alpha-synuclein antibodies

Abstract

The present invention relates to alpha-synuclein antibodies, particularly but not exclusively antibodies targeting the P1 motif of alpha-synuclein. The invention also encompasses compositions comprising said antibodies, including pharmaceutical compositions and kits. Furthermore, the invention also encompasses methods of making and using said antibodies, for example for the treatment and diagnosis of neurodegenerative diseases, such as Parkinson's disease.

Description

[0001] This invention relates to α-synuclein antibodies, particularly but not limited to antibodies targeting the α-synuclein P1 motif. The invention also covers compositions comprising said antibodies, including pharmaceutical compositions and kits. Furthermore, the invention covers methods for preparing and using said antibodies, for example, for the treatment and diagnosis of neurodegenerative diseases such as Parkinson's disease.

[0002] Antibodies are characterized by high binding affinity and specificity to their targets, making them ideal probes for biomedical research and highly attractive clinical molecules. 1,2 Over the past two decades, antibody discovery technology has developed rapidly, and there are now numerous in vitro screening methods for generating such molecules, including cell display and cell-free display. 3,4 However, these methods are generally limited to stable and soluble proteins, with only a few reported cases of successful targeting of challenging proteins such as membrane proteins and amyloid. 5-7 .

[0003] Currently, an increasing number of research efforts are dedicated to developing antibodies that can inhibit the self-assembly of toxic proteins, such as the formation of amyloid fibrils, which are associated with neurodegenerative diseases. 8 Studies have shown that targeting aggregates rather than monomers is an effective strategy for achieving aggregation inhibition. 6 However, these proteins are highly hydrophobic, and their interactions with other proteins are nonspecific. 9 Therefore, developing antibodies that can specifically bind to aggregates is extremely challenging.

[0004] The aggregation of amyloid protein in the protein α-synuclein is associated with pathologies called synucleinopathy, including Parkinson's disease (PD) and multiple system atrophy (MSA). 11 α-syn is 140 residues long and is highly expressed in dopaminergic neurons. 11 Under physiological conditions, its function is to regulate the size and activity of the presynaptic terminal by controlling the distribution of neurotransmitter vesicles. 12 It consists of three regions: the N-terminal domain (residues 1-60), which is crucial for interactions with other biomolecules. 13-15 The non-amyloid β fraction (NAC) region (residues 61-95) has a high tendency to form amyloid proteins. 16 ; and the C-terminal tail (residues 96-140), whose truncation promotes aggregation. 17 Recently, the 7-residue-long P1 motif (residues 36-42) has been identified as a key regulator of α-syn aggregation. 18Furthermore, the absence of this motif has been shown to prevent α-syn aggregation under in vitro physiological pH conditions. Additionally, in *C. elegans* (…),… C. elegans In this study, the absence of P1 has been shown to prevent age-dependent α-syn aggregation and toxicity. 18 .

[0005] However, to date, there are no available therapies capable of inhibiting the aggregation of toxic amyloid-α-synuclein. This is because α-synuclein aggregates are highly hydrophobic and interact with a variety of other proteins in a non-specific manner, making the development of antibodies that specifically bind to the aggregates extremely challenging. Therefore, there is an urgent need to develop novel therapies that can inhibit the aggregation of toxic amyloid-α-synuclein by binding to the aggregates in order to treat a variety of neurodegenerative diseases, including Parkinson's disease.

[0006] The inventors performed ribosome display on single-domain antibodies (sdAbs) targeting peptides containing the P1 region of α-synuclein, and then conducted multi-parameter screening, ranking candidate antibodies based on their ability to specifically bind to the aforementioned peptides and their ability to inhibit the aggregation of full-length α-synuclein. Figure 1 Unexpectedly, the inventors identified an antibody that can inhibit the aggregation of toxic α-synuclein amyloid protein by specifically binding to the P1 motif.

[0007] Therefore, a first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the P1 motif of α-synuclein or a variant or fragment thereof.

[0008] As illustrated in the examples, the inventors have developed an antibody that inhibits the toxic aggregation of α-synuclein by binding to the P1 motif of the protein. For example, as described in Example 5 and... Figure 4 As shown in A, the inventors have demonstrated that the antibody of the present invention strongly inhibits the aggregation of α-synuclein, one embodiment of which is referred to herein as "αSP1". Furthermore, the inventors unexpectedly discovered that the antibody works effectively in vitro at extremely low concentrations, as only one antibody molecule is required for every 100 α-synuclein molecules to provide a strong inhibitory effect.

[0009] The inventors have demonstrated that the antibody of this invention binds to the P1 motif of α-synuclein aggregates. For example, as described in Example 6, the binding of αSP1 is enhanced with the aggregation of α-synuclein, indicating that the antibody is specific to the aggregation form of α-synuclein (see Example 6). Figure 5 A).

[0010] Therefore, in one embodiment, the antibody or its antigen-binding fragment specifically binds to the P1 motif of aggregated α-synuclein. In some embodiments, the antibody or its antigen-binding fragment specifically binds to the P1 motif of α-synuclein aggregates. It should be understood that some of these aggregates are formed in the early stages of aggregation.

[0011] In some embodiments, the antibody or its antigen-binding fragment is capable of inhibiting the aggregation of α-synuclein. In one embodiment, the antibody or its antigen-binding fragment is capable of inhibiting the aggregation of α-synuclein to form toxic amyloid fibrils.

[0012] In one embodiment, the amino acid sequence of the α-synuclein can be represented by Gene ID: 6622 (Genome Accession Number (Homo sapiens α-synuclein (SNCA), RefSeqGene on chromosome 4): NG_011851), which is provided herein as SEQ ID No: 1, as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA [SEQ ID No: 1] Therefore, the antibody or its antigen-binding fragment can specifically bind to the region between amino acid positions 36 and 42 of SEQ ID No: 1 (as shown in bold above), which corresponds to the P1 motif of α-synuclein. It should be understood that SEQ ID No: 1 is the sequence of the wild-type protein, but the antibody or its antigen-binding fragment can bind to variants or fragments of SEQ ID No: 1.

[0013] In one embodiment, the amino acid sequence of the P1 motif of the α-synuclein may be provided herein as SEQ ID No: 2, as follows: GVLYVGS [SEQ ID No: 2] Therefore, in some embodiments, the antibody or its antigen-binding fragment binds to the P1 motif, which comprises or consists of an amino acid sequence as shown in SEQ ID No: 2 or a variant or fragment thereof.

[0014] The present invention also relates to complete antibodies (i.e. immunoglobulins) with immune specificity to the P1 motif of α-synuclein, and antigen-binding fragments or regions of the corresponding full-length antibodies.

[0015] The antibody or its antigen-binding fragment can be monovalent, divalent, or multivalent. A monovalent antibody is a dimer (HL) comprising a heavy chain (H) and a light chain (L) linked by a disulfide bridge. A divalent antibody is a tetramer (H2L2) comprising two dimers linked by at least one disulfide bridge. Multivalent antibodies can also be prepared, for example, by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains, which are non-covalently linked and connected by disulfide bonds. Each heavy and light chain contains an amino-terminal variable region of approximately 110 amino acids, and a constant sequence for the remainder of the chain. The variable region contains multiple hypervariable regions, or complementarity-determining regions (CDRs), which constitute the antigen-binding site of the antibody molecule and determine its specificity for an antigen, namely the P1 motif or a variant or fragment thereof (e.g., an epitope). Flanking the CDRs of the heavy and light chains are framework regions, which are relatively conserved amino acid sequences used to anchor and position the CDRs. Antibody fragments may include bispecific antibodies (BsAb) or chimeric antigen receptors (CAR).

[0016] Each heavy chain typically contains a heavy chain variable region (V). H The heavy-chain constant region typically contains three structural domains C. H1 C H2 and C H3 Each light chain typically contains a light chain variable region (V). L ( ) and the light chain constant region. The light chain constant region typically contains a structural domain, abbreviated as C. L .

[0017] Each heavy and light chain typically contains three cored receptors (CDRs) and four frame regions (FRs), arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. CDRs participate in antigen binding, conferring antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated herein by reference in its entirety.

[0018] Heavy chains in any vertebrate species can be classified into five distinct categories (or isotypes): IgA, IgD, IgE, IgG, and IgM. These categories are also denoted by α, δ, ε, γ, and µ, respectively. The IgG and IgA categories are further subdivided based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The IgG antibody category can be preferred.

[0019] Based on the sequence of constant structural domains, light chains in any vertebrate species can be classified into one of two types, called κ and λ.

[0020] The constant region consists of one of five heavy chain sequences (μ, γ, ζ, α, or ε) and one of two light chain sequences (κ or λ). The heavy chain constant region sequence determines the antibody isotype and the effector function of the molecule.

[0021] In some embodiments, the antibody or its antigen-binding fragment is isolated or purified.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be generated in rabbits, mice, or rats.

[0023] In some embodiments, the antibody or its antigen-binding fragment is obtained by immunizing a host animal with P1 protein or a variant or fragment thereof, and then collecting the antibody or its antigen-binding fragment. The host animal may be a rabbit.

[0024] In another embodiment, the antibody or its antigen-binding fragment comprises a monoclonal antibody or its antigen-binding fragment. The antibody or fragment may be of mammalian origin. In some embodiments, the antibody of the present invention is a human antibody. As used herein, the term "human antibody" can refer to an antibody, such as a monoclonal antibody, containing a sequence substantially identical to the heavy and light chain CDR amino acid sequence found in a specific human antibody having immunospecificity to the α-synuclein P1 motif or a variant or fragment thereof. The substantially identical amino acid sequence to the heavy or light chain CDR has a considerably high sequence identity compared to a reference sequence. This identity is clearly known or identifiable, representing the amino acid sequence of a specific human antibody. The substantially identical heavy and light chain CDR amino acid sequence may have, for example, minor modifications or conserved amino acid substitutions. Such a human antibody or fragment thereof retains the function of selectively binding to the α-synuclein P1 motif or a variant or fragment thereof. The antibody or its antigen-binding fragment may be produced using a cell-free screening method (e.g., ribosome display).

[0025] The term “human monoclonal antibody” can include monoclonal antibodies that have a basic or complete human CDR amino acid sequence, produced, for example, by recombinant methods such as phage libraries, lymphocytes, or hybridoma cells.

[0026] The term "monoclonal antibody" refers to antibodies that are a substantially homogeneous group of antibodies derived from the same B cell. A substantially homogeneous antibody group contains antibodies that are substantially similar and bind to the same epitopes, except for variants that may occur during monoclonal antibody production. These variants are typically present in very low amounts. Monoclonal antibodies are usually obtained through a process that involves screening a single antibody from a pool of multiple antibodies. For example, the screening process may involve selecting a unique clone from multiple clones, such as a hybridoma clone pool, a phage clone pool, a yeast clone pool, a bacterial clone pool, or other recombinant DNA clone pool. The selected antibody can be further modified, for example, to increase its affinity for the target (through so-called "affinity maturation"), humanize the antibody, increase its yield in cell culture, and / or decrease its immunogenicity in a subject.

[0027] The term "humanized antibody" can refer to antibodies derived from non-human species (such as mice or rabbits) whose protein sequences have been modified to increase their similarity to naturally occurring human antibodies.

[0028] The antibody may be a recombinant antibody. The term "recombinant human antibody" may include human antibodies produced using recombinant DNA technology.

[0029] The term "antigen-binding fragment" can refer to a region of an antibody that has a specific binding affinity for its target antigen, such as the P1 motif of α-synuclein or a variant or fragment thereof. In some embodiments, this fragment is an epitope. The epitope can be linear or conformational. The antigen-binding region can be a hypervariable CDR or its functional portion. The term "functional portion" of a CDR can refer to a sequence within the CDR that has a specific affinity for the target antigen. The functional portion of a CDR may include a ligand that specifically binds to the α-synuclein P1 motif or a fragment thereof.

[0030] The term "CDR" can refer to the hypervariable region in the variable heavy and light chains. Each heavy and light chain of an antibody may have one, two, three, or more CDRs. Typically, each chain has at least three CDRs, which together constitute the antigen-binding site, i.e., the three-dimensional confluence site for antigen binding or specific reaction. However, some studies have inferred that the heavy chain of certain antibodies may have four CDRs.

[0031] The definition of a CDR also includes overlaps or subsets of amino acid residues when compared with each other. The exact residue numbers that constitute a particular CDR or its functional portion will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine the residues that constitute a particular CDR based on the amino acid sequence of the variable region of an antibody.

[0032] The amino acid sequence boundaries of a CDR can be determined using any of many known numbering schemes, including Kabat et al. (as described above) (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. , 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol. , 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol. Biol. , 2001, 309:657-70 (“AHo” numbering scheme).

[0033] The term "functional fragment" of an antibody can refer to the portion of the antibody that retains its functional activity. Functional activity can be, for example, antigen-binding activity or specificity. Functional activity can also be, for example, the effector function provided by the antibody's constant region. The term "functional fragment" is also intended to include fragments produced, for example, by protease digestion or reduction of human monoclonal antibodies and by recombinant DNA methods known to those skilled in the art. Functional fragments of human monoclonal antibodies include, for example, single heavy or light chains and their fragments, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-chain Fv (scFv); and Fc fragments. Alternatively, the effector function of an antibody can be silenced or reduced by introducing amino acid substitutions into the Fc region to inactivate the Fc fragment.

