Α-synuclein-targeting antibody and use thereof

Abstract

The present application relates to an antibody specifically targeting α-synuclein and a use of the antibody. The present application also relates to a humanized antibody involved in the antibody and a use of the humanized antibody.

Description

Antibodies targeting α-synuclein and their applications Technical Field

[0001] This application relates to antibodies that specifically target α-synuclein and their uses, and also to humanized antibodies of said antibodies and their uses. Background Technology

[0002] Alpha-synuclein (α-syn) is a protein primarily expressed in nerve cells, playing a crucial role in synapse formation and neural signal transduction. However, α-synuclein is closely associated with the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). In these diseases, α-syn misfolds, accumulating within nerve cells to form α-syn aggregate fibers that propagate between neurons, triggering neuroinflammation and disrupting protein homeostasis. This leads to a series of lesions from the brainstem to the cerebral cortex, resulting in diverse symptoms ranging from motor impairment to cognitive impairment. α-syn aggregates eventually combine with other components to form Lewy bodies (LBs), leading to neuronal damage and dysfunction. The formation of Lewy bodies is considered a hallmark pathological change in PD. Therefore, the abnormal aggregation and propagation of α-syn is a key driver of PD development, making it a focus of current PD treatment strategies and research.

[0003] α-Syn consists of 140 amino acids, comprising a positively charged N-terminus, a hydrophobic NAC domain associated with aggregation, and a negatively charged C-terminus. Studies have found that α-syn mediates the intercellular propagation of α-syn aggregates and induces neuroinflammation through interactions between its C-terminus and various receptor proteins.

[0004] Currently, there are no effective drugs to halt the progression of Parkinson's disease (PD) and thus cure it. Among numerous treatment strategies, monoclonal antibody therapy targeting α-synuclein (αsyn) is particularly noteworthy. Currently, five monoclonal antibody drugs targeting α-synuclein are in clinical trials, such as the humanized antibody Prasinezumab jointly developed by Roche and Prothena. However, the Phase II clinical trial of Prasinezumab failed to meet its primary endpoint. Other antibodies, such as AbbVie's ABBV-0805 and Biogen's BIIB054, have either terminated or cancelled their clinical trials. It is evident that the development of antibody drugs targeting α-synuclein is still in its early stages, with no antibody drugs yet on the market. There remains an urgent need in this field to develop antibodies targeting α-synuclein for the treatment of diseases such as PD. This invention addresses these needs to some extent. Summary of the Invention

[0005] Given the crucial role of the C-terminal domain of α-syn in Parkinson's disease (PD), the inventors have developed monoclonal antibodies targeting the C-terminus of α-syn and the C-terminus of α-syn aggregate fibers for PD treatment. This antibody drug not only directly targets the pathological process of the disease, potentially altering the progression of PD, but may also block the spread of this toxicity, thereby slowing disease development.

[0006] In a first aspect, the present invention provides an antibody targeting α-syn and its antigen-binding fragment, which has the following advantages:

[0007] (1) High affinity binding of wild-type α-syn monomers, wild-type α-syn preformed fibrils (PFFs), and α-syn aggregates;

[0008] (2) It binds to a variety of mutant α-syn PFFs that cause neurological diseases with high affinity;

[0009] (3) Block the binding of α-syn to its membrane surface receptors;

[0010] (4) Recognize new antigenic epitopes, namely, recognize the C-terminal fragments (residues 131-140) of α-syn fibers.

[0011] In one embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn or α-syn fibers and its antigen-binding fragment, comprising:

[0012] 1) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:2 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:7.

[0013] 2) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:15 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:16; or

[0014] 3) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:15 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:17.

[0015] In one embodiment, the present invention provides an anti-α-synuclein antibody that specifically binds to α-synuclein or α-synuclein filaments and its antigen-binding fragment, comprising:

[0016] 1) Containing sequences as shown in SEQ ID NO:3, 4 and 5, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences, or HCDR1, HCDR2, HCDR3 composed of said sequences; and containing sequences as shown in SEQ ID NO:8, 9 and 10, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences, or LCDR1, LCDR2, LCDR3 composed of said sequences;

[0017] 2) Sequences comprising the sequences shown in SEQ ID NO:3, 6, and 5, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions relative to said sequences, or HCDR1, HCDR2, and HCDR3 composed of said sequences; and sequences comprising the sequences shown in SEQ ID NO:11, 9, and 10, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions relative to said sequences, or LCDR1, LCDR2, and LCDR3 composed of said sequences; or

[0018] 3) Containing sequences as shown in SEQ ID NO:3, 6 and 5, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences, or HCDR1, HCDR2, HCDR3 composed of said sequences; and containing sequences as shown in SEQ ID NO:12, 9 and 10, or sequences containing one or more amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences, or LCDR1, LCDR2, LCDR3 composed of said sequences.

[0019] In one embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn or α-syn fibers and its antigen-binding fragment, comprising:

[0020] 1) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 4 and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:8, 9 and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences;

[0021] 2) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 6, and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:11, 9, and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences; or

[0022] 3) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 6 and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:12, 9 and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences.

[0023] In one embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn or α-syn fibers and its antigen-binding fragment, comprising a heavy chain variable region, wherein:

[0024] 1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 2, or is composed of SEQ ID NO: 2;

[0025] 2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 13, or is composed of SEQ ID NO: 13; or

[0026] 3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 15, or is composed of SEQ ID NO: 15.

[0027] In one embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn or α-syn fibers and its antigen-binding fragment, comprising a light chain variable region, wherein:

[0028] 1) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 7, or is composed of SEQ ID NO: 7;

[0029] 2) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 14, or is composed of SEQ ID NO: 14;

[0030] 3) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 16, or is composed of SEQ ID NO: 16; or

[0031] 4) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17, or is composed of SEQ ID NO: 17;

[0032] In another embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn and its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein:

[0033] 1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 2, or is composed of SEQ ID NO: 2; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 7, or is composed of SEQ ID NO: 7;

[0034] 2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 13, or is composed of SEQ ID NO: 13; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 14, or is composed of SEQ ID NO: 14;

[0035] 3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 15, or is composed of SEQ ID NO: 15; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 16, or is composed of SEQ ID NO: 16; or

[0036] 4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 15, or is composed of SEQ ID NO: 15; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17, or is composed of SEQ ID NO: 17.

[0037] In some embodiments, the antibody or its antigen-binding fragment further comprises heavy chain and / or light chain constant region sequences derived from human antibody germline common sequences. The light chain constant region is preferably a human κ or λ chain constant region. The heavy chain constant region can be a γ, μ, α, δ, or ε chain, and in some embodiments, the heavy chain constant region is preferably derived from the constant region sequences of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the light chain constant region comprises, or is composed of, the sequence shown in SEQ ID NO: 19. In another embodiment, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 18.

[0038] It should be understood that sequence variants of these constant region structural domains may also be used, for example, to contain one or more amino acid modifications, wherein the amino acid sites are identified by the EU indexing system of Kabat et al. (1991).

[0039] In one specific embodiment, the present invention provides an anti-α-syn antibody that specifically binds to α-syn or α-syn fibers and its antigen-binding fragment, comprising a heavy chain and a light chain, wherein the heavy chain is composed of the aforementioned heavy chain variable region and SEQ ID NO: 18, and the light chain is composed of the aforementioned light chain variable region and SEQ ID NO: 19.

[0040] In some embodiments of the antibody described above, the antibody is monoclonal.

[0041] In some embodiments of the antibody described in any of the foregoing embodiments, the antibody is a full-length antibody. In a preferred embodiment, the antibody is a humanized antibody.

[0042] In one embodiment, the anti-α-syn antibody of the present invention is a complete antibody, such as IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the anti-α-syn antibody of the present invention covers only its antigen-binding portion, such as Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment.

[0043] In a second aspect, the present invention provides a pharmaceutical composition comprising (1) an antibody or an antigen-binding fragment thereof of the first aspect, and (2) a pharmaceutically acceptable carrier.

[0044] In one embodiment, the present invention provides a kit comprising the pharmaceutical composition, or comprising an antibody or antigen-binding fragment thereof of the first aspect.

[0045] Thirdly, the present invention provides isolated polynucleotide molecules that encode any of the antibodies or antigen-binding fragments described in the first aspect.

[0046] Fourthly, the present invention provides a vector comprising the nucleic acid molecule of the third aspect. In one embodiment, the vector is an expression vector.

[0047] Fifthly, the present invention provides a host cell comprising the vector of the fourth aspect or the nucleic acid molecule of the third invention. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as HEK293 cells, CHO cells, yeast cells, or plant cells.

[0048] In a sixth aspect, the present invention provides a method for treating neurodegenerative diseases, reducing the severity of neurodegenerative diseases, delaying the progression of neurodegenerative diseases, and / or delaying the onset of neurodegenerative diseases in subjects in need, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0049] In one implementation, the neurodegenerative disease is Parkinson's disease, Parkinson's dementia, Lewy body dementia, Lewy body disease, or multiple system atrophy.

[0050] In some implementations, the sample is cerebrospinal fluid, brain tissue extract, urine, or blood.

[0051] In some implementations, the reference amount is the amount of complex formed by the antibody and a sample from a healthy subject.

[0052] In one embodiment, the present invention provides a method for inhibiting the formation or increase of insoluble or soluble α-synuclein fibrillary aggregates in cells, comprising contacting the cells with an effective amount of the antibody of the present invention or an antigen-binding fragment thereof.

[0053] In a seventh aspect, the present invention provides the use of anti-α-syn antibody or its antigen-binding fragment or pharmaceutical composition in the preparation of medicaments for diagnosing or treating neurodegenerative diseases, reducing the severity of neurodegenerative diseases, delaying the progression of neurodegenerative diseases, and / or delaying the onset of neurodegenerative diseases.

[0054] In one embodiment, the present invention provides an anti-α-syn antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof for the diagnosis or treatment of neurodegenerative diseases.

[0055] In one implementation, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease (AD), Parkinson's dementia, Lewy body dementia, Lewy body disease, or multiple system atrophy. Attached Figure Description

[0056] Figure 1 shows an image of α-syn fibers examined using transmission electron microscopy.

[0057] Figure 2 shows images of α-syn PFFs detected using transmission electron microscopy.

[0058] Figure 3 shows the statistical analysis of the binding activity of different antibody mixtures with α-syn PFFs on the surface of primary rat neurons. Nine to 13 images were randomly taken for each sample. Tukey's post-hoc test was performed after one-way ANOVA. Data are presented as mean ± standard deviation. ns indicates no significant difference; * indicates p < 0.05, *** indicates p < 0.001.

[0059] Figure 4 shows a quantitative analysis of the effects of mixtures of different antibodies with α-syn PFFs on the induction of endogenous α-syn aggregation in primary neurons. The intensity of pS129α-syn in each sample was normalized to the intensity of DAPI. Six images were randomly taken for each sample. Tukey's post-hoc test was performed after one-way ANOVA. Data are presented as mean ± standard deviation. ** indicates p < 0.01.

[0060] Figure 5 shows the co-localization results of primary neuronal endogenous α-syn aggregation (pS129α-syn) induced by antibody 30H21 and α-syn PFFs. DAPI (blue), MAP2 (gray), antibody 30H21 (green), and pS129α-syn (red) are stained. Scale bar: 20 μm. Dashed boxes indicate magnified views of the areas within solid boxes, at a magnification of 4x.

[0061] Figure 6 shows tissue sections from three patients: (A) a medullary tissue section from a Parkinson's disease (PD) patient, (B) a frontal cortex tissue section from a Lewy body dementia (DLB) patient, and (C) a frontal cortex tissue section from an Alzheimer's disease (AD) patient with Lewy pathology. Merge indicates the colocalization of 30H21 and phosphorylated α-syn (pS129α-syn). DAPI (blue), pS129α-syn (red), and 30H21 (green) are stained. Scale bar: 20 μm. The dashed box indicates a magnified view of the area within the solid box, at a magnification of 6.25x.

[0062] Figure 7 shows representative immunofluorescence images of endogenous α-syn aggregation induced by α-syn PFFs in primary neurons of WT and Snca KO mice. DAPI (blue), MAP2 (green), and pS129α-syn (red) are stained. Scale bar: 20 μm.

[0063] Figure 8 shows the co-localization results of primary neuronal endogenous α-syn aggregation (pS129α-syn) induced by 30H21 and α-syn PFFs under different staining conditions. DAPI (blue), MAP2 (gray), antibody 30H21 (green), and pS129α-syn (red) were stained. The scale bar is 20 μm, and the dashed box indicates a magnified view of the area within the solid box, with a magnification of 4x.

[0064] Figure 9 shows the binding affinity of antibody 30H21 to α-syn monomers (A) and α-syn PFFs (B). ELISA absorbance values ​​were normalized and plotted against α-syn concentrations. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0065] Figure 10 shows the effects of different concentrations of H21 on α-syn fiber formation, observed by ThT kinetics (Figure 10A) and negative staining transmission electron microscopy (Figure 10B). The concentration of α-syn monomer used was 50 μM; the concentration of α-syn PFFs was 0.5 μM; the α-syn PFFs:H21 ratio represents their molar ratio. Fiber images were acquired by negative staining transmission electron microscopy at the endpoint of ThT kinetics (60 hours). In the ThT kinetic curves, data are presented as mean ± standard deviation, n = 3 independent samples. Scale bar: 200 nm.

[0066] Figure 11 shows the effect of different concentrations of ThT on the inhibition of α-syn fiber formation by H21, with the left graph using 5 μM ThT and the right graph using 10 μM ThT. The concentration of α-syn monomer used was 50 μM, and the concentration of α-syn PFFs was 0.5 μM. The ratio of α-syn PFFs to H21 represents their molar ratio (e.g., 1:0 means the molar ratio of PFFs to H21 is 1:0). In the ThT kinetic curves, the data are displayed as mean ± standard deviation, with n = 3 independent samples.

[0067] Figure 12 shows the competitive binding of antibody 30H21 (H21) to L3D1 (A), FAM171A2D1 (B), vRAGE (C), and LC3B (D) to α-syn PFFs. ELISA absorbance values ​​were normalized and plotted against the concentration of 30H21. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0068] Figure 13 shows the dot blot results of antibody 30H21(H21) recognizing α-syn monomers and PFF epitopes. Here, 140, 130, 110, and 100 represent the full-length α-syn, the truncated form of the first 130 amino acids of α-syn, the truncated form of the first 110 amino acids of α-syn, and the truncated form of the first 100 amino acids of α-syn, respectively.

[0069] Figure 14 shows the NMR spectral results of antibody 30H21 (H21) recognizing the α-syn monomeric epitope. The left image shows the two-dimensional NMR spectra of α-syn (black) and the result after adding antibody 30H21 (red). 1 H- 15 N HSQC overlay plot. Amino acids with significant changes in NMR signal intensity are marked with black boxes and enlarged in the upper right corner of the plot. The lower right corner shows the NMR signal intensity changes (I / I0) specific to α-syn amino acids in the left plot, where I is the NMR signal intensity of α-syn amino acids after antibody addition, and I0 is the NMR signal intensity of α-syn amino acids alone.

[0070] Figure 15 shows the binding of 30H21 to the neuronal cell membrane. DAPI (blue), cell membrane (CellMask, gray), antibody 30H21 (green), and α-syn (red) are stained. Arrows indicate 30H21 staining. The scale bar is 20 μm, and the dashed boxes indicate magnified views of the areas within the solid boxes, at a magnification of 4x.

[0071] Figure 16 shows the effect of 30H21 on the binding activity of α-syn PFFs to the surface of primary rat neurons. DAPI (blue), cell membrane (CellMask, gray), antibody 30H21 (green), and α-syn (red) were stained. ns indicates no significant difference; ** indicates p < 0.01, *** indicates p < 0.001.

[0072] Figure 17 shows the results of a Western blot experiment demonstrating the knockdown of the Fam171a2 gene in N2a cells. "NC" represents the negative control group; "KD" represents the knockdown experimental group. The experiment detected the internal reference protein β-actin and the target protein FAM171A2.

[0073] Figure 18 shows the binding of a mixture of 30H21 and α-syn PFFs on the surface of WT and Fam171a2 KD N2a cells. DAPI (blue) and α-syn (green) are stained. Scale bar: 20 μm; dashed boxes indicate magnified views of the areas within solid boxes, at a magnification of 4x. Tukey's post-hoc test was performed after one-way ANOVA. Data are presented as mean ± standard deviation. *** indicates p < 0.001.

