RU2798399C2 - Antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates in particular, to antibodies and their fragments capable of binding alpha-synuclein in the form of a monomer and in fibrils, preventing alpha-synuclein aggregation induced by alpha-synuclein fibrils and a method for their production. Also presented are the following: an isolated polynucleotide encoding an antibody or its antigen-binding fragment, a cloning vector and an expression vector, a host cell for producing an antibody, and a method for treating Parkinson's disease (PD) in a patient, which includes administering to the said patient a therapeutically effective amount of the antibody or its antigen-binding fragment.

EFFECT: invention is used for the treatment of alpha-synucleinopathies, including Parkinson's disease.

23 cl, 11 tbl, 14 dwg, 10 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to antibodies against alpha-synuclein and to a method of using these antibodies for the treatment of synucleinopathies. In particular, the present invention relates to antibodies against alpha-synuclein and their use in the treatment of Parkinson's disease.

BACKGROUND OF THE INVENTION

Alpha-synuclein is a small, 140 amino acid long, soluble protein that exists in many different forms. Alpha-synuclein is primarily found in presynaptic nerve endings, and although its exact function is unknown, researchers believe it plays a central role in many neurodegenerative processes.

Over the past 15 years, alpha-synuclein has been shown to play a key role in the pathogenesis of all forms of Parkinson's disease. Genetic mutations or multiplications of the alpha-synuclein gene cause familial early-onset Parkinson's disease (PD). It is interesting to note that when the locus of gene families is multiplied, the pathogenic effect undoubtedly depends on the dose of the gene. Gene duplications cause Parkinson's disease with a relatively early onset (~47 years), which is characterized by a normal course of the disease, while gene tripplications are associated with a very early age of onset (~33 years) and a very rapid course of the disease. In all forms of Parkinson's disease, alpha-synuclein is the main structural component of Lewy bodies, which are a key pathological sign of the disease.

The Lewy body pathology spreads over the course of the disease, and it is suggested that alpha-synuclein acts as a prion-like protein that misfolds to form toxic oligomers and aggregates that can spread from affected neurons to intact neurons (Olanow C.W et al. Movement Disorders, Vol 28, No. 1, 2013). Current treatments fail to stop the spread of the disease and only alleviate the symptoms associated with the progressive loss of motor neuron-dependent activity. In 2014, in a publication by Tran H.T. et al, Cell Reports 7, 2054-2065, June 26, 2014, intraperitoneal administration of a monoclonal antibody against misfolded alpha-synuclein to mice previously intrastriatally injected with fibrils preformed from alpha-synuclein was shown to reduce Lewy body pathology , reduce the loss of dopaminergic neurons in the substantia nigra and alleviate motor impairment. In this regard, there is still a need for passive immunotherapy, which would allow to achieve therapeutic effects in Parkinson's disease and other synucleinopathies.

SUMMARY OF THE INVENTION

The present invention addresses the above need by generating anti-alpha-synuclein antibodies according to the following embodiments.

Embodiment 1. An antibody or antigen-binding fragment thereof that binds to alpha-synuclein, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 selected from SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3; And

b. a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 selected from SEQ ID NO: 5 and CDR-H3 selected from SEQ ID NO: 6.

Embodiment 2 The antibody or antigen-binding fragment thereof of Embodiment 1 wherein the glycine amino acid residue (Gly; G) at position 6 of SEQ ID NO: 3 is replaced with alanine (Ala; A).

Embodiment 3. The antibody or antigen-binding fragment thereof according to embodiments 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to two or more alpha-synuclein amino acid residues between position 113 and 129 relative to SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof binds to at least amino acid residues D119, N122 and Y125.

Embodiment 4 An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding fragment prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils.

Embodiment 5. The antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding fragment is capable of binding alpha-synuclein in monomer form and in fibril form.

Embodiment 6. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, which has a higher binding affinity for alpha-synuclein in fibrils than for alpha-synuclein in monomer form, characterized by a dissociation constant (K _D ), which is at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

Embodiment 7 An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, which has a dissociation constant (K _D ) for alpha-synuclein in fibrils of 60 pM or less.

Embodiment 8. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody is a chimeric, humanized, or human antibody.

Embodiment 9 An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody is a full length antibody.

Embodiment 10. The antibody or antigen-binding fragment thereof of Embodiment 9, wherein the full-length antibody is selected from IgG1, IgG4, or IgG4P.

Embodiment 11 An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ , scFv, dAb, or V _HH .

Embodiment 12. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region corresponding to SEQ ID NO: 13 and a heavy chain variable region selected from SEQ ID NO: 25; or

b. a light chain variable region corresponding to SEQ ID NO: 17 or a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain variable region corresponding to SEQ ID NO: 21; and a heavy chain variable region selected from SEQ ID NO: 25.

Embodiment 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain corresponding to SEQ ID NO: 14 and a heavy chain corresponding to SEQ ID NO: 26; or

b. a light chain corresponding to SEQ ID NO: 18 and a heavy chain corresponding to SEQ ID NO: 26; or

c. a light chain according to SEQ ID NO: 22; and a heavy chain according to SEQ ID NO: 26.

Embodiment 14. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of Embodiments 1-13.

Embodiment 15. The isolated polynucleotide of Embodiment 14, wherein the polynucleotide encodes:

a. the variable region of the light chain, where the polynucleotide:

i. at least 90% identical to the sequences of SEQ ID NO: 15, 19 or 23; or

ii. includes SEQ ID NO: 15 or 19 or 23; or

iii. consists essentially of SEQ ID NO: 15, 19 or 23; or

b. heavy chain variable region, where the polynucleotide:

iv. at least 90% identical to the sequence of SEQ ID NO: 27; or

v. includes SEQ ID NO: 27; or

vi. consists mainly of SEQ ID NO: 27; or

c. light chain, where the polynucleotide:

vii. at least 90% identical to the sequences of SEQ ID NO: 16, 20 or 24; or

viii. includes SEQ ID NO: 16, 20 or 24; or

ix. consists essentially of SEQ ID NO: 16, 20 or 24;

d. heavy chain, where the polynucleotide:

x. at least 90% identical to the sequence of SEQ ID NO: 28; or

xi. includes SEQ ID NO: 28; or

xi. consists essentially of SEQ ID NO: 28.