[0034] The term "VL fragment" can refer to a fragment of the light chain of a human monoclonal antibody that contains all or part of the variable region of the light chain, including the CDR. A VL fragment can also contain the constant region sequence of the light chain.

[0035] The term "VH fragment" can refer to a fragment of the heavy chain of a human monoclonal antibody that contains all or part of the variable region of the heavy chain, including the CDR.

[0036] The term "Fd fragment" can refer to the variable region of the heavy chain connected to the first heavy chain constant region, namely VH and CH-1. The "Fd fragment" does not include the light chain, nor does it include the second and third constant regions of the heavy chain.

[0037] The term "Fv fragment" can refer to a monovalent antigen-binding fragment of a human monoclonal antibody, containing all or part of the variable regions of the heavy and light chains, but excluding the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, CDRs. For instance, an Fv fragment contains all or part of the variable regions of approximately 110 amino acids from the N-terminus of both the heavy and light chains.

[0038] The term "Fab fragment" can refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than the Fv fragment. For example, a Fab fragment contains variable regions of both the heavy and light chains, as well as all or part of the first constant region. Thus, a Fab fragment also contains, for example, about 110 to about 220 amino acid residues of the heavy and light chains.

[0039] The term "Fab' fragment" can refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment contains the entire light chain, the entire heavy chain variable region, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also contain part or all of amino acid residues from position 220 to 330 of the heavy chain.

[0040] The term "F(ab')2 fragment" can refer to a bivalent antigen-binding fragment of a human monoclonal antibody. For example, the F(ab')2 fragment may contain all or part of the variable regions of two heavy chains and two light chains, and may also contain all or part of the first constant domains of two heavy chains and two light chains.

[0041] The term “single-chain Fv (scFv)” can refer to a fusion of the heavy chain (VH) and light chain (VL) variable regions linked by short linker peptides.

[0042] The term "bispecific antibody (BsAb)" can refer to a bispecific antibody that contains two scFvs linked together by a short linker peptide.

[0043] However, in one embodiment, the antigen-binding fragment is a single-domain antibody (sdAb) (also known as a nanobody). Those skilled in the art will understand that an sdAb is an antibody fragment composed of a single monomeric variable antibody domain (referred to as VHH).

[0044] Alternatively, in another embodiment, the antigen-binding fragment is a single-chain antibody, an intracellular antibody, a peptide (e.g., a bicyclic peptide), or any other type of fragment or protein scaffold.

[0045] Those skilled in the art will recognize that the exact boundaries of an antibody fragment are not important as long as it retains its functional activity. Using known recombination methods, those skilled in the art can engineer polynucleotide sequences to express functional fragments with arbitrary ends required for a specific application. The functional fragment of an antibody may contain or be composed of fragments substantially identical to or composed of the variable regions of the heavy and light chains of human antibodies.

[0046] In some embodiments, with respect to a first aspect of the invention, the antibody or its antigen-binding fragment has immune specificity for an epitope within the α-synuclein P1 motif. The antigen-binding fragment may comprise or consist of any fragment selected from or composed of the VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab')2, and Fc fragments.

[0047] Therefore, the antigen-binding fragment may comprise any VL antigen-binding region sequence of a human antibody, any VH antigen-binding region sequence, or a combination or composition thereof of VL and VH antigen-binding regions. Those skilled in the art can determine the appropriate number and combination of VH and VL antigen-binding region sequences based on the desired affinity, specificity, and intended use of the antigen-binding fragment. The functional fragment or antigen-binding fragment of the antibody can be readily prepared and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolysis, recombinant methods, and chemical synthesis. Proteolysis for isolating functional fragments includes using human antibodies as starting material. Enzymes suitable for the proteolysis of human immunoglobulins may include, for example, papain and pepsin. Those skilled in the art can readily select a suitable enzyme based on, for example, whether the desired fragment is a monovalent or bivalent fragment. For example, papain cleavage can produce two monovalent Fab' fragments (which bind antigens) and one Fc fragment. Pepsin cleavage, for example, can produce a bivalent F(ab') fragment. The F(ab')2 fragment of the present invention can be further reduced, for example using DTT or 2-mercaptoethanol, to generate two monovalent Fab' fragments.

[0048] Antibody functional fragments or antigen-binding fragments generated from protein hydrolysis can be purified using affinity chromatography and column chromatography. For example, undigested antibodies and Fc fragments can be removed by binding to protein A. Furthermore, functional fragments can be purified based on their charge and size, for example, using ion exchange chromatography and gel filtration chromatography. These methods are well known to those skilled in the art.

[0049] The antibody or its antigen-binding fragment can be prepared by recombinant methods. In some embodiments, polynucleotides encoding desired regions of the antibody heavy and light chains are first isolated. These regions may, for example, include all or part of the variable regions of the heavy and light chains. In some embodiments, these regions may in particular include antigen-binding regions of the heavy and light chains, such as antigen-binding sites, especially CDRs.

[0050] The polynucleotide encoding the antibody or its antigen-binding fragment of the present invention can be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or its antigen-binding fragment can be directly synthesized using oligonucleotide synthesis methods known in the art. Alternatively, smaller fragments can be synthesized first, and then these smaller fragments can be linked together using recombination methods known in the art to form a larger functional fragment.

[0051] As used herein, the term "immunospecificity" refers to the ability of the binding region of an antibody or its antigen-binding fragment to specifically bind to the P1 motif of α-synuclein or its variants or fragments, resulting in an immune response. The antibody or its antigen-binding fragment selectively interacts with the antigen (the P1 motif of α-synuclein) with an affinity constant of approximately 10. -5 Up to 10 - ¹³ M - ¹, usually 10 -6 Up to 10 -9 M - ¹, more typically 10 - ¹ 0 Up to 10 - ¹² M - ¹.

[0052] The term "immune response" can refer to the immune response triggered when the binding region binds to the P1 motif of α-synuclein or its epitope.

[0053] The term "epitope" can refer to any region on an antigen that can induce and bind to an antibody or its antigen-binding fragment. Epitopes can be linear. This means that an antibody can interact with multiple consecutive amino acids on the antigen, and therefore an epitope can be composed of these specific amino acids.

[0054] Alternatively, epitopes can be conformational, i.e., non-linear or discontinuous. This means that antibodies can interact with multiple different segments of the primary amino acid sequence of the antigen. (See example...) Figure 5 As shown in A), the inventors unexpectedly discovered that the antibody or its antigen-binding fragment of the present invention binds to α-synuclein aggregates more effectively than α-synuclein monomers. Therefore, in one embodiment, the antibody or its antigen-binding fragment binds to a conformational epitope.

[0055] As described in the examples, the inventors have unexpectedly demonstrated that the antibody αSP1, as described herein, can specifically bind to the P1 motif of aggregated α-synuclein aggregates and inhibit the toxic amyloid aggregation of α-synuclein.

[0056] Therefore, in one embodiment, the antibody or its antigen-binding fragment is referred to herein as αSP1. The antibody or its antigen-binding fragment may include the CDR1 domain of SEQ ID No: 3, as shown below: RHQWME [SEQ ID No: 3] Therefore, the antibody or its antigen-binding fragment typically contains a CDR1 domain, which contains a sequence substantially as shown in SEQ ID No: 3 or a variant or fragment thereof, or is composed of such a sequence.

[0057] The antibody or its antigen-binding fragment may contain the CDR2 domain of SEQ ID No: 4, as shown below: SSFGQWTK [SEQ ID No: 4] Therefore, the antibody or its antigen-binding fragment typically contains a CDR2 domain, which contains a sequence substantially as shown in SEQ ID No: 4 or a variant or fragment thereof, or is composed of such a sequence.

[0058] The antibody or its antigen-binding fragment may contain the CDR3 domain of SEQ ID No: 5, as shown below: VVEVGYEYY [SEQ ID No: 5] Therefore, the antibody or its antigen-binding fragment typically contains a CDR3 domain, which contains a sequence substantially as shown in SEQ ID No: 5 or a variant or fragment thereof, or is composed of such a sequence.

[0059] In some embodiments, the antibody or its antigen-binding fragment comprises at least one, at least two, or at least three CDRs. In some embodiments, the antibody or its antigen-binding fragment comprises at least one CDR3.

[0060] In some embodiments, the antibody or its antigen-binding fragment comprises: a CDR1 domain comprising or consisting of SEQ ID No: 3, a CDR2 domain comprising or consisting of SEQ ID No: 4, and / or a CDR3 domain comprising or consisting of SEQ ID No: 5. However, in some embodiments, the antibody or its antigen-binding fragment comprises: a CDR1 domain comprising or consisting of SEQ ID No: 3, a CDR2 domain comprising or consisting of SEQ ID No: 4, and a CDR3 domain comprising or consisting of SEQ ID No: 5.

[0061] The antibody or its antigen-binding fragment may contain a variable domain (VHH) sequence as shown in SEQ ID No: 19, as follows: QVQLVESGGGLVQAGGSLRLSCAASGFPVRHQWMEWYRQAPGKEREWVAAISSFGQWTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVVEVGYEYYGQGTQVTVS [SEQ ID No: 19] In some embodiments, the antibody or its antigen-binding fragment includes a variable domain (VHH) region that contains or consists of a sequence substantially as shown in SEQ ID No: 19 or a variant or fragment thereof.

[0062] An embodiment of the nucleotide sequence encoding the variable domain (VHH) region is referred to herein as SEQ ID No: 20, and is shown below: CAGGTTCAGCTGGTTGAGCGGTGGTGGCCTGGTCCAAGCTGGCGGTTCGCTGCGTCTGAGCTGCGCCGCAAGCGGTTTCCCGGTGAGGCATCAGTGGATGGAATGGTATCGTCAGGCCCCGGGCAAAGAACGTGAGTGGGTCGCGGCGATTTCTAGCTTCGGTCAATGG ACGAAATACGCAGATTCTGTTAAGGGCCGCTTTACCATCAGCCGCGACAACGCGAAGAATACGGTCTATTTGCAGATGAATAGCCTGAAACCGGAAGATACCGCGGTTTACTACTGTGTTGTGGAAGTGGGTTACGAATACTACGGCCAAGGTACCCAAGTGACTGTGAGC [SEQ ID No: 20] In some embodiments, the antibody or its antigen-binding fragment includes a variable domain (VHH) region encoded by a nucleic acid sequence, essentially as shown in SEQ ID No: 20, or a variant or fragment thereof.

[0063] In one embodiment, the antibody or its antigen-binding fragment comprises the PelB sequence. In some embodiments, the PelB sequence is located at the N-terminus of a CDR domain (typically a CDR1 domain). In one embodiment, the amino acid sequence of PelB is provided herein as SEQ ID No: 6, as follows: MSKYLLPTAAAGLLLLAAQPAMA [SEQ ID No: 6] In one embodiment, the antibody or its antigen-binding fragment comprises a Myc tag. In some embodiments, the Myc sequence is located at the C-terminus of a CDR domain (e.g., a CDR3 domain). In one embodiment, the amino acid sequence of the Myc tag is provided herein as SEQ ID No: 7, as follows: EQKLISEEDL [SEQ ID No: 7] In one embodiment, the antibody or its antigen-binding fragment comprises a His tag. In some embodiments, the His tag sequence is located at the C-terminus of a CDR domain (e.g., a CDR3 domain), and more commonly, a Myc tag. In one embodiment, the amino acid sequence of the His tag is provided herein as SEQ ID No: 8, as follows: HHHHHH [SEQ ID No: 8] The antibody or its antigen-binding fragment may contain a variable domain (VHH) sequence as shown in SEQ ID No: 9, as follows: MSKYLLPTAAAGLLLLAAQPAMAGSSSQVQLVESGGGLVQAGGSLRLSCAASGFPVRHQWMEWYRQAPGKEREWVAAISSFGQWTKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCVVEVGYEYYGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHHH [SEQ ID No: 9] In some embodiments, the antibody or its antigen-binding fragment includes a variable domain (VHH) region that contains or consists of a sequence substantially as shown in SEQ ID No: 9 or a variant or fragment thereof.

[0064] An embodiment of the nucleotide sequence encoding the variable domain (VHH) region is provided herein as SEQ ID No:10, as follows: ATGAGTAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCCCAGCCTGCAATGGCCGGCTCTTCAAGTCAGGTTCAGCTGGTTGAGAGCGGTGGTGGCCTGGTCCAAGCTGGC GGTTCGCTGCGTCTGAGCTGCGCCGCAAGCGGTTTCCCGGTGAGGCATCAGTGGATGGAATGGTATCGTCAGGCCCCGGGCAAAGAACGTGAGTGGGTCGCGGCGATTTCTAGCTTCGGTCAATGG ACGAAATACGCAGATTCTGTTAAGGGCCGCTTTACCATCAGCCGCGACAACGCGAAGAATACGGTCTATTTGCAGATGAATAGCCTGAAACCGGAAGATACCGCGGTTTACTACTGTGTTGTGGAA GTGGGTTACGAATACTACGGCCAAGGTACCCAAGTGACTGTGAGCGCAGGAAAGAGCTGGCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA [SEQ ID No: 10] In some embodiments, the antibody or its antigen-binding fragment comprises a variable domain (VHH) region encoded by a nucleic acid sequence or a variant or fragment thereof, as shown in SEQ ID No: 10.