[0074] Figure 19 shows the inhibition of inflammatory cytokine expression in primary microglia by antibody 30H21 (H21) after treatment with α-syn PFFs. Mouse microglia were treated with PBS (black), LPS (purple), α-syn monomers (green), α-syn PFFs (orange), a mixture of α-syn PFFs-H21 (blue), and a mixture of α-syn PFFs-FPS-ZM1 (pink), respectively. Data are presented as mean ± standard deviation, n = 4 independent samples. Tukey's post-hoc test was performed after one-way ANOVA. ns indicates no significant difference; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0075] Figure 20 shows cryo-electron microscopy images of α-syn fibers (left), α-syn fibers incubated at a molar ratio of α-syn:H21 = 1:0.2 (middle), and α-syn fibers co-incubated at a molar ratio of α-syn:H21 = 1:0.5 (right). Scale bar: 50 nm.

[0076] Figure 21 shows representative two-dimensional classification images after co-incubation of α-syn fibers and H21 at molar ratios of 1:0.2 and 1:0.5 (α-syn:H21), respectively. In the magnified two-dimensional classification images, the positions of α-syn fibers are marked with black arrows, and the locations of density changes around the fibers caused by H21 are marked with green arrows. The images also show the distances between α-syn fibers and H21, as well as the distances between adjacent H21 particles.

[0077] Figure 22 shows the overall resolution evaluation results after co-incubation of α-syn fibers with H21. The left figure shows the Fourier shell correlation (FSC) curve of α-syn fibers with a frame size of 600 pixels. The overall resolution was calculated based on the 0.143 cutoff value of the FSC curve, and the result is as follows. The right figure shows the FSC curve of the α-syn fiber when using a 360-pixel frame size. The overall resolution is also calculated based on a 0.143 cutoff value, and the result is...

[0078] Figure 23 shows three-dimensional density images (A and B) of α-syn fibers after co-incubation with H21. The N-terminal and C-terminal domains of the α-syn fibers are indicated by arrows. The lower figure shows the cryo-electron microscopy density map (C) and reconstructed cross-sectional view (D) of the α-syn fibers after co-incubation with H21. The increased density around the α-syn fibers after co-incubation with H21 is shown in orange.

[0079] Figure 24 shows images of α-syn-H21 composite fibers (α-syn-H21) reconstructed by cryo-electron tomography (cryo-ET) after incubation at a molar ratio of α-syn:H21 = 1:0.2. A side and top view of a representative α-syn fiber are also shown. Scale bar: 50 nm.

[0080] Figure 25 shows the results of three-dimensional segmentation and spatial distribution analysis of the α-syn-H21 complex using cryo-ET. α-Syn fibers are pink, H21 is shown as green in the experimental group and orange in the simulated group. The right figure shows the histogram analysis results of the closest distance between H21 and α-syn fibers. The data are presented as mean ± standard deviation.

[0081] Figure 26 shows the structural modeling and prediction of the binding mode between H21 and α-syn fibers, based on AlphaFold3 predictions and cryo-EM data analysis results.

[0082] Figure 27 shows the prediction of full-length H21 dimer and α-syn using AlphaFold 3. 131-140 The binding model (above), and the H21 antigen-binding fragment dimer and α-syn 131-140 The combined model (see figure below). Different colors indicate the predicted Local Distance Difference Test (pLDDT) scores, with the confidence score intervals represented by each color marked above the structure diagram. Below each predicted structure, the prediction template modeling score (pTM) and the interface prediction template modeling score (ipTM) are displayed, respectively. α-Syn 131-140 The structure is indicated by an arrow. α-Syn 131-140 This represents the truncated form of α-syn from position 131 to position 140.

[0083] Figure 28 shows that at 15 and 21 weeks after stereotactic injection of α-syn PFFs (PFFs+PBS) into the brain and after intraperitoneal injection of antibody 30H21 (H21) (PFFs+H21), there was no difference in body weight between mice and the control group (PBS+PBS and PBS+H21).

[0084] Figure 29 shows the results of the open field experiment in mice at week 15.

[0085] Figure 30 shows the results of the average movement time (left) and maximum movement time (right) of mice on the rotarod at week 15.

[0086] Figure 31 shows the results of the average movement time (left) and maximum movement time (right) of mice on the rotarod at week 21.

[0087] Figure 32 shows the results of the climbing test in mice at week 15. The left figure shows the results of the climbing test, and the right figure shows the results of the turning test.

[0088] Figure 33 shows representative images of mouse brain tissue stained with immunofluorescence at week 15. Left image: striatum (STR). Right image: substantia nigra (SN). Antibodies were used to label pS129α-syn (red) and DAT (green). Scale bar: 100 μm.

[0089] Figure 34 shows representative images of mouse brain tissue stained with immunofluorescence at week 21. Left image: striatum (STR). Right image: substantia nigra (SN). Antibodies were used to label pS129α-syn (red) and DAT (green). Scale bar: 100 μm.

[0090] Figure 35 shows the statistical results of pS129α-syn fluorescence intensity in the striatum (STR) and substantia nigra (SN) regions of mice at week 21 (left and middle panels), and the statistical results of the number of dopaminergic neurons in the substantia nigra (SN) region (right panel). Data were analyzed using GraphPad Prism 9.5.1, and Tukey's post-hoc test was performed after one-way ANOVA. Data are presented as mean ± standard error, n = 3 mice per group. ns indicates no significant difference; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0091] Figure 36 shows the monomer rate results detected by the humanized antibody SEC-HPLC system. Figure 36A shows the results of hz30H21-H3L0, Figure 36B shows the results of hz30H21-H13L4, and Figure 36C shows the results of hz30H21-H13L8.

[0092] Figure 37 shows the hydrophobic properties of the humanized antibody. Figure 37A shows the results of hz30H21-H3L0, Figure 37B shows the results of hz30H21-H13L4, and Figure 37C shows the results of hz30H21-H13L8.

[0093] Figure 38 shows the thermostability results of the humanized antibodies of the present invention. Figure 38A shows the real-time fluorescence quantitative results of the thermodenaturation of antibody 30H21-H3L0, Figure 38B shows the real-time fluorescence quantitative results of the thermodenaturation of antibody 30H21-H13L4, and Figure 38C shows the real-time fluorescence quantitative results of the thermodenaturation of antibody 30H21-H13L8.

[0094] Figure 39 shows the binding activity of humanized antibodies to α-syn PFFs.

[0095] Figure 40 shows the binding activity of different antibodies to wild-type α-syn PFFs in the in vitro binding assay. ELISA absorbance values ​​were normalized and plotted against α-syn PFF concentrations. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0096] Figure 41 shows the effects of different antibodies on inhibiting the aggregation of neuronal endogenous α-syn induction by α-syn PFFs in the neuronal endogenous α-syn aggregation experiment.

[0097] Figure 42 shows the effects of different antibodies on inhibiting the release of inflammatory factors.

[0098] Figure 43 shows the binding activity of different antibodies against early-onset α-syn PFFs (JOS-like PFFs) (top) and multisystem atrophic α-syn PFFs (MSA-like PFFs) in in vitro binding assays (bottom). ELISA absorbance values ​​were normalized and plotted against the concentrations of α-syn JOS-like PFFs and α-syn MSA-like PFFs, respectively. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0099] Figure 44 shows the effect of different antibodies on inhibiting the aggregation of neuronal endogenous pS129α-syn in early-onset α-syn PFFs (JOS-like PFFs) and multisystem atrophic α-syn PFFs (MSA-like PFFs).

[0100] Figure 45 shows the competitive binding of antibody H21L4 to L3D1 (A), FAM171A2D1 (B), vRAGE (C), and LC3B (D) and α-syn PFFs. ELISA absorbance values ​​were normalized and plotted against H21L4 concentration. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0101] Figure 46 shows the competitive binding of the antibody Prasinezumab to L3D1 (A), FAM171A2D1 (B), vRAGE (C), and LC3B (D) and α-syn PFFs. ELISA absorbance values ​​were normalized and plotted against Prasinezumab concentrations. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0102] Figure 47 shows the competitive binding of antibody BIIB054 to L3D1 (A), FAM171A2D1 (B), vRAGE (C), and LC3B (D) to α-syn PFFs. ELISA absorbance values ​​were normalized and plotted against BIIB054 concentration. Data are presented as mean ± standard deviation, n = 3 independent samples.

[0103] Figure 48 shows the PK activity of the humanized antibody of the present invention in mice.

[0104] Figure 49 shows the half-life of the humanized antibody of the present invention in mice.

[0105] Figure 50 shows the serum concentrations of H21L4 (pink) and Prasinezumab (blue) at 11 time points after antibody injection. Data were analyzed using an unpaired two-tailed t-test. Data are presented as mean ± standard error, n = 3 mice per group. ns indicates no significant difference; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0106] Figure 51 shows the concentrations of H21L4 (pink) and Prasinezumab (blue) in CSF at six time points after antibody injection. At the 2-day post-injection time point, the antibody concentration in CSF was below the detection threshold and could not be detected. Data were analyzed using an unpaired two-tailed t-test. Data are presented as mean ± standard error, n = 3 mice per group. ** indicates p < 0.01.

[0107] Figure 52 shows the changes in the CSF / Serum concentration ratios of H21L4 (pink) and Prasinezumab (blue) in mice at six time points after antibody injection. The results at each time point are expressed as the average values ​​of antibody concentrations measured in the serum and cerebrospinal fluid of three mice.

[0108] Figure 53 shows a comparison of the pharmacokinetic parameters of H21L4 and Prasinezumab in serum. CL: Clearance, the volume of drug cleared from plasma per unit time. Measures the ability of a drug to be cleared from the body. Vss: The extent, i.e., the volume, of the drug apparently distributed in tissues at steady state in the body. T 1 / 2 Half-life: Indicates the duration of drug exposure. AUC: Represents systemic drug exposure. MRT: Mean residence time, measures the average time the drug remains in the body. n = 3 mice. Results are expressed as the average of 3 mice.

[0109] Figure 54 shows the daily body weight records of mice in the H21L4-treated group. Data are presented as mean ± standard error, n = 3 mice per group.

[0110] Figure 55 shows the daily food consumption of mice in the H21L4-treated group. n = 3 mice per group.

[0111] Figure 56 shows the serum H21L4 concentration at different doses at nine time points following H21L4 injection. Data are presented as mean ± standard error, n = 3 mice per group.

[0112] Figure 57 shows the daily body weight records of mice in the Prasinezumab-treated group. Data are presented as mean ± standard error, n = 3 mice per group.

[0113] Figure 58 shows the daily food consumption records per cage of mice in the Prasinezumab-treated group. Data are presented as mean ± standard error, n = 3 mice per group.

[0114] Figure 59 shows the serum concentrations of Prasinezumab at different doses at nine time points following Prasinezumab injection. Data are presented as mean ± standard error, n = 3 mice per group.

[0115] Invention Details

[0116] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as these methods and conditions can be modified. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be restrictive.

[0117] I. Definition

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0119] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

[0120] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0121] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0122] The terms “α-synuclein” and “α-syn” are used interchangeably herein to refer to a 140-amino acid protein primarily expressed in nerve cells. α-Syn is physiologically soluble and comprises three domains: a positively charged N-terminus (approximately amino acids 1-60), a hydrophobic aggregation-associated NAC domain (approximately amino acids 61-95), and a negatively charged C-terminal domain (approximately amino acids 96-140). Unless otherwise stated, α-synuclein refers to wild-type α-synuclein, preferably derived from humans, such as the α-synuclein having the sequence shown in UniProt accession number P37840 (SEQ ID NO:1).

[0123] In some implementations, α-synuclein also includes variant forms containing mutations at different sites, such as α-syn variants containing mutations of A30P, E46K, A53T, A76T, E83Q, and A90V.

[0124] In pathological conditions, α-synuclein molecules form α-synuclein fibers through electrostatic and hydrophobic interactions. Abnormally aggregated α-synuclein can act as "seeds," inducing the aggregation of soluble α-synuclein and leading to lesion proliferation. As Parkinson's disease progresses, α-synuclein fibers can propagate and spread pathologically between neurons via membrane receptors on the neuronal surface. Furthermore, by binding to membrane receptors on microglia, α-synuclein fibers can induce cellular inflammatory responses. Studies have found that post-translational modifications of α-synuclein (e.g., phosphorylation or ubiquitination) are associated with α-synuclein aggregation and neurotoxicity.

[0125] The term "α-syn PFFs," also known as α-syn preformed fibrils, refers to preformed fibrils that can be taken up by cells and induce the aggregation of endogenous α-syn to form pathological α-syn aggregates. Pathological α-syn fibers within cells can be broken down into smaller fiber fragments by protease hydrolysis and other processes, and then spread between cells. Specifically, the "α-syn PFFs" used in this article refer specifically to preformed α-syn fibrils formed by the ultrasonic disruption of in vitro-prepared α-syn fibers, which can be taken up into cells and induce the formation of pathological α-syn aggregates from soluble endogenous α-syn monomers.

[0126] The term "alpha-synucleinopathy" or "synucleinopathy" refers to a group of neurodegenerative diseases characterized by the abnormal aggregation and intercellular proliferation and spread of alpha-synuclein, ultimately forming pathological inclusion bodies. These diseases mainly include Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple system atrophy (MSA).

[0127] Existing research indicates that the structure of amyloid fibril aggregates exhibits polymorphism, and this polymorphism can be used to explain the clinicopathological heterogeneity of neurodegenerative diseases. Different fibril structures of the same pathogenic protein are likely related to different types of diseases or different time points in disease progression. For example, the structures of α-synucleinic fiber aggregates from patients with Parkinson's disease (PD), multiple system atrophy (MSA), and Juvenile-onset synucleinopathy (JOS) are different. MSA is also an α-synucleinopathy, and its pathological feature is mainly the deposition of α-synucleinic aggregates in oligodendrocytes, forming oligodendrocyte inclusions (GCIs). JOS is an early-onset synucleinopathy, usually starting in adolescence, with rapid disease progression. In 2023, Michel Goedert et al. used cryo-electron microscopy to resolve the structures of α-synucleinic fibers from MSA and JOS patients (PMID: 32461689; PMID: 36847833). These two fiber structures differ significantly from the α-syn fiber structures derived from PD patients. PD-derived α-syn fibers consist of a single fibril, while MSA-derived α-syn fibers comprise two types (MSA-I and MSA-II), each composed of two fibrils with different conformations. Compared to PD-derived fibrils, MSA fibrils not only have a higher amino acid composition and more complex folding patterns, but also contain cofactors. JOS-derived α-syn fibers include two subtypes: one composed of a single fibril and the other of two fibrils, with each fibril having the same conformation. Their C-terminal structure is very similar to one fibril in MSA-II, while their N-terminal structure is similar to the other fibril in MSA-II. These differences likely affect their binding affinity to drugs such as antibodies, thus impacting drug efficacy. Therefore, we recombinantly constructed α-syn mutants to form α-syn fibers with structures similar to those from MSA or JOS patients under in vitro conditions. We then processed these fibers using ultrasound to prepare them into pre-formed fiber seeds (PFFs) for subsequent experiments. Based on their origin, these pre-formed fiber seeds were named “Multi-system atrophic α-syn PFFs (MSA-like PFFs)” or “JOS-like PFFs”.

[0128] The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. A complete antibody will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains; for example, naturally occurring antibodies in camels may contain only heavy chains.

[0129] The terms "anti-α-synuclein antibody," "anti-α-syn antibody," or "α-synuclein antibody" refer to antibodies that specifically bind to α-synuclein. In some embodiments of the present invention, the anti-α-synuclein antibody is a murine anti-α-synuclein antibody. In preferred embodiments, the anti-α-synuclein antibody of the present invention is a chimeric antibody containing a human constant region, a human antibody constant region, or a humanized antibody. In some embodiments, the anti-α-synuclein antibody of the present invention binds to human α-synuclein with high affinity and can effectively block or inhibit the binding of human α-synuclein to its receptor. In some embodiments of the present invention, the anti-α-syn antibody of the present invention can recognize specific types or structures of α-synuclein or aggregates thereof, such as α-syn monomers, α-syn pre-formed fibrous seeds, or α-syn aggregates, such as MSA-like PFFs or JOS-like PFFs.

[0130] The term "antibody fragment" refers to a molecule distinct from the intact antibody, which contains a portion of the intact antibody and is capable of binding to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain antibodies (e.g., scFv); single-domain antibodies; and camelid antibodies (heavy chain antibodies).

[0131] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to provide antigen-binding specificity.

[0132] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. CDRs within the variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known schemes or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and nearest neighbor propagation clustering based on a large number of crystal structures. North CDR definition of propagation clustering) (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0133] For example, different schemes are used to define different ranges of CDR areas using the Kabat and Chothia numbering systems.

[0134] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined by any of the above schemes.

[0135] CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention). Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position determined according to 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)).

