two VL antibody genes obtained by screening in Example 3 were cloned into pGS003-hIgG1CH and pGS003-hIgKCL or pGS003-hIgLCL using an in vitro recombination method (Suzhou Hongxun, iMulli multi-fragment recombination cloning kit). The amino acid sequences of the VH and VL of the two antibodies and the CDRs defined by the Kabat, IMGT and Chothia numbering systems are shown in Table 1 and the sequence table; the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NOs: 28 and 29, respectively. After sequencing confirmed the correct insertion of the antibody gene, the recombinant expression vector was transformed into Escherichia coli TOP10F'. A single colony was picked and inoculated into LB medium containing 100 μg / ml ampicillin and cultured with shaking at 37°C for 16 hours. The plasmid was extracted using a Zymo Research endotoxin-free extraction kit and dissolved in 1 ml of ultrapure water. The plasmid concentration and OD260 / 280 ratio were measured using a spectrophotometer. An OD260 / 280 ratio of 1.8 to 1.9 indicates high-purity plasmid DNA.

[0210] 4.2 Transfection, expression and purification in mammalian 293E cells

[0211] Using FreeStyle TM 293E cells were transiently transfected and expressed with two antibodies in Freestyle medium. 24 hours before transfection, 0.5×10 6 300 ml of 293E cells at a concentration of 10 cells / ml were cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm. For transfection, 300 μl of 293fectin was added to 5.7 ml of OPtiMEM, mixed thoroughly, and incubated at room temperature for 2 minutes. Simultaneously, 300 μg each of the heavy and light chain expression plasmids were diluted to 6 ml in OPtiMEM. The diluted transfection reagent and plasmids were thoroughly mixed and incubated at room temperature for 15 minutes. The entire mixture was then added to the cells, mixed thoroughly, and cultured in a 37°C, 5% CO2 incubator with a shaker at 120 rpm for 7 days.

[0212] 4.3 Antibody Purification and Detection

[0213] The cell culture medium was centrifuged at 2000g for 20 minutes, and the supernatant was collected. The antibody expression level in the supernatant was detected by Octet, as shown in Table 2. The supernatant was filtered through a 0.22 μm filter membrane and then purified by MabSelect SuRe affinity chromatography column (GE), eluted with 20 mM citric acid-sodium citrate, pH 3.0, and adjusted to neutral pH with 1 M Tris base. The purified protein was detected by SDS-PAGE with a 4-20% gradient gel (GenScript Biotechnology Co., Ltd.). The specific test results are shown in Figure 1 E.

[0214] 4.4 Physical and chemical testing of fully human antibodies

[0215] After purification, the fully human antibody was subjected to ELISA testing for species cross-reactivity with human, mouse, and monkey α-Synuclein (abbreviated as α-Syn) and binding activity to family proteins β / γ-Synuclein (abbreviated as β / γ-Syn) (wherein, the amino acid sequence of human α-Synuclein is shown in SEQ ID NO: 1 (Uniprot ID: P37840), and the amino acid sequences of mouse and monkey α-Synuclein are shown in NCBI Sequence ID: AAC00521.1 and Uniprot ID: P61142, respectively; the amino acid sequence of human β-Synuclein is shown in Uniprot ID: Q16143, and the amino acid sequence of human γ-Synuclein is shown in Uniprot ID: Q6FHG5 (wherein the E at positions 13 and 68 are mutated to K)). The results are shown in Table 2.

[0216] Table 2 Physical and chemical test results of fully human antibodies

[0217]

[0218] 4.5 Fully human antibody epitope detection

[0219] After purification, the fully human antibodies were subjected to epitope binding ELISA tests for α-Synuclein (aa 1-60), α-Synuclein (aa 1-95), α-Synuclein (aa 61-140), and α-Synuclein (aa 96-140). The results showed that P21-hIgG1 and P22-hIgG1 only bound to α-Synuclein (aa 96-140). To obtain further epitope information, the 96-140 amino acids at the C-terminus of the α-Synuclein monomer were fragmented into multiple fragments. The binding ability of the two preferred antibodies to the peptides was tested by binding ELISA to further determine the epitope range of their binding to α-Synuclein (96-140). The specific results are shown in Table 3:

[0220] Table 3 Fully human antibody epitope mapping results

[0221]

[0222] 4.6 Affinity testing of fully human antibodies

[0223] The affinity (K) of the selected full-length antibody to the α-Synuclein monomer was detected by SPR technology (Biacore). D The full-length antibody to be detected was captured using a Protein A chip. α-Synuclein monomer was diluted in HBS-EP+Buffer, starting at 800 nM and followed by a two-fold serial dilution to eight concentration points. The assay was run at a flow rate of 30 μl / min: α-Synuclein monomer was allowed to flow through the chip to bind to the antibody to be detected for 90 seconds; HBS-EP+Buffer was allowed to flow through the chip to dissociate for 120 seconds; the chip was treated with Gly-HCl pH 1.5 at 30 μl / min for 30 seconds to regenerate for the next round of detection. After the assay was completed, K was calculated using analysis software. D value.