Embodiment 16 A cloning or expression vector comprising one or more of the polynucleotides of any one of Embodiments 14 or 15.

Embodiment 17. A host cell comprising:

a. one or more polynucleotides according to any one of embodiments 14 or 15 or

b. one or more expression vectors of embodiment 16.

Embodiment 18. A method for producing an antibody or antigen-binding fragment thereof according to any one of Embodiments 1-13, comprising culturing the host cells of Embodiment 17 under appropriate conditions to produce the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

Embodiment 19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 and 13 and one or more pharmaceutically acceptable carriers, excipients, or diluents, wherein the pharmaceutical composition comprises one or more additional active ingredients.

Embodiment 20 An antibody or antigen-binding fragment thereof according to any one of Embodiments 1-13 or a pharmaceutical composition according to Embodiment 19 for use in therapy.

Embodiment 21 An antibody or antigen-binding fragment thereof according to any one of Embodiments 1-13 or a pharmaceutical composition according to Embodiment 19 for use in the treatment of one or more synucleinopathies.

Embodiment 22. The antibody or antigen-binding fragment thereof used in Embodiment 21, wherein the synucleinopathy is selected from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD) , Lewy body variant Alzheimer's disease (LBVAD), mixed type Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation type 1 (NBIA-1).

Embodiment 23 The antibody or antigen-binding fragment thereof used in Embodiment 22 wherein the synucleinopathy is Parkinson's disease.

Embodiment 24. A method of treating synucleinopathy in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 or 13 or a pharmaceutical composition according to Embodiment 19.

Embodiment 25. The method of Embodiment 24 wherein the synucleinopathy is selected from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), body variant Alzheimer's disease Lewy disease (LBVAD), mixed type of Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1), preferably from Parkinson's disease.

Embodiment 26 An antibody or antigen-binding fragment thereof according to any one of Embodiments 1-13 or a pharmaceutical composition according to Embodiment 19 for use in the diagnosis of synucleinopathy, preferably in the diagnosis of Parkinson's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of alpha-synuclein expression samples. His-tagged alpha-synuclein (1) and after removing the His-tag with highly specific tobacco etch virus (TEV) cysteine protease (2), gel permeation chromatography on a Superdex 75 column of human alpha-synuclein treated with TEV protease (3 ). SeeBluePlus2 protein molecular weight marker (Invitrogen) (M). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of human alpha-synuclein purified from the supernatant of Expi293 expression medium as an unlabeled wild-type protein. (4) SeeBluePlus2 protein molecular weight marker (Invitrogen) (M).

Figure 2. (A) Analysis of fibrils by conducting JC-1 examination of monomer without fluorescence and fibrils with maximum fluorescence at 540 nm. (B) Typical example of the spectrum of random helix of monomeric human alpha-synuclein (wavelength 1646 cm-1) and formation of a β-pleated intermediate layer in recombinant human alpha-synuclein in fibrils (wavelength 1625-1630 cm-1).

Figure 3. Enzyme immunoassay (ELISA) binding assay. ELISA binding analysis of (A) mouse 5811 Fab10HIS with recombinant human alpha-synuclein monomer and fibrils and human alpha-synuclein PVDPDNEAYE peptide and (B) mouse 5811 IgG1 with recombinant human alpha-synuclein monomer and fibrils.

Figure 4. (A) Western blot showing binding of mouse IgG1 5811 to human alpha-synuclein and human beta-synuclein. 1 - human alpha-synuclein; 2 - human alpha-synuclein (rPeptide); 3 - human beta-synuclein (rPeptide); marker, MagicMark XP. (B) NMR chemical shift changes showing the expected mouse Fab 5811 epitope on human alpha synuclein.

Figure 5. Inhibition of 5811 Fab binding to immobilized alpha-synuclein (bars on the left - monomer, and bars on the right - fibrils, respectively, for each of the tested peptides).

Figure 6. Western blotting of an alanine scan for epitope characterization. (A) Analysis of wild-type human (His-labeled) alpha synuclein and its unidirectional amino acid mutants on a 4-12% Bis/Tris NuPage gel. Bands: M, SeeBluePlus2; 1, h a-syn V118A; 2, h a-syn D119A; 3, h a-syn P120A; 4, h a-syn D121A; 5, h a-syn N122A; 6, h a-syn E123A; 7, h a-syn A124S; 8, h a-syn Y125A; 9, h a-syn E126A; 10, h a-syn M127A; 11, h a-syn P128A; 12 and 13 h a-syn wild type. Western blotting on polyvinylidene difluoride (PVDF) membranes using (B) 5811 mFab and (C) 5811 mIgG as first antibodies. Bands: M, SeeBluePlus2; 1, h a-syn V118A; 2, h a-syn D119A; 3, h a-syn P120A; 4, h a-syn D121A; 5, h a-syn N122A; 6, h a-syn E123A; 7, h a-syn A124S; 8, h a-syn Y125A; 9, h a-syn E126A; 10, h a-syn M127A; 11, h a-syn P128A; 12 h a-syn wild-type (His-labeled); 13, Magic Mark XP; 14, h a-syn wild-type (no label).

Figure 7 Humanization of the light chain. 5811 denotes a rat light chain variable sequence. 5811gL5, 5811gL8, and 5811gL14 denote humanized grafts of the 5811 antibody variable light chain using the human IGKV1-39 germline gene as an acceptor framework. Hypervariable regions (CDRs) are in bold/underlined. The donor residue is shown in bold/italic and shaded: Y71. A mutation in CDR-L3 to modify a potential deamidation site is shown in bold/underlined and shaded: G94A.

Figure 8. Humanization of the heavy chain. 5811 denotes a rat light chain variable sequence. 5811gH4 denotes humanized grafting of the 5811 antibody heavy chain variable using the human IGHV3-15 germline gene as the acceptor framework. Hypervariable regions (CDRs) are in bold/underlined. Donor remains are shown in bold/italic and shaded: A49 and A100.