[0136] The term "chimeric antibody" refers to an antibody molecule in which a constant region, or a portion thereof, is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered constant region of a class and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug), which endows the chimeric antibody with new properties. For example, a mouse antibody can be formed by replacing its constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original mouse antibody.

[0137] The term "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody have been replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible, as long as they do not eliminate the antibody's ability to bind to a given antigen. "Humanized" antibodies retain antigen specificity similar to that of the original antibody.

[0138] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by conventional methods known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0139] The term "apo-α-syn" refers to an α-syn protein in a state where it is not bound by ligands, cofactors, substrates, or binding molecules (such as DNA, RNA, drugs, metal ions, etc.).

[0140] The term "half-maximal effective concentration (EC50)" 50 "" refers to the concentration of a drug, antibody, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time.

[0141] The term "half-inhibitory concentration (IC50)" refers to the concentration at which a test drug or substance inhibits the measured biological response, biological function, or activity by 50% relative to an untreated control.

[0142] The term "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, conditions, ailments, or diseases. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of this invention are used to delay disease development or to slow disease progression.

[0143] The term "effective amount" refers to the quantity or dose of the antibody, conjugate, or composition of the present invention, which, when administered to a patient in single or multiple doses, produces the intended effect in a patient requiring treatment. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health condition; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0144] The term "therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. Therapeutic effective amounts of antibodies, antibody fragments, or combinations thereof can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or antibody fraction to elicit the desired response in the individual. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody, antibody fragment, or its conjugate or composition are less than the beneficial therapeutic effect. Relative to an untreated subject, a "therapeutic effective amount" preferably inhibits a measurable parameter by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%.

[0145] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0146] II. The Composition of the Invention

[0147] In some embodiments, the present invention provides compositions comprising any anti-α-synuclein antibody or antigen-binding fragment thereof described herein, preferably pharmaceutical compositions. In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-α-synuclein antibody or antigen-binding fragment thereof of the present invention, and a combination of one or more other therapeutic agents.

[0148] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0149] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0150] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0151] The compositions of the present invention can be administered by known methods, such as orally, intravenously, intraperitoneally, intracerebral (internal parenchyma), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, or intralesionally; via a continuous release system or via an implanted device. In some embodiments, the compositions can be administered by bolus injection, continuous infusion, or via an implanted device.

[0152] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., a neurodegenerative disease).

[0153] A pharmaceutical preparation comprising the antibody described herein can be prepared by mixing the anti-α-synuclein antibody of the present invention, or its antigen-binding fragment, having the desired purity, with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0154] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0155] III. Preparation of the antibody of the present invention

[0156] In one embodiment, the present invention provides a method for preparing an anti-α-synuclein antibody or an antigen-binding fragment thereof, wherein the method comprises culturing a host cell containing a nucleic acid encoding an anti-α-synuclein antibody or an antigen-binding fragment thereof, or an expression vector containing said nucleic acid, under conditions suitable for expressing a nucleic acid encoding said anti-α-synuclein antibody or an antigen-binding fragment thereof, and optionally isolating said anti-α-synuclein antibody or antigen-binding fragment thereof. In one embodiment, the method further comprises recovering the anti-α-synuclein antibody or antigen-binding fragment thereof from said host cell (or host cell culture medium).

[0157] To recombinantly generate the anti-α-synuclein antibody or its antigen-binding fragment of the present invention, the nucleic acid encoding the anti-α-synuclein antibody or its antigen-binding fragment of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the anti-α-synuclein antibody or its antigen-binding fragment of the present invention.

[0158] The anti-α-synuclein antibody or its antigen-binding fragment prepared as described herein can be purified using known prior art techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the anti-α-synuclein antibody or its antigen-binding fragment of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. Example

[0159] The following embodiments further illustrate the present invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0160] Unless otherwise expressly stated, the present invention will be practiced using conventional chemical, biochemical, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology methods within the art. Unless otherwise specified, all experimental materials used are commercially available products. Where specific techniques or conditions are not specified in the examples, they shall be performed according to the techniques or conditions described in the literature in the art, or according to the corresponding product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0161] Example 1. Expression and purification of antigen α-synuclein

[0162] 1. Preparation of N-terminal acetylated α-syn (Ac-α-syn) protein

[0163] Following standard procedures, nucleic acids encoding both wild-type α-syn (UniProt accession number P37840) and mutant α-syn were constructed into an expression plasmid. This plasmid, along with a plasmid containing nucleic acid encoding yeast N-acetyltransferase complex B (NatB), was co-transformed into *E. coli* BL21(DE3) competent cells. After transformation, the cell culture was plated on a double-antibiotic solid medium containing ampicillin and chloramphenicol and incubated overnight at 37°C. Single colonies were picked and amplified in 10 mL of LB broth (containing the corresponding antibiotics) at 37°C, 220 rpm for 6–8 h. Then, 10 mL of the bacterial culture was inoculated into 1 L of LB broth for further amplification. When the OD600 of the bacterial culture reached 0.8–1, IPTG was added to a final concentration of 1 mM, and expression was induced at 37°C for 5 h. The culture medium was centrifuged at 4,000 rpm and 4°C for 20 min, and the E. coli that formed the precipitate were collected to obtain the expressed Ac-α-syn.

[0164] 2. Preparation of α-syn protein and its variants

[0165] For the preparation and expression of non-acetylated α-syn proteins, such as the full-length α-syn protein, tagged α-syn (e.g., FLAG-α-syn), and various truncated forms of α-syn, such as α-syn130 (containing amino acids 1-130 of α-syn), α-syn110 (containing amino acids 1-110 of α-syn), and α-syn100 (containing amino acids 1-100 of α-syn), BL21(DE3) competent cells were transformed with plasmids containing nucleic acids encoding the corresponding polypeptides. After transformation, the corresponding cell culture medium was plated on a solid medium containing only ampicillin resistance and incubated overnight at 37°C. Expression was then induced according to the method described in Section 1.

[0166] for 15 The expression method for N-labeled α-syn protein is the same as described above, except that N-labeled α-syn protein is used in the amplification and expression stage. 15 Escherichia coli were cultured in M9 medium containing NH4Cl (1 g / L).

[0167] 3. Protein purification

[0168] i. Ac-α-syn for cell and mouse processing; FLAG-α-syn for ELISA assays; α-syn140 and α-syn130 for dot blot assays; and MRI for nuclear magnetic resonance assays. 15 Purification of N-labeled α-syn protein

[0169] Preprocessing:

[0170] The collected *E. coli* were resuspended in 70-80 mL of bacterial lysis buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM PMSF). Cells were then lysed using an autoclave. The lysate was boiled in a water bath for 10 min. After centrifugation (14,000 rpm, 4°C, 30 min), streptomycin sulfate (20 mg / mL, wt / vol) was added to the supernatant, and the solution was continuously stirred at 4°C for 30 min. The lysate was further centrifuged (14,000 rpm, 4°C, 30 min), and the pH of the supernatant was adjusted to 3.5. Finally, after centrifugation (14,000 rpm, 4°C, 30 min), the supernatant was dialyzed overnight at 4°C using 25 mM Tris-HCl, pH 8.0, to obtain the appropriate dialysate.

[0171] Preliminary purification using anion exchange column (Q column):

[0172] First, wash the Q column with Buffer B (25mM Tris-HCl, pH 8.0, 1M NaCl), then equilibrate the Q column with Buffer A (25mM Tris-HCl, pH 8.0) (5-10 column volumes). Filter the protein dialysate through a 0.22μm filter and load it onto the Q column using the AKTA protein purification system. Then, wash away unbound proteins with Buffer A. Perform gradient elution with 0-60% Buffer B for 40 minutes. Collect the components using the AKTA automated collection system.

[0173] Purification using a gel size exclusion column (Superdex 75):

[0174] Equilibrate the Superdex 75 column (2 column volumes) with molecular sieve buffer (50mM Na2HPO4-NaH2PO4, pH 7.0, 50mM NaCl) beforehand; concentrate and filter the target protein fraction eluted from the Q column, and then load it through a sample loop; elute the molecular sieve with molecular sieve buffer, and collect the eluent using the AKTA automated collection system.

[0175] ii. Purification of α-syn100 and α-syn110 proteins for dot blot assays

[0176] Preprocessing:

[0177] The collected *E. coli* were resuspended in 70-80 mL of bacterial lysis buffer (100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM PMSF). Cells were then lysed using an autoclave. The lysis buffer was boiled in a water bath for 10 min and then centrifuged (14,000 rpm, 4°C, 30 min). After centrifugation, the supernatant was dialyzed overnight at 4°C to obtain the corresponding dialysate, in which the dialysate buffer was 25 mM Na₂HPO₄-NaH₂PO₄, pH 6.0.

[0178] Cation exchange column (SP column) purification:

[0179] First, the SP column was washed with Buffer B (25mM Na2HPO4-NaH2PO4, pH 6.0, 1M NaCl), and then equilibrated with Buffer A (25mM Na2HPO4-NaH2PO4, pH 6.0). The pretreated protein dialysate was filtered through a 0.22μm filter and then loaded onto the SP column using the AKTA protein purification system. Unbound protein was then washed away with Buffer A. Gradient elution was performed using 0-60% Buffer B for 40 minutes. The eluent was collected using the AKTA automated collection system.

[0180] Superdex 75 column purification:

[0181] The Superdex 75 column was equilibrated with molecular sieve buffer (50 mM Na2HPO4-NaH2PO4, pH 7.0, 50 mM NaCl) (2 column volumes). The target protein solution eluted from the SP column was collected, concentrated, filtered, loaded onto a sample loop, and eluted with molecular sieve buffer. Automated collection was performed using the AKTA sample collection system.

[0182] iii. Purification of receptor proteins L3D1, FAM171A2D1, vRAGE, and LC3B

[0183] Plasmid transformation:

[0184] The plasmids pET28a-L3D1, FAM171A2D1, vRAGE, and LC3B were transformed into BL21(DE3)E. coli competent cells, respectively. The transformation solution was plated on kanamycin-resistant solid culture plates and cultured at 37°C. Single colonies were picked and amplified in 10 mL of LB broth (containing kanamycin antibiotic) at 37°C, 220 rpm for 6–8 h. Then, 10 mL of the bacterial culture was inoculated into 1 L of LB broth for further amplification. When the OD600 of the bacterial culture reached 0.8–1, IPTG was added to a final concentration of 1 mM, and expression was induced at 25°C for 12 h. The culture medium was centrifuged at 4,000 rpm, 4°C for 20 min, and the E. coli precipitate was collected to obtain the expressed receptor protein.

[0185] Preprocessing:

[0186] The collected E. coli were resuspended in 70-80 mL of bacterial lysis buffer (50 mM Tris, 500 mM NaCl, pH 8.0, 1 mM PMSF). The cells were then lysed using a high-pressure homogenizer, and the cell lysis buffer was centrifuged (14,000 rpm, 4°C, 30 min) and the precipitate was collected. The precipitate was then washed in three steps: (1) The precipitate was resuspended in Triton X-100 buffer (50mM Tris, 5% Triton X-100, pH 8.0), sonicated at 40% power for 20min until the precipitate was completely dissolved, and then centrifuged (14,000rpm, 4℃, 30min) to collect the precipitate; (2) The precipitate was resuspended in high-salt buffer (50mM Tris, 1M NaCl, pH 8.0), sonicated at 40% power for 30min until the precipitate was completely dissolved, and then centrifuged (14,000rpm, 4℃, 30min) to collect the precipitate; (3) The precipitate was resuspended in guanidine hydrochloride buffer (6M guanidine hydrochloride, 50mM Tris, pH 8.0, 50mM NaCl), sonicated at 40% power for 30min until the precipitate was completely dissolved.

[0187] Nickel affinity column (Ni column) purification:

[0188] First, the contaminating proteins on the Ni column were washed away with a solution of 6M guanidine hydrochloride, 50mM Na2HPO4, pH 2.0, and then the Ni column was equilibrated with 6M guanidine hydrochloride, 50mM Na2HPO4, pH 8.0. The protein solution obtained from the pretreatment was filtered through a 0.22μm filter membrane and then loaded onto the Ni column using the AKTA protein purification system. Gradient elution was then performed using guanidine hydrochloride buffers with different pH values. The buffers used were: (1) 6M guanidine hydrochloride, 50mM Tris, pH 8.0, 50mM NaCl, used to equilibrate the Ni column and wash away unbound proteins; (2) 6M guanidine hydrochloride, 50mM Na2HPO4, pH 6.0, used to wash away some contaminating proteins; (3) 6M guanidine hydrochloride, 50mM Na2HPO4, pH 4.0, used to elute the target protein; (4) 6M guanidine hydrochloride, 50mM Na2HPO4, pH 2.0, used to further elute the Ni column, with occasional small amounts of the target protein remaining.

[0189] Receptor protein refolding:

[0190] After diluting the target protein to 0.5 mg / mL, a three-step gradient renaturation was performed. (1) 2 M guanidine hydrochloride, 50 mM Na2HPO4, pH 7.0, 50 mM NaCl, 5% glycerol, dialyzed at 4℃ for 12 h; (2) 1 M guanidine hydrochloride, 50 mM Na2HPO4, pH 7.0, 50 mM NaCl, 2.5% glycerol, dialyzed at 4℃ for 12 h; (3) 50 mM Na2HPO4, pH 7.0, 50 mM NaCl, dialyzed at 4℃ for 12 h.

[0191] Superdex 75 column purification:

[0192] The Superdex 75 column was equilibrated with molecular sieve buffer (50 mM Na₂HPO₄-NaH₂PO₄, pH 7.0, 50 mM NaCl) (2 column volumes). The refolded dialysis solution was collected, concentrated, filtered, loaded through a loop, and eluted with molecular sieve buffer. The eluent was automatically collected using an AKTA collection system. The eluent with a peak position of 60-70 mL was collected to obtain the target protein.

[0193] Example 2. Preparation of α-Syn fibers and fiber seeds

[0194] a. Preparation of spontaneously assembled α-Syn fibers:

[0195] α-syn protein monomers (200 μM, in 50 mM Tris, pH 7.5, 150 mM KCl buffer) were placed on a ThermoMixer shaker and incubated at 37°C and 900 rpm for 5–7 days. The monomers spontaneously aggregated to form α-syn amyloid filaments. The morphology of mature α-syn filaments was finally examined using transmission electron microscopy (TEM). This preparation method is applicable to other tagged α-syn monomers, JOS-like α-syn, MSA-like α-syn, and truncated α-syn fragments.

[0196] b. α-Syn fiber concentration correction:

[0197] α-syn fibers with a concentration of Ct and a volume of Vt were centrifuged (14,462 × g, 25 °C, 45 min), the supernatant was removed, and the concentration (Cs) and volume (Vs) of the supernatant were measured. The concentration (Cp) of the α-syn fibers was then calculated using the following formula:

[0198] c. Preparation of α-Syn pre-fabricated fiber seeds:

[0199] The spontaneously formed α-syn fibers from step a were quantified to 100 μM. Longer fibers were then ultrasonically treated using an ultrasonic disruptor to prepare α-syn pre-fiber seeds. The ultrasonic parameters were: 20% power, 1 s for both ultrasonic time and interval, for a total time of 50 s. Ultrasonic disruption resulted in α-syn PFFs with lengths of 50-100 nm. The morphology of the α-syn PFFs was observed using transmission electron microscopy (TEM).

[0200] For α-syn PFFs used in cell and mouse experiments, a sterile environment must be maintained during preparation, and the α-syn protein monomers and buffer solution must be sterilized by filtration through a 0.22 μm filter membrane. For inflammation experiments using primary microglia, the required α-syn PFFs must first undergo endotoxin removal column treatment to remove endotoxins from the protein monomers before fiber preparation. The endotoxin removal procedure should be performed according to the kit instructions. The prepared α-syn PFFs are shown in Figure 2.

[0201] d. Preparation of α-syn fibers induced by PFFs using α-syn PFFs:

[0202] 0.5% (molar concentration) of α-syn PFFs was added to the full-length α-syn protein monomer (200 μM, in 50 mM Tris, pH 7.5, 150 mM KCl buffer), and the mixture was placed on a ThermoMixer shaker and incubated at 37 °C and 900 rpm for 3-5 days to prepare induced α-syn fibers with more uniform fiber morphology and structure. The results are shown in Figure 1.

[0203] Example 3. Preparation and preliminary screening of hybridoma monoclonal antibodies using ELISA

[0204] Six- to eight-week-old female Balb / c mice were immunized with recombinant human α-syn PFFs protein in groups of six. Specifically, each mouse received a primary immunization via subcutaneous injection of a mixture of 50 μg of recombinant α-syn PFFs protein and Freund's complete adjuvant (CFA). A total of four immunizations were administered. The second and third immunizations were performed using mixtures of 50 μg and 30 μg of recombinant α-syn PFFs protein and IFA, respectively. The fourth immunization consisted of only 25 μg of recombinant α-syn PFFs protein. The intervals between the second, third, and fourth immunizations were 2-3 weeks. Starting with the third immunization, orbital blood samples were collected one week after immunization. The obtained serum was used for antigen-specific binding and binding blockade tests to select suitable mice for hybridoma fusion.