[0224] BLI technology (Octet) was used to detect the affinity (K) of the selected full-length antibody to α-Synuclein PFF. DThe full-length antibody to be detected was captured using a Protein A probe. α-Synuclein PFF protein was diluted in PBST buffer, starting at 500 nM and followed by a 2-fold gradient dilution to seven concentration points. The probe bound to the antibody to be detected was inserted into the α-Synuclein PFF protein solution for 240 seconds to allow antibody-antigen binding. After binding, the probe was inserted into PBST buffer to allow the antibody-antigen complex to dissociate for 300 seconds. The probe was treated with Gly-HCl pH 1.5 for 30 seconds to regenerate the probe for the next round of detection. After detection, K was calculated using analysis software. D value.

[0225] The specific results are shown in Table 4:

[0226] Table 4 Affinity test results of fully human antibodies

[0227]

[0228] Example 5: Detection of the inhibition of α-Synuclein polymerization by fully human anti-α-Synuclein antibodies

[0229] Use DPBS diluent to dilute the fully human anti-α-Synuclein antibodies P21-hIgG1 and P22-hIgG1 prepared in Example 4, starting from 1.3μM (2×), 2-fold dilution, 10 concentration gradients, and 280μl for each concentration well. 50μl of antibody needs to be added to each well. Mix equal volumes of α-Synuclein PFF (Abcam / ab218819) and monomer with DPBS, dilute them to 16μg / ml (4×) and 300μg / ml (4×), respectively, and add 50μl to each well, that is, the final concentrations of the two are 4μg / mL and 75μg / ml, respectively. Take 50μl of antibody from each well and incubate with the mixture of PFF and monomer at 37°C, 800rpm, and incubate for 72h. Set the microplate reader to 37°C in advance, remove 50 μl of supernatant and transfer to the corresponding wells of a new 96-well plate. Add 50 μl of ThT (50 μM), mix thoroughly, and analyze on a microplate reader (Ex 440 nm, Em 485 nm). Measure ThT fluorescence intensity (Ex / Em = 440 / 485). A negative control is a non-synuclein-related control antibody of the same isotype.

[0230] ThT test results are shown in Figure 2 The horizontal axis represents the different sample concentrations, and the vertical axis represents ThT fluorescence intensity. The results show that the ED50 values ​​for antibodies P21-hIgG1 and P22-hIgG1 are 20.78 nM and 19.49 nM, respectively. This indicates that both antibodies P21-hIgG1 and P22-hIgG1 inhibit α-synuclein aggregation.

[0231] Example 6: Fully human anti-α-Synuclein antibody inhibits α-Synuclein phosphorylation detection

[0232] 5000 HEK293-GFP-syn cells (obtained from Wuhan University) were plated / well in a 96-well cell culture plate (Corning, 3603) and cultured for 48 h.

[0233] Antibody was diluted 5-fold in three dilution steps starting at 50 μg / ml (330 nM). Antibody was incubated with PFF (10 μg / ml) at 37°C for 15 minutes. Transfection reagent Fugene HD (1 μl / well) was then added and incubated at 37°C for 15 minutes. A 10 μl solution (2 μl PFF + 5 μl antibody + 1 μl Fugene HD + 2 μl OPTI-MEM) was added directly to cells and incubated for 48 hours. Supernatant was discarded, and cells were fixed with 50 μl of fixative for 30 minutes. Cells were permeabilized with 50 μl of 0.1% Triton X-100 for 10 minutes, and then blocked with 50 μl of blocking solution for 30 minutes at room temperature. Anti-p-syn (phosphorylated α-Synuclein) directly conjugated primary antibody (BioLegend, 825708) was diluted 1:700 (v / v), and 50 μl was added to the plate. The plate was incubated at 37°C for 1.5 h and then washed three times with PBST (5 min each). The supernatant was discarded, and 10 μg / ml Hoechst nuclear stain (1:10,000, v / v) was added and incubated for 5 min before high-content detection.