Figure 9. Immunohistochemistry. Immunoreactivity in brain sections of patients with Parkinson's disease (A-E) and non-Parkinson's patients (F-H). (A-C) In the temporal cortex of patients with Parkinson's disease, the 5811 mIgG1 antibody labeled the neuropil and the cytoplasm of some cells; Lewy body-like structures were sometimes observed (white arrows). (D, E) The 5811 mIgG1 antibody labeled Lewy body-like characteristic components (white arrows) in the substantia nigra of patients with Parkinson's disease. (F, G) In temporal cortex tissues of non-Parkinson's patients, the 5811 mIgG1 antibody also labeled the neuropil, but no Lewy body-like structures were observed. (H) No Lewy body-like structures were observed in the substantia nigra of a non-Parkinsonian patient; black arrows indicate non-specific labeling corresponding to neuromelanin-containing neurons and neuronal fibers. Scale bar = 50 µm.

Figure 10. Cell aggregation analysis (HEK cell line); the antibodies of the present invention were able to inhibit alpha-synuclein aggregation induced by alpha-synuclein in fibrils with an IC ₅₀ value below 5 nM. The line segments represent the standard error of the mean of the measurement (SEM, N=4, n=12).

Figure 11. Analysis of cell aggregation (primary neurons). The antibodies of the present invention were able to inhibit fibril-induced alpha-synuclein aggregation on mouse primary neurons expressing endogenous levels of alpha-synuclein with an IC ₅₀ value below 5 nM. The line segments represent the standard error of the mean (SEM, N=5, n=17).

Figure 12. Immunohistochemical images of alpha-synuclein pathology (arrows) in different brain regions of SNCA-OVX transgenic mice injected with mouse preformed fibrils (PFF) of alpha-synuclein.

Figure 13. Quantification of alpha-synuclein pathology in various brain regions of SNCA-OVX transgenic mice in the cerebral cortex, striatum, and substantia nigra.

Figure 14 Pharmacokinetic profiles of anti-alpha-synuclein antibodies 5811 in wild-type mice.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in relation to specific non-limiting aspects and variants of its implementation and with reference to specific figures and examples.

Technical terms are used according to their generally accepted meaning, unless otherwise indicated. If certain terms are given a specific meaning, then the definitions of the terms will be given taking into account the terms of use of these terms.

When the term "comprising" is used in the present description and claims, this does not exclude the presence of other elements. For the purposes of the present invention, it is believed that the term "consisting of" is the preferred version of the term "comprising".

When used in the invention of a noun in the singular, it is understood that this form includes this noun in the plural, unless specifically indicated otherwise.

Used in the invention, the term "treatment" and other similar terms refer to the achievement of the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, and/or may be therapeutic in terms of partially or completely curing a disease and or an adverse effect associated with the disease. Thus, the term "treatment" encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease but who has not yet been diagnosed as having the disease; (b) suppression of the disease, that is, stopping its development; and (c) alleviating the disease, ie achieving remission of the disease.

The term "therapeutically effective amount" refers to the amount of anti-alpha-synuclein antibody, or antigen-binding fragment thereof, which, when administered to a mammal or other subject for the purpose of treating a disease, is sufficient to effect such treatment of the disease. The therapeutically effective amount will vary depending on the particular anti-alpha-synuclein antibody or antigen-binding fragment used, the disease and its severity, and the age, body weight, and other factors of the subject being treated.

The term "isolated" means in this invention that the antibody, antigen-binding fragment or polynucleotide, as the case may be, exists in a physical environment other than that in which it may occur naturally.

The present invention provides an antibody or antigen-binding fragment thereof that binds to alpha-synuclein, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 selected from SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3; And

b. a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 selected from SEQ ID NO: 5 and CDR-H3 selected from SEQ ID NO: 6.

In one embodiment, the glycine amino acid residue (Gly; G) at position 6 of SEQ ID NO: 3 is replaced with alanine (Ala; A).

Alpha-synuclein (or alpha syn; a-synuclein; a-syn or any other known synonym) refers to the common name for this protein and includes, but is not limited to, alternative splicing variants, mutants, and alpha-synuclein from other individuals (mice, monkeys, and so on). Unless otherwise indicated, when human alpha synuclein is meant or explicitly referred to, such alpha synuclein includes the sequence represented by SEQ ID NO: 8 or Uniprot P37840.

E A YEMPSEE GYQDYEPEA (SEQ ID NO: 8).

Used in the invention, the term "antibody" usually refers to intact (whole) antibodies, that is, including elements of two heavy chains and two light chains. The antibody may further comprise additional binding domains, for example as in the DVD-Ig molecule disclosed in WO 2007/024715 or in the so-called (FabFv) ₂ Fc described in WO2011/030107. Thus, the antibody used in the present invention includes bi-, tri- or tetravalent full length antibodies.

Antigen binding fragments of antibodies include single chain antibodies (ie, full length heavy chain and light chain); Fab, Fab modified, Fab', Fab' modified, F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. V _H or V _L or V _HH ), scFv, two-, three - or tetravalent antibodies, Bis-scFv, diabodies, tribodies, triabodies, tetrabodies and epitope-binding fragments of any of the above antibodies (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson , 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and obtaining these antibody fragments are well known (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in patent document WO2009/040562 and its disulfide-stabilized version, Fab-dsFv, was first disclosed in patent document WO2010/035012. Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International patent applications WO2005/003169, WO2005/003170 and WO2005/003171. Polyvalent antibodies may include polyspecificities, for example, may be bispecific, or may be monospecific (see, for example, patent documents WO 92/22583 and WO05/113605). One such example of the last mentioned antibody is the Tri-Fab (or TFM) described in patent document WO92/22583.

An alternative antigen-binding fragment comprises a Fab linked to two scFvs or dsscFvs, with each scFv or dsscFv binding to the same or different target (e.g., one scFv or dsscFv that binds the therapeutic target and one scFv or dsscFv that increases the half-life by binding, for example, albumin). Such antibody fragments are described in International Patent Application Publication No, WO2015/197772, the entire content of which is incorporated herein by reference and, in particular, in relation to the discussion of antibody fragments.