[0205] Two mice were ultimately selected, and after booster immunization, their spleens were harvested. Splenic cells were then electrofused with mouse myeloma cells SP2 / 0 (ATCC, Cat#CRL-1581). The fused cells were diluted in DMEM (Corning-cellgro, Cat#10-013-CVR) medium containing HT (Corning-cellgro, Cat#25-047-Cl), at a ratio of 1 x 102 spleen cells. 8 Spread 40 plates at a ratio into 96-well plates and incubate overnight in a 5% CO2, 37°C incubator. After 24 hours, add DMEM medium containing 2×HAT (Sigma, Cat#H-0262) and incubate for 10-14 days. Then perform hybridoma screening.

[0206] First, clone screening was performed using ELISA. 1 μg / mL human α-syn PFFs protein was coated onto 384-well plates (40 μL / well), incubated overnight, washed, and blocked. Then, 30 μL of hybridoma supernatant was added for incubation. Next, a 1:30000 dilution of goat anti-mouse IgG F(ab')2 fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#115-036-146) was added, and the binding results were detected using OD0.05. 650nm A value >0.5 was used as the positive screening criterion, and 473 clones were finally obtained through screening.

[0207] Biofilm layer interferometry (BLI) was used to detect the activity of the positive clones in blocking the α-syn binding ligand LAG3 (Lymphocyte activation gene 3). Hybridoma supernatant was mixed with 14.4 μg / mL α-syn PFF at a 1:1 ratio and incubated at room temperature for 1 h. A SA sensor was used to capture 3 μg / mL of biotinylated human LAG3 to a height of 3 nm, and the binding signal between the immobilized LAG3 sensor and the α-syn PFF was detected. Using the control antibody MEDI1341 as a control, hybridoma clones with similar or lower dissociation rates were screened, resulting in 46 clones with blocking activity. These 46 positive clones underwent further subcloning, and after 7 days of culture, the binding of mouse anti-α-syn PFFs and the blocking activity against human LAG3 were further confirmed, ultimately yielding 46 hybridomas expressing monoclonal antibodies specifically binding to α-syn.

[0208] Example 4. Preparation and expression of chimeric antibodies

[0209] 1. Extraction of candidate cloned genes

[0210] 1) Extract mRNA from hybridoma cells and synthesize the corresponding cDNA.

[0211] The positive monoclonal hybridoma cells obtained in Example 3 were cultured in 1640 medium at 37°C for 10 days until the logarithmic growth phase, and the cells (approximately 1 × 10⁻⁶) were collected. 6 RNA was extracted from cells / clones using the TRIzol method according to the manufacturer's instructions. Then, following the manufacturer's instructions, the RNA was reverse transcribed into cDNA using the SMART RACE method.

[0212] 2) PCR amplification of cDNA yielded the antibody VH and VL sequence encoding genes.

[0213] Using synthesized cDNA as a template, high-fidelity DNA polymerase and degenerate primers were used to specifically amplify the cDNA to obtain the heavy / light chain variable region gene.

[0214] 3) Construct T-vector clones, sequence them, and obtain antibody sequences.

[0215] The recovered fragment was cloned into the pMD19-T (TaKaRa, Cat#3271) vector using Solution I (TaKaRa, Cat#6022Q), and then transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL). Positive clones were selected and sequenced, and the sequence of the target antibody was obtained by bioinformatics analysis of the sequencing results.

[0216] 2. Construction, expression, and purification of chimeric antibodies

[0217] 1) Constructing a vector for expressing chimeric antibodies

[0218] The nucleic acid sequences expressing antibodies VH and VL were inserted into corresponding linearized vectors containing the human heavy chain constant region (pTT5_hIgG1.G1m3) and the light chain constant region (pTT5_hKappa.Km3), respectively; then plasmid amplification and sequence sequencing were performed.

[0219] 2) Expression and purification of chimeric antibodies

[0220] The sequenced plasmids containing the chimeric heavy and light chains were transfected into HEK293 cells at a 2:3 ratio using PEI reagent (1 μg plasmid: 4 μg PEI). The transfected cells were cultured for 5–7 days to express the chimeric antibody (the chimeric antibody was named with the prefix 'ch' followed by the corresponding clone number). The antibody in the culture supernatant was then purified using a Protein G affinity purification column, and the concentration of the purified antibody was determined for later use.

[0221] Example 5. Activity of chimeric antibodies binding to the surface of primary neurons

[0222] 1. Primary neuron culture

[0223] Place slides in 24-well plates, add 0.01 mg / mL poly-L-Lysine (PLL) solution to each well, and incubate overnight in an incubator. The next day, wash three times with deionized water and sterilize with UV light for half an hour. Dissect SD rats (Shanghai Bikai Keyi Biotechnology Co., Ltd.) at gestation days 15-18, remove the fetal rats, separate the left and right hemispheres, and place them in HBSS buffer containing HEPES. Rinse the fetal rat cortical tissue with approximately 50 mL of HBSS buffer, and digest the cortical tissue using papain and DNase. Filter the tissue digest using a 40 μm cell filter, and rinse the filter with approximately 10 mL of plating medium (DMEM containing 10% FBS and 1% PS and pyruvate-free). Centrifuge at 900 rpm, filter the collected cell solution for 5 min, resuspend the cell pellet in plating medium, and count the cells. Perform cell counting at 15 × 10⁻⁶ cells / well. 4 Add 0.5 mL of plating medium per well to a 24-well plate containing a glass slide. After 1-2 hours, replace the plating medium in the well with 1 mL of Neurobasal medium per well (containing 1% penicillin and streptomycin (PS), B27 supplement, and 0.5 mM GlutaMAX Neurobasal medium). Culture in a cell culture incubator until rat primary neurons mature (7-10 days).

[0224] 2. Primary neuron surface binding experiment

[0225] α-syn PFFs or a mixture of α-syn PFFs and chimeric antibodies were added to culture wells containing mature rat primary neurons. The final concentration of α-syn PFFs was 400 nM, and the molar ratio of α-syn PFFs to chimeric antibody in the 400 nM mixture was 1:1. Cells were cultured at 37°C for 2 h. The cell culture medium was removed, and the cells were washed once with PBS containing 5 mM MgCl2 and 1 mM CaCl2. Fixative (PBS solution containing 4% PFA and 8% sucrose) was added, and the cells were incubated at room temperature for 15 min. After fixation, the cells were washed three times with PBS solution containing 0.1% Tween-20, 10 min each time. The cell membrane was permeabilized with PBS solution containing 0.15% Triton X-100, and the cells were incubated at room temperature for 15 min. After permeabilization, the cells were washed three times with PBS solution containing 0.1% Tween-20, 10 min each time. Cells were blocked with PBS solution containing 3% goat serum and incubated at room temperature for 30 min. Then, rabbit-derived anti-α-syn antibody (Abcam, ab138501) and chicken-derived anti-MAP2 antibody (Abcam, ab5392) were diluted 1:1000 and 1:2500 respectively with PBS solution containing 3% goat serum, and incubated overnight at 4°C. Cells were washed three times with PBS solution containing 0.1% Tween-20 for 10 min each time. Goat-derived anti-chicken AlexaFluor-488 (Invitrogen, A-11039) and goat-derived anti-rabbit AlexaFluor-568 (Invitrogen, A-11041) fluorescent secondary antibodies diluted 1:1000 with PBS solution containing 3% goat serum were added, and the cells were incubated at room temperature for 1 h in the dark. The sample was washed three times with PBS solution containing 0.1% Tween-20, 10 min each time. The coverslips with attached neurons were then mounted on glass slides using a mounting medium containing DAPI (4',6-Diamidino-2-Phenylindole) (ProLong Gold Antifade reagent) and stored in the dark. Neuronal fluorescence signals were observed and photographed using an SP8 laser confocal microscope, and the results were processed and analyzed using ImageJ software. The results showed that a large number of α-syn PFFs were adsorbed onto the neuronal surface after the addition of α-syn PFFs. After the addition of different chimeric antibody strains, nine chimeric antibodies inhibited the binding of α-syn PFFs to the neuronal surface, while antibodies 31B21 and 1H1 showed almost no reduction in the binding of α-syn PFFs to the neuronal surface (see Figure 3).

[0226] Example 6. The antibody of this application inhibits the aggregation of endogenous α-synuclein in primary neurons and its intercellular propagation induced by α-synuclein PFFs.

[0227] 1. This application describes an antibody that inhibits the aggregation of endogenous α-synuclein in primary neurons induced by α-synuclein PFFs and its intercellular propagation.

[0228] Under normal conditions, intracellular α-synuclein exists in monomeric form with a very low phosphorylation rate. α-synuclein recruited into pathological inclusion bodies undergoes extensive phosphorylation at serine 129 (Ser129) (pS129), thus antibodies targeting pS129 can selectively recognize pathological α-synuclein aggregates (PMID: 11813001). In 2011, Laura et al. published an article in *Neuron* (PMID: 21982369), finding that in vitro recombinant α-synuclein progenitor cells (PFFs) could enter neurons after being added to cell culture medium and promote the aggregation of soluble endogenous α-synuclein within neurons, forming insoluble Lewy bodies and Lewy neurites similar to those in neurodegenerative diseases (PD). Since then, this has become a widely used cell model in the field for discovering treatments for neurodegenerative diseases mediated by pathological α-synuclein fibers. This embodiment uses this model to examine the effect of chimeric antibodies on the aggregation of endogenous α-synuclein in primary neurons induced by α-synuclein PFFs and its intercellular propagation. The results showed that the antibody in this application inhibited the aggregation of endogenous α-syn in primary neurons and its intercellular propagation induced or induced by α-syn PFFs.

[0229] α-syn PFFs or a mixture of α-syn PFFs and chimeric antibodies (made by mixing α-syn PFFs and chimeric antibodies selected in Experiment 5 that effectively inhibit the binding of α-syn PFFs to the neuronal surface at a molar ratio of 1:1) were added to culture wells containing mature rat primary neurons. The final concentration of α-syn PFFs was 100 nM; the α-syn PFFs-chimeric antibody mixture was 100 nM. Cells were cultured at 37°C for 14 days. The cell culture medium was removed, and the cells were washed once with PBS containing 5 mM MgCl2 and 1 mM CaCl2. Fixative (PBS solution containing 4% PFA and 8% sucrose) was added, and the cells were incubated at room temperature for 15 min. After fixation, the cells were washed three times with PBS solution containing 0.1% Tween-20, 10 min each time. The cell membrane was permeabilized with PBS solution containing 0.15% Triton X-100, and the cells were incubated at room temperature for 15 min. After permeabilization, the cells were washed three times with PBS containing 0.1% Tween-20, 10 min each time. The cells were then blocked with PBS containing 3% goat serum and incubated at room temperature for 30 min. Rabbit anti-pS129α-syn antibody (Abcam, ab51253) and chicken anti-MAP2 antibody (Abcam, ab5392), diluted 1:1000 and 1:2500 with PBS containing 3% goat serum, respectively, and incubated overnight at 4°C. The cells were washed three times with PBS containing 0.1% Tween-20, 10 min each time. Anti-chicken AlexaFluor-488 (Invitrogen, A11039) and anti-rabbit AlexaFluor-568 fluorescent secondary antibodies (Invitrogen, A11036), diluted 1:1000 with PBS containing 3% goat serum, respectively, and incubated at room temperature in the dark for 1 h. Wash three times with PBS solution containing 0.1% Tween-20, 10 min each time. Mount the coverslips with attached neurons onto a glass slide using a mounting medium containing DAPI, and store in the dark. Observe and photograph the neuronal fluorescence signals using an SP8 laser confocal microscope, and process and analyze the results using ImageJ software.

[0230] The results showed that the addition of α-syn PFFs significantly induced the aggregation and propagation of endogenous α-synesthesia in primary neurons. The inhibitory effects of the mixtures of α-syn PFFs and different chimeric antibodies on the aggregation and propagation of α-synesthesia in primary neurons varied (see Figure 4). Specifically, the mixture of α-syn PFFs and 30H21 antibody significantly inhibited the induced aggregation and propagation of endogenous α-synesthesia in primary neurons; the other antibodies had little effect and even led to the appearance of fluorescent patches.

[0231] 2. The chimeric antibody in this application co-localizes with phosphorylated α-syn (pS129α-syn).

[0232] The co-localization of 30H21 antibody and pS129α-syn was detected by immunofluorescence co-localization method.

[0233] Except for the different primary and secondary antibodies used as described below, the remaining method steps are the same as those in Experiment 1 of Example 6 above. Primary antibody: Rabbit-derived anti-pS129α-syn antibody, chicken-derived anti-MAP2 antibody, and the 30H21 (H21) antibody of this application were diluted with PBS solution containing 3% goat serum at ratios of 1:1000, 1:2500, and 1:100, respectively. Secondary antibody: Anti-chicken AlexaFluor-488, anti-rabbit AlexaFluor-568, and anti-mouse AlexaFluor-647 (Invitrogen, A-21235) fluorescent secondary antibodies were diluted with PBS solution containing 3% goat serum at a ratio of 1:1000.

[0234] Regarding the co-staining experiment of H21 and pS129α-syn in patient brain tissue samples.

[0235] Human brain tissue samples were fixed overnight in 4% paraformaldehyde (PFA). After fixation, the tissues were dehydrated sequentially at 4°C with 20%, 30%, and then 30% (w / v) sucrose solution (diluted with PBS) until the tissues settled. After dehydration, the tissues were embedded in OCT tissue freezing medium (Leica), rapidly frozen, and stored at -80°C. Subsequently, the tissues were cut into 15 μm thick sections using a cryostat (Leica, CM3050s-1-1-1), mounted on glass slides, and air-dried overnight at room temperature. The sections were stored at -80°C until staining. Before staining, the sections were rinsed once with PBS and then incubated with primary antibody overnight at 4°C. After incubation, the sections were washed three times with PBST (PBS containing 0.1% Tween-20), 10 minutes each time. Then, the sections were incubated with secondary antibody at room temperature for 2 hours in the dark. The slides were then stained with DAPI (1:10,000, Yeasen) at room temperature for 15 minutes, followed by washing three times with PBST for 10 minutes each time, and then rinsing once with PBS. To reduce lipofuscin autofluorescence, after DAPI staining, the slides were treated with a solution of 3% Sudan Black in 70% ethanol for 1 minute, followed by rinsing with deionized water (ddH2O) for 10 minutes. Finally, the slides were mounted with Prolong Gold anti-fluorescence quenching mounting medium (Thermo, catalog number: P36930) to preserve the fluorescence signal.

[0236] Primary antibodies: Rabbit anti-pS129α-syn antibody and the H21 antibody of this application were diluted with PBS solution containing 3% donkey serum at ratios of 1:2000 and 1:25, respectively. Secondary antibodies: Donkey anti-rabbit Alexa Fluor 594 (Invitrogen, A-21207) and donkey anti-mouse Alexa Fluor 488 (Invitrogen, A32766) fluorescent secondary antibodies were diluted with PBS solution containing 3% donkey serum at a ratio of 1:1000. Patient information involving human brain tissue is shown in Table 4.

[0237] Table 4 Basic Patient Information

[0238] The results showed that the 30H21(H21) antibody and pS129α-syn had good fluorescence co-localization, indicating that the 30H21(H21) antibody recognizes phosphorylated α-syn, as shown in Figures 5 and 6.

[0239] 3. Verification of the specificity of the chimeric antibody against endogenous pathological α-syn aggregates in this application.

[0240] To verify the recognition specificity of the chimeric antibody of this application for endogenous pathological pS129α-syn aggregates induced by α-syn PFFs, primary cortical neurons derived from wild-type (WT) and Snca knockout (KO) mice were cultured under the same experimental conditions. PBS or 200 nM α-syn PFFs were added to culture wells containing mature WT and Snca KO primary neurons, respectively, and cultured at 37°C for 14 days.

[0241] Subsequently, the pS129α-syn signal generated in this aggregation model was detected using immunofluorescence. Except for the different primary and secondary antibodies used as described below, all other experimental steps were performed according to the first experiment in Example 6 above. Primary antibody: Rabbit-derived anti-pS129α-syn antibody and chicken-derived anti-MAP2 antibody were diluted with PBS solution containing 3% goat serum at ratios of 1:1000 and 1:2500, respectively. Secondary antibody: Anti-chicken Alexa Fluor-488 and anti-rabbit Alexa Fluor-568 fluorescent secondary antibodies were diluted with PBS solution containing 3% goat serum at a ratio of 1:1000.