[0234] See the results Figure 3 The horizontal axis represents the different sample loading groups, and the vertical axis represents relative fluorescence intensity. The maximum inhibition rate of P22-hIgG1 phosphorylation was 109.97%. The maximum inhibition rate was calculated as follows: ("PFF group fluorescence intensity" - "P22-hIgG1 group fluorescence intensity") / ("PFF group fluorescence intensity" - "blank cell group fluorescence intensity") × 100%. This indicates that the α-synuclein antibody prepared by the present invention is capable of inhibiting α-synuclein phosphorylation.

[0235] Example 7: Detection of α-Synuclein Propagation Inhibited by Fully Human Anti-α-Synuclein Antibodies

[0236] Experiment 1:

[0237] Donor SHSY5Y cells (BNCC) (5000 cells / well) were infected with 50 μg / ml α-Synuclein PFF, 500 μl per well. After 24 h of infection, the infection supernatant was discarded, the cells were washed twice with DPBS, and the culture medium was replaced with blank experimental medium. After 24 h, the supernatant was collected and centrifuged at 1000 rpm and 4°C for 5 min to remove cell debris.

[0238] The fully human anti-α-synuclein antibody P22-hIgG1 prepared in Example 4 was serially diluted three-fold, starting at a concentration of 667 nM and spanning six dilutions. The diluted antibody was incubated with the infection supernatant at 37°C for 1 hour. The sample was then added to recipient SHSY5Y cells and incubated for 16 hours.

[0239] After the culture was completed, the cells were fixed with 4% formaldehyde at 4°C overnight, permeabilized with 0.1% Triton-100X for 10 min, blocked for 30 min, and incubated with a 1:500 (v / v) dilution of anti-α-synuclein antibody (Abcam / ab27766) at room temperature for 2 h. The primary antibody was washed, and a 1:1000 (v / v) dilution of mouse 647 fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 h. Then, a 1:2500 (v / v) dilution of Hoechst was added and incubated at room temperature in the dark for 5 min. The cells were washed, and high-content photography was used to detect intracellular α-Synuclein expression.

[0240] See the results Figure 4A The horizontal axis represents the different sample loading groups, and the vertical axis represents fluorescence intensity. After adding different concentrations of the antibody P22-hIgG1, fluorescence intensity decreased compared to the PFF supernatant alone group. This was dose-dependent, with increasing antibody concentration leading to a decrease in fluorescence intensity. This demonstrates that the anti-α-synuclein antibodies prepared by the present invention can inhibit α-synuclein transmission.

[0241] Experiment 2:

[0242] This study used an optimized method to detect the inhibition of α-Synuclein transmission by fully human anti-α-Synuclein antibodies, as follows:

[0243] SH-SY5Y cells (Chinese Academy of Sciences) were seeded in 24-well plates, with 50,000 cells per well, and cultured overnight. These were the recipient cells. Another SH-SY5Y cell was seeded in a 6 cm culture dish (2.4 × 10 6The cells were infected with 33.3 μg / ml α-Synuclein PFF for 24 hours, digested, and transferred to a transwell (150,000 cells / transwell). These served as donor cells. The fully human anti-α-Synuclein antibodies P21-hIgG1 and P22-hIgG1 were preincubated with recipient cells for 30 minutes. The transwell containing the donor cells was then transferred to a plate containing recipient cells and incubated for 48 hours. The final antibody concentrations were 300 nM, 30 nM, and 3 nM.

[0244] After the culture was completed, the cells were fixed with 4% formaldehyde for 15 min, washed three times with DPBS, permeabilized with 0.1% TritonX-100 for 10 min, blocked at room temperature for 1 h, and incubated at room temperature for 2 h with 300 μL of 1:700 (v / v) diluted anti-α-synuclein antibody (Abcam / ab195025). The cells were then washed twice with DPBS, and 400 μL of 1:5000 (v / v) diluted DAPI antibody (Merck / D9542) was added and incubated for 10 min. The cells were washed twice with DPBS, and the amount of intracellular α-Synuclein was detected by high-content photography.