A typical Fab' molecule includes a pair of heavy and light chains, wherein the heavy chain includes a VH variable region, a CH1 constant domain and a natural or modified hinge region, and a light chain includes a VL variable region and a CL constant domain. The Fab' dimers of the present invention form F(ab') ₂ where, for example, the dimerization may terminate at the hinge region.

An antibody or antigen-binding fragment thereof of the present invention binds to an alpha-synuclein epitope. Within the scope of the present invention, the term "epitope" is used interchangeably for both conformational and linear epitopes, where the conformational epitope consists of discontinuous sections of the antigen's primary amino acid sequence and the linear epitope is formed by a sequence formed by contiguous amino acids.

An epitope can be identified by known appropriate epitope mapping in combination with any one of the antibodies of the present invention. Examples of such methods include screening peptides of various lengths derived from full-length alpha-synuclein for binding to an antibody or fragment thereof of the present invention and identifying the smallest fragment that can specifically bind to an antibody containing an epitope sequence recognized by the antibody. Alpha-synuclein peptides can be prepared synthetically or by hydrolytic cleavage of the alpha-synuclein protein. Peptides that bind an antibody can be identified, for example, by mass spectrometric analysis. In another example, nuclear magnetic resonance spectroscopy or X-ray diffraction analysis can be used to identify an epitope bound by an antibody of the present invention. Once identified, an epitope can be used to prepare fragments that bind an antibody of the present invention and, if necessary, can be used as an immunogen to generate additional antibodies that bind the same epitope.

In one embodiment, an antibody or antigen-binding fragment thereof of the present invention binds two or more alpha-synuclein amino acid residues between positions 113 and 129 with respect to SEQ ID NO: 8. In particular, the antibody or antigen-binding fragment thereof binds at least amino acids residues D119, N122 and Y125 in SEQ ID NO: 8.

Preferably, the antibody or antigen-binding fragment thereof of the present invention prevents alpha-synuclein aggregation caused by alpha-synuclein fibrils.

Within this particular context, the term "prevent" (and its various grammatical variants) is used interchangeably with the term "inhibit" in the invention, and this term indicates that the antibodies of the present invention have an effect on fibril-induced alpha-synuclein aggregation. alpha synuclein. The action can be preventive, which is expressed in the complete or partial prevention of aggregation; or in total or partial reduction, i.e. blocking of aggregation, in order to prevent its further progression, or in total or partial reduction in the occurrence of further aggregation; or in the total or partial elimination of aggregation that has already occurred.

Without resorting to any theory as evidence, however, it can be assumed that the antibody or antigen-binding fragment of the present invention binds to alpha-synuclein:

i) in monomeric form and prevents the formation of oligomers and aggregates of alpha-synuclein; and/or

ii) in oligomeric and fibrillar form and prevents the spread of alpha-synuclein from one neuron to another neuron; and/or

iii) in oligomeric and fibrillar form and prevents alpha-synuclein aggregation caused by alpha-synuclein fibrils, preferably endogenous alpha-synuclein aggregation.

The term "fibrils", "fibrillar form", or "in fibrils" as used herein in relation to alpha-synuclein refers to non-monomeric forms of alpha-synuclein, including alpha-synuclein oligomers, which can form substances that circulate within and between brain structures. brain.

In one embodiment, the antibody or antigen-binding fragment of the present invention is capable of binding alpha-synuclein in monomer form and in fibrils. In one embodiment, the antibody, or antigen-binding fragment thereof, has a higher binding affinity for alpha-synuclein in fibrils compared to monomeric alpha-synuclein. This is characterized by a dissociation constant (K _D ) which is at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

In one embodiment, the antibody or antigen-binding fragment of the present invention has a dissociation constant (K _D ) of less than 60 pM for monomeric alpha-synuclein. In another embodiment, the antibody or antigen-binding fragment of the present invention, the antibody or antigen-binding fragment of the present invention has a dissociation constant (K _D ) of less than 30 pM for alpha-synuclein in fibrils.

Used in the invention, the term "K _D "refers to the dissociation constant, which is calculated in relation to K _d to K _a (ie K _d /K _a ) and expressed as a molar concentration (M). K _d and K _a denote the dissociation rate and the association rate, respectively, for a particular antigen-antibody (or antigen-binding fragment thereof) interaction. The K _D values for antibodies can be determined by well-known methods. As a method for determining the K _D value of an antibody, a surface plasmon resonance method is used, for example, on the Biacore ^® system described in the examples of the invention, using isolated natural or recombinant alpha-synuclein, its suitable fusion protein/polypeptide, or its fibrils. In one example, affinity is measured using recombinant human alpha-synuclein described in the examples of the invention. For surface plasmon resonance, target molecules are immobilized on the solid phase and exposed to the ligand in the mobile phase moving along the flow cell. If the ligand binds to the immobilized target, then the local refractive index changes, resulting in a change in the surface plasmon resonance (SPR) angle, which can be continuously monitored in real time by detecting changes in reflected light intensity. Rates of change of the SPR signal can be determined to calculate apparent rate constants for the association and dissociation phases of the coupling reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al, Cancer Res. 53:2560-65 (1993)).

In one embodiment, an antibody or antigen-binding fragment thereof of the present invention has a higher binding affinity (ie, a lower K _D ) for alpha-synuclein in fibrils compared to monomeric alpha-synuclein. The term "affinity" refers to the strength of the interaction between an antibody or antigen-binding fragment and alpha-synuclein.

In one embodiment, the antibody or antigen-binding fragment of the present invention blocks or prevents or reduces alpha-synuclein-induced aggregation, preferably alpha-synuclein-induced aggregation in fibrils.

In one embodiment, the antibody or antigen-binding fragment of the present invention has an IC ₅₀ value of less than 10 nM to block alpha-synuclein induced aggregation in fibrils, preferably the antibody or antigen-binding fragment of the present invention has an IC ₅₀ value of less than 5 nM to block aggregation caused by alpha-synuclein in fibrils.