[0242] The results showed that significant pS129α-syn signals were detected only in WT neurons, while no identifiable pS129α-syn signals were detected in Snca KO neurons, indicating that the generation of pS129α-syn in this neuronal aggregation model depends on the presence of endogenous α-syn. This also further demonstrates that the chimeric antibody of this application can specifically recognize endogenous pathological α-syn aggregates induced by α-syn PFFs. The results are shown in Figure 7.

[0243] It should be noted that when co-staining with the chimeric antibody H21 and anti-pS129α-syn antibody simultaneously, slight changes in the fluorescence intensity and aggregate morphology of pS129α-syn were observed. Based on this phenomenon, it is speculated that the above changes may be related to competitive binding or steric hindrance between the chimeric antibody and anti-pS129α-syn antibody. For further observation and explanation, a sequential staining method was used for detection. The results showed that regardless of whether anti-pS129α-syn antibody staining was performed before H21 staining or vice versa, changes in aggregate morphology could not be avoided, but co-localization signals between H21 and pS129α-syn aggregates could still be observed. The results are shown in Figure 8.

[0244] Example 7. Affinity of 30H21 antibody with α-syn monomers and α-syn PFFs

[0245] This embodiment uses enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of antibody 30H21 with different forms of α-syn. Specifically, 100 μL of 50 μg / mL 30H21 (H21) PBS solution was added to a 96-well ELISA plate and coated overnight at 4°C. The plate was washed five times with PBS solution containing 0.1% Tween-20, and then 200 μL of blocking buffer (PBS solution containing 5% milk and 0.1% Tween-20) was added to each well for blocking at 4°C for 1 h. After blocking, the plate was washed five times with PBS solution containing 0.1% Tween-20, and then 100 μL of serially diluted FLAG-α-syn monomer prepared according to the method of Example 1 or FLAG-α-syn PFFs prepared according to the method of Example 2 was added to each well, and the plate was incubated at 4°C for 1 h. The FLAG-α-syn monomer was serially diluted 4-fold from 0 μM to 20 μM; the FLAG-α-syn PFFs were serially diluted 10-fold from 0 μM to 2 μM. After completion, the samples were washed 5 times with PBS containing 0.1% Tween-20. 100 μL of anti-FLAG-tagged antibody (Abcam, 49763) conjugated with horseradish peroxidase (HRP) (dissolved in PBS containing 0.1% Tween-20) was added to each well and incubated at 4°C for 1 h. After washing 5 times with PBS containing 0.1% Tween-20, 100 μL of substrate TMB was added to each well for color development. The color development reaction was terminated with 100 μL of 2M HCl.

[0246] The absorbance (A450) of each well was measured at 450 nm. The relationship between ELISA absorbance A450 and α-syn concentration was plotted using Graphpad Prism 9 software. A nonlinear regression was used to fit the binding curve to obtain the antibody-bound EC50. 50 value.

[0247] The results showed that the EC50 of 30H21(H21) bound to α-syn monomers was 45.8 nM; the EC50 of 30H21(H21) bound to α-syn PFFs was 45.8 nM. 50 The molecular weight was 3.7 nM, which is 12 times the binding capacity to α-syn monomers, as shown in Figure 9. This demonstrates that the 30H21(H21) of this invention more specifically recognizes α-syn aggregates.

[0248] Example 8. Antibody 30H21 inhibits α-syn fibrillation after co-incubation with α-syn PFFs.

[0249] This embodiment uses thiosulfate T (ThT) fluorescence kinetics to detect the inhibition of α-syn fibrillation by co-incubating different concentrations of antibody 30H21 with α-syn PFFs. First, different concentrations of H21 and 0.5 μM α-syn PFFs were incubated in 50 mM Tris and 150 mM KCl (pH 7.5) buffer at room temperature for 10 minutes. Then, the PFFs-H21 mixture was added to a system containing 50 μM α-syn monomer, and finally, 30 μM ThT was added to the reaction system for detection. The solution was added to a 384-well plate (Thermo Scientific, 142761) and detected using a Varioskan Flash spectral scanner (Thermo Scientific). Detection parameters: excitation wavelength 440 nm, emission wavelength 485 nm; 900 rpm, 37 °C. Data are displayed as mean ± standard deviation, n = 3 independent samples. The α-syn fibers at the endpoint of the ThT kinetic experiment (60 h) were observed using transmission electron microscopy (TEM). The results are shown in Figure 10.

[0250] The results showed that co-incubation of α-syn PFFs with different concentrations of 30H21 exhibited a concentration-dependent aggregation inhibition effect; that is, the higher the concentration of 30H21, the stronger the inhibitory effect on the aggregation of α-syn monomers into fibers. This indicates that the 30H21 (H21) of the present invention can bind to α-syn PFFs and effectively block their in vitro induction of α-syn monomers into fibers. Furthermore, it demonstrates that 30H21 has a good in vitro intervention effect in blocking the pathological aggregation induced by α-syn PFFs.

[0251] Furthermore, this application investigated the effect of different concentrations of ThT on the inhibition of α-syn aggregation by 30H21. Fluorescence kinetics were performed as described above using 5 μM and 10 μM ThT, respectively. The results obtained under the 5 μM and 10 μM ThT conditions were consistent with those obtained under the 30 μM ThT condition, as shown in Figure 11. This indicates that the inhibitory effect of 30H21 on α-syn aggregation is independent of the ThT concentration used. The differences observed under different ThT concentrations were mainly reflected in changes in fluorescence signal intensity, while the hysteresis phase in the α-syn aggregation kinetics process did not change significantly. These results demonstrate that 30H21 can effectively block the in vitro induction of α-syn monomers into fibers by α-syn PFFs, and this result is unaffected by changes in ThT concentration.

[0252] Example 9. Identification of the epitope of α-syn recognized by antibody 30H21

[0253] Studies have shown that the membrane surface receptor LAG3 is a key receptor for the intercellular propagation of α-synucleinic pathological fibers. RAGE (Receptor for Advanced Glycation Endproducts) is an important membrane receptor for microglia, which can bind to α-synuclein aggregates, mediating the activation of microglia by α-synucleinic progenitor cells (PFFs) and further triggering an inflammatory response. Research has found that LAG3 and RAGE utilize positively charged pockets on their D1 domain (L3D1) and V-domain (vRAGE) to bind to the negatively charged C-terminal domain of α-synuclein, respectively. Therefore, inhibiting the binding of α-synucleinic PFFs to the LAG3 receptor can inhibit their intercellular propagation, and inhibiting the binding of α-synucleinic PFFs to the RAGE receptor can significantly attenuate the inflammatory response in microglia induced by α-synucleinic fibers. Correspondingly, the key domains L3D1 and vRAGE for binding to α-synucleinic PFFs by LAG3 and RAGE can serve as competitive agents for screening antibodies that specifically bind to α-synucleinic fibers.

[0254] 1. The epitopes of α-syn bound by antibody 30H21 were detected using an ELISA competitive binding method.

[0255] A gradient concentration mixture of FLAG-α-syn PFFs and 30H21(H21) was prepared at a concentration of 1 μM, using a 30H21(H21):PFFs concentration ratio of 10:1, 5:1, 2.5:1...0.005:1. The mixture was incubated at 4°C for 30 min. Detection was performed according to the ELISA method disclosed in Example 7.

[0256] The results, as shown in Figure 12, indicate that 30H21(H21) can inhibit the binding of the C-terminal domain of α-syn to L3D1, FAM171A2D1, vRAGE, and LC3B, thus demonstrating that antibody 30H21(H21) binds to the C-terminal domain of α-syn.

[0257] 2. Detection of epitopes of α-syn bound by antibody 30H21 using the dot blot method

[0258] The dot blot method was performed according to the manufacturer's instructions, with each sample concentration on the cellulose acetate membrane being 10 μM. The primary antibody used was the 30H21 (H21) antibody of this application, and the commercial antibody Ab 138501 (Abcam, ab138501) targeting the C-terminus 118-123 of α-syn; the secondary antibodies were horseradish peroxidase (HRP) conjugated anti-rabbit and anti-mouse secondary antibodies.

[0259] The results, shown in Figure 13, indicate that for the control antibody Ab 138501, it only binds to the truncated form of α-syn containing amino acids 118-123. Antibody 30H21 (H21) does not bind to monomers containing truncated forms of α-syn, including α-syn130, α-syn110, and α-syn100, but only to the full-length α-syn, indicating that the epitope for H21 binding to α-syn is at amino acids 131-140. Furthermore, for the same concentration of α-syn monomers and α-syn PFFs, the biomarker from H21 binding to α-syn PFFs is darker, indicating a stronger binding affinity between H21 and α-syn PFFs.

[0260] 3. Detection of the epitope of α-syn bound by antibody 30H21 using liquid-state NMR spectroscopy.

[0261] Detection was performed using a 900 MHz (Bruker) nuclear magnetic resonance spectrometer equipped with a cryogenic probe, and results were acquired at 298 K. The buffer solution used was 50 mM Na₂HPO₄-NaH₂PO₄, pH 7.0, 50 mM NaCl, and 10% D₂O. 15 The N-α-syn concentration was collected at 20 μM, and the system volume was 500 μL. 30H21 titration experiment: in the mixed sample... 15 The N-α-syn concentration was 20 μM, the H21 concentration was 10 μM, and the system volume was 500 μL. NMR data were processed using NMRPipe and Sparky software.

[0262] The results, as shown in Figure 14, indicate that the addition of H21 causes a significant decrease in the strength of amino acid sites 131-140 at the C-terminus of α-syn. For example, the strength of amino acid sites G132, Y133, Y136, E137, E139, and A140 decreases by about 80%, indicating that H21 interacts with amino acid sites 131-140 at the C-terminus of α-syn.

[0263] Example 10. The chimeric antibody of this application competitively blocks the binding of α-syn PFFs to neuronal receptors on the cell surface.

[0264] To further investigate the mechanism by which the chimeric antibody 30H21 inhibits the binding of α-syn PFFs on the cell surface, we introduced cell membrane staining into the neuronal surface binding assay. First, we examined whether H21 could enter neurons; then, we assessed whether H21 reduced the number of α-syn PFFs already bound to the cell surface through competitive inhibition; finally, we analyzed the interaction between the cell receptor and H21 using a Fam171a2 gene knockdown (KD) assay.

[0265] 1. Antibody 30H21 cannot enter cells.

[0266] A neuronal surface binding assay was performed. Primary neurons were treated with 100 nM H2I and incubated at room temperature for 2 hours. Immunofluorescence was used to detect the binding of 30H2I to the neuronal surface. Except for the use of different primary and secondary antibodies, the remaining experimental steps were performed as described in Experiment 1 of Example 6 above. Primary antibodies: Rabbit anti-α-syn antibody and 30H2I were diluted 1:1000 and 1:100, respectively, with PBS solution containing 3% goat serum. Secondary antibodies: Anti-mouse Alexa Fluor-647 and anti-rabbit Alexa Fluor-488 (Invitrogen, A-11008) fluorescent secondary antibodies were diluted 1:1000 with PBS solution containing 3% goat serum. Cell membrane dye: CellMask (Thermo, C10046) was used, diluted 1:1000 with PBS.

[0267] The results showed that no signal of 30H21 was observed on the surface of neurons, indicating that H21 does not bind to the neuronal surface or enter the cell. See Figure 15 for the results.

[0268] 2. Antibody 30H21 competitively inhibits the binding of α-syn PFFs to the neuronal surface.

[0269] A neuronal surface binding assay was performed. Primary neurons were treated with a mixture of α-syn PFFs and H21 and incubated at room temperature for 2 hours; or neurons were first treated with 100 nM α-syn PFFs for 1 hour, followed by incubation with 100 nM H21 for 2 hours. Immunofluorescence was then used to detect the binding of α-syn PFFs to the neuronal surface. The antibodies and dyes used were the same as in the first experiment of this embodiment, and the remaining experimental procedures were performed as described in the first experiment of Example 6 above. Each group had three biological replicates, with six images taken per replicate, for a total of n = 18 images for statistical analysis. One-way ANOVA was performed followed by Tukey's post-hoc test. Data are presented as mean ± standard deviation. ns indicates no significant difference; ** indicates p < 0.01, *** indicates p < 0.001.

[0270] The results showed that, compared with the α-syn PFFs alone treatment group, the mixture of PFFs and H21 co-incubated significantly reduced the binding of PFFs to the neuronal surface; in addition, H21 was also able to reduce the PFFs already bound to the neuronal surface by competing with neuronal surface receptors. See Figure 16 for the results.

[0271] 3. The inhibitory effect of antibody 30H21 is independent of specific cell receptors.

[0272] a. Constructing Fam171a2 knockdown (Fam171a2 KD) neuroblastoma cells (Neuro-2a, N2a)

[0273] N2a cells were cultured in DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 10099-141) and 1% PS (Gibco, 15140122) and incubated in a cell culture incubator at 37°C and 5% CO2.

[0274] N2a cells were seeded in 12-well plates and transfected with siRNA when the cell density reached approximately 70–80%. The transfection conditions per well were: 100 nM siRNA; 2 μL of Lipofectamine 2000 transfection reagent (Thermo, 11668030). The specific transfection procedure was as follows: the required siRNA and Lipofectamine 2000 were added separately to 100 μL of DMEM medium (without FBS and PS) and gently mixed by pipetting; then the medium containing siRNA was mixed with the medium containing Lipofectamine 2000, and incubated at room temperature for 10 minutes to form a transfection complex. The formed transfection complex was added to the wells containing the cultured N2a cells and incubated for another 48 hours for subsequent Western blot or cell surface binding experiments.

[0275] b. Western blot verification of KD effect

[0276] N2a cells were lysed using RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor (Roche, 04693116001) and a phosphatase inhibitor (Roche, 5892970001). After adding SDS-PAGE protein loading buffer, the protein samples were boiled at 100°C for 15-20 minutes, then separated by electrophoresis. The proteins were then transferred to a PVDF membrane (Millipore, ISEQ00010) using the eBlot L1 rapid transfer system (Genescript).

[0277] The PVDF membrane was blocked with protein-free rapid blocking buffer (EpiZyme) at room temperature for 10 minutes. Subsequently, it was incubated with primary antibodies. Mouse anti-β-actin antibody (Proteintech, 66009-1-Ig) and rabbit anti-FAM171A2 antibody (Signalway Antibody Technology, 47788) were diluted with PBS containing 5% BSA at ratios of 1:3000 and 1:500, respectively, and incubated overnight at 4°C. After incubation, the membrane was washed three times with TBST for 10 minutes each time.

[0278] Secondary antibody incubation was then performed. The HRP-conjugated goat anti-rabbit IgG antibody (Abcam, ab6721) and goat anti-mouse IgG antibody (Abcam, ab6789) were diluted 1:5000 with TBST solution containing 5% skim milk and incubated at room temperature for 1 hour. After incubation, the membrane was washed three times with TBST for 10 minutes each time.

[0279] Finally, development was performed using ECL chemiluminescence colorimetric reagent (Yeasen), and imaging was performed using an iBright CL1500 imaging system (Invitrogen).

[0280] The results showed that FAM171A2 was successfully knocked down in cells, with a significant reduction in expression level exceeding 50%, providing a basis for subsequent cell membrane surface binding experiments. See Figure 17 for the results.

[0281] c. Immunofluorescence detection of antibody 30H21 inhibiting the binding of α-syn PFFs to the cell surface

[0282] Cell surface binding assays were performed using a mixture of α-syn PFFs and H21 to treat WT and Fam171a2 KD N2a cells, incubating at room temperature for 2 hours. Immunofluorescence was then used to detect the binding of α-syn PFFs to the cell surface. The primary antibody used was rabbit anti-α-syn antibody, and the secondary antibody was goat anti-rabbit Alexa Fluor-568. The remaining experimental procedures were performed as described in Experiment 1 of Example 6 above. Three biological replicates were set for each group, with eight images taken per replicate, for a total of n = 24 images for statistical analysis. One-way ANOVA was performed followed by Tukey's post-hoc test. Data are presented as mean ± standard deviation. *** indicates p < 0.001.

[0283] The results showed that Fam171a2 KD significantly reduced the binding of α-synuclear fiber filaments (PFFs) to the cell surface, indicating that the FAM171A2 receptor plays an important role in mediating the binding of α-synuclear fibers to the cell surface. Compared with the α-synuclear PFFs-only treatment group, the mixture of α-synuclear PFFs co-incubated with H21 significantly reduced the binding of α-synuclear PFFs to the surface of WT and Fam171a2 KD cells. 30H21 effectively blocked the binding of α-synuclear fibers to cells in the absence of specific cell receptors, highlighting the broad therapeutic potential of 30H21. See Figure 18 for the results.