[0245] See the results Figure 4B The horizontal axis represents the different sample loading groups, and the vertical axis represents the ratio of the number of intracellular fluorescent spots in the test antibody group to the negative control (unrelated hIgG1). The results show that as the antibody concentration increases, the number of intracellular fluorescent spots decreases. P22-hIgG1 exhibits significant inhibition of α-synuclein propagation, and P21-hIgG1 also exhibits significant inhibitory effects at high concentrations. This indicates that the anti-α-synuclein antibodies prepared by the present invention can inhibit α-synuclein propagation.

[0246] Example 8: Detection of the effect of fully human anti-α-Synuclein antibodies on the phagocytosis of α-Synuclein by microglia

[0247] Experiment 1:

[0248] 10,000 BV2 (BNCC) cells were cultured overnight. A mixture of 25 μg / ml PFF and 667 nM antibody was added and exposed to the cells for 3 hours. Then, 10 μg / ml Hoechst nuclear stain (1:1000, v / v) was added and incubated for 5 minutes. High-content imaging was used to detect the antibody's ability to promote phagocytosis. To detect inflammatory cytokine secretion, PFF were sonicated for 1 hour at a concentration of 50 μg / ml. The antibody concentration was 667 nM, and the positive control, LPS, was 1 μg / ml. Two replicates were set up. Cells were treated for 24 hours in a final volume of 100 μl. Cell supernatants were collected, diluted 2-fold, and ELISA was used to detect mTNF-α release. The results of high-content imaging and ELISA are shown in Figures 5A and 5B, respectively. As can be seen, P21-hIgG1 strongly promoted microglial phagocytosis of PFF without inducing excessive TNF-α production during phagocytosis.

[0249] Experiment 2:

[0250] This experiment uses an optimized method to detect whether fully human anti-α-Synuclein antibodies promote the phagocytosis of α-Synuclein by microglia, as follows:

[0251] PFF was labeled with pHrodo Red (Gibco, catalog number: P36600), 4 μM PFF and antibodies (5× system; fully human anti-α-Synuclein antibodies P21-hIgG1 and P22-hIgG1) were pre-mixed for 30 min; 5000 BV2 (procell) cells were cultured per well overnight. 40 μL of 62.5 μg / ml Fucioden (purchased from Sigma, catalog number: F8315) was added to each well to act on the cells for 30 minutes. After the end, 10 μL of the above-mentioned pre-mixed mixture (containing PFF / fully human anti-α-Synuclein antibody complex) was added and treated for 1 hour (final antibody concentrations were 300 nM, 100 nM, 30 nM, 10 nM, and 3 nM; PFF was 0.8 μM; Fucoiden was 50 μg / mL). Hoechst nuclear staining solution (1:5000, v / v) was incubated for 5 minutes, and 50 μL of DPBS was replaced. High-content photography was used to detect the effect of the antibody on promoting phagocytosis.

[0252] Test results such as Figure 5C As shown, the horizontal axis represents the different antibody treatment groups, and the vertical axis represents the intracellular fluorescence intensity. The results show that P21-hIgG1 has a strong ability to promote microglial cells to phagocytose PFFs, and P22-hIgG1 also has a certain promoting effect on phagocytosis.

[0253] Example 9: In vivo efficacy testing of murine anti-α-synuclein antibodies in PD model mice (induced by intracranial stereotaxic injection of α-synuclein)

[0254] Murine-derived antibodies were prepared from P21-hIgG1 and P22-hIgG1 for in vivo efficacy evaluation in PD model mice and immunohistochemical testing of PD brain tissue sections. The murine-derived antibodies were prepared by replacing the heavy chain constant region of the fully human P21-hIgG1 and P22-hIgG1 antibodies with a murine IgG1 constant region (NCBI Sequence ID: AAK53870.1); replacing the light chain constant region of the fully human P21-hIgG1 antibody with a murine kappa light chain constant region (Uniprot ID: P01837); and replacing the light chain constant region of the fully human P22-hIgG1 antibody with a murine lambda light chain constant region (Uniprot ID: A0A0G2JE99, where X is G).

[0255] 1. Experimental methods and steps:

[0256] 1.1 PD mouse model establishment and antibody injection

[0257] Wild-type C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Laboratory Animal Quality Certificate No.: 110011210102978614). Mice were randomly assigned to each group in a blinded manner. The groups were as follows:

[0258]

[0259] According to the different injected drugs, the PBS group was the complete blank control group, the PFFs group was the PD model group, and the P21-mIgG1, P22-mIgG1 and negative control mlgG1 (anti-HEL, Biointron, B118301) were the drug groups. All drugs were injected intraperitoneally. Antibody injection began one week before modeling and was administered once a week at a dose of 30 mg / kg. The total administration period was twenty-six weeks.