The term "IC ₅₀ " as used herein refers to half the maximum inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a specific biological or biochemical function, in relation to the present invention, aggregation caused by alpha-synuclein, preferably alpha-synuclein. in fibrils. The IC ₅₀ value is a quantitative measure that indicates how much of a particular substance is required to inhibit half of a given biological process.

In one embodiment, the antibody or antigen-binding fragment of the present invention does not bind beta-synuclein and/or gamma-synuclein and is specific for alpha-synuclein.

As used herein, the term "specific" is intended to refer to an antibody that only recognizes the antigen for which it is specific, or to refer to an antibody that has a significantly higher binding affinity for the antigen for which it is specific (e.g., alpha- synuclein), compared with binding to antigens to which it is non-specific (gamma and beta synucleins), for example, at least a higher binding affinity value of 5, 6, 7, 8, 9, 10 times.

The antibodies of the present invention can be obtained by any conventional method. Alpha-synuclein polypeptide/protein, including fusion proteins, cells expressing (recombinantly or naturally) the polypeptide can be used to produce antibodies that specifically recognize alpha-synuclein. The polypeptide may be a mature polypeptide or a biologically active fragment or derivative thereof.

In one embodiment, the polypeptide (ie, antigen) is a human alpha-synuclein monomer or fragment thereof, preferably produced as described in the examples below.

The polypeptides that are used to immunize the host may be prepared by well-known methods from genetically engineered host cells, including expression systems, or they may be derived from natural biological sources. In the present invention, the term "polypeptides" includes peptides, polypeptides and proteins. They are used interchangeably unless otherwise noted. The alpha-synuclein polypeptide or fragment thereof may, in some cases, be part of a larger protein, such as a fusion protein, eg, fused to an affinity marker or the like.

Antibodies generated against the alpha-synuclein polypeptide can be obtained, in cases where immunization of the animal is needed, by administering the polypeptides to the animal, preferably a lower animal, using well-known and accepted protocols, see for example the Handbook of Experimental Immunology, D.M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, cows, camels, or pigs. However, mice, rabbits, pigs and rats are usually the most suitable for this purpose.

Monoclonal antibodies can be prepared by any known method such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, hybridoma technology using human B cells (Kozbor et al., 1983, Immunology Today , 4:72) and Epstein-Barr virus hybridoma technology (Cole et al., Monoclonal antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention can also be generated using methods for producing antibodies from single lymphocytes by cloning and expressing immunoglobulin variable region cDNA generated from single lymphocytes selected to produce specific antibodies, for example, the methods described in Babcook, J. et al. ., 1996, Proc. Natl. Acad. sci. USA 93(15):7843-7848l; in patent documents WO92/02551; WO2004/051268 and WO2004/106377.

Antibody screening can be performed by measuring alpha-synuclein binding and/or by measuring inhibition of alpha-synuclein during fibril formation in the presence of an antibody or fragment thereof.

An antibody or antigen-binding fragment thereof of the present invention includes hypervariable regions (CDRs), three from the heavy chain and three from the light chain. Typically, hypervariable regions are found in a framework region and together form a variable region. By convention, the hypervariable regions (CDRs) in the heavy chain variable regions of an antibody or antigen-binding fragment thereof are referred to as CDR-H1, CDR-H2, and CDR-H3, and in the light chain variable regions as CDR-L1, CDR-L2, and CDR-L3. . They are numbered sequentially from the N-terminus to the C-terminus of each strand.

Hypervariable regions (CDRs) are numbered by convention according to the system proposed by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al. (supra)"). This numbering system is used in the present invention unless a different numbering system is specified.

The designation of residues according to the Kabat system does not always correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or more amino acids than strictly following the Kabat numbering system, due to the shortening of the structural component or the insertion into the structural component, whether it be a framework region or a hypervariable region (CDR), of the basic structure of the variable domain. The correct Kabat residue numbering can be determined for a given antibody by using a procedure to align homology residues in the antibody sequence with a "standard" Kabat numbered sequence.

The heavy chain variable domain hypervariable regions (CDRs) are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia, C. and Lesk, A.M. J. Mol. Biol., 196, 901-917 (1987), the CDR-H1 equivalent loop extends from residue 26 to residue 32. Therefore, unless otherwise indicated, "CDR-H1" used in the invention is assumed to be refers to residues 26-35, described by using a combination of the Kabat numbering system and the topological definition of the loop according to the Chothia numbering system.

The light chain variable domain hypervariable regions (CDRs) are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system.

In one embodiment, the antibody may be a chimeric, humanized or human antibody or fragment thereof.

In one embodiment, the antibody or antigen-binding fragment of the present invention may include framework regions of the animal in which the antibody was induced. For example, if an antibody is induced in a rat, it will include the hypervariable regions (CDRs) as defined and claimed in the invention and the framework regions of a rat antibody, such as an antibody or antigen-binding fragment thereof, comprising the light chain variable region corresponding to SEQ ID NO: 9 ( the nucleotide sequence of which is shown in SEQ ID NO: 11) and the heavy chain variable region corresponding to SEQ ID NO: 10 (the nucleotide sequence of which is shown in SEQ ID NO: 12).

Chimeric antibodies are usually produced using recombinant DNA techniques. The DNA can be modified by replacing the coding sequence of the H and L constant regions of a lower animal (eg, rodent) with the corresponding coding sequence of the L and H chains of a human (Morrison; PNAS 81, 6851 (1984)).

Human antibodies include heavy or light chain variable regions or full-length heavy or light chains that are "the product of" or "derived from" a particular germline sequence, if the antibody variable regions or full-length chains are derived from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest, or screening a human immunoglobulin gene library by phage display using an antigen of interest. A human antibody or fragment thereof that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting a human germline immunoglobulin sequence that closest in terms of sequence (ie, has the highest % identity) to the sequence of a human antibody. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to natural somatic mutations or the deliberate introduction of a site-directed mutation. However, the selected human antibody is typically 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequences of other mammalian species (e.g., sequences mouse germ line). In certain cases, a human antibody can be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, in its amino acid sequence, 97%, 98% or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will show no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In specific instances, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

Human antibodies can be produced using a variety of methods known to those skilled in the art. Human antibodies can be generated by hybridoma technology using a human myeloma cell line or a mouse-human heteromyeloma cell line (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, each using human variable region gene repertoires (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:1 1- 23).