[0284] Example 11. Antibody 30H21 alleviates neuroinflammation caused by α-syn PFFs.

[0285] α-syn fibrous aggregates can induce microglial cell activation and inflammatory responses. This example tested the inhibitory activity of antibody 30H21 on microglial inflammation.

[0286] 1. Primary microglia culture

[0287] a. Add 5 mL of 0.01 mg / mL poly-L-lysine (PLL) solution to a T25 culture flask and incubate overnight in an incubator. The next day, wash three times with deionized water, then open the flask and sterilize under UV light in a laminar flow hood for half an hour.

[0288] b. Dissect newborn C57 / BL6 mice that are 1-2 days old, separate their left and right hemispheres, and place them in HBSS buffer containing HEPES.

[0289] c. Rinse the tissue with 50 mL of HBSS buffer, then mechanically fragment the tissue.

[0290] d. Filter the cell tissue using a 40 μm cell filter and rinse the filter with approximately 15 mL of DMEM medium (Gibco, 11995065) containing 10% FBS and 1% PS.

[0291] e. Centrifuge the cell solution at 500×g for 3 min. Resuspend the cell pellet in DMEM medium. Seed the cells in a T25 culture flask. Change the medium after 3 days.

[0292] After 14 days, place the culture flasks in a shaker and shake at 220 rpm and 37°C for 60-90 minutes. (Note: Only glial cells can survive for a long time under these culture conditions; and microglia are less adherent than astrocytes, so under these conditions, microglia can be shaken off but astrocytes will not). Collect the cell culture medium and centrifuge (500×g, 3 min). Resuspend the cell pellet in DMEM medium and seed the microglia evenly into PLL-coated 24-well plates. After the microglia have fully adhered, perform subsequent inflammation experiments.

[0293] 2. Real-time reverse transcription PCR to measure the expression levels of inflammatory factors in primary microglia.

[0294] a. Cell treatment: Primary microglia were treated for 4 h with PBS, lipopolysaccharide (LPS, Thermo, 00-4976-03) at a final concentration of 1 μg / mL, α-syn monomers, α-syn PFFs, a mixture of α-syn PFFs and 30H21 (H21) at a final concentration of 5 μM (PFFs:H21 = 1:1 molar ratio), a mixture of α-syn PFFs and the inhibitor FPS-ZM1 (PFFs:FPSZM1 = 1:1 molar ratio), and an inhibitor control group (inhibitor FPS-ZM1 targets RAGE receptors on the microglia membrane, blocking the spread of α-syn through receptors and significantly reducing neuroinflammation caused by α-syn PFFs).

[0295] b. RNA extraction: Collect cells, wash once with pre-cooled PBS, and then add RNA lysis buffer to lyse the cells. Follow the instructions on the RNA extraction kit (Zymo Research, R1055) to obtain RNA. Measure RNA concentration using Nanodrop.

[0296] c. Reverse transcription: The 20 μL system included: 4 μL 5×All-in-one qRT SuperMix; 1 μL Enzyme Mix; 1 μg RNA; the remaining volume was made up with RNase-free deionized water. Parameter settings: 50℃, 15 min; 85℃, 5 s. After reverse transcription, the cDNA concentration was measured using Nanodrop.

[0297] d. Perform real-time quantitative PCR amplification using a qPCR instrument. The 10 μL system includes: 5 μL 2*SYBR Green qPCR Master Mix; 0.4 μL 5 μM forward primer; 0.4 μL 5 μM reverse primer; 0.2 μL ROX Reference Dye; 100 ng cDNA; the remaining volume is brought to the nearest whole number with deionized water. The parameters are set as follows: 95℃, 10 min; 95℃, 15 s (40 cycles); 60℃, 1 min.

[0298] The results, as shown in Figure 19, indicate that the addition of α-syn PFFs to cells alone significantly increased the expression levels of cytokines IL-6, IL-1β, and TNFα; while the addition of antibodies H21 and FPS-ZM1 significantly reduced the expression levels of these three cytokines, suggesting that H21 has a certain effect on alleviating neuroinflammation caused by α-syn PFFs.

[0299] Example 12 utilizes cryo-electron microscopy (Cryo-EM), cryo-electron tomography (Cryo-ET), and AlphaFold3 to analyze the structure of α-syn fibers co-incubated with H21.

[0300] 1. Cryo-electron microscopy data collection

[0301] For the preparation of α-syn-H21 fiber samples, apo-α-syn fibers were diluted to 10 μM and incubated with 2 μM or 5 μM H21 at 25 °C for 1 hour. Simultaneously, apo-α-syn fiber samples of the same concentration were prepared as controls. 3 μL of the α-syn-H21 solution or apo-α-syn fiber solution incubated with H21 was dropped onto a copper mesh (Quantifoil R1.2 / 1.3, 300 mesh) coated with a perforated carbon film after glow discharge treatment. Samples were then prepared using a Vitrobot Mark IV (FEI, Thermo) in a sealed chamber at 16 °C and 95% humidity, followed by rapid freezing in liquid ethane. The Krios... TM Cryo-electron microscopy (Thermo Scientific) was used to acquire images in super-resolution mode at 300 kV using a BioContinuum K3 direct detector (Gatan, Inc.). Inelastic scattered electrons were removed using a GIF Quantum energy filter (Gatan, Inc.) with an energy slit width of 20 eV. The photomicrographs were captured at a magnification of 105,000, with 40 frames per second, and the image pixel size was [missing information]. The total dose is approximately The total exposure time was 2 seconds, and the defocus value ranged from -1.0 to -2.0 μm. It was then analyzed by Thermo Scientific. TM EPU software was used for automatic data acquisition. Cryo-electron microscopy images of Apo-α-syn fibers incubated with different concentrations of H21 are shown in Figure 20.

[0302] Cryo-electron microscopy results showed that, compared with α-syn fibers incubated with H21, the α-syn fibers treated with H21 antibody exhibited a significant increase in diameter, suggesting that H21 can bind to the fiber surface. The increase in α-syn fiber diameter became more pronounced with increasing molar ratio of H21 to α-syn, indicating that H21 can bind along the axial direction of the α-syn fibers, thus providing preliminary morphological verification of H21's binding ability to α-syn aggregates.

[0303] 2. Cryo-electron microscopy image preprocessing, helical reconstruction, and model building

[0304] In the image preprocessing process, the collected cryo-EM data were first preprocessed, and MotionCorr 2 was used to correct ice surface drift caused by the electron beam. Each frame was then aligned and overlaid. Subsequently, the contrast transfer function was estimated using CTFFIND-4.1.8. Next, the coordinates of all fibers were manually selected using the manual picking program of RELION 3.1. In helical reconstruction, 10,924 fibers were manually selected from 5,000 micrographs, and these fibers were cut into 360×360 pixel particles. High-quality particles were selected through multiple rounds of two-dimensional classification screening, and an initial three-dimensional model was constructed using the average image of the selected two-dimensional classifications. The average two-dimensional classification images of α-syn fibers incubated at different concentrations of H21 are shown in Figure 21. The results show that when α-syn fibers were co-incubated with H21 at molar ratios of 1:0.2 (left) and 1:0.5 (right), images were acquired showing a blurred additional density around the fiber core structure, presumably representing bound H21 antibodies.

[0305] Then, the initial helical twist angle was determined by combining the two-dimensional classification results and cryo-electron microscopy images. This serves as the initial value for the spiral ascent height. In the 3D classification, the final spiral twist angle and ascent height are determined through a local symmetry search. In the initial 3D classification, the initial spiral twist angle is set to 179.64°, and the spiral ascent height is... After several rounds of 3D classification with K=3, the optimal 3D category containing 61,085 particles was selected for 3D refinement, followed by automatic 3D refinement using optimized helical twist angles and rise heights. Finally, using the standard post-processing procedure of RELION 3.1, the density map was sharpened by adding a mask to its edges, and the Fourier shell correlation (FSC) between the two half-density maps was calculated. Based on an FSC value of 0.143, the final overall resolution of the density map was estimated. An atomic model was built using the WinCOOT program and refined using the real_space_refine procedure in the PHENIX program. For reconstructions without applying helical symmetry parameters, manually selected fibers were cut into 600×600 pixel particles. Multiple rounds of 2D classification were performed, without forcing repetitive units, and selecting particles exhibiting layered density around the fibers. Subsequently, automatic 3D refinement was performed using the optimized helical parameters of the apo-α-syn fiber. Then, a new round of automatic 3D refinement was performed. This round of refinement did not force the application of helical symmetry. The density map output from the previous round of automatic 3D refinement was used as the reference density map, and the input data was the data.star file generated from the previous round of automatic 3D refinement. More information about helical reconstruction and model construction can be found in Figure 22 and Table 5. The reconstruction results show that the α-syn fibers incubated with the H21 antibody have a double-stranded fiber conformation, and their overall structural characteristics are highly similar to the conformation of α-syn fibers without H21 antibody incubation, suggesting that the binding of H21 did not significantly affect the original conformation of the α-syn fiber core.

[0306] Table 5. Relevant data from cryo-electron microscopy data acquisition, image processing, spiral reconstruction, and model building.

[0307] Cryo-electron microscopy images have been submitted to the Electron Microscopy Data Bank (EMDB), with the structure number EMD-61500 for the α-syn-H21 fiber. Additionally, its corresponding atomic model has been submitted to the Protein Data Bank (PDB), with the number 9JI8. The apo-α-syn fiber structure used in this application can be found and obtained from the PDB database, with the number 6A6B.

[0308] The results show that the binding of H21 to the C-terminal region of the fiber induces a conformational change in α-syn, causing its N-terminal segment to be stably embedded in the fiber core, thereby altering the fiber's surface structure and variable regions. See Figure 23 for the results.

[0309] 3. Cryo-electron computed tomography (Cryo-ET) data acquisition and 3D reconstruction

[0310] Sample preparation was performed using the same method as for cryo-electron microscopy (cryo-EM). Vitrified frozen samples were imaged on a Titan Krios cryo-transmission electron microscope equipped with an energy filter (20 eV slit width) and a BioContinuum K3 direct electron detector (Gatan). Multiple tilt series were acquired at 3° intervals within a tilt range of -60° to 60° using the dose-symmetric scheme in Tomography software (v5.12), with pixel sizes of [missing information]. The total electron dose is approximately The defocusing amount is set to 4μm.

[0311] In tomographic image processing, motion correction and contrast transfer function (CTF) estimation were performed on the original tilt series images using Warp. After manually filtering out tilt images of poor quality, the tilt series were automatically aligned using the patch-tracking method in the AreTomo software package (v2.0) and reconstructed into tomograms via back-projection. To enhance the contrast of the target region, CTF deconvolution was performed using IsoNet (v0.2). The results showed that under the condition of co-incubation of α-syn fibers and H21 at a molar ratio of 1:0.2, electron density signals generated by antibody binding could be observed around almost every α-syn fiber, further demonstrating that H21 can stably bind to the surface of α-syn fibers. The results are shown in Figure 24.

[0312] 4. Segmentation and quantification of minimum nearest neighbor distance for α-Syn-H21 fibers

[0313] During segmentation, the three-dimensional coordinates of amyloid fibrils and antibodies were first obtained. For fibrils, the coordinates were manually selected using Dynamo software (v1.1.532), followed by resampling (2 pixels in bin8 tomographic images). For antibody coordinates, template matching was performed using PyTom software, using the Fab region targeting the H21 antibody as the template. The template was low-pass filtered to... Resolution was determined, and after template matching using PyTom software, 4,994 coordinate points were finally selected using an LCCmax (Local Cross-Correlation Maximum) threshold of 0.45. The initial model was generated in bin8 using the relion_reconstruct command, and further segmentation and 3D rendering were performed in Chimera X.

[0314] The minimum nearest neighbor distance between antibodies and amyloid fibrils was calculated using a custom script (available on GitHub). In short, the antibody-fibril nearest distance for each antibody point is defined as the minimum Euclidean distance from that point to the nearest fibril. As a control, an equal number of random points were generated within the boundaries of the complete tomographic map, and their nearest distances to fibrils were calculated in the same manner. Data analysis and plotting were performed using GraphPad Prism 9. Comparing the observed H21 distribution in the experiment with simulated results of random antibody distribution showed that the average H21 density is located approximately [distance missing from fibril axis]. At that point, the peak value of the random distribution is approximately The distribution is more dispersed. This shows that H21 does not bind randomly on the fiber surface, but rather exhibits a clear binding preference. See Figure 25 for the results.

[0315] Scripts for fiber reconstruction, segmentation, and closest distance quantification are available at the following URL:

[0316] https: / / github.com / DavidH2024 / Ab-Fibrils-Distribution-Analysis.git.

[0317] 5. Log in to https: / / alphafoldserver.com and use AlphaFold3 to predict the full length of H21 and α-syn. 131-140 The dimer structure of the peptide, and the antigen-binding fragment on H21 and α-syn 131-140 The binding structure of the peptides. The results are shown in Figures 26 and 27.

[0318] In summary, the structural analysis of this embodiment reveals the molecular basis for H21's selective recognition of α-syn fibers. H21 binds to the C-terminal segments (residues 131-140) of α-syn fibers through its CDR region. After binding, H21 exhibits a specific orientation relative to the fiber, adhering to the fiber surface at a fixed interval and inducing a slight conformational remodeling of the α-syn fiber, including the ordering of N-terminal residues.

[0319] Example 13 Effects of Antibody on PD Mice

[0320] 1. Construction of PD mice

[0321] In 2012, Luk et al. discovered that a single injection of synthetic α-syn fibers into the striatum of wild-type non-transgenic mice led to intercellular transmission of pathological α-synergies and the development of Lewy pathology, a Parkinson's disease-like condition, in anatomically interconnected regions. The accumulation of Lewy pathology resulted in the gradual loss of dopamine neurons in the substantia nigra pars compacta, a phenomenon not observed in the adjacent ventral tegmental area, accompanied by a decrease in dopamine levels, ultimately leading to motor dysfunction (PMID: 23161999). Subsequently, the method published by Luk et al. became one of the most widely used methods in the field for constructing PD mouse models and has been used to study therapeutic approaches targeting neurodegenerative diseases mediated by pathological α-synergies at the mouse level.

[0322] Following this method, we selected 8-10 week old male C57BL / 6 mice for the experiment. Anesthetized mice were fixed on a stereotaxic apparatus, and the position of the brain was adjusted to ensure it was roughly aligned and symmetrical. The skull was exposed, and the location of the Bregma point was determined, with the origin at (0, 0, 0). The injection site was determined (±2.0, 0.2), and a microcranial drill was used to make the incision. Finally, using a microinfusion pump, 2.5 μL of α-syn PFFs (2 g / L) or 2.5 μL of PBS was injected into each side of the brain at a depth of 2.6 mm. After the injection, the mice were placed on a 37°C heated pad. After the mice recovered, they were returned to their original cages for continued care.

[0323] 2. Intraperitoneal injection of antibodies

[0324] Once a week for 13 consecutive weeks, each mouse was intraperitoneally injected with antibody 30H21 (H21) or an equivalent volume of PBS. Each mouse was weighed before each weekly injection, and the antibody dosage was calculated based on the mouse's body weight, with a standard calculation of 20 mg / kg.

[0325] Based on the different types of stereotactic injection (before) and intraperitoneal injection (after), a total of 4 groups were set up: PBS+PBS; PBS+Ab; α-syn PFFs+PBS; α-syn PFFs+Ab.

[0326] 3. Mouse behavioral experiments

[0327] Following 13 consecutive weeks of intraperitoneal antibody injections, behavioral experiments were conducted at the first time point starting at week 15, 15 weeks after the completion of stereotactic brain injections. To observe whether 30H21 (H21) also had an effect over a longer period, behavioral experiments were conducted at the second time point at week 21 after the completion of stereotactic brain injections. The mouse behavioral experiments conducted in this study included the open-field test (OFT), rotador test, and pole test. Mouse behavioral results and subsequent immunostaining results were plotted using GraphPad Prism 9. One-way ANOVA was used for analysis, and the error bars were calculated as mean ± standard error. ns (no significance); * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0328] Before each behavioral experiment cycle, each mouse was weighed to ensure that the two injection methods, stereotactic injection and intraperitoneal injection, would not affect the mice's physical condition, and to ensure that the behavioral differences among the mice were not caused by differences in weight.

[0329] The results, as shown in Figure 28, indicate that there were no significant differences in body weight among the groups of mice at two time points: week 15 and week 21 after the completion of stereotactic injection.