[0260] The PD model group was created by injecting α-synuclein preformed fibrils into the right striatum of 2-month-old female wild-type C57BL / 6J mice. Stereotaxic injections were performed using a standard brain stereotaxic apparatus (RWD) at the injection site 0.2 mm anterior, 2.0 mm lateral, and 2.7 mm ventral to the bregma. Using a 10 μl microinjection needle (Hamilton) and a microinjection pump (KD Scientific), 14 μg (7 μl, 2 mg / ml) of α-synuclein preformed fibrils were injected into the designated site at a rate of 300 nl / min.

[0261] 1.2 Antibody efficacy testing in PD model mice: mouse brain immunohistochemistry test

[0262] 1.2.1 Mouse Brain Immunohistochemistry Procedure: Mouse brain tissue was fixed with 4% paraformaldehyde and embedded in paraffin. 4 μm paraffin sections were cut and stained using a universal immunohistochemistry kit (ZSGB-BIO, PV-6000) and horseradish enzyme-conjugated streptavidin (ZSGB-BIO, ZB-2404). Images were captured using an inverted microscope (Olympus, IXplore Standard) and analyzed using Image J software.

[0263] 1.2.2 Results of immunohistochemistry test on rat brain (immunohistochemistry results and quantitative evaluation results of α-Synuclein deposition in rat brain are shown in Figure 2. Figure 6 A and Figure 6 B): Immunohistochemistry of mouse brain showed that the α-Synuclein deposition on the injection side and the contralateral side of the PD model mice treated with P21-mIgG1 and P22-mIgG1 was significantly reduced compared with the PD model mice (PFFs group), and the results were statistically significant ( Figure 6 B). Dopamine neuron staining showed that the P21-mIgG1 and P22-mIgG1 antibody treatment groups could reduce neuronal loss on the injected side ( Figure 7 A), the results showed significant difference ( Figure 7 B).

[0264] 1.3 Antibody efficacy testing in PD model mice: HPLC neurotransmitter content testing

[0265] 1.3.1 High-Performance Liquid Chromatography (HPLC) Neurotransmitter Content Assay Procedure: Mouse brain tissue was lysed with 0.1 M perchloric acid + 0.1% cysteine ​​lysis buffer and centrifuged at 16,000 g for 30 minutes at 4°C. The supernatant was collected for HPLC sample preparation. Dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) content in the samples was measured using a high-performance liquid chromatograph (Waters, e2695, e2465).

[0266] 1.3.2 High-performance liquid chromatography (HPLC) neurotransmitter content test results: From the levels of the neurotransmitter dopamine and its metabolite 3,4-dihydroxyphenylacetic acid, it can be seen that the P21-mIgG1 and P22-mIgG1 antibody treatment groups can significantly increase the levels of dopamine and 3,4-dihydroxyphenylacetic acid, and reduce the damage to the dopaminergic pathway ( Figure 8 ).

[0267] 1.4 Antibody efficacy testing in PD model mice: behavioral experimental testing in mice

[0268] 1.4.1 Mouse behavioral test: Rotarod test

[0269] Mice were placed on the rotating rod of a horizontal rotarod apparatus (Taimeng). The rotation speed was gradually increased from 5 rpm to 40 rpm over 3 minutes. Mice were trained three times daily, with a 30-minute rest period between each training session. The formal experiment began after three consecutive days of training. The time from the start of the rotarod rotation until the mouse fell was recorded.

[0270] 1.4.2 Mouse behavioral test: Wire hang test

[0271] Place a mouse on a 1.5m x 1.5m metal mesh with 5mm pores. Swing the mesh horizontally several times, then flip it over so that the mouse hangs upside down by its paws. Measure the time from the moment the mouse begins to hang upside down until it falls.

[0272] 1.4.3 Mouse behavioral test: Pole test

[0273] Mice were placed head-up on top of a vertical rough wooden pole (45 cm long, 1 cm diameter) and allowed to descend voluntarily to the ground. Mice were trained three times daily for two consecutive days before the formal experiment began. The time it took for the mice to turn and crawl to the ground was recorded.

[0274] 1.4.4 Mouse behavioral test: Balance beam test

[0275] Mice were placed at one end of an 80 cm long, 1.6 cm wide wooden pole and allowed to crawl to the other end. Mice were trained three times daily for two consecutive days before the formal experiment began. The formal experiment used an 80 cm long, 0.9 cm wide wooden pole. The time it took the mice to crawl 50 cm on the pole was recorded.