In one preferred embodiment of the invention, the antibody or antigen-binding fragment of the invention is humanized.

As used herein, the term "humanized antibody molecule" refers to an antibody molecule wherein the heavy and/or light chain contains one or more hypervariable regions (CDRs) (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a non-human antibody, such as a mouse or rabbit monoclonal antibody) grafted into the heavy and/or light chain variable region framework of an acceptor antibody (eg, a human antibody). An overview can be found in Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment, not the entire CDR is transferred, but only one or more of the specificity-determining residues from any one of the CDRs described in the invention above. transferred to a human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity-determining residues from one or more of the CDRs described above are transferred to a human antibody framework. In another embodiment, only the specificity-determining residues from each of the CDRs described above are transferred to a human antibody framework.

When grafting hypervariable regions (CDRs), any suitable variable region acceptor framework sequence may be used, taking into account the class/type of donor antibody from which the hypervariable regions (CDRs) are derived, including murine, primate, and human framework regions.

Accordingly, a humanized antibody of the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the hypervariable regions (CDRs) specifically provided by the invention. Therefore, in one embodiment, a blocking humanized antibody is provided that binds alpha synuclein, preferably human alpha synuclein, wherein the variable domain includes human acceptor framework regions and non-human donor hypervariable regions (CDRs).

Examples of human scaffolds that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences can be used; they are available at http://www.imgt.org/.

In a humanized antibody or antigen-binding fragment thereof of the present invention, the acceptor heavy and light chains need not be derived from the same antibody and may, if desired, include multiple chains having framework regions derived from different chains.

A suitable framework region for the light chain of a humanized antibody of the present invention is derived from the human IGKV1-39 germline gene having SEQ ID NO:29 and the nucleotide sequence of which is shown in SEQ ID NO:31.

Accordingly, in one embodiment, a humanized antibody or antigen-binding fragment thereof is provided, comprising the sequence shown in SEQ ID NO: 1 for CDR-L1, the sequence shown in SEQ ID NO: 2 for CDR-L2, and the sequence shown in SEQ ID NO: 2 for CDR-L2, and in SEQ ID NO: 3 or SEQ ID NO: 7, for CDRL3, wherein the light chain framework region is derived from the human IGKV1-39 germline gene.

A suitable framework region for the heavy chain of a humanized antibody or antigen-binding fragment thereof of the present invention is derived from the IGHV3-15 germline gene having the sequence shown in SEQ ID NO: 30 and the nucleotide sequence of which is shown in SEQ ID NO: 32.

In one embodiment, a humanized antibody or antigen-binding fragment thereof is provided, comprising the sequence shown in SEQ ID NO: 4 for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2, and the sequence shown in SEQ ID NO: 5 for CDR-H2. ID NO: 6, for CDR-H3, wherein the heavy chain framework region is derived from the IGHV3-15 germline gene.

In another embodiment, a humanized antibody, or antigen-binding fragment thereof, is provided, comprising:

a light chain variable region comprising a CDR-L1 corresponding to the sequence shown in SEQ ID NO: 1, a CDR-L2 corresponding to the sequence shown in SEQ ID NO: 2, and a CDR-L3 corresponding to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7, wherein the light chain framework is derived from the human IGKV1-39 germline gene, and

a heavy chain variable region comprising a CDR-H1 corresponding to the sequence shown in SEQ ID NO: 4, a CDR-H2 corresponding to the sequence shown in SEQ ID NO: 5, and a CDR-H3 corresponding to the sequence shown in SEQ ID NO: 6 wherein the heavy chain framework is derived from the IGHV3-15 germline gene.

In a humanized antibody or antigen-binding fragment thereof of the present invention, the framework regions may not share the same sequence as the acceptor antibody sequences. For example, atypical residues can be replaced with more common residues for the class or type of acceptor chain. Alternatively, selected residues in the acceptor framework regions can be changed to match residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in acceptor frameworks that can be modified is described in patent document WO91/09967.

Thus, in one embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 residues in the framework are replaced with an alternative amino acid residue.

Accordingly, in one embodiment, a humanized antibody or antigen-binding fragment thereof is provided, wherein the residue at position 71 (Phe (F) 71) of the light chain variable domain (corresponding to SEQ ID NO: 17 or 18) is a donor residue (Tyr (Y) 71), see for example the sequence shown in SEQ ID NOs: 13 and 14.

In another embodiment, a humanized antibody or antigen-binding fragment thereof is provided, wherein the residues at each of positions 49 and 100 (Gly (G) 49 and Thr (T) 100) corresponding to SEQ ID NO: 30 of the heavy chain variable domain are donor residues (Ala 49 and Ala 100), see for example the sequence shown in SEQ ID NOS: 25 and 26.

In one embodiment, the invention provides an antibody, or an antigen-binding fragment thereof, comprising

1. a light chain variable region corresponding to SEQ ID NO: 13 and a heavy chain variable region selected from SEQ ID NO: 25; or

2. a light chain variable region corresponding to SEQ ID NO: 17 and a heavy chain variable region selected from SEQ ID NO: 25; or