[0065] Advantageously, the ability of the antibody or its antigen-binding fragment according to the first aspect of the invention to inhibit α-synuclein aggregation means that it itself has important use as a therapeutic agent for treating, improving or preventing conditions caused by α-synuclein aggregation, such as Parkinson's disease.

[0066] Therefore, in a second aspect of the invention, an antibody or antigen-binding fragment thereof according to the first aspect is provided for therapeutic purposes.

[0067] A third aspect of the invention provides an antibody or antigen-binding fragment thereof according to the first aspect of the invention, which is used to treat, prevent or improve conditions associated with or caused by α-synuclein aggregation.

[0068] According to a fourth aspect of the invention, a method is provided for treating, preventing, or improving a condition in a subject associated with or caused by α-synuclein aggregation, the method comprising administering, or having already administered, a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect to a patient requiring such treatment.

[0069] Conditions associated with or caused by α-synuclein aggregation may be termed synucleinopathy. In some embodiments, conditions associated with or caused by α-synuclein aggregation are neurodegenerative diseases. Furthermore, in some embodiments, conditions associated with or caused by α-synuclein aggregation are selected from Parkinson's disease, Lewy body dementia (DLB), multiple system atrophy (MSA), Alzheimer's disease, and pure autonomic failure (PAF).

[0070] It should be understood that antibodies or antigen-binding fragments thereof according to the invention (referred to herein as "pharmaceuticals") can be used as monotherapy (e.g., antibody alone) to treat, improve, or prevent conditions associated with or caused by α-synuclein aggregation. Alternatively, pharmaceuticals according to the invention can be used as adjunctive therapy or in combination with known therapies for treating, improving, or preventing conditions associated with or caused by α-synuclein aggregation, such as levodopa and other dopaminergic drugs, dopamine metabolism inhibitors, and dopamine receptor agonists (these drugs are designed to control or delay symptoms). Pharmaceuticals according to the invention can also be used as adjunctive therapy or in combination with potential disease-modifying therapies, including drugs for reducing α-synuclein levels, promoting mitochondrial functional recovery, and reducing cellular oxidative stress, microglial activation, and neuroinflammation.

[0071] The pharmaceutical agents according to the invention can be combined into a variety of different forms of compositions, depending particularly on the manner of application. Thus, for example, the composition can be a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, or any other form suitable for administration to humans or animals requiring treatment. It should be understood that the vehicle for the pharmaceutical agent according to the invention should be a vehicle well-tolerated by the subject to which it is administered.

[0072] The pharmaceuticals comprising the agents of the present invention can be used in a variety of ways. For example, oral administration may be required, in which case the agent may be contained in a composition that can be taken orally, for example, in the form of tablets, capsules, or liquids. Compositions comprising the agents and pharmaceuticals of the present invention can be administered by inhalation (e.g., intranasal). The compositions can also be formulated for external use. For example, a cream or ointment may be applied to the skin. Alternatively, the agent may be delivered via gene therapy, for example, via an AAV carrier.

[0073] The agents and drugs of the present invention can also be encapsulated in sustained-release or delayed-release devices. For example, such devices can be implanted subcutaneously or epidermally, allowing for continuous release of the drug over weeks or even months. The device can be located at least near the treatment site. Such devices are particularly advantageous when long-term treatment with the formulations prepared according to the present invention is required, and frequent administration (e.g., at least daily injections) is typically necessary.

[0074] In one embodiment, the agents and drugs of the present invention can be administered to a subject by injection into the bloodstream or by direct injection into the site of treatment. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

[0075] It should be understood that the required amount of antibody or its antigen-binding fragment (i.e., the agent) depends on its biological activity and bioavailability, which in turn depend on the route of administration, the physicochemical properties of the agent, and whether it is used as monotherapy or in combination therapy. The frequency of administration is also affected by the agent's half-life in the treated subject. Those skilled in the art can determine the optimal dosage, which will vary depending on the specific agent used, the strength of the pharmaceutical composition, the route of administration, and the progression of the neurodegenerative disease. Other factors, including the subject's age, weight, sex, diet, and timing of administration, may also necessitate dosage adjustments depending on the specific circumstances of the treated subject.

[0076] Typically, the pharmaceutical agents of the present invention, at daily doses ranging from 0.01 µg / kg body weight to 100 mg / kg body weight, can be used to treat, alleviate, or prevent neurodegenerative diseases, depending on the specific agent used. In some embodiments, the daily dose of the agent is from 1 µg / kg body weight to 100 mg / kg body weight, more commonly from 10 µg / kg body weight to 10 mg / kg body weight, and most commonly from about 100 µg / kg body weight to 10 mg / kg body weight.

[0077] The agent may be administered before, during, or after an attack of a neurodegenerative disease. The daily dose may be given as a single administration (e.g., once-daily injection). Alternatively, the agent may need to be administered two or more times a day. For example, the agent may be administered twice daily (or more times, depending on the severity of the neurodegenerative disease being treated), with each dose ranging from 0.07 µg to 700 mg (i.e., assuming a body weight of 70 kg). Patients receiving treatment may receive the first dose upon waking, followed by the second dose at night (if a two-dose regimen is used), or thereafter every 3 or 4 hours. Alternatively, a sustained-release device may be used to deliver the optimal dose of the agent of the invention to the patient without the need for repeated doses. Known procedures, such as those commonly used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to formulate specific formulations and precise treatment regimens (e.g., daily doses and frequency of administration) of the agent of the invention.

[0078] A fifth aspect of the invention provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to a first aspect of the invention, and optionally a pharmaceutically acceptable mediator.

[0079] A sixth aspect of the invention also provides a method for preparing a pharmaceutical composition according to the fifth aspect, the method comprising conjugating a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as defined in the first aspect with a pharmaceutically acceptable mediator.

[0080] The “subject” can be a vertebrate, mammal, or livestock. Therefore, the medicine according to the invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary purposes. In some embodiments, the subject is a human.

[0081] The "therapeutic effective amount" of an antibody or its antigen-binding fragment refers to a dose of medicine that, when administered to a subject, is sufficient to treat a neurodegenerative disease or produce the desired effect.

[0082] For example, the therapeutically effective amount of the antibody or its antigen-binding fragment used may be from about 0.1 ng / kg to about 100 mg / kg, typically from about 1 ng / kg to about 10 mg / kg. It is believed that the amount of the antibody or its antigen-binding fragment is from about 10 ng / kg to about 10 mg / kg, with the most common amount being from about 50 ng / kg to about 5 mg / kg.

[0083] The term "pharmaceutically acceptable mediator" as used herein refers to any known compound or combination of known compounds that can be used to formulate pharmaceutical compositions, as known to those skilled in the art.

[0084] In one embodiment, the pharmaceutically acceptable medium can be a solid, and the composition can be in powder or tablet form. Pharmaceutically acceptable solid mediums can include one or more substances that can also be used as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, tableting aids, inert binders, sweeteners, preservatives, dyes, coating agents, or tablet disintegrants. The medium can also be an encapsulating material. In powders, the medium is a finely dispersed solid mixed with the finely dispersible active agent of the present invention. In tablets, the active agent can be mixed with a medium having the necessary tableting properties in an appropriate proportion and compressed into the desired shape and size. Powders and tablets typically contain up to 99% active agent. Suitable solid mediums include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins. In another embodiment, the pharmaceutical medium can be a gel, and the composition can be a cream or other similar form.

[0085] However, the drug carrier can be a liquid, while the pharmaceutical composition is in solution form. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active agent according to the invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, organic solvents, mixtures of the two, or pharmaceutically acceptable oils or fats. Liquid carriers may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid carriers suitable for oral and parenteral administration include water (partially containing the above-mentioned additives, such as cellulose derivatives, typically sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the carrier can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are suitable for compositions in sterile liquid form for parenteral administration. For pressurized compositions, the liquid medium may be a halocarbon or other pharmaceutically acceptable propellant.

[0086] Liquid pharmaceutical compositions, such as sterile solutions or suspensions, can be used for various routes of administration, including intramuscular, intrathecal, epidural, intraperitoneal, and intravenous injection, especially subcutaneous injection. Pharmaceuticals can also be prepared as sterile solid compositions, which can be dissolved or suspended in sterile water, physiological saline, or other suitable sterile injection media before administration.

[0087] The pharmaceutical preparations and compositions of the present invention can be administered orally in the form of sterile solutions or suspensions containing other solutes or suspending agents (e.g., sufficient physiological saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (an oleate copolymer of sorbitan and its anhydrides with ethylene oxide), etc. The pharmaceutical preparations used according to the present invention can also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms suitable for parenteral administration include sterile solutions, emulsions, and suspensions.

[0088] The present invention also relates to a method for producing an antibody or antigen-binding fragment of the first aspect, and the antibody or antigen-binding fragment produced in this manner.

[0089] Therefore, the seventh aspect provides an antibody or an antigen-binding fragment thereof, which is obtained by a method comprising the following: (i) Immunizing a host organism with the P1 motif of α-synuclein or a fragment or variant thereof; and (ii) Collect antibodies or their antigen-binding fragments from the host.

[0090] The host can be a mammal, such as a human, rabbit, mouse, or chicken. In some implementations, the host is a rabbit.

[0091] The method may include immunizing the host with at least 50 µg, 75 µg, or 100 µg of immunogen. In some embodiments, the method includes subsequently administering a first booster immunization to the host with at least 25 µg, 35 µg, or 50 µg of immunogen. In some embodiments, the first booster immunization is administered at least 20, 30, or 40 days after the initial immunization. In some embodiments, the method includes subsequently administering a second booster immunization to the host with at least 25 µg, 35 µg, or 50 µg of immunogen.

[0092] In some embodiments, the method includes collecting blood from a host animal and then typically collecting antibodies or antigen-binding fragments from the blood (most commonly serum). In some embodiments, the method includes collecting blood from the host animal at least 30, 40, or 50 days after the initial immunization.

[0093] In some embodiments, serum is passed through a gravity column carrying a covalently bound peptide carrier. After washing, antibodies or antigen-binding fragments thereof are typically eluted with a buffer (usually an acidic buffer), and the solution is then neutralized. The method may also include dialysis with a suitable buffer (e.g., PBS) and optionally lyophilization.

[0094] Alternatively, in another aspect, an antibody or antigen-binding fragment thereof is provided, which is obtained by a method comprising: selecting, using directed evolution or computational methods, an antibody or antigen-binding fragment thereof that specifically binds to the α-synuclein P1 motif.

[0095] The eighth aspect of the present invention provides a polynucleotide sequence encoding an antibody or antigen-binding fragment as defined in the first aspect.

[0096] A ninth aspect of the present invention provides an expression cassette comprising the polynucleotide sequence described in the eighth aspect.

[0097] The polynucleotide sequence encoding the antibody of the present invention or its antigen-binding fragment is typically present in a recombinant vector, such as a recombinant vector for delivery to a target host cell, to produce the antibody or its antigen-binding fragment.

[0098] Therefore, a tenth aspect of the present invention provides a recombinant vector comprising an expression cassette according to the ninth aspect.

[0099] The vector encoding the antibody or antigen-binding fragment described in the first aspect can be, for example, a plasmid, a granule, or a bacteriophage, and / or a viral vector. Such recombinant vectors are very useful in the delivery system of the present invention for converting nucleotide sequences into cells. The nucleotide sequence is typically a DNA sequence, and it is this DNA sequence that encodes the antibody or antigen-binding fragment sequence constituting the antibody or antigen-binding fragment described in the first aspect.

[0100] Recombinant vectors encoding the antibody or antigen-binding fragments described in the first aspect may also contain other functional elements. For example, they may further contain a variety of other functional elements, including suitable promoters to initiate transgenic expression after the vector is introduced into a host cell. For example, the vector is typically capable of autonomous replication within the host cell nucleus. In this case, elements that induce or regulate DNA replication may be needed in the recombinant vector. Alternatively, the recombinant vector may be designed to integrate into the host cell's genome. In this case, DNA sequences that facilitate targeted integration (e.g., through homologous recombination) may be considered. Suitable promoters may include the SV40 promoter, CMV, EF1α, PGK, viral long terminal repeat sequences, and inducible promoters, such as the tetracycline-inducible system. The cassette or vector may also contain terminators, such as β-globin, SV40 polyadenylated sequences, or synthetic polyadenylated sequences. The recombinant vector may also contain promoters, regulators, or enhancers to control nucleic acid expression as needed.

[0101] The vector may also contain DNA encoding a gene that can be used as a selection marker during the cloning process, i.e., to screen transfected or transformed cells, and to screen cells carrying vectors containing heterologous DNA. For example, ampicillin, neomycin, puromycin, or chloramphenicol resistance genes could be considered. Alternatively, the selection marker gene may be located in another vector and used concurrently with a vector containing the transgene. The cassette or vector may also contain DNA involved in regulating nucleotide sequence expression or targeting expressed polypeptides to specific sites on the host cell.