[0276] 1.4.5 Results of mouse behavioral experiments

[0277] The test results are as follows Figure 9 As shown in the results, the PFFs group mice showed significant motor behavior defects in all behavioral experiments, indicating that the model successfully manifested the motor phenotype of PD; Figure 9 A) and Wire hang test ( Figure 9 B) In the experiment, the mice in the P21-mIgG1 and P22-mIgG1 antibody groups had significantly longer time on the rotating rod and metal mesh than those in the PFFs model group and the negative control antibody group. Figure 9 C) and balance beam ( Figure 9D) In ​​the experiment, mice in the P21-mIgG1 and P22-mIgG1 antibody groups took significantly less time to complete the test than the PFFs model group and the negative control antibody group, indicating that the P21-mIgG1 and P22-mIgG1 treatment groups had stronger motor abilities. Overall, all behavioral experimental results were consistent, indicating that P21-mIgG1 and P22-mIgG1 treatment significantly restored the motor impairment caused by PFFs and improved the motor behavioral ability simulated by PFFs, indicating that treatment with P21-mIgG1 and P22-mIgG1 has the potential to improve motor ability in Parkinson's disease.

[0278] Example 10: In vitro immunohistochemical detection of PD patients with murine anti-α-Synuclein antibodies

[0279] 1. Experimental methods and steps:

[0280] 1.1 Main instruments, equipment and reagents:

[0281] Cryostat (Thermo Scientific), 4-8°C refrigerator (Haier), water bath thermostat (YLE-1000-80°C), ultra-low temperature refrigerator (Thermo), fluorescence confocal microscope (Nikon, Japan), digital virtual section microscope (MOTIC); Paraformaldehyde (Sinopharm Chemical Reagent), sodium chloride (Sinopharm Chemical Reagent), OTC embedding medium (SAKURA), phosphate buffered saline (PBS) (Sinopharm Chemical Reagent), Tween-20 (Solarbio), sodium citrate (SSC) (Fisher Scientific), disodium hydrogen phosphate (Sinopharm Group Chemical Reagent), sodium dihydrogen phosphate (Sinopharm Group Chemical Reagent), ethylene glycol (Sinopharm Group Chemical Reagent), polyvinyl pyrrolidone / PVP (Yuanye Biological), formic acid (Sinopharm Group Chemical Reagent), 30% hydrogen peroxide (Xilong Scientific Chemical Reagent), diaminobenzidine (DAB) (Vectorlab), bisbenzimide (Sigma), Sudan Black (Sigma), anhydrous ethanol (Sinopharm Group Chemical Reagent), dimethylbenzene (Sinopharm Group Chemical Reagent), neutral resin (Sigma). PD patient brain tissue was obtained from the brain bank of Xiangya Medical College.

[0282] 1.2 Pathological section processing

[0283] 1.2.1 Tissue Fixation and Sugar Sedimentation: Fix the tissue in 4% paraformaldehyde (for human brains, fix for 2-3 weeks). After the fixed tissue is settled in 15% sucrose solution, it is then placed in 30% sucrose solution and then in 40% sucrose solution. Sedimentation is considered complete when any tissue floating on the surface of the liquid before settling has settled to the bottom, using a 50ml centrifuge tube as an example.

[0284] 1.2.2 Embedding and Sectioning: First, prepare a mold based on the desired tissue size. Place the tissue flatly into the mold, add embedding medium, and freeze in a -80°C freezer. Pre-cool the microtome to -20°C. Remove the foil mold surrounding the embedded tissue. Add embedding medium between the bottom of the tissue and the slicer, then freeze in the microtome for 10 minutes. Once the tissue is on the slicer, install the slicer, attach the blade, and adjust the tissue thickness for sectioning. Generally, slice human brain tissue at 40 μm. Wash the cut tissue with PBS, unfold it, and store in the refrigerator.

[0285] 1.2.3 Antibody Incubation

[0286] 1.2.3.1 Rewarming and rinsing: Take the tissue out of the -20°C freezer and rewarm it for 30 minutes. Then, select the target tissue and rinse it with 0.01M PBS (pH=7.4) buffer three times, each time for 6-10 minutes.

[0287] 1.2.3.2 Antigen repair and antigenic determinant exposure: Perform acid repair or heat repair on the tissue. Antibodies that do not require a repair step can skip the repair step and buffer treatment and proceed directly to the removal of endogenous peroxidase.