[0330] A. Open field experiment

[0331] Before the experiment, the mice needed to be trained to adapt to the environment and become familiar with the experimenter. The specific procedure was as follows: the mouse was placed in the palm of the hand and allowed to explore freely within the open palm area for one minute at a time. This training was continued for three days. After the training period, the mouse would remain calmly in the hand. During the experiment, the mouse was placed in the center of a 40×40cm box, and the light intensity inside the box was adjusted to 30-35 lux. The mouse's movement trajectory within the box was recorded using a camera over 10 minutes. The experimental results were analyzed using EthoVision XT (Noldus 11.5) software.

[0332] The open field experiment results at the first time point showed no significant difference in spontaneous movement ability among the groups of mice, indicating that the mice's spontaneous movement ability was not impaired by the two injection methods, and they could proceed with subsequent behavioral experiments. However, at the second time point, because the interval was too short (only one month apart), the mice still remembered the experimental environment, so the open field experiment at the second time point was not conducted. The results are shown in Figure 29.

[0333] B. Rotating bar experiment

[0334] Before the formal experiment, all mice underwent three consecutive days of training. During training, the mice were placed on a rotundus. For the first two rounds, the rotundus was rotated at a constant speed of 4 rpm / min. In the third round, the rotundus was accelerated from 0 to 40 rpm / min within 90 seconds. Each round lasted 2 minutes, with a 3-minute rest period between rounds. After the three-day training period, the mice rested for one day before the formal test, which consisted of two rounds. The experimental setup for both rounds was identical: the rotundus was accelerated to 40 rpm / min within 90 seconds and then rotated continuously at 40 rpm / min for 3.5 minutes. The average and maximum movement times of the mice on the rotundus during the formal experiment were analyzed based on the results of the two rounds to assess the mice's horizontal coordination ability.

[0335] The results are shown in Figure 30. At the first time point, the mice in the PFFs+PBS group showed significant behavioral impairment compared to the PBS+PBS group, indicating that the PD mice were successfully constructed. Although the mice in the PFFs+Ab group injected with H21 did not show significant behavioral improvement compared to the PFFs+PBS group, their behavioral abilities were comparable to those of the control group PBS+PBS group, indicating that H21 can alleviate the behavioral disorders of PD mice to some extent.

[0336] At the second time point, mice in the PFFs+Ab group injected with H21 showed significant behavioral improvements compared to the PFFs+PBS group, and their behavioral abilities were comparable to those of the control group (PBS+PBS group), indicating that H21 can improve the horizontal movement ability of PD mice. See Figure 31 for the results.

[0337] C. Pole Climbing Experiment

[0338] First, place the mouse head-down on top of a 50cm long metal pole wrapped with sponge tape. Record the time it takes for the mouse to climb from the top to the bottom of the pole, not exceeding 60 seconds. Perform one preliminary experiment, followed by five consecutive formal experiments, recording the climbing time for each of the five experiments. After a 30-minute rest, perform a rotation test. Place the mouse head-up on top of the pole and record the time it takes for the mouse to rotate until its head and body are completely facing down. Perform one preliminary experiment, followed by five consecutive formal experiments, recording the time required for each of the five rotations. For data analysis, remove the maximum value from the five data points and calculate the average of four sets to assess the mouse's vertical motor coordination ability. Because the two time points were too close together, the mouse would remember the experimental behavior, so the second time point pole-climbing experiment was not performed.

[0339] The experimental results at the first time point are shown in Figure 32, indicating that the PFFs+PBS group mice showed significant behavioral impairment compared to the PBS+PBS group. In the climbing test, the PFFs+Ab group mice injected with 30H21 (H21) did not show statistically significant behavioral improvement compared to the PFFs+PBS group, but their behavioral ability was comparable to that of the control group PBS+PBS group. However, in the turning test, the PFFs+Ab group mice injected with H21 showed significant behavioral improvement compared to the PFF+PBS group, indicating that H21 can alleviate the vertical motor coordination ability of PD mice to some extent.

[0340] 4. Immunohistochemical detection of mouse brain tissue sections

[0341] After the behavioral experiments, mice underwent corresponding immunohistochemical tests. First, cardiac perfusion was performed. After anesthesia and euthanasia, mouse brain tissue was fixed using pre-cooled PBS and 4% PFA. The brain was completely dissected and then dehydrated three times. The first dehydration was performed using 20% ​​sucrose solution (dissolved in PBS) for at least one full day. The subsequent two dehydration treatments used 30% sucrose solution (dissolved in PBS) for at least one full day each. After dehydration, the brain tissue was embedded using tissue cryopreservation medium and sectioned into 30 μm thick sections using a cryostat. Sections from the striatum and substantia nigra, representing their maximum volume, were selected for subsequent immunostaining. After washing (using 0.1% Tween-20 dissolved in PBS) and permeabilization (using 0.15% Triton-100 dissolved in PBS), the sections were blocked at non-specific sites with 3% goat serum. The sections were then incubated overnight at 4°C with primary antibody. The primary antibodies used were anti-pS129-α-syn antibody (Abcam, ab51253; 1:1000 dilution) and anti-dopamine transporter antibody (DAT; Abcam, ab5990; 1:500 dilution). The sections were incubated with the corresponding secondary antibodies at room temperature for 1 hour the following day. After staining, the sections were mounted with mounting media and observed and detected using a confocal microscope. Fluorescence intensity statistical analysis was performed using ImageJ software, and dopaminergic neuron counting was performed using Imaries software.

[0342] Since the mice injected with 30H21 (H21) did not show significant improvement in motor function at the first time point, only one mouse in each group was perfused for immunostaining observation, making statistical analysis impossible. However, the results showed that the fluorescence intensity of pathological pS129α-syn was reduced to some extent in the striatum and substantia nigra regions, as shown in Figure 33.

[0343] Immunostaining and statistical results at the second time point showed that in the striatum, H21 significantly reduced the pathological α-syn accumulation caused by α-syn PFF injection; in the substantia nigra, although H21 did not significantly reduce the spread of pathological α-syn caused by α-syn PFF injection, it rescued the death of dopaminergic neurons, as shown in Figures 34 and 35.

[0344] Based on the combined results of behavioral experiments and immunostaining in mice, antibody H21 can effectively improve behavioral disorders in PD mice and reduce the aggregation and spread of pathological α-synuclein between brain regions.

[0345] 5. Applicability of antibodies to DLB and MSA

[0346] Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA) all belong to synucleinopathies. Their common core pathological mechanism is the abnormal amyloid aggregation of α-syn, and the proliferation and spread of pathological aggregates between cells in a prion-like manner. Based on the different cell types in which α-syn pathological deposits, synucleinopathies can be further divided into two main categories: one is Lewy body disease (LBD), which mainly includes PD, Parkinson's disease dementia (PDD), and DLB. Its pathological feature is the aggregation of α-syn within neurons, forming Lewy bodies (LBs) and Lewy neurites (LNs); the other is MSA, whose main pathological feature is the aggregation of α-syn within oligodendrocytes, forming glial cytoplasmic inclusions (GCIs). Although different diseases differ in the types of cells affected, the distribution of brain regions, and the conformation of fibers, they all share the common pathogenic basis of the formation, amplification, and spread of α-syn pathological aggregates (PMID: 9804538, 9857966, 36859484).

[0347] Recent cryo-electron microscopy studies have further revealed that α-synuclein fibers have different pathological conformations in different synucleinopathies. For example, the research groups of Michel Goedert and Sjors HWScheres resolved the cryo-electron microscopy structure of isolated α-synuclein fibers from the brains of MSA patients (PMID: 32461689); and the cryo-electron microscopy structure of isolated α-synuclein fibers from the brains of LBD patients (including PD, PDD, and DLB), respectively. They found that the C-terminal region of α-synuclein fibers was exposed on the fiber surface in different types of pathological fibers, thus proving that α-synuclein mainly functions through its C-terminus.

[0348] Recent research indicates that the C-terminus of α-synuclein fibers is not only a structurally exposed region but also a core functional hub mediating pathological propagation and neurotoxicity. This region can specifically interact with various pathogen-related receptors and proteins, including neuronal receptors FAM171A2 and LAG3, microglia receptor RAGE, and the autophagy key protein LC3B (PMID: 39977508, 34172566, 3679499, 36130498, 41575853). These interactions directly mediate the intercellular propagation of α-synucleinic aggregates, the neuroinflammatory response triggered by microglia activation, and autophagy dysfunction—all common pathological amplification mechanisms in synucleinopathies (PMID: 39609631).

[0349] Antibody 30H21 (H21) specifically recognizes the C-terminal fragment (residues 131-140) of α-synuclein fibers and binds with higher affinity to α-synuclein pathological fibers present in various synucleinopathies. Therefore, the therapeutic value of antibody 30H21 (H21) targeting the C-terminus of α-synuclein fibers lies not only in "recognizing pathological aggregates" but also in directly blocking multiple pathogenic signals mediated by this "pathological interaction hub," thereby simultaneously intervening in the downstream pathological processes of different synucleinopathies.

[0350] The cross-indication development of immunotherapies targeting α-synucleinopathies in the field of synucleinopathies has become an industry consensus. For example, AstraZeneca's α-synuclein antibody MEDI1341, after completing a Phase I clinical trial in PD, is now being developed for MSA (synucleinopathic leukemia). This fact demonstrates that antibodies targeting α-synuclein pathological aggregates are not only applicable to a single disease, but also provide a realistic basis for expanding indications to different synucleinopathies.

[0351] This embodiment has demonstrated in PD-related models that 30H21 (H21) can reduce α-synuclein pathological burden, inhibit pathological spread, and alleviate neuroinflammatory responses. Based on the shared α-synucleinogenic mechanism of synucleinopathies and the importance of the epitopes recognized by 30H21 (H21), it is reasonable to infer that H21 is also applicable to delaying, preventing, or treating other synucleinopathies, such as Lewy body dementia (DLB) and multiple system atrophy (MSA). Although the actual therapeutic efficacy in different disease subtypes may vary due to differences in aggregation conformation, brain region distribution, and target tissue accessibility, this difference does not affect its application as a multi-indication therapeutic antibody under the same inventive concept.

[0352] Example 14. Humanization of candidate molecules

[0353] For the candidate molecule 30H21, the closest human antibody sequence was searched in the IMGT database, and the CDR of the mouse candidate antibody was completely transplanted into the corresponding human template sequence. Simultaneously, to ensure that the humanized antibody retains its affinity activity for α-syn, point-by-point / multi-point reversion mutations were performed to obtain the heavy chain variable region and light chain variable region of the humanized antibody molecule.

[0354] The variable region sequences of the aforementioned humanized antibodies were codon-optimized and finally synthesized and constructed into corresponding linearized vectors containing human heavy chain constant regions (pTT5_hIgG1.G1m3) and light chain constant regions (pTT5_hKappa.Km3).

[0355] The synthesized plasmid was amplified and prepared, and the heavy and light chain plasmids were transfected at a ratio of 2:3 using PEI reagent (Polysciences, Cat#24885) (1 μg plasmid: 4 μg PEI) at a density of 2 × 10⁻⁶. 6 HEK293 cells were cultured at 37°C and 5% CO2 for 5-7 days. The culture supernatant was collected, purified, and the concentration of the purified antibody was determined using Nanodrop (Thermo Fisher Scientific Inc.) for later use. Humanized antibodies are indicated by the prefix "hz" followed by the clone number.

[0356] Three humanized antibodies were obtained: hz30H21-H3L0, hz30H21-H13L4, and hz30H21-H13L8.

[0357] Example 15. Physicochemical property analysis and characterization of humanized antibody molecules

[0358] 1. The monomer content of antibody samples was determined using a SEC-HPLC system (Waters UPLC system).

[0359] Using pH 7.4 PBS solution (GIBCO, Cat No. A12586-01) as the mobile phase, the antibody samples to be analyzed were diluted with PBS solution to a concentration of 1 mg / mL and injected into a TSKGel G3000SWXL (Tosoh#808541; 5 μm; 7.8 mm × 300 mm) separation column at a flow rate of 0.75 mL / min. The total injection volume for each sample was 50 μg. The samples were detected using a Waters high-performance liquid chromatography system, and the A280 nm absorbance was collected to analyze the monomer content of the samples.

[0360] The experimental results are shown in Figure 36, where the monomer yield of hz30H21-H3L0 was 99.06%, that of hz30H21-H13L4 was 99.24%, and that of hz30H21-H13L8 was 100%. The high monomer yield indicates that the candidate derived antibodies have good drug-like properties.

[0361] 2. Determination of the hydrophobic properties of antibody samples using a HIC-HPLC system.

[0362] The antibody sample was injected into a Butyl-NPR (Tosoh #014947; 4.6 mm × 35 mm) column at a flow rate of 0.7 mL / min, initially using solution A (20 mM Histidine, 1.6 M (NH4)2SO4, pH 6.0) as the mobile phase. The total sample volume was 25 μg. Immediately after injection, a gradient increase of solution A / B was set. After 16 min, solution A was replaced by 100% solution B (20 mM Histidine, pH 6.0). At the same time, the characteristic absorption peak signal of protein sample A at 280 nm was detected. The hydrophobic properties of the antibody sample were calculated and analyzed by combining parameters such as peak time and area.

[0363] The experimental results are shown in Figure 37. The HIC of hz30H21-H3L0 exhibits good hydrophobic properties with a coefficient of 1.09M(NH4)2SO4, the HIC of hz30H21-H13L4 exhibits good hydrophobic properties with a coefficient of 1.09M(NH4)2SO4, and the HIC of hz30H21-H13L8 exhibits good hydrophobic properties with a coefficient of 1.08M(NH4)2SO4.

[0364] 3. CEX-HPLC system for analyzing the modification / degradation of antibody sample components.

[0365] Solution system:

[0366] (1) Mobile phase stock solution (40mM Tris, 40mM imidazole, 40mM piperazine)

[0367] Weigh 4.8g Tris, 2.7g imidazole, and 3.4g piperazine into a 1000ml beaker, add about 800mL of ultrapure water, stir on a magnetic stirrer until dissolved, transfer the solution to a 1000mL graduated cylinder, add ultrapure water to 1000mL, filter through a 0.2μm filter membrane, sonicate for 10min, and store at 2-8℃. It is effective for 2 weeks from the date of preparation.

[0368] (2) Mobile phase A (4 mM Tris, 4 mM imidazole, 4 mM piperazine, pH 5.0)

[0369] Measure 100 mL of the mobile phase stock solution into a 1000 mL beaker, add about 800 mL of ultrapure water, stir on a magnetic stirrer to dissolve, then adjust the pH of the solution to 5.0 with 6 mol / L HCl solution, transfer the solution to a 1000 mL graduated cylinder, add ultrapure water to 1000 mL, filter through a 0.2 μm filter membrane, sonicate for 10 min, and store at 2–8 °C. It is effective for 2 weeks from the date of preparation.

[0370] (3) Mobile phase B (4 mM Tris, 4 mM imidazole, 4 mM piperazine)

[0371] Measure 100 mL of the mobile phase stock solution into a 1000 mL graduated cylinder, add ultrapure water to 1000 mL, filter through a 0.2 μm filter membrane, sonicate for 10 min, and store at 2–8 °C. It is effective for 2 weeks from the date of preparation.

[0372] (4) Mobile phase C (500mM NaCl)

[0373] Weigh 14.6g NaCl into a 500mL beaker, add about 400mL of ultrapure water, stir on a magnetic stirrer to dissolve, transfer the solution to a 500mL graduated cylinder, add ultrapure water to 500mL, filter through a 0.2μm filter membrane, sonicate for 10min, store at 2-8℃, and it is effective for 2 weeks from the date of preparation.

[0374] Instrument: Waters High Performance Liquid Chromatography System

[0375] How to run:

[0376] The experimental results are shown in Table 1, indicating that the three humanized antibody samples have good drug-like properties.

[0377] Table 1

[0378] 4. RT-PCR method for detecting the thermostability of antibody samples

[0379] Real-time quantitative PCR was performed on a 96-well white PCR sample analysis plate (Bio-Rad, catalog number HSP-9655). Following the manufacturer's instructions, 5 μg of the antibody sample was mixed with a protein dye (Sigma's Sypro Orange, catalog number S5692-50UL), and then the mixture was added to the sample analysis plate. The plate was incubated at 25°C for 10 min, with the temperature incremented by 0.5°C every 30 s from 25°C to 95°C. The plate was then incubated at 15°C for 30 min. The sample detection optical path was set to SYBR / FAM only.

[0380] The results, shown in Figure 38, indicate that the thermal denaturation temperature of antibody hz30H21-H3L0 is 77.5℃, that of hz30H21-H13L4 is 73.5℃, and that of hz30H21-H13L8 is 73.0℃. This demonstrates that the three candidate antibody molecules exhibit good thermal stability.