[0102] The purified vector can be directly introduced into host cells by appropriate methods, such as direct endocytosis. The vector can be directly introduced into host cells (e.g., eukaryotic or prokaryotic cells) via transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate method, cationic liposome transfection, polymer or dendritic polymer method, or bead bombardment method. Alternatively, the vector of the present invention can also be directly introduced into host cells using a particle gun.

[0103] Alternatively, delivery systems can deliver polynucleotides to host cells without integrating them into a vector. For example, nucleic acid molecules can be integrated into liposomes or viral particles. Alternatively, "naked" polynucleotides can be introduced into host cells via suitable pathways, such as direct endocytosis.

[0104] The eleventh aspect of the present invention provides a host cell comprising a polynucleotide sequence according to the eighth aspect, an expression cassette according to the ninth aspect, or a vector according to the tenth aspect.

[0105] The host cell can be a eukaryotic or prokaryotic host cell. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is a mammalian host cell, such as NSO mouse myeloma cells, PER.C6® human cells, human embryonic kidney 293 cells, or Chinese hamster ovary (CHO) cells. In some embodiments, the host cell is a CHO cell.

[0106] The twelfth aspect provides a method for preparing an antibody or antigen-binding fragment according to the first aspect, the method comprising: a) Introducing the vector described in aspect ten into a host cell; and b) Culture host cells under conditions that enable the production of the antibodies or antigen-binding fragments described in the first aspect.

[0107] The host cell in step a) can be a eukaryotic or prokaryotic host cell. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is a mammalian host cell, such as NSO mouse myeloma cells, PER.C6® human cells, human embryonic kidney 293 cells, or Chinese hamster ovary (CHO) cells. In some embodiments, the host cell is a CHO cell.

[0108] The method may further include (c) collecting, centrifuging and / or filtering cell culture medium to obtain a cell culture supernatant containing antibody or antigen-binding fragments.

[0109] The method may further include (d) isolating and purifying the antibody or its antigen-binding fragment from the cell culture supernatant. In some embodiments, purification is performed by at least one chromatographic step.

[0110] Suitable chromatographic procedures include affinity chromatography and / or ion exchange chromatography. In some embodiments, affinity chromatography is protein A chromatography. Ion exchange chromatography can be anion exchange chromatography and / or cation exchange chromatography.

[0111] In some implementations, step (d) includes isolating and purifying the antibody or its antigen-binding fragment from the cell culture supernatant by: i) Protein A chromatography; ii) Anion exchange chromatography; and / or iii) Cation exchange chromatography.

[0112] The method may further include (e) filtering the purified antibody or its antigen-binding fragment obtained from step (d). In some embodiments, step (e) includes viral filtration. Therefore, viral filtration membranes are typically used to filter the purified antibody or its antigen-binding fragment obtained from step (d). Suitable membranes are known to those skilled in the art.

[0113] As discussed herein, α-synuclein aggregates are increased in many conditions, including neurodegenerative diseases such as Parkinson's disease and multiple system atrophy (MSA). Therefore, given that the antibody of the present invention is capable of binding to the P1 motif of α-synuclein aggregates, the antibody or its antigen-binding fragment can serve as a reliable diagnostic tool for detecting the presence of α-synuclein aggregates and determining their concentration.

[0114] Therefore, the thirteenth aspect provides for antibodies or antigen-binding fragments thereof based on the first aspect, which are used for diagnosis or prognosis.

[0115] According to the fourteenth aspect of the invention, an antibody or antigen-binding fragment thereof according to the first aspect is provided for the diagnosis or prognosis of conditions associated with or caused by α-synuclein aggregation.

[0116] According to the fifteenth aspect, a method is provided for diagnosing or prognosing conditions in a subject that are associated with or caused by α-synuclein aggregation, the method comprising detecting α-synuclein aggregates in a biological sample obtained from the subject using an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0117] Conditions associated with or caused by α-synuclein aggregation may be termed synucleinopathy. In some embodiments, conditions associated with or caused by α-synuclein aggregation are neurodegenerative diseases. Furthermore, in some embodiments, conditions associated with or caused by α-synuclein aggregation are selected from Parkinson's disease, Lewy body dementia (DLB), multiple system atrophy (MSA), Alzheimer's disease, and pure autonomic failure (PAF).

[0118] The method can be an in vitro method or an ex vivo method. In some embodiments, the method is an in vitro method.

[0119] The use or method may include measuring the level of α-synuclein aggregates in a subject, wherein an increase in the concentration of α-synuclein aggregates in a biological sample, compared to a reference concentration in a healthy control population, indicates the presence of a condition or poor prognosis caused by α-synuclein aggregation.

[0120] In one implementation, a 1-fold increase in α-synuclein aggregates compared to reference values ​​in healthy controls indicates a condition or poor prognosis caused by α-synuclein aggregation. In another implementation, a 2-fold, 3-fold, 4-fold, or 5-fold increase in α-synuclein aggregates compared to reference values ​​in healthy controls indicates a condition or poor prognosis caused by α-synuclein aggregation. In yet another implementation, a 10-fold, 50-fold, or 100-fold increase in α-synuclein aggregates compared to reference values ​​in healthy controls indicates a condition or poor prognosis caused by α-synuclein aggregation.

[0121] According to a sixteenth aspect of the invention, a kit is provided for diagnosing a subject suffering from a condition associated with or caused by α-synuclein aggregation, or for providing a prognosis for the subject's condition, the kit comprising an antibody or an antigen-binding fragment thereof as described in the first aspect for detecting α-synuclein aggregates from a sample of a test subject.

[0122] The kit may also include instructions for use and / or containers for obtaining biological samples from subjects.

[0123] In some implementations, conditions caused by α-synuclein aggregation are defined as above.

[0124] Prognosis may be related to treatment outcomes in subjects diagnosed with α-synuclein aggregation-related conditions. Prognosis may be associated with the rate and / or duration of progression or improvement of the α-synuclein aggregation-related condition, survival probability, and / or the efficacy of various treatment regimens. Therefore, a poor prognosis may indicate that the α-synuclein aggregation-related condition is progressing, with a low survival probability and reduced treatment efficacy. A good prognosis may indicate that the α-synuclein aggregation-related condition is resolving, with a high survival probability and enhanced treatment efficacy.

[0125] In some implementations, the sample includes a biological sample. The sample can be any material available to the subject from which proteins can be obtained.

[0126] Biological samples can be tissues or bodily fluids. A biological sample can be any substance obtained from a subject from which plasma, endothelial cells, megakaryocytes, and platelets can be obtained. Furthermore, samples can be blood, plasma, serum, cerebrospinal fluid, urine, sweat, saliva, tears, breast aspiration, milk, prostatic fluid, semen, vaginal fluid, feces, cervical smears, cells, amniotic fluid, intraocular fluid, mucus, respiratory moisture, animal tissue, cell lysates, tumor tissue, hair, skin, oral smears, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone meal, earwax, lymph, granulomas, cancer biopsy tissue, or combinations thereof.

[0127] The sample can be a liquid aspirate. For example, the sample can be bronchoalveolar lavage fluid (BAL), ascites, pleural lavage fluid, or pericardial lavage fluid.

[0128] Samples may include blood, urine, tissue, etc. In one embodiment, the biological sample includes a blood sample. The blood may be venous or arterial blood. The blood sample can be tested immediately. Alternatively, the blood sample may be cryogenically stored before the method is performed, such as in a refrigerator or even frozen. Alternatively, the blood sample may also be stored at room temperature before the method is performed, such as between 18 and 22 degrees Celsius. The blood sample may include serum. The blood sample may include plasma. However, in some embodiments, the test is performed on whole blood, and the most common blood sample is peripheral blood.

[0129] Before being used in the fifteenth aspect, the blood may require further processing. For example, anticoagulants such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Therefore, the sample collection container may contain anticoagulants to prevent the blood sample from clotting.

[0130] In some implementations, the sample may include plasma, endothelial cells, megakaryocytes, and / or platelets.

[0131] It should be understood that this invention relates to any nucleic acid or peptide or its variants, derivatives or analogs, which substantially comprises an amino acid or nucleic acid sequence of any sequence described herein, including its variants or fragments. The terms “basic…amino acid / nucleotide / peptide sequence,” “variant” and “fragment” can refer to a sequence having at least 40% sequence identity with an amino acid / nucleotide / peptide sequence of any sequence described herein, for example, having 40% identity with the sequence of SEQ ID No: 1-20, and so on.

[0132] The present invention also considers amino acid / polynucleotide / peptide sequences that have a sequence identity greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and more preferably greater than 80% with any of the stated sequences. In some embodiments, the amino acid / polynucleotide / peptide sequence has a sequence identity of at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99%.

[0133] Skilled technicians know how to calculate the percentage of identity between two amino acid / polynucleotide / peptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / peptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The percentage of identity between two sequences may vary depending on the following factors: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment based on 3D comparison; (ii) the parameters used in the alignment method, such as local versus global alignment, the pairing score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, such as functional form and constants.

[0134] After alignment, there are many ways to calculate the percentage of identity between two sequences. For example, the number of identity sequences can be divided by: (i) the length of the shortest sequence; (ii) the alignment length; (iii) the average length of the sequences; (iv) the number of non-empty positions; or (v) the number of equivalent positions excluding protrusions. Furthermore, it should be understood that the percentage of identity is also closely related to sequence length. Therefore, the shorter the sequence pair, the higher the probability of coincidental sequence identity.

[0135] Therefore, it should be understood that accurate alignment of protein or DNA sequences is a complex process. Commonly used multiple alignment programs include ClustalW (Thompson). et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al. The preferred method for generating multiple alignments of the proteins or DNA of this invention is described in *Nucleic Acids Research* (1997, 24, 4876-4882). Suitable parameters for ClustalW can be as follows: For DNA alignment: vacancy opening penalty = 15.0, vacancy extension penalty = 6.66, matrix = Identity. For protein alignment: vacancy opening penalty = 10.0, vacancy extension penalty = 0.2, matrix = Gonnet. For both DNA and protein alignment: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will understand that these and other parameters may need to be changed to obtain optimal sequence alignment.

[0136] In some implementations, the percentage of identity between two amino acid / polynucleotide / peptide sequences can be calculated as (N / T) using such alignments. 100, where N is the number of positions of identical residues in the sequence, and T is the total number of positions compared, including vacancies and may or may not include overhangs. In some embodiments, overhangs are included in the calculation. Therefore, the most common method for calculating the percentage of identity between two sequences includes: (i) preparing the sequence alignment using the ClustalW program with a suitable set of parameters (e.g., as described above); and (ii) substituting the values ​​of N and T into the following formula: - Sequence Identity = (N / T) 100.

[0137] Those skilled in the art are aware of other methods for identifying similar sequences. For example, substantially similar nucleotide sequences can be encoded by sequences that hybridize with a DNA sequence or its complementary sequence under stringent conditions. Stringent conditions refer to the hybridization of nucleotides with DNA or RNA bound to a filter membrane in a 3× sodium chloride / sodium citrate (SSC) solution at about 45°C, followed by washing at least once in a 0.2×SSC / 0.1% SDS solution at about 20–65°C. Alternatively, substantially similar polypeptides may differ from, for example, the amino acid sequences shown in SEQ ID Nos: 1 to 20 by at least one amino acid, but less than 5, 10, 20, 50, or 100 amino acids.

[0138] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or mutated without substantially affecting the protein sequence it encodes, thus providing its functional variants. Suitable nucleotide variants refer to changes resulting from the substitution of different codons encoding the same amino acid in the sequence, thus producing a silent (synonymous) alteration. Other suitable variants refer to sequences with homologous nucleotide sequences, but containing all or part of the sequence, which are altered by substituting different codons encoding side-chain amino acids with similar biophysical properties to the substituted amino acid, thus producing a conserved alteration. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, people should be able to understand which amino acids can be replaced by amino acids with similar biophysical properties, and skilled technicians should know the nucleotide sequences that encode these amino acids.

[0139] All features described herein (including any appended claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined with any of the foregoing aspects in any combination, except that at least some of the features and / or steps are mutually exclusive combinations.

[0140] To better understand the present invention and to show how to implement embodiments of the present invention, the following description will be given with reference to the accompanying drawings, in which: Figure 1 This paper demonstrates a process combining ribosome display technology with multi-parameter screening to discover sdAbs that inhibit α-synuclein (α-syn) aggregation. A) Ribosome display panning cycle: Three rounds of ribosome display panning were performed on synthetic peptides containing the α-synuclein P1 motif. B) The ribosome display products were cloned into a vector with an N-terminal PelB signal sequence that guides the sdAbs into the E. coli periplasmic space. sdAbs from 95 monoclonal strains were expressed in the E. coli periplasmic space and screened. To identify sdAbs interacting with the P1 motif, periplasmic extracts containing sdAbs and control periplasmic extracts without sdAbs were prepared in 96-well deep-plates. These extracts were then used for binding assays against the P1 motif and the out-of-order peptide S1 (containing P1 residues in a different sequence), as well as ThT assays to assess α-syn aggregation inhibition.