[0288] 1.2.3.3 Rinse: Buffer treatment, 0.1M Tris-HCl treatment for 10 minutes, then rinse three times with 0.01M PBS (pH=7.4) buffer, each time for 6-10 minutes.

[0289] 1.2.3.4 Blocking: First, prepare 5% (v / v) animal serum (blocking serum is generally from the same source as the secondary antibody), that is, mix the animal serum stock solution with 0.01M PBS-T (pH = 7.4) buffer at a ratio of 1:19 (v / v) to make 5% animal serum. Then, pick the tissue into an EP tube containing 5% animal serum and incubate on a shaker at room temperature for 2 hours to achieve the purpose of blocking nonspecific antigens in the tissue.

[0290] 1.2.3.5 Incubation with primary antibody: According to the optimal concentration recommended in the antibody manual and found in actual experiments, add the primary antibody (antibody P21-mIgG1, P22-mIgG1, P01-mIgG1) in proportion to the sealed EP tube in the above step, incubate on a shaker at room temperature for 2 hours, and then incubate in a 4°C refrigerator overnight.

[0291] 1.2.3.6 Incubation with secondary antibody: Remove the EP tube from the refrigerator and rewarm it at room temperature on a shaker for 20 minutes. Rinse it three times with 0.01M PBS (pH=7.4) buffer, each time for 6-10 minutes. Then, place the tissue in a biotinylated broad-spectrum IgG secondary antibody or a specific secondary antibody and incubate it at room temperature on a shaker for 2 hours. The secondary antibody concentration is 1:250 (v / v).

[0292] 1.2.3.7 Incubation with tertiary antibody: Rinse three times with 0.01M PBS (pH 7.4) buffer for 6-10 minutes each time. Place the tissue in an EP tube containing the tertiary antibody (i.e., biotin-avidin-horseradish peroxidase complex) and incubate on a shaker at room temperature for 2 hours.

[0293] 1.2.3.8 Color Development: Rinse three times with 0.01M PBS (pH=7.4) buffer, each time for 6-10 minutes. Place the tissue into a well plate and add an appropriate amount of 0.05% diaminobenzidine (DAB) to each well, enough to cover all tissue and allow it to float freely. After a 3-minute reaction, add 0.03% H2O2 sequentially. Monitor the color development time under a microscope; be careful to avoid light throughout the color development process. Terminate color development immediately when the desired effect is achieved under the microscope by placing the tissue into 0.01M PBS (pH=7.4) to terminate color development.

[0294] 1.2.3.9 Sealing: Use neutral resin to seal the slides. When sealing, special attention should be paid to ensure that no bubbles are generated between the cover slip and the slide and there is no excess neutral resin around the slide.

[0295] 1.3 Antibody immunohistochemistry test results of brain tissue sections of PD patients

[0296] Test results such as Figure 10 As shown, Figure 10 A, 10C, and 10E show the immunohistochemical results of brain tissues from PD patients using antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1, respectively. The results show that antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1 can recognize Lewy bodies and Lewy neurofilaments in the brain tissues of PD patients; Figure 10B, 10D and 10F show the immunohistochemistry results of hippocampal tissues of PD patients using antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1, respectively. The results showed that antibodies P21-mIgG1, P22-mIgG1, and P01-mIgG1 can recognize Lewy bodies and Lewy neurofilaments in hippocampal tissues of PD patients.

[0297] In summary, antibodies P21-mIgG1 and P22-mIgG1, like the positive control antibody P01-mIgG1, can recognize α-synuclein deposits, namely Lewy bodies and Lewy neurofilaments, in patient tissues with relatively strong specificity. In terms of specificity and clarity of recognition, antibodies P21-mIgG1 and P22-mIgG1 both outperformed the positive control antibody P01-mIgG1.