3. a light chain variable region corresponding to SEQ ID NO: 21 and a heavy chain variable region selected from SEQ ID NO: 25.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof, comprising a sequence that is 80% similar or identical to the sequence disclosed in the invention, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99%, relative to part or all of the corresponding sequence, eg, a variable domain sequence, a CDR sequence, or a variable domain sequence, excluding the CDR. In one embodiment, the corresponding sequence is SEQ ID NO: 25. In one embodiment, the corresponding sequence is SEQ ID NO: 13, SEQ ID NO: 17, or SEQ ID NO: 21.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds human alpha synuclein, comprising a light chain, wherein the light chain variable domain comprises a sequence having at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identity or similarity to the sequence shown in SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21, and/or wherein the heavy chain variable domain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99 % identity or similarity to the sequence shown in SEQ ID NO:25.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds human alpha synuclein, wherein the antibody or antigen-binding fragment thereof has a light chain variable domain that is at least 90%, 91%, 92% 93%, 94%, 95% 96%, 97%, 98% or 99% similar or identical to the sequence shown in SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21, but where the antibody or its antigen-binding the fragment has the sequence shown in SEQ ID NO: 1 for CDR-L1, the sequence shown in SEQ ID NO: 2 for CDR-L2, and the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7 for CDR-L3.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds human alpha synuclein, wherein the antibody or antigen-binding fragment thereof has a heavy chain variable domain that is at least 90%, 91%, 92% 93%, 94%, 95% 96%, 97%, 98%, or 99% similar or identical to the sequence shown in SEQ ID NO: 25, but where the antibody or antigen-binding fragment has the sequence shown in SEQ ID NO: 4, for CDR-H1, the sequence shown in SEQ ID NO: 5 for CDR-H2, and the sequence shown in SEQ ID NO: 6 for CDR-H3.

Used in the invention, the term "identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical for these sequences. Used in the invention, the term "similarity" indicates that at any particular position in aligned sequences, the amino acid residue is similar in type to these sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that can often substitute for each other include, but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

- lysine, arginine and histidine (amino acids having side chains with basic properties);

- aspartate and glutamate (amino acids having side chains with acidic properties);

- asparagine and glutamine (amino acids having amide side chains); And

- cysteine and methionine (amino acids with sulfur-containing side chains). The degree of identity and similarity can be easily calculated (see Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York , 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, the BLAST™ software available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215: 403-410 Gish, W. & States, D. J. 1993, Nature Genet 3:266-272 Madden, T. L. et al., 1996, Meth Enzymol 266:131-141 Altschul, S. F. et al., 1997 , Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L. 1997, Genome Res. 7:649-656,).

In one embodiment, the antigen binding fragment may be, but is not limited to, Fab, Fab modified, Fab', Fab' modified, F(ab')2, Fv, single domain antibodies (e.g., VH or VL or VHH), scFv , dsscFv, bi-, tri-, or tetravalent antibodies, Bis-scFv, diabodies, tribodies, tetrabodies, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126- 1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and obtaining these antibody fragments are well known (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in patent documents WO2005/003169, WO2005/003170 and WO2005/003171. Polyvalent antibodies may include polyspecificities, such as bispecificity, or may be monospecific (see, for example, patent documents WO 92/22853, WO05/113605, WO2009/040562 and WO2010/035012).

An alternative antigen binding fragment comprises a Fab bound to two scFvs or dsscFvs, whereby each scFv or dsscFv binds to the same or different target (e.g., one scFv or dsscFv binds to a therapeutic target and one scFv or dsscFv increases half-life by binding, e.g. , albumin). Such antibody fragments are described in International Patent Application Publication No, WO2015/197772, the entire content of which is incorporated into the present invention by reference and, in particular, when discussing antibody fragments.

The constant region domains of an antibody molecule of the present invention, if any, may be selected based on their intended function of the antibody molecule, and in particular on the effector functions that may be desired. For example, constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, human IgG constant region domains, in particular IgG1 and IgG3 isotypes, can be used when the antibody molecule is intended to be used for therapeutic purposes and effector functions of the antibody are required. Alternatively, the IgG2 and IgG4 isotypes can be used when the antibody molecule is intended to be used for therapeutic purposes and the effector functions of the antibody are not required. It should be appreciated that sequence variations of these constant region domains may also be used. For example, IgG4 molecules in which the serine at position 241 has been replaced by a proline can be used, as described in Angal et al., Molecular Immunology, 1993, 30(1), 105-108. It is also obvious to any person skilled in the art that antibodies can be subject to various post-translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the heavy chain of the antibody may be absent.

In one embodiment, the C-terminal amino acid of the antibody is cleaved during post-translational modifications.

In one embodiment, the N-terminal amino acid of the antibody is cleaved during post-translational modifications.

In another embodiment, an antibody of the present invention may comprise a complete antibody having full length heavy and light chains, or an antigen-binding fragment thereof. For example, the full length antibody is selected from IgG1, IgG4, or IgG4P.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises:

1. light chain according to SEQ ID NO: 14 and heavy chain according to SEQ ID NO: 26; or

2. light chain according to SEQ ID NO: 18 and heavy chain according to SEQ ID NO: 26; or

3. light chain according to SEQ ID NO: 22 and heavy chain according to SEQ ID NO: 26.

In one embodiment, an antibody of the present invention is provided in the form of an alpha-synuclein-binding recombinant antibody protein that comprises an immunoglobulin fragment, e.g., a Fab or Fab' fragment, and one or two single-domain antibodies (dAbs) linked directly or indirectly to it, e.g. , described in patent documents WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492 and WO2011/086091, the contents of which are incorporated into the present invention by reference.

In one embodiment, the recombinant protein comprises dual-domain antibodies, eg, in the form of a resulting heavy chain variable domain (VH) and light chain variable domain (VL) pair, optionally linked via a disulfide bond.

In one embodiment, the Fab or Fab' element of the recombinant protein has the same or similar specificity for the single domain antibody or antibodies. In one embodiment, the Fab or Fab' have different specificity for a single domain antibody or antibodies, ie the recombinant protein is multivalent. In one embodiment, the polyvalent recombinant protein of the present invention has an albumin-binding site, for example, the VH/VL pair present therein provides an albumin-binding site.

In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region corresponding to SEQ ID NO: 13 or 17 or 21 and a heavy chain variable region selected from SEQ ID NO: 25. For example, the antibody is a full length IgG4 antibody, comprising a light chain variable region corresponding to SEQ ID NO: 13 or 17 or 21 and a heavy chain variable region selected from SEQ ID NO: 25. In another example, the antibody is a full length IgG4 antibody comprising a light chain variable region corresponding to SEQ ID NO: 14, 18, or 22, and a heavy chain corresponding to SEQ ID NO: 26. In yet another example, the antigen binding fragment is a Fab' comprising a light chain variable region corresponding to SEQ ID NO: 13, 17, or 21, and a heavy chain variable region selected from SEQ ID NO: 25.