[0141] Figure 2The results show the screening of the antibody, referred to as "αSP1" in this invention, by ELISA and ThT assays. A) ELISA screening results. Data are expressed as the ratio of the optical density (OD) at 650 nm for P1 and S1 in the ELISA, i.e., the binding score. sdAbs with a binding score higher than 1.5 times (gray line) are considered potential "hit". B) ThT assay results. Data are expressed as the α-syn aggregation half-life (t) in the presence of sdAb. 50 A) The aggregation score is represented by the ratio of the aggregation half-life to that of the control without sdAb. C) Binding and aggregation score plot, with αSP1 (clone A6) highlighted. Black arrows in A, B, and C indicate the values ​​of αSP1. D) Schematic diagram of the original library, showing the frame region (FWR) and CDR of the antibody, as well as the shared CDR sequence and the CDR sequence of αSP1. E) AlphaFold2 structure of αSP1 [Jumper, J., Evans, R., Pritzel, A.] et al. Highly accurate protein structure prediction with AlphaFold. Nature 596,583–589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2] Prediction. Randomized residues are shown in dark gray. PelB signal sequences and tags are not included in this schematic.

[0142] Figure 3 Displayed based on Figure 2 Raw screening data for the ability of the antibody to inhibit α-syn aggregation. αSP1 (clone A6) and control experiments using non-expressing crude extracts are marked with rectangles.

[0143] Figure 4 The purification and structural characterization of αSP1 are shown. A) SDS-PAGE of the purified αSP1 by immobilized metal affinity chromatography (IMAC). FT: flow path, W1: wash 1, W2: wash 2, E1: elution 1, E2: elution 2. B) Characterization of each step of size exclusion chromatography (SEC). Representative chromatograms (left) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, right) are shown. C) Far-UV circular dichroism (CD) spectrum of antibody αSP1 (10 μM) at pH 7.4 and 25°C.

[0144] Figure 5The inhibitory effect of αSP1 on α-syn aggregation is shown. A) ThT measurements of α-syn alone (squares) and in the presence of different concentrations of αSP1 (from light gray to dark gray, [αSP1]: [α-syn] ratios of 1:100, 1:50, 1:20, and 1:10, respectively). n = 3, and the figure shows the results of individual replicate experiments. B) Negative staining TEM of α-syn in the absence of αSP1 (left) and with αSP1 (right) ([αSP1]: [α-syn] = 1:100). C) Analysis of α-syn soluble fractions. Western blot (left) and quantification of the bands using ImageJ software (right).

[0145] Figure 6 The figure shows the inhibitory effect of αSP1 on α-syn aggregation under conditions that promote α-syn oligomer formation. This process is called secondary nucleation. A) ThT of WT α-syn under secondary nucleation conditions at different concentrations of αSP1 ([αSP1]:[α-syn] 1:500, 1:250, 1:100, 1:50, 1:20, and the black curve 0:1), N=3. The figure shows the results of individual replicate experiments. B) Far-UV CD spectra of antibody αSP1 (15 μM) were collected hourly after incubation at low pH 4.8 and 37 °C for 15 hours. Spectral overlap indicates that αSP1 is stable under low pH conditions. C) DLS of pre-formed fiber seeds under low pH conditions after ultrasonic treatment. The figure shows representative data from the number distribution (C) and correlation plot (D).

[0146] Figure 7 This study demonstrates the specificity of αSP1 for α-syn oligomers relative to other protein types. A) α-syn aggregation was monitored using a ThT aggregation assay (left panel). ELISA analysis was performed at multiple time points (right panel). αSP1 binding increased with α-syn aggregates, indicating that the antibody is specific for the aggregation forms of α-syn (oligomers and amyloid fibrils). B) αSP1 binding to monomeric, oligomeric, and fibril-like α-syn was detected using an ELISA assay. Negative controls were performed in α-syn-free wells using BSA to report nonspecific binding levels. Error bars represent the standard deviation of the technical replicate mean, N=3. Statistical analysis was performed using one-way ordinary ANOVA and Tukey's multiple comparison test, where P > 0.05 (ns), 0.001 < P ≤ 0.01 (ns). P ≤ 0.001 C) Förster resonance energy transfer (FRET)-based titration experiments were used to measure the binding of αSP1 to monomers (black), oligomers (dark gray), and protofibrils (light gray) α-syn. Two biological replicates (labeled as solid or hollow circles, respectively) are shown in the figure for each condition. Error bars represent the standard deviation of the technical replicate mean, N=3. Enlarged views of the portions in the figure are shown within the boxes. D) Native Page and Western blot analyses were performed on samples containing α-syn monomers and aggregates used in the FRET-based titration experiments.

[0147] Figure 8 This study demonstrates the specificity of αSP1 for the P1 sequence of α-syn relative to other proteins and biomolecules. A) ELISA assays were used to verify the binding of αSP1 to the P1 region. Monomers of WT α-syn (light gray) or the α-syn variant lacking the P1 region (referred to as α-syn ΔP1, dark gray) were detected using incremental concentrations of αSP1. Statistical analysis was performed using two-way ANOVA and Sidak multiple comparison tests, where P > 0.05 (ns), 0.001 < P ≤ 0.01 (ns). P ≤ 0.001 B) ThT determination of 1 μM Aβ42 in the absence (solid symbol) or presence (hollow symbol) of 1:10 [αSP1]: [Aβ42], N=3. C) ThT determination of E. coli in the absence and presence of 1:100 [αSP1]: [α-syn] (…). E. coli ThT determination of α-syn in cytoplasmic extract, N=3.

[0148] Figure 9This study demonstrates how αSP1 protects cells from syn-induced toxicity. (A) SH-SY5Y cells were treated with α-syn aggregates in the presence or absence of αSP1 (1:20 [αSP1]:[α-syn] molar ratio) for 48 or 72 hours, followed by MTS cell viability assays. Data were normalized to absorbance values ​​of cells treated with α-syn in the absence of αSP1. The figures show the mean of 4 to 7 biological replicates. Error bars represent the standard error (SEM) of the mean of the biological replicates. Each biological replicate is the mean of 4 to 10 technical replicates. (B) Caspase 3 / 7 activation was detected by exposing SH-SHY5Y cells to α-syn aggregates formed in the presence or absence of αSP1. Cells were treated with aggregated α-syn in the presence or absence of αSP1 (1:20 molar ratio, [αSP1]:[α-syn]) for 72 hours, and caspase 3 / 7 activation was detected as a readout for apoptosis. The luminescence values ​​under different conditions were standardized for cells incubated with α-syn alone. The figures show the mean of three biological replicates. Error bars represent SEM values ​​of biological replicates. Each biological replicate is the mean of 4–10 technical replicates. Statistical analyses shown in (A) and (B) were performed using one-way ANOVA and Tukey's multiple comparison test. P > 0.05 (ns), 0.05 ≥ P > 0.01 ( ), 0.01 ≥ P > 0.001 ( ), 0.001 ≥ P > 0.0001 ( P ≤ 0.0001 ).

[0149] Example

[0150] The inventors have developed an antibody that inhibits the aggregation of α-syn by binding to aggregates. This antibody specifically binds to the P1 motif of α-syn aggregates, thereby inhibiting the toxic aggregation of α-syn protein into amyloid fibrils through binding to its P1 motif.

[0151] Materials and Methods

[0152] reagents

[0153] For cloning purposes, *E. coli* strain XL1-Blue (Agilent) was used. *E. coli* BL21(DE3)(NEB) was used for protein overexpression. Target peptides (P1 and S1) were synthesized from Genescript. These peptides were biotinylated at the N-terminus and amidated at the C-terminus.

[0154] plasmid construction

[0155] The DNA primers used for cloning recombinant DNA are listed in Table 1. All enzymes used for cloning recombinant DNA were purchased from New England Biolabs. All plasmids used are listed in Table 2. The TAC136TAT mutation was previously introduced into the pT7-7 α-syn WT plasmid (a gift from Hilal Lashuel, Addgene plasmid number 36046). The resulting plasmid was named pT7-7 α-syn WT.

[0156] Table 1. Primers used in this study

[0157] Table 2. Plasmids used in this study

[0158] Ribosome display for P1

[0159] The first round of ribosome display was performed as previously described. 20 The second and third rounds of ribosome display were performed as previously described. 7 With slight modifications, in vitro translation was performed using the PUREfrex 2.1 kit and DS supplemental translation mixture (GeneFrontier Corporation, Kashiwa, Japan). Kit kits were dispensed to a total volume of 9.3 µl and incubated at 37 °C for 5 minutes. The concave RNA library was added to the translation mixture (0.7 µl, equivalent to 1.6 × 10⁻⁶). 12(One mRNA strand) was incubated at 37 °C for 30 minutes. After ribosome complex formation, 100 µl of ice-cold WTB buffer (50 mM Tris-acetic acid pH 7.5, 150 mM NaCl, 50 mM magnesium acetate) was added, followed by 40 units of RNase inhibitor (Promega), 0.5% BSA, and 5 mg / ml heparin. 12 µl of Dynabeads MyOne streptavidin T1 (Life Technologies) was washed three times, followed by 500 µl of WTB. Dynabeads were then blocked with 500 µl of WTB containing 0.5% BSA for 1 hour, followed by three washes with 500 µl of WTB-BSA (0.5% BSA). To coat Dynabeads, they were incubated with 100 µl of WTB-BSA containing 500 nM biotinylated P1 for 20 minutes. After washing three times with 500 µl WTB-BSA, the ribosome complex was added to the beads and incubated for 20 minutes. The beads were then washed twice with 500 µl WTB and transferred to a new centrifuge tube. A second wash with 500 µl WTB was then performed, followed by the addition of 100 µl of 50 mM Tris / acetic acid buffer (pH 7.4) supplemented with 150 mM NaCl, 50 mM EDTA (pH 8), and 100 µg / ml yeast RNA, and incubated at room temperature for 10 minutes for RNA elution. RNA was purified using the RNeasy micro kit (Qiagen) by elution with 14 µl of RNase-free water. For reverse transcription, the eluted RNA was mixed with 2 µl of 100 µM RT-Primer and 4 µl of 10 mM dNTPs and heated at 65 °C for 5 minutes. The samples were then cooled on ice, and a 40 µl RT reaction mixture was prepared according to the manufacturer's instructions (AffinityScript, Agilent). The reaction was incubated at 37°C for 1 hour, followed by incubation at 95°C for 5 minutes. cDNA was purified using a PCR purification kit (Macherey Nagel) and eluted with 30 µl of elution buffer. PCR amplification was then performed using 29 µl of purified cDNA, Q5 high-fidelity DNA polymerase (NEB), and primers Med_FX_for and Med_FX_rev for concave and circular libraries. The PCR products were purified by gel electrophoresis and used as templates for PCR assembly using giant primers for connecting flanking regions in the in vitro transcription step. The preparation of the giant primers was as previously described.To facilitate the lateral attachment of concave and circular sybody, a pRDV5 plasmid containing circular synthetic nanobodies (Sybody) was used as a template, and giant primers were amplified using primer pairs 5'_flank_for / Medium_ORF_5'_rev and Medium_ORF_3'_for / tolAk_rev.

[0160] The target DNA fragment was assembled by PCR, as previously described. 7 In short, the sybody pool (200 ng), 5' flanking sequence (120 ng), 3' flanking sequence (360 ng), and 5 mM primers 5'_flank_for and tolAk_2 obtained from RT-PCR were added to a 100 μl reaction mixture. The assembled PCR product was purified by gel electrophoresis and translated into RNA using a 10 μl reaction with a T7 RiboMAX large-scale RNA production system (Promega). The RNA was then purified using an RNeasy kit (Qiagen) and used as input material for the next round of ribosome display. The second round was performed according to the first round using Dynabeads MyOne streptavidin C1. For the third round, Dynabeads MyOne streptavidin T1 was used. After cDNA amplification, the PCR product was cloned into the pSB_init vector via FX cloning for further analysis, as previously described. 20 .

[0161] The hit FX is cloned into the expression vector pSB_init.

[0162] 300 ng of the ribosome display product and 100 ng of pSB_init were used to clone the antibody into the expression vector pSB_init. Cloning was performed as previously described. 20 The process was performed, but the ligation and transformation steps differed. Ligation was carried out using T4 DNA ligase (400 units, NEB, MO202S) at 16°C for 16 hours. Electroporation was performed using BL21(DE3) cells.

[0163] Peripheral extract and hit analysis using ELISA

[0164] The analysis was performed using a 96-well plate, as previously described. 20The procedure was modified slightly. In short, a single antibody clone was expressed in BL21(DE3) cells in 2 ml 96-well deep-plate media, using 1 ml of terrific broth supplemented with 25 mg / ml chloramphenicol. To prepare the periplasmic extract, the culture was centrifuged, and the precipitate was resuspended in 100 μl of periplasmic extraction buffer (20% (w / v) sucrose, 50 mM Tris, 25 mM EDTA, pH 8.0, 0.5 μg / ml lysozyme). The lysis buffer was diluted with 900 μl of Tris-buffered saline (TBS) supplemented with 1 mM MgCl2 and centrifuged to precipitate cell debris. The supernatant was used as the periplasmic cell extract for subsequent ELISA and aggregation assays.