[0298] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to α-synuclein, wherein the antibody or antigen-binding fragment thereof comprises: (a) VH CDR1, VH CDR2, and VH CDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 11; and VL CDR1, VL CDR2, and VL CDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO: 15; or (b) VH CDR1, VH CDR2 and VH CDR3 contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 19; and VL CDR1, VL CDR2 and VL CDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO:

23.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1)(a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 12, VH CDR2 of SEQ ID NO: 13, and VH CDR3 of SEQ ID NO: 14; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 16, VL CDR2 of SEQ ID NO: 17, and VL CDR3 of SEQ ID NO: 18; or (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 20, VH CDR2 of SEQ ID NO: 21, and VH CDR3 of SEQ ID NO: 22; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 24, VL CDR2 of SEQ ID NO: 25, and VL CDR3 of SEQ ID NO: 26; in, The CDRs are defined by the Kabat numbering system; or, (2) (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 36, VH CDR2 of SEQ ID NO: 37, and VH CDR3 of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 39, VL CDR2 of SEQ ID NO: 40, and VL CDR3 of SEQ ID NO: 41; or (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 42, VH CDR2 of SEQ ID NO: 43, and VH CDR3 of SEQ ID NO: 44; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 45, VL CDR2 of SEQ ID NO: 46, and VL CDR3 of SEQ ID NO: 47; wherein the CDRs are defined by the IMGT numbering system; or, (3) (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 48, VH CDR2 of SEQ ID NO: 49, and VH CDR3 of SEQ ID NO: 50; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 51, VL CDR2 of SEQ ID NO: 52, and VL CDR3 of SEQ ID NO: 53; or (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 54, VH CDR2 of SEQ ID NO: 55, and VH CDR3 of SEQ ID NO: 56; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 57, VL CDR2 of SEQ ID NO: 58, and VL CDR3 of SEQ ID NO: 59; Wherein, the CDRs are defined by the Chothia numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 11 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 15 or a variant thereof; or (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 19 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 23 or a variant thereof; in, Said variants have one or several amino acid substitutions, deletions or additions compared to the sequence from which they are derived, or have a sequence identity of at least 80%.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 19 and a VL comprising the sequence shown in SEQ ID NO:

23.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 11 and a VL comprising the sequence shown in SEQ ID NO:

15. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a constant region derived from human immunoglobulin.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein The heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG1, IgG2, IgG3 or IgG4.

9. The antibody or antigen-binding fragment thereof according to claim 7 or 8, wherein The heavy chain constant region comprises the sequence shown in SEQ ID NO:

27.

10. The antibody or antigen-binding fragment thereof according to claim 6, wherein The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein The light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin κ or λ.

12. The antibody or antigen-binding fragment thereof according to claim 10 or 11, wherein The light chain constant region comprises the sequence shown in SEQ ID NO: 28 or 29.

13. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 32 and a light chain as shown in SEQ ID NO:

33.

14. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO:

31.

15. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single-domain antibody (sdAb); and / or, the antibody is a murinized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

16. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the heavy chain variable region and light chain variable region thereof, or the heavy chain and light chain thereof.

17. A vector comprising the isolated nucleic acid molecule of claim 16.

18. A host cell comprising the isolated nucleic acid molecule of claim 16 or the vector of claim 17.

19. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, comprising culturing the host cell according to claim 18 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier and / or excipient.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the isolated nucleic acid molecule according to claim 16, the vector according to claim 17, the host cell according to claim 18, or the pharmaceutical composition according to claim 20 for preparing a medicament for preventing and / or treating an α-synuclein-related disease in a subject, wherein the α-synuclein-related disease is Parkinson's disease.

22. The use according to claim 21, wherein The antibodies or antigen-binding fragments thereof are used alone or in combination with another pharmaceutically active agent.

23. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and a detectable label linked to the antibody or antigen-binding fragment thereof.

24. The conjugate according to claim 23, wherein The detectable label is selected from an enzyme, a chemiluminescent reagent, a fluorescent dye, a radionuclide or biotin.

25. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the conjugate according to claim 23 or 24.

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the conjugate according to claim 23 or 24, in the preparation of a detection reagent for detecting the presence or level of α-Synuclein in a sample and / or diagnosing a disease associated with α-Synuclein, wherein the disease associated with α-Synuclein is Parkinson's disease.

27. A method for detecting the presence or level of α-Synuclein in a sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the conjugate according to claim 23 or 24, wherein the method is for non-diagnostic purposes.

28. The method of claim 27, wherein The method is an immunological assay.

29. The method of claim 27, wherein The method is immunoblotting, enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

30. The method of claim 27, wherein The method comprises using the conjugate of claim 23 or 24.

31. The method of claim 27, wherein The method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label.

32. The method of claim 31, wherein The detectable label is selected from an enzyme, a chemiluminescent reagent, a fluorescent dye, a radionuclide or biotin.

33. The method of claim 27, comprising: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the conjugate according to claim 23 or 24; (2) Detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of α-Synuclein or cells expressing α-Synuclein.

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