[0381] Example 16 Binding activity of humanized antibody to α-syn PFFs

[0382] Coat 96-well plates with 100 μL / well of 0.5 μg / mL α-syn PFFs solution and incubate overnight at 4°C. Wash the plates three times with PBST solution (Hegao Biotechnology, PBT-2050), add 100 μL / well of serially diluted antibody solution (initial concentration 0.1 μg / mL, 3-fold dilution), and incubate at room temperature for 1 h. Wash the plates three times with PBST, add 100 μL / well of HRP-labeled goat anti-human IgG, Fcγ fragment antibody (Jackson Immune Research, catalog number 109-035-098), and incubate at room temperature for 1 h. Wash the plates three times with PBST, add 100 μL / well of TMB chromogenic buffer, incubate at room temperature for 8 min, and stop the chromogenic reaction by adding 100 μL / well of 1N HCl. Read the OD at 450 nm using a microplate reader.

[0383] The results, as shown in Figure 39, indicate that all three humanized antibodies of 30H21 retained good affinity for binding human α-syn PFFs antigen.

[0384] Example 17: Blocking activity of humanized antibody against binding to human LAG3 and α-syn PFFs

[0385] The BLI assay method was used for detection. The specific experimental steps are as follows:

[0386] 1. Biotinylated human LAG3 / CD223 protein (Avitag) TM , catalog number LA3-H82E5); SA sensor (Forte Bio, catalog number 18-5019) cured LAG3 (5μg / mL), sample added for 200s.

[0387] 2. Dilute α-Syn PFFs to a concentration of 1000 nM with 50 mM phosphate buffer and sonicate (start for 1 second; stop for 1 second; 15 times).

[0388] 3. Dilute the candidate-derived antibody molecules and control antibody to 18.75 μg / mL, mix them with α-syn PFFs in an equal proportion, and react at room temperature for 30 min.

[0389] 4. OCTET (Forte Bio, Serial No. FB-50482) was used to detect the antibody's ability to block the binding of α-syn PFFs to human LAG3.

[0390] The results are shown in Table 2, indicating that the humanized antibodies all retained good activity in blocking the binding of human LAG3 to α-syn PFFs.

[0391] Table 2 Note: Biotinylated human LAG3 was purchased from ACRO Biosystems, catalog number: Cat. No. LA3-H82E5, Lot. No. CBV95P1-8BPF1-NU.

[0392] Example 18. Comparison of humanized antibodies with other commercially available antibodies

[0393] This embodiment compares the humanized antibodies hz30H21-H13L4 (L4) and hz30H21-H13L8 (L8) with three monoclonal antibodies currently undergoing clinical trials. The antibodies in clinical trials include Prasinezumab (Roche), which targets amino acids 118-126 of the α-synuclein C-terminal domain; BIIB054 (Biogen), which targets amino acids 1-10 of the α-synuclein N-terminal domain; and MEDI1341 (AstraZeneca), which targets amino acids 102-130 of the α-synuclein C-terminal domain. Specifically, the binding affinity to wild-type α-syn PFFs (α-syn unmutated), early-onset α-syn PFFs (JOS-like PFFs), and multiple system atrophy α-syn PFFs (MSA-like PFFs) was compared in vitro. The ability of H21L4, Prasinezumab, and BIIB054 to inhibit the binding of different receptor proteins to α-syn PFFs was also tested in vitro. Furthermore, the activity of inhibiting aggregation and neuroinflammation induced by α-syn PFFs was compared at the cellular level.

[0394] For specific experimental methods, please refer to the corresponding embodiments described above in this application.

[0395] 1. Activity against wild-type α-syn PFFs

[0396] In in vitro binding experiments, humanized antibodies 30H21L4 (L4) and 30H21L8 (L8) showed stronger binding affinity to wild-type α-syn PFFs, approximately twice as strong as Prasinezumab, approximately four times as strong as MEDI1341 (452), and comparable to BIIB054; see Figure 40 for the results.

[0397] In experiments inhibiting the aggregation and propagation of endogenous α-synuclei (PAFs) in neurons, L4 and L8 were more effective than the other two antibodies, 452 and BIIB054, in inhibiting the aggregation and interneuronal propagation of endogenous α-synuclei induced by PFFs. Prasinezumab, however, induced unexplained fluorescent patches on neurons, which could not be statistically analyzed. See Figure 41 for the results.

[0398] In the experiment of inhibiting the release of inflammatory factors induced by α-syn PFFs, L4 and L8 were generally comparable in their effectiveness in inhibiting the release of inflammatory factors induced by α-syn PFFs, and were superior to the other three antibodies. See Figure 42 for the results.

[0399] 2. For early-onset α-synuclear PFFs (JOS-like PFFs) and multisystem atrophic α-synuclear PFFs (MSA-like PFFs):

[0400] In in vitro binding assays, the binding abilities of L4 and L8 to JOS-like PFFs and MSA-like PFFs were comparable to or higher than those of the three control antibodies, indicating that antibodies L4 and L8 may be effective against a variety of α-syn PFFs, representing multiple types of diseases. See Figure 43 for the results.

[0401] In experiments inhibiting the aggregation of endogenous α-synesthesia and the propagation of α-synesthesia fibers in neurons, L8 showed better inhibitory effects than MEDI1341(452) on the intercellular propagation of pathological α-synesthesia aggregates induced by JOS-like PFFs, suggesting that L8 may be more effective for early-onset PD. See Figure 44 for the results.

[0402] 3. Regarding the competitive binding of Prasinezumab and BIIB054 to different receptor proteins and α-syn PFFs.

[0403] In in vitro binding assays, the humanized antibody 30H21L4 (L4) was able to compete for binding with different receptors and α-syn PFFs at lower concentrations, while the N-terminal-targeting antibody BIIB054 could not compete for binding with different receptor proteins and α-syn PFFs. See Figures 45, 46, and 47 for the results.

[0404] Example 19. Determination of PK activity of humanized antibody molecules in mice.

[0405] The humanized antibody of this application was administered to female Balb / C mice via tail vein or intraperitoneal injection, with 4 mice in each group and each mouse receiving 200 μg. Blood samples were collected from the tail vein at the experimental design time points. The samples were incubated at room temperature for at least 30 minutes, and serum was collected at 4000 rpm for 15 minutes. The serum samples were stored at -20°C, and the last serum collection was frozen at -20°C for at least 24 hours.

[0406] ELISA was used for detection. 96-well ELISA plates were coated with antigen and anti-IgG Fab monoclonal antibody, 0.2 μg / ml, 100 μL / well, and incubated overnight at 4°C. The plates were washed three times with 0.1% Tween + PBS, 300 μL / well. Blocking buffer (5% BSA + PBS), 200 μL / well, was added, and the plates were blocked at 37°C for 1 h. Antibody serum was diluted to a suitable concentration range with antibody dilution buffer (5% BSA + PBS + 20% blank mouse serum), 100 μL / well, and added to the ELISA plate, incubated at 37°C for 1 h. The plates were washed three times with PBST. Anti-HuIgG Fab monoclonal antibody (Sigma, I5260-1 mL) (1:5000 dilution), 100 μL / well, was added, and the plates were incubated at 37°C for 40 min. The plates were washed three times with PBST. TMB was added for color development in the dark for 10 min. The reaction was stopped with 50 μL of 2M HCl, and the reading was taken at 450 nm.

[0407] The results are shown in Table 3 and Figures 48-49. The experimental results indicate that the metabolic levels of the two humanized antibodies in mice are within the normal range for antibody drugs, thus demonstrating good drug-like properties.

[0408] Table 3

[0409] Example 20. PK activity and toxicological study of humanized antibody molecules in mice.

[0410] This embodiment conducted pharmacokinetic and toxicological studies of H21L4 to evaluate its in vivo stability, safety, and brain tissue distribution. C57BL / 6 mice were used in the experiment, receiving a single 20 mg / kg injection, and antibody concentrations in plasma and cerebrospinal fluid were monitored. Toxicological responses after multiple doses were also assessed. For control analysis, Prasinezumab developed by Roche was used as the reference antibody, and parallel comparisons were performed using the same dosage and administration regimen. The experimental methods were essentially the same as in Example 19, with only minor adjustments, which are adjustments that could be determined by those skilled in the art based on the actual situation.

[0411] For pharmacokinetic experiments, H21L4 and Prasinezumab were administered to 6-week-old mice via tail vein injection. Blood and cerebrospinal fluid (CSF) samples were collected from the mice at 11 time points after antibody injection to detect antibody concentration.

[0412] Regarding serum antibody concentrations, H21L4 generally showed slightly higher levels than Prasinezumab, especially at five time points: 10 minutes, 4 hours, 24 hours, 2 days, and 7 days post-injection. The serum concentrations of H21L4 were 26%, 25%, 75%, 59%, and 26% higher than those of Prasinezumab, respectively, indicating stronger metabolic stability and higher effective plasma concentrations. This suggests a significant advantage in achieving therapeutic effects. See Figure 50 for the results.

[0413] Regarding antibody concentrations in cerebrospinal fluid (CSF), since most antibodies in CSF are free antibodies, they may bind to cell surfaces or tissue components, leading to significant fluctuations in the measurable concentration of free antibodies. Therefore, antibody concentrations in CSF exhibit fluctuating changes. Four hours after injection, the concentration of H21L4 in CSF was significantly higher than that of Prasinezumab at the same time point, exceeding it by 164%. The concentration ratio between CSF and serum was not constant but dynamically changed at different time points. See Figures 51 and 52 for the results.

[0414] Regarding pharmacokinetic parameters, the calculation results show that the half-life (T) of H21L4 is... 1 / 2 Key parameters such as mean residence time (MRT) and area under the curve (AUC) are comparable to, or even slightly better than, Prasinezumab. Overall, pharmacokinetic studies indicate that H21L4 can cross the blood-brain barrier and exhibits good metabolic stability in vivo, comparable to Roche's Prasinezumab. These results further highlight the potential advantages of H21L4 as a therapeutic antibody. See Figure 53 for the results.

[0415] In the toxicological study of H21L4, we selected three male and three female C57BL / 6 mice to ensure comprehensive assessment across genders. The experiment lasted 28 days and was designed with three dose gradients: 20 mg / kg, 60 mg / kg, and 100 mg / kg, covering low, medium, and high dose ranges. A solvent control group (0 mg / kg, vehicle) was also included to systematically evaluate the dose-dependent toxicity characteristics of L4.

[0416] During the administration period, animals were monitored daily, with changes in weight and food intake recorded. Clinical observation was also conducted, focusing on any abnormal behaviors or physiological manifestations, such as reduced activity, kyphosis, drooling, and lethargy, as indicators of early toxicity. All animals behaved normally during the experiment, and no drug-related adverse behaviors or physiological changes were observed. Results are shown in Figures 54 and 55.

[0417] After the experiment, all experimental animals underwent systematic necropsy, and samples were collected for hematological and serum chemistry analysis to assess whether H21L4 induced organ toxicity, immune system abnormalities, or metabolic disorders in vivo. The results showed that all indicators were within the normal range, and no obvious signs of toxicity or organ damage were found.

[0418] Furthermore, pharmacokinetic studies of H21L4 in serum showed that even at high doses of up to 100 mg / kg, H21L4 maintained good metabolic clearance efficiency in mice, without drug accumulation. This indicates that H21L4 has a rapid in vivo metabolic rate and controllable drug exposure levels, which helps reduce the risk of adverse reactions caused by long-term use and high-dose administration, and provides strong support for subsequent dose optimization and the development of clinical medication strategies. These results further validate that H21L4 possesses good in vivo stability and safety. See Figure 56 for the results.

[0419] We also conducted a combined toxicological and toxicokinetic evaluation of Roche's antibody Prasinezumab using the same design, and the results showed no administration-related adverse reactions. All parameters, including body weight, food intake, clinical manifestations, organ necropsy findings, and hematological and serum biochemical indicators, were within the normal range, with no signs of toxicity. In terms of pharmacokinetics, Prasinezumab also exhibited good in vivo stability and metabolic characteristics at different dose levels, with no significant accumulation or abnormal drug exposure observed, further supporting its safety evaluation results. In summary, the safety and tolerability of our developed H21L4 antibody are comparable to Prasinezumab in vivo, further supporting its feasibility and potential as a therapeutic antibody candidate for clinical development. See Figures 57-59 for the results.

[0420] In summary, based on in-depth analysis of the pathogenic conformation of α-syn aggregates, a monoclonal antibody, H21, targeting its C-terminus, was developed. The non-humanized H21 antibody demonstrated good inhibitory effects on pathological spread, inflammation relief, and motor improvement in cell and animal models, validating the biological rationale and intervention potential of this target. Building upon this, we completed the humanization process, obtaining the candidate clinical antibody H21L4. We systematically compared its function with representative clinical antibodies in various α-syn aggregate conformation models, demonstrating good recognition specificity and broad-spectrum intervention capabilities.

[0421] sequence list

Claims

1. An anti-α-syn antibody that specifically binds to α-syn or α-syn fibrils and its antigen-binding fragment, comprising: 1) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:15 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:

16. 2) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:15 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:17; or 3) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:2 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:

7.

2. An anti-α-syn antibody that specifically binds to α-syn or α-syn fibrils and its antigen-binding fragment, comprising: 1) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 6 and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:11, 9 and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences; 2) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 6, and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:12, 9, and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences; or 3) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:3, 4 and 5, or HCDR1, HCDR2, HCDR3 composed of said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:8, 9 and 10, or LCDR1, LCDR2, LCDR3 composed of said sequences.

3. The anti-α-syn antibody and its antigen-binding fragment according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein: 1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15, or is composed of SEQ ID NO: 15; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16, or is composed of SEQ ID NO: 16; 2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15, or is composed of SEQ ID NO: 15; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 17, or is composed of SEQ ID NO:

17. 3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, or is composed of SEQ ID NO: 2; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 7, or is composed of SEQ ID NO: 7; or 4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 13, or is composed of SEQ ID NO: 13, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14, or is composed of SEQ ID NO:

14.

4. The isolated anti-α-syn antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from Fab, Fab'-SH, Fv (e.g., scFv) or (Fab')2 fragment.

5. The isolated anti-α-syn antibody or its antigen-binding fragment according to any one of claims 1 to 4, comprising a constant region sequence, wherein at least a portion of the constant region sequence is a human common constant region sequence.

6. The anti-α-syn antibody or its antigen-binding fragment as described in any one of claims 1-5, wherein the heavy chain constant region of the antibody comprises or is composed of the amino acid sequence shown in SEQ ID NO:18, and the light chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:

19.

7. The anti-α-syn antibody and its antigen-binding fragment according to any one of claims 1-6, comprising a heavy chain and a light chain, wherein: 1) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 30, or is composed of SEQ ID NO: 30; the light chain comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 31, or is composed of SEQ ID NO:

31. 2) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 30, or is composed of SEQ ID NO: 30; the light chain comprises the amino acid sequence shown in SEQ ID NO: 32, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 32, or is composed of SEQ ID NO:

32. 3) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 26, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 26, or is composed of SEQ ID NO: 26; the light chain comprises the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 27, or is composed of SEQ ID NO: 27; or 4) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 28, or is composed of SEQ ID NO: 28, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 29, or is composed of SEQ ID NO:

29.

8. A pharmaceutical composition comprising: (1) The anti-α-syn antibody or its antigen-binding fragment as described in any one of claims 1-7, and; (2) Medicinal carrier.

9. An isolated polynucleotide molecule encoding an anti-α-syn antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

10. A vector comprising the nucleic acid molecule of claim 9, preferably, the vector being an expression vector.

11. A host cell comprising the vector of claim 10 or the polynucleotide molecule of claim 9.

12. Use of the anti-α-syn antibody or its antigen-binding fragment as described in any one of claims 1-7, or the pharmaceutical composition as described in claim 8, in the preparation of a medicament for treating neurodegenerative diseases, reducing the severity of neurodegenerative diseases, delaying the progression of neurodegenerative diseases, and / or delaying the onset of neurodegenerative diseases.

13. A method for treating neurodegenerative diseases, reducing the severity of neurodegenerative diseases, delaying the progression of neurodegenerative diseases, and / or delaying the onset of neurodegenerative diseases in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, or a therapeutically effective amount of a pharmaceutical composition as described in claim 7.

14. The use or method as described in claim 12 or 13, wherein the neurodegenerative disease is Parkinson's disease, Parkinson's dementia, Lewy body dementia, Lewy body disease, or multiple system atrophy.

15. A method for inhibiting the formation or increase of insoluble or soluble α-synuclein fibrillary aggregates in cells, comprising contacting the cells with an effective amount of an antibody or an antigen-binding fragment thereof as described in any one of claims 1-7.

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