[0165] For ELISA, after coating with 100 μl / well of 5 μg / ml Protein A in TBS overnight, Maxi-Sorp immunoplates (Nunc) were used. After washing three times with 250 μl TBS, the plates were blocked with 250 μl TBS-BSA (TBS containing 0.5% BSA). Each incubation step was performed in 100 μl TBS-BSA for 20 minutes, including the sequential addition of 1:1000 diluted anti-c-Myc antibody (Abcam, ab206486), diluted antibody lysis buffer, 500 nM biotin-labeled target peptide or biotin-labeled disordered peptide, and finally 1:5000 diluted streptavidin-HRP (Sigma Aldrich, S2438). Figure 1 After each incubation step, the sample was washed three times with 250 μl of TBS. ELISA was performed as follows: 100 μl / well of 0.1 mg / ml TMB (Tokyo Chemical Industry UK T2573) containing 50 mM Na2HPO4, 25 mM citric acid, and 0.006% H2O2 was added. The absorbance at 650 nm was measured using a CLARIOstar Plus microplate reader.

[0166] wild-type α-syn expression and purification

[0167] The pT7-7 α-syn WT plasmid was transformed into BL21(DE3) (NEB) and cultured in LB medium supplemented with ampicillin (100 μg / ml) at OD... 600At a concentration of 0.7, wild-type or mutant α-syn expression was induced at 37°C with 1 mM IPTG for 4 hours. Cells were pelleted and resuspended in buffer A (20 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing a protease inhibitor tablet (without EDTA, Roche). Then, proceed as previously described. 25Purification of α-syn. Briefly, the crude extract was incubated at 80°C for 30 min to induce misfolding and precipitation of all proteins except α-syn. The sample was then centrifuged at 35,000 xg for 30 min, and the supernatant was incubated at 4°C for 20 min in the presence of 10 mg / mL streptomycin sulfate, followed by centrifugation at 35,000 xg to precipitate and remove nucleic acids. Finally, the proteins were separated by precipitation with 360 mg / mL ammonium sulfate. Anion exchange chromatography was then performed using a HiPrep Q HP 16 / 10 column (Cytiva) and a linear gradient of 0 to 1 M NaCl. Monomeric α-syn was purified by size exclusion chromatography using a HiLoad 16 / 600 Superdex 75 pg (GE Healthcare) in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, pH 7.4). For the ΔP1 α-syn variant, the same expression and purification steps were used, and the plasmid created in the reference [Doherty, CPA; Ulamec, SM; Maya-Martinez, R.; Good, SC; Makepeace, J.; Khan, GN; vanOosten-Hawle, P.; Radford, SE; Brockwell, DJ, A short motif in the N-terminal region of alpha-synuclein is critical for both aggregation and function. Nat Struct Mol Biol 2020, 27 (3), 249-259.] was used to determine the protein concentration using the molecular weight coefficient (4200 M-1 cm-1).α-syn oligomers were obtained as previously described [Chen, SW; Drakulic, S.; Deas, E.; Ouberai, M.; Aprile, FA; Arranz, R.; Ness, S.; Roodveldt, C.; Guilliams, T.; De-Genst, EJ; Klenerman, D.; Wood, NW; Knowles, TP; Alfonso, C.; Rivas, G.; Abramov, AY; Valpuesta, JM; Dobson, CM; Cremades, N., Structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation. Proc Natl Acad Sci US A2015, 112 (16), E1994-2003]. In short, 800 μM α-syn was lyophilized overnight, resuspended in PBS, and incubated at 37°C for 20–24 hours. The sample was then ultracentrifuged at 50,000 rpm for 90 minutes (using a Beckman Coulter Optima MAX-XP ultracentrifuge equipped with a TLA-55 rotor), and the supernatant was filtered four times through a 100 kDa molecular weight cutoff filter to remove monomers. Protein concentration was determined by measuring absorbance at 275 nm and using an extinction coefficient of 7000 M⁻¹ cm⁻¹ [Xu, CK; Castellana-Cruz, M.; Chen, SW; Du, Z.; Meisl, G.; Levin, A.; Mannini, B.; Itzhaki, LS; Knowles, TPJ; Dobson, CM; Cremades, N.; Kumita, JR, The Pathological G51D Mutation in Alpha-SynucleinOligomers Confers Distinct Structural Attributes and Cellular Toxicity. Molecules 2022, 27 (4).]. Oligomer formation was confirmed by native-pAGE.

[0168] Antibody expression and purification

[0169] Antibody αSP1 was expressed in BL21(DE3) cells (NEB) cultured at 37°C in lysogenic medium containing 25 μg / ml chloramphenicol until OD500. 600 The target concentration was 0.7. Protein expression was induced for 18 hours at 22°C with 0.02% (w / v) L-arabinose. Cells were lysed using an ultrasonic homogenizer in TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl) supplemented with 30 mM imidazole and one tablet of protease inhibitor (Roche) per liter of culture. Cell debris was then removed by centrifugation at 10,000 g for 15 minutes, and Ni was used to treat the cells. 2+ Proteins were purified by affinity chromatography. A 1.5 ml bed volume Ni-NTA column slurry (Qiagen) was used for each purification. Beads were washed twice with 25 ml TBS (pH 7.5) containing 30 mM imidazole, followed by elution twice with 10 ml TBS (pH 7.5) containing 300 mM imidazole. The Ni-NTA-purified antibody was dialyzed into TBS to remove imidazole. The antibody was concentrated using ultrafiltration centrifuge tubes with a molecular weight cutoff of 3 kDa (Amicon Ultra-4) and separated by SEC in PBS pH 7.4 using Superdex 200 Increase 10 / 300 GL (GE Healthcare, Glattbrugg, Switzerland). A Superdex 75 Increase pg column (GE Healthcare) was used. Protein concentration was determined by measuring absorbance at 280 nm, and the theoretical extinction coefficient was calculated using ExPASy ProtParam.

[0170] Western blot analysis

[0171] Protein samples were analyzed on NuPAGE Bis-Tris protein gels (Invitrogen). For Western blotting, proteins were transferred to nitrocellulose (NC) membranes (Invitrogen) and transferred at 20 V for 7 minutes on an iblot2 gel transfer device (Invitrogen). The membranes were blocked for 1 hour at room temperature with 5% skim milk powder prepared in Tris-buffered saline (TBST) containing 0.1% Tween-20. After washing three times with TBST, the membranes were incubated overnight in TBST with rabbit monoclonal anti-α-syn antibody (Abcam, ab138501) diluted 1:1000. After washing three times with TBST, the membranes were incubated for 1 hour with goat anti-rabbit Alexa Fluor 555-labeled secondary antibody (Invitrogen) diluted 1:2000 in TBST. This was followed by three TBST washes and then fluorescence scanning using a Typhoon Variable Mode Imager 9500 (GE Healthcare) via Cy3(λ) ex 532 nm, λ em Proteins are visualized by detecting Alexa Fluor 555 fluorophores using a 610 nm filter.

[0172] CD chromatography

[0173] Far-UV CD spectra of 20 µM αSP1 in PBS (137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 2.7 mM KCl, pH 7.4) and 15 µM αSP1 in PBS adjusted to pH 4.8 were measured at 37 °C using a Chirascan V100 circular dichroism chromatograph (Applied Photophysics). In 0.1 cm cuvettes, at 0.5 nm increments and a 1-second dwell time at each point, the spectral range was 200–250 nm. Accumulated values ​​were collected five times per hour at pH 7.4 or once per hour at pH 4.8 for 15 hours. The buffer spectrum was subtracted from the spectrum at each time point.

[0174] Aβ42 purification

[0175] The purification of Aβ42 was as previously described [Abelein, A.; Chen, G.; Kitoka, K.; Aleksis, R.; Oleskovs, F.; Sarr, M.; Landreh, M.; Pahnke, J.; Nordling, K.; Kronqvist, N.; Jaudzems, K.; Rising, A.; Johansson, J.; Biverstal, H., High-yield Production of Amyloid-beta Peptide Enabled by a Customized Spider Silk Domain. Sci Rep2020, 10 (1), 235.]. In short, to improve solubility, the Aβ42 peptide was coupled to a spider silk domain (referred to as a fusion protein) and expressed in BL21 E. coli cells. Cultures were grown in Lennox medium supplemented with 50 μg / ml kanamycin at 37°C with shaking at 200 rpm until an OD value of 0.8 was reached. Protein expression was induced overnight with 1 mM IPTG at 20°C with shaking at 200 rpm. Cells were collected by centrifugation, and the pellet was resuspended in 20 mM Tris-HCl and 8 M urea at pH 8. After sonication (15 sec on, 45 sec off pulse, 20% amplitude), cell debris was removed by centrifugation. The filtered supernatant was loaded onto two tandem HisTrap 5 mL columns (Cytiva, Little Chalfont, UK), which had been pre-equilibrated with binding buffer (20 mM Tris-HCl and 8 M urea, pH 8, supplemented with 15 mM imidazole). After washing several column volumes with binding buffer, the fusion protein was eluted from the column with 5 column volumes of elution buffer (20 mM Tris-HCl and 8 M urea, pH 8, with 300 mM imidazole added). The eluted fusion protein was dialyzed overnight against 20 mM Tris-HCl, pH 8 to remove imidazole. Then, TEV protease was added to the fusion protein at a 1:15 molar ratio (TEV protease: fusion protein to cleave the spider silk domain), and incubated overnight at 4°C. 7 M guanidine hydrochloride was then added to the sample, and the sample was incubated on ice for at least 2 hours, followed by size exclusion chromatography on a Superdex 75 Increase pg 10 / 600 column (Cytiva) in 20 mM phosphate buffer (pH 8) supplemented with 200 μM EDTA. Monomer concentrations (in μM) were determined from the size exclusion chromatography using the following calculation method:

[0176] Where A280 is the absorbance of the Aβ42 elution peak at 280 nm, 0.2 is the optical path length (cm) of ATKA Pure (Cytiva, UK), and 1490 M–1 cm–1 is the molecular coefficient of Aβ42.

[0177] At neutral pH, ThT α-syn and Aβ42 aggregation were determined using periplasmic extracts or purified protein.

[0178] For α-syn, a 20 µM protein monomer solution (dissolved in PBS at pH 7.4) was aggregated in the presence of 12 µM ThT and 0.02% NaN3. 170 µL of each sample (3 replicates) was added to a 96-well full-area plate (non-binding, clear bottom) and incubated at 37°C for approximately 140 hours using a CLARIOstar Plus microplate reader (BMG Labtech, Germany). For the ThT α-syn aggregation assay using periplasmic extract, a 30 µM α-syn protein monomer solution in PBS at pH 7.4 was aggregated in the presence of approximately 75% v / v periplasmic extract, 12 µM thioflavin T (ThT), and 0.02% NaN3. 150 µL of each sample (one replicate only) was added to a 96-well full-area plate (non-binding, clear bottom) and incubated at 37°C for approximately 40 hours using a CLARIOstar Plus microplate reader (BMG Labtech, Germany). All experiments were performed to stimulate aggregation using linear oscillation (300 rpm, 300 s oscillations before each cycle), with a single (3 mm diameter) glass bead added to each well. Fluorescence intensity was measured every 606 seconds using a spiral averaging method (5 mm diameter) with an excitation wavelength of 440 nm, a dichroic wavelength of 460 nm, and an emission wavelength of 480 nm. The filter had a gain of 3 and 50 flashes per well. For Aβ42, aggregation was performed using a 1 µM protein monomer solution in PBS pH 7.4 in the presence of 20 µM ThT. 180 µL of each sample (3 replicates) was added to a 96-well full-area plate (non-binding, clear bottom) and incubated at 37°C for 22.5 h in a CLARIOstar Plus microplate reader (BMG Labtech, Germany).

[0179] To assess the efficacy of αSP1, aggregation was performed using 20 μM α-syn in the presence of incremental BL21 *E. coli* cell cytoplasmic extracts and different concentrations of αSP1. Briefly, cells were cultured overnight in antibiotic-free LB medium at 37°C with shaking at 200 rpm. Cells were collected by centrifugation, resuspended in buffer A (20 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing protease inhibitor tablets (EDTA-free, Roche), and sonicated for 5 minutes with 15-second pulses followed by 45-second intervals. After centrifugation to remove cell debris, the supernatant was boiled at 80°C for 20 minutes. Then, the supernatant was collected by centrifugation at the highest speed (approximately 18,000 rpm) for 20 minutes. Streptomycin was gradually added to a final concentration of 10 mg / ml to precipitate DNA, and the mixture was incubated at 4°C for 20 minutes. Finally, the supernatant was collected as the cytoplasmic extract after centrifugation at the highest speed for 30 minutes. After sample preparation, aggregation experiments were performed according to the above method.

[0180] Negative staining and transmission electron microscopy

[0181] The sample was spotted onto a Formvar / Carbon-coated 300-mesh copper grid for 1 minute, followed by blotting dry with Whatman filter paper. The copper grid was then washed with water and stained with 2% w / v uranyl acetate solution. The copper grid was imaged using a T12 Spirit electron microscope (Thermo Fisher Scientific–FEI).

[0182] Enzyme-linked immunosorbent assay (ELISA) assesses the binding of αSP1 to α-syn aggregates.