In one preferred embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for use in therapy is an antibody or antigen-binding fragment thereof, comprising:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody, or antigen-binding fragment thereof, binds to alpha-synuclein with an epitope of at least D119, N122, and Y125 in SEQ ID NO: 8.

Even more preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein at a dissociation constant (K _D ) value of at least 10 times higher for monomeric alpha-synuclein compared to alpha-synuclein. synuclein in fibrils.

Biological molecules, such as antibodies or fragments, contain acidic and/or basic functional groups, resulting in a net positive or negative charge on the molecule. The value of the total "recorded" charge will depend on the absolute amino acid sequence of the molecule, the local environment of charged groups in the three-dimensional structure, and the conditions surrounding the molecule. The isoelectric point (pI) is the pH value at which a particular molecule or its solvent-accessible surface carries no net electrical charge. In one example, an anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention can be engineered to have an appropriate isoelectric point. This allows the creation of antibodies and/or fragments with more stable properties, in particular with suitable solubility and/or stability profile and/or improved purification characteristics.

Accordingly, in one aspect, the invention provides a humanized anti-alpha-synuclein antibody, or antigen-binding fragment thereof, designed to have a different isoelectric point than the isoelectric point of the antibody originally identified. An antibody may, for example, be constructed by substituting an amino acid residue, eg, substituting an amino acid residue with acidic properties, with one or more amino acid residues with basic properties. Alternatively, basic amino acid residues may be introduced, or acidic amino acid residues may be removed. Alternatively, if the molecule has an unacceptably high pI value, then, if necessary, acid residues can be introduced to reduce the pI value. It is important that when manipulating the pI value, particular attention is paid to maintaining the desired activity of the antibody or fragment. Thus, in one embodiment, the engineered antibody or antigen-binding fragment thereof has the same or substantially the same activity as the "unmodified" antibody or fragment.

Programs such as ** ExPASY http://www.expasy.ch/tools/pi_tool.html and http://www.iut-arles.up.univ-mrs can be used to predict the isoelectric point of an antibody or fragment. fr/w3bb/d_abim/compo-p.html.

It should be borne in mind that the affinity of the antibodies of the present invention can be changed by using a suitable known method. Therefore, the present invention also relates to variants of the antibody molecules of the present invention which have improved affinity for alpha-synuclein, in particular human alpha-synuclein. Such variants can be generated using a variety of affinity maturation protocols, including hypervariable region (CDR) mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand shuffling (Marks et al. ., Bio/Technology, 10, 779-783, 1992), the use of E. coli mutator strains (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al. , Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexually mimic PCR (Crameri et al. ., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses these "affinity maturation" methods.

Optionally, the antibody or antigen-binding fragment thereof of the present invention may be conjugated to one or more effector molecule(s). It is to be understood that an effector molecule may include a single effector molecule or two or more such molecules linked to form a single fragment that can be attached to the antibodies or antigen-binding fragment thereof of the present invention. When it is desired to obtain an antibody fragment bound to an effector molecule, it can be prepared using standard chemical or recombinant DNA techniques in which the antibody fragment is linked, either directly or via a binding agent, to the effector molecule. Methods for conjugating such effector molecules to antibodies are well known (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical techniques include, for example, those described in patent documents WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 03/031581. Alternatively, when the effector molecule is a protein or polypeptide, the binding may be accomplished using recombinant DNA techniques, such as those described in patent documents WO 86/01533 and EP0392745.

As used herein, the term "effector molecule" includes, for example, antitumor agents, drugs, toxins, biologically active proteins, such as enzymes, other antibody or antibody fragments, synthetic or natural polymers, nucleic acids and their fragments, such as DNA, RNA and their fragments, radionuclides, specific radioiodide, radioisotopes, metal chelates, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or EPR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is detrimental to cells (eg, leads to cell death). Examples include combrestatins, dolastatins, epothilones, staurosporine, maytansinoids, spongistatins, risoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxor ubicin, daunorubicin , dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof.

Effector molecules also include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannite, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (eg, dactinomycin ( formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins) and antimitotic agents (eg vincristine and vinblastine).

Other effector molecules may include chelated radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , bismuth ²¹³ , californium ²⁵² , iridium ¹⁹² and tungsten ¹⁸⁸ /rhenium ¹⁸⁸ ; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin or diphtheria toxin, protein such as insulin, tumor necrosis factor, interferon α, interferon β, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, antithrombotic agent or antiangiogenic agent such as angiostatin or endostatin, or biological response modifier such as lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2) , granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins.

Other effector molecules may include detectable substances used, for example, in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron active metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See in general terms US Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescine dichlorotriazinylamine, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; and suitable radioactive nuclides include ¹²⁵ I, ¹³¹ I, ¹¹¹ In and ⁹⁹ Tc.

In another example, an effector molecule can increase the in vivo half-life of an antibody and/or reduce the immunogenicity of an antibody and/or enhance delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds such as those described in WO05/117984.

If the effector molecule is a polymer, this polymer may in most cases be a synthetic or natural polymer, for example an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, for example a homo- or hetero-polysaccharide.

Specific optional substituents that may be present on the synthetic polymers mentioned above include one or more hydroxyl, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted linear or branched polyethylene glycol, polypropylene glycol, polyvinyl alcohol or derivatives thereof, in particular optionally substituted polyethylene glycol such as methoxy polyethylene glycol or derivatives thereof.

Specific natural polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

In one embodiment, the polymer is a collection of albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

The term "derivatives" as used herein is intended to include reactive derivatives, for example thiol selective reactive groups such as maleimides and the like. The reactive group may be linked directly or via a linker segment to the polymer. It will be appreciated that the remainder of such a group will in some cases form part of the product in the form of a linking group between the antibody fragment and the polymer.

If desired, the size of the polymer can be changed, but, as a rule, it is in the range of average molecular weights from 500 Da to 50,000 Da, for example, from 5