[0183] As described above, 20 μM α-syn was aggregated, and 20 μl aliquots were collected at T0, 5 h, 10 h, 24 h, and 48 h. These samples were triplicated and immobilized on 96-well Maxisorp ELISA plates (Nunc) and incubated at 4°C. The plates were then washed three times with TBS (20 mM Tris, pH 7.4, 100 mM NaCl) and blocked overnight with 5% bovine serum albumin (BSA) in TBS. Afterward, the plates were washed six times with TBS and incubated with 30 μl of 2 μM αSP1 in each well at room temperature with continuous shaking for 1 hour. Subsequently, the plates were washed six times with TBS and incubated with 30 μl of rabbit polyclonal 6x His-tagged horseradish peroxidase (HRP) conjugate (Abcam) diluted 1:4000 in 5% BSA-TBS with continuous shaking for 1 hour at room temperature. Finally, the plate was washed three times with TBS, twice with TBS supplemented with 0.02% Tween 20, and then three times with TBS again. Following the manufacturer's instructions, the amount of αSP1 bound was quantified using 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific), and the absorbance at 450 nm was read using a CLARIOstar microplate reader (BMG Labtech).

[0184] To evaluate the binding of αSP1 to P1, 30 μM of WT and ΔP1 α-syn monomers were immobilized on 96-well Maxisorp ELISA plates (Nunc) and incubated at 4 °C. Subsequent steps were performed as described above using 1–10 μM of αSP1.

[0185] Generation of pre-formed protofibril seeds by α-syn

[0186] Aggregation was carried out in Eppendorf tubes with 100–200 µM WT α-syn (PBS, pH 7.4) at 37 °C (400–500 rpm) for approximately 4 days with shaking in the presence of 0.02% NaN3. The fibers were precipitated by centrifugation (14,000 rpm, 30 min) and resuspended in PBS at pH 7.4. After repeated centrifugation and resuspending, 0.02% NaN3 was added, and the mixture was denatured in 4 M guanidine hydrochloride solution. The fiber concentration (ε) was estimated at 275 nm using a UV-Vis spectrophotometer. 275 = 5600 cm -1 M -1If necessary, the fibers were diluted to ~100 µM and their concentration was reassessed. The fibers were further diluted to 5 µM in PBS, pH 4.8, and then sonicated on ice at 20% power using a probe-type sonicator, with 5-second pulses followed by 5-second rests for 3 cycles. [Flagmeier, P.; Meisl, G.; Vendruscolo, M.; Knowles, TP; Dobson, CM; Buell, AK; Galvagnion, C., Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of alpha-synuclein aggregation. Proc Natl Acad Sci USA 2016, 113 (37), 10328-33.] The size distribution of the fibers was measured by DLS using a Zetasizer Ultra (Malvern Panalytical, UK).

[0187] Secondary nucleation and aggregation assay

[0188] αSP1 was aggregated in the presence of 20 µM monomeric WT α-syn, 20 nM WT α-syn pre-formed fibrous seeds, 20 µM ThT, and 0.02% NaN3 at varying molar ratios of αSP1 to monomeric α-syn (0:1, 1:20, 1:50, 1:100, 1:250, and 1:500). Experiments also included control groups with no fibrous seeds (all proportions) and no monomeric α-syn (highest proportion), and control groups with only fibrous seeds and only αSP1 (highest proportion). Reactions were performed in PBS at pH 4.8, and all protein stock solutions were dialyzed or diluted in PBS at pH 4.8 immediately before use. 170 µL of each sample (3 replicates) was added to 96-well full-area plates (non-binding, clear bottom) and incubated at 37 °C for approximately 100 hours using a FLUOstar Omega microplate reader (BMG Labtech, Germany). A 440 nm excitation, 460 nm dichroic, and 480 nm emission filter was used with a gain of 4. Fifty flashes were performed per aperture, and a spiral-averaged (3 mm diameter) fluorescence intensity measurement was conducted every 420 seconds. All data were background corrected.

[0189] FRET-based titration

[0190] To obtain the binding K of αSP1 with α-syn monomers, oligomers, and fibrils d The A140C α-syn variant was constructed and purified for Alexa Fluor 488 C5-maleimide (Thermo Fisher Scientific) conjugation. Protein labeling was performed according to the manufacturer's instructions. Alexa Fluor 488-labeled oligomers were obtained by lyophilizing a 1:10 labeled:unlabeled α-syn monomer solution overnight. The lyophilized protein was then resuspended in the same volume of PBS as before lyophilization to maintain a 1:10 labeled:unlabeled molar ratio (80:800 μM, respectively) and incubated at 37°C for 20–24 hours. After ultracentrifugation as described above, the monomer was removed by four centrifugations using a 100 kDa molecular weight cutoff filter. 488-labeled fibers were obtained by incubating 100 μM 488 monomer with 10 μM PFF at pH 7.4 for 48 hours. The labeled fibers were collected by centrifugation at the highest speed (approximately 17,000 g) for 30 minutes and washed with PBS to remove any soluble substances. For both labeled oligomers and fibers, concentrations were determined as described above, taking into account fluorescence absorption at 494 nm and a correction factor of 0.11, according to the manufacturer's instructions. αSP1 was labeled using the NTA Alexa Fluor 647 kit (Thermo Fisher Scientific) according to the manufacturer's instructions. A series of different concentrations of labeled α-syn monomers and fibers were added to 0.5 μM of labeled αSP1 in triplicate, and fluorescence intensity was measured using excitation and emission wavelengths at 493 nm and 672 nm, respectively. Measurements were performed using a ClarioStar Plus microplate reader (BMG Labtech). Fluorescence data were plotted and analyzed using GraphPad Prism version 9.3.1 (GraphPad Software). The fluorescence data were fitted using the following single-site specific binding model.

[0191] MTS cell viability assay

[0192] SH-SY5Y neuroblastoma cells were cultured in RPMI medium supplemented with 10% (v / v) fetal bovine serum at 5% CO2. Cells were passaged to passage 19 and then discarded. For the MTS assay, 10,000 cells / well were seeded into 96-well plates and incubated with protein samples after 24 hours. α-syn (diluted with serum-free cell culture medium) at a final concentration of 10 μM was aggregated at a sybody:α-syn molar ratio of 1:20 for 48 and 72 hours, with and without αSP1. After 24 hours of incubation, the medium was removed and replaced with fresh serum-free medium. Cell viability was assessed using the MTS assay (Promega) according to the manufacturer's instructions. Absorbance was measured at 490 nm using a ClarioStar Plus microplate reader (BMG Labtech). For data analysis, absorbance values ​​for medium-only conditions were subtracted from the absorbance values ​​for each condition, and then normalized for cells treated with α-syn aggregates without αSP1 supplementation. Data were plotted using GraphPad Prism version 9.3.1. Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test.

[0193] Caspase-Glo 3 / 7 assay

[0194] SH-SY5Y cells were cultured, plated, and incubated according to the MTS cell viability assay described above, and incubated with samples from the 72-hour aggregation assay. After 24 hours of incubation, apoptosis was assessed by detecting caspase 3 / 7 activation using the Caspase-Glo 3 / 7 Assay System (Promega) according to the manufacturer's instructions. Illumination values ​​were read using a ClarioStarPlus microplate reader (BMG Labtech). As described above, for data analysis, absorbance values ​​under each condition were subtracted from those containing only the culture medium, and the data were then expressed and normalized for cells incubated with α-syn aggregates without αSP1 supplementation. Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test.

[0195] Results and Discussion

[0196] Example 1 - Experimental Strategy

[0197] The inventors aimed to develop an antibody capable of inhibiting α-syn aggregation by binding to aggregates. They chose the P1 region of α-syn (GVLYVGS; SEQ ID No: 2) as the target because this region is a key regulator of α-syn aggregation. Indeed, the absence of this region inhibited α-syn aggregation and toxicity in vitro and in animal models.18 Furthermore, cryo-electron microscopy (CryoEM) shows that the P1 region is difficult to access in mature fibrils. 19 Therefore, antibodies targeting the P1 region may preferentially bind to aggregation intermediates rather than mature fibrils, which are considered to be less toxic.

[0198] The inventor developed a concave structure targeting this point. 7 A large library of synthesized sdAbs was screened. Three rounds of ribosome display techniques were performed, combined with enzyme-linked immunosorbent assay (ELISA) and protein aggregation inhibition screening. Specifically, these sdAbs were expressed in the periplasm of *E. coli* and their specificity for binding to P1 was detected directly in the periplasmic extract by: 1) ELISA; and 2) thioflavin-T (ThT) fluorescence assay to detect their inhibitory effect on α-syn aggregation.

[0199] Example 2 - Ribosome Display and Screening

[0200] Synthetic sdAb library of anti-GFP antibody based on concave structure 7,20 These synthetic sdAbs possess a 6-residue complementarity-determining region 3 (CDR3). The library contains 15 random amino acids, with a theoretical diversity of 8.3 x 10⁻⁶. 17 For the ribosome display experiment, the inventors used a commercially available PURE system. 21 Purified E. coli translational components. Three consecutive rounds of ribosome display were performed on the synthetic peptide P1 derived from α-syn. Figure 1 ).

[0201] Subsequently, the ribosome display was compared with the previously described sdAb for the synthesis of membrane proteins. 7,20 This method involves combining a single antibody FX clone. 22 The antibody is overexpressed in the expression vector pSB_init, which generates a fusion protein of the PelB leader sequence and the antibody. After overexpression, the antibody is targeted to the periplasmic space of *E. coli*, where disulfide bonds can be readily formed. Furthermore, the vector incorporates C-terminal Myc and His tags to the antibody for detection by ELISA, followed by immobilized metal affinity chromatography (IMAC) purification.

[0202] Example 3 - Binding of antibody to P1 motif

[0203] To screen for binding, the inventors used E. coli periplasmic extracts containing each candidate antibody to perform ELISA on peptide P1. Figure 1 , 2And 3). To test specificity for the P1 region, an additional ELISA was performed on the disordered peptide S1 (YSGGLVV; SEQ ID No: 11) containing P1 residues but in a different sequence. Antibodies with a P1 binding ELISA signal to S1 binding ELISA signal ratio greater than 1.5 were considered "hit" (i.e., Figure 2 (Antibody above the gray line in A).

[0204] ELISA data provide important information indicating the specificity and binding affinity of antibodies for peptide P1. However, importantly, the screening method is based on the property of interest: inhibition of α-syn aggregation. Therefore, as a preliminary step in assessing the effect of the antibody on α-syn aggregation, the inventors performed a ThT aggregation assay, the results of which are as follows: Figure 2 B and Figure 3 As shown. To omit the purification step and obtain as much preliminary data as possible about the antibody, we induced the aggregation of recombinant wild-type α-syn in the presence of E. coli periplasmic extract containing the antibody. Given its highly specific binding, we chose A6 for further characterization. This antibody will be referred to as αSP1 below.

[0205] Example 4 - Structural Characterization of αSP1

[0206] αSP1 was overexpressed in the periplasm of *E. coli* and purified using IMAC and subsequent size exclusion chromatography (SEC). Figure 4 A, 4B). The final purified yield was 0.033 mg / L culture. Circular dichroism (CD) spectroscopy was performed to assess the structural integrity of the purified protein. Data showed that the CD spectrum of αSP1 conformed to the characteristic β-sheet of the immunoglobulin domain. Figure 4 C) 23 .

[0207] Example 5 - Inhibitory effect of αSP1 on α-syn aggregation

[0208] To determine whether αSP1 affects the in vitro aggregation of α-syn, ThT fluorescence measurements were performed on α-syn solutions containing different concentrations of αSP1. The results showed that αSP1 strongly inhibited the aggregation of α-syn, and this inhibitory effect depended on the concentration of αSP1. Figure 5 A). Negative staining and transmission electron microscopy also confirmed the inhibitory effect of αSP1. Figure 5 B). Electron microscopy images showed that in the presence of α-synuclein alone, fibrillary aggregates formed at the end of aggregation; however, when α-synuclein aggregated in the presence of αSP1 antibody at a 1:100 molar ratio, fibrillary aggregates did not form. As an additional control, soluble protein fractions at the aggregation endpoint were separated by centrifugation, and the amount of soluble α-synuclein was quantified by Western blotting and density analysis. Figure 5 As shown in C, the amount of soluble α-syn increases with increasing αSP1 concentration, which is consistent with the ThT staining results.

[0209] These aggregation experiments demonstrate that αSP1 effectively inhibits the aggregation of α-syn even at very low substoichiometric concentrations (e.g., even at a molar ratio of αSP1 to α-syn of 1:100). This observation suggests that the inhibition of αSP1 is achieved through the preferential interaction between the antibody and the α-syn aggregates.

[0210] The effect of αSP1 on one of the key forms of oligomer formation (i.e., secondary nucleation) was also investigated. To this end, ThT aggregation assays were performed under low pH conditions with different molar ratios of αSP1 to WT α-syn in the presence of pre-formed WT α-syn fibrils. Under these conditions, αSP1 at substoichiometric concentrations (i.e., a molar ratio of αSP1 to α-syn as low as 1:50) significantly inhibited secondary nucleation of α-syn. Figure 6 A). CD spectroscopy was used to confirm that αSP1 is stable under these buffer conditions. Figure 6 B). The uniformity of seed size was ensured by brief ultrasonic treatment, and this was verified by dynamic light scattering (DLS). Figure 6 C-6D).

[0211] Example 6 - Specificity of αSP1 antibody against α-syn aggregates

[0212] To further investigate the hypothesis that αSP1 preferentially interacts with α-syn aggregates, the binding of αSP1 to different aggregate states of α-syn was examined. For this purpose, samples were collected at different incubation time points during the in vitro α-syn aggregation process, and then analyzed using αSP1 as the primary antibody via ELISA. Figure 7 A). The results showed that αSP1 reactivity was higher in α-syn samples collected in the later stages of aggregation, demonstrating that the antibody preferentially binds to α-syn aggregates. Using a solution rich in stabilized α-syn aggregates, the antibody was analyzed by ELISA (…). Figure 7 B) and FRET-based titration experiments ( Figure 7 C) Further evaluation of the specificity for aggregates ( Figure 7 D). These experiments show that αSP1 interacts preferentially with oligomers of α-syn rather than monomers and fibrils.

[0213] To verify whether αSP1 binds to P1, ELISA was used to evaluate αSP1 with WT α-syn and the α-syn variant (α-syn ΔP1) lacking the P1 region. 18 The combination ( Figure 8A). In an indirect ELISA assay, ELISA wells were coated with the same concentration of protein and incrementally increased amounts of αSP1 (as primary antibody). Results showed that when wells were coated with α-syn ΔP1, the ELISA signal was significantly reduced. This observation is consistent with the binding of αSP1 to P1.

[0214] To further test the specificity of αSP1, another known target that forms amyloid protein was targeted ( Figure 8 B) - A 42-residue-long β-amyloid peptide (Aβ42) 24 ThT assays were performed. These assays were conducted at a molar ratio of αSP1 to Aβ42 of 1:10, which is the maximum molar ratio for α-syn detection. The results showed that αSP1 did not significantly inhibit the in vitro aggregation of Aβ42, indicating that αSP1 specifically inhibits the aggregation of α-syn. Finally, the effects of ThT assays on the aggregation of α-syn were evaluated in the presence of E. coli protein extracts (…). Figure 8 C) The ability of αSP1 to affect α-syn aggregation in the absence of a buffer solution. The inventors found that even under these conditions, αSP1 can inhibit α-syn aggregation at substoichiometric concentrations (i.e., a molar ratio of αSP1 to α-syn of 1:100).

[0215] Example 7 - αSP1 protects cells from α-syn-induced toxicity

[0216] α-syn aggregates have been reported to cause cytotoxicity. Therefore, the inventors evaluated the ability of αSP1 to prevent this mechanism in SH-SY5Y neuroblastoma cells. To this end, they performed cell viability (MTS) and apoptosis (caspase 3 / 7 activation) assays on cells incubated with α-syn aggregates in the presence or absence of αSP1 ([αSP1]:[α-syn] molar ratio of 1:20). The results showed that cells incubated only with α-syn had significantly lower viability than cells incubated with PBS, and exhibited higher levels of caspase 3 / 7 activation, confirming the cytotoxicity of α-syn aggregates.

[0217] Therefore, compared with cells incubated with α-syn alone, cells incubated with α-syn aggregates in the presence of αSP1 exhibited higher cell viability (similar to PBS-treated cells) and lower caspase 3 / 7 activation (partially restored to PBS-treated cell levels). In summary, these data demonstrate that αSP1 can inhibit the toxicity of α-syn in cell experiments.

[0218] in conclusion

[0219] The inventors have surprisingly discovered an antibody capable of specifically binding to the P1 motif of α-synuclein aggregates. Advantageously, this antibody is able to inhibit the toxic clustering of α-synuclein into amyloid fibrils by binding to the P1 motif of α-synuclein. Figure 4 and Figure 5 As shown, the inventors have demonstrated that αSP1 (i.e., the antibody described in this invention) can strongly inhibit the aggregation of α-synuclein into toxic fibrils through preferential interaction with α-synuclein aggregates. Furthermore, the inventors have surprisingly discovered that this antibody is effective even at very low concentrations in vitro (only one antibody molecule is required per 100 α-synuclein molecules to produce a strong inhibitory effect). Therefore, advantageously, the antibody of this invention can be used to treat a variety of conditions associated with the toxic amyloid aggregation of α-synuclein, including Parkinson's disease (PD) and multiple system atrophy (MSA).

[0220] References

[0221] 1. Delidakis, G., Kim, JE, George, K. & Georgiou, G. ImprovingAntibody Therapeutics by Manipulating the Fc Domain: Immunological and Structural Considerations. Annual Review of Biomedical Engineering 24, (2022).

[0222] 2. Gan, SK-E., Phua, S.-X. & Yeo, JY Sagacious epitopeselection for vaccines, and both antibody-based therapeutics and diagnostics: tips from virology and oncology. Antibody Therapeutics 5, 63–72 (2022).

[0223] 3. Almagro, J. C., Pedraza-Escalona, M., Arrieta, H. I. & Pérez-Tapia, S. M. Phage Display Libraries for Antibody Therapeutic Discovery andDevelopment. Antibodies 8, 44 (2019).

[0224] 4. Li, R., Kang, G., Hu, M. & Huang, H. Ribosome Display: A PotentDisplay Technology used for Selecting and Evolving Specific Binders withDesired Properties. Molecular Biotechnology 61, 60–71 (2019).

[0225] 5. Munke, A. et al. Phage display and kinetic selection of antibodiesthat specifically inhibit amyloid self-replication. Proceedings of theNational Academy of Sciences 114, 6444–6449 (2017).

[0226] 6. Linse, S., Sormanni, P. & O’Connell, D. J. An aggregationinhibitor specific to oligomeric intermediates of Aβ42 derived from phagedisplay libraries of stable, small proteins. Proceedings of the NationalAcademy of Sciences 119, (2022).

[0227] 7. Zimmermann, I. et al. Synthetic single domain antibodies for theconformational trapping of membrane proteins. Elife 7, (2018).

[0228] 8. Giorgetti, S., Greco, C., Tortora, P. & Aprile, F. TargetingAmyloid Aggregation: An Overview of Strategies and Mechanisms. InternationalJournal of Molecular Sciences 19, 2677 (2018).

[0229] 9. Aprile, F. A. et al. Rational design of a conformation-specificantibody for the quantification of Aβ oligomers. Proceedings of the NationalAcademy of Sciences 117, 13509–13518 (2020).

[0230] 10. Chia, S. et al. SAR by kinetics for drug discovery in proteinmisfolding diseases. Proceedings of the National Academy of Sciences 115,10245–10250 (2018).

[0231] 11. Goedert, M., Jakes, R. & Spillantini, M. G. TheSynucleinopathies: Twenty Years On. Journal of Parkinson’s Disease 7, S51–S69(2017).

[0232] 12. Vargas, K. J. et al. Synucleins Have Multiple Effects onPresynaptic Architecture. Cell Reports 18, 161–173 (2017).

[0233] 13. Sulzer, D. & Edwards, R. H. The physiological role of α‐synucleinand its relationship to Parkinson’s Disease. Journal of Neurochemistry 150,475–486 (2019).

[0234] 14. Burré, J., Sharma, M. & Südhof, T. C. Cell Biology andPathophysiology of α-Synuclein. Cold Spring Harbor Perspectives in Medicine8, a024091 (2018).

[0235] 15. Davidson, W. S., Jonas, A., Clayton, D. F. & George, J. M.Stabilization of α-Synuclein Secondary Structure upon Binding to SyntheticMembranes. Journal of Biological Chemistry 273, 9443–9449 (1998).

[0236] 16. Li, H.-T., Du, H.-N., Tang, L., Hu, J. & Hu, H.-Y. Structuraltransformation and aggregation of humanα-synuclein in trifluoroethanol: Non-amyloid component sequence is essential andβ-sheet formation is prerequisiteto aggregation. Biopolymers 64, 221–226 (2002).

[0237] 17. Crowther, R. A., Jakes, R., Spillantini, M. G. & Goedert, M.Synthetic filaments assembled from C-terminally truncated α-synuclein. FEBSLetters 436, 309–312 (1998).

[0238] 18. Doherty, C. P. A. et al. A short motif in the N-terminal regionof α-synuclein is critical for both aggregation and function. NatureStructural & Molecular Biology 27, 249–259 (2020).

[0239] 19. Schweighauser, M. et al. Structures of α-synuclein filaments frommultiple system atrophy. Nature 585, 464–469 (2020).

[0240] 20. Zimmermann, I. et al. Generation of synthetic nanobodies againstdelicate proteins. Nature Protocols 15, 1707–1741 (2020).

[0241] 21. Osada, E., Shimizu, Y., Akbar, B. K., Kanamori, T. & Ueda, T.Epitope Mapping Using Ribosome Display in a Reconstituted Cell-Free ProteinSynthesis System. The Journal of Biochemistry 145, 693–700 (2009).

[0242] 22. Geertsma, E. R. & Dutzler, R. A Versatile and Efficient High-Throughput Cloning Tool for Structural Biology. Biochemistry 50, 3272–3278(2011).

[0243] 23. Dumoulin, M. et al. Single-domain antibody fragments with highconformational stability. Protein Science 11, 500–515 (2009).

[0244] 24. Matiiv, A. B. et al. Amyloid and Amyloid-Like Aggregates:Diversity and the Term Crisis. Biochemistry (Moscow) 85, 1011–1034 (2020).

[0245] 25. Arter, W. E. et al. Rapid Structural, Kinetic, and ImmunochemicalAnalysis of Alpha-Synuclein Oligomers in Solution. Nano Letters 20, 8163–8169(2020).

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to the P1 motif of α-synuclein or a variant or fragment thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to the P1 motif of aggregated α-synuclein.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to the P1 motif of α-synuclein aggregates.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof specifically binds to the region between amino acid positions 36 and 42 of the α-synuclein substantially as shown in SEQ ID No:

1.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to the P1 motif, the P1 motif comprising or consisting of an amino acid sequence substantially as shown in SEQ ID No: 2, or a variant or fragment thereof.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a single-domain antibody (sdAb).

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: (i) Contains a sequence basically as shown in SEQ ID No: 3 or a variant or fragment thereof or a CDR1 domain composed thereof; (ii) Contains a sequence essentially as shown in SEQ ID No: 4, or a variant or fragment thereof, or a CDR2 domain composed thereof; and / or (iii) Contains a CDR3 domain consisting of a sequence basically as shown in SEQ ID No: 5 or a variant or fragment thereof, or composed of such a sequence. Optionally, the antibody or its antigen-binding fragment comprises at least one, at least two, or at least three CDRs.

8. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a variable structural domain (VHH) region comprising or consisting of a sequence substantially as shown in SEQ ID No: 19 or a variant or fragment thereof.

9. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a variable structural domain (VHH) region encoded by a nucleic acid sequence substantially as shown in SEQ ID No: 20 or a variant or fragment thereof.

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, used for treatment.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, for the treatment, prevention or improvement of conditions related to or caused by α-synuclein aggregation.

12. The antibody or antigen-binding fragment thereof for the use described in claim 11, wherein the condition associated with or caused by α-synuclein aggregation is a neurodegenerative disease.

13. The antibody or antigen-binding fragment thereof for use according to claim 11 or 12, wherein the condition associated with or caused by α-synuclein aggregation is selected from Parkinson's disease, Lewy body dementia (DLB), multiple system atrophy (MSA), Alzheimer's disease, and pure autonomic failure (PAF).

14. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 9, and optionally a pharmaceutically acceptable mediator.

15. A method for preparing the pharmaceutical composition of claim 14, the method comprising conjugating a therapeutically effective amount of an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 9 with a pharmaceutically acceptable mediator.

16. An antibody or an antigen-binding fragment thereof, obtained by a method comprising: (i) Immunizing a host organism with the P1 motif of α-synuclein or a fragment or variant thereof; and (ii) Collect antibodies or their antigen-binding fragments from the host.

17. A polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 9.

18. An expression cassette comprising the polynucleotide sequence of claim 17.

19. A recombinant vector comprising the expression cassette of claim 18.

20. A host cell comprising the polynucleotide sequence of claim 17, the expression cassette of claim 18, or the vector of claim 19.

21. A method for preparing an antibody or antigen-binding fragment according to any one of claims 1 to 9, the method comprising: a) Introducing the vector of claim 19 into a host cell; as well as b) Culture host cells under conditions that enable the production of the antibody or antigen-binding fragment as described in any one of claims 1 to 9.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, used for diagnosis or prognosis.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, used for the diagnosis or prognosis of conditions associated with or caused by α-synuclein aggregation.

24. A method for diagnosing or predicting conditions in a subject associated with or caused by α-synuclein aggregation, the method comprising detecting α-synuclein aggregates in a biological sample obtained from the subject using an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 9.

25. A kit for diagnosing a subject with a condition associated with or caused by α-synuclein aggregation, or for providing a prognosis for the subject's condition, the kit comprising an antibody or an antigen-binding fragment thereof for detecting α-synuclein aggregates in a sample from a test subject, as described in any one of claims 1 to 9.

26. The use as described in claim 23, the method as described in claim 24, or the kit as described in claim 25, wherein the condition associated with or caused by α-synuclein aggregation is a neurodegenerative disease, optionally, wherein the condition associated with or caused by α-synuclein aggregation is selected from Parkinson's disease, Lewy body dementia (DLB), multiple system atrophy (MSA), Alzheimer's disease, and pure autonomic failure (PAF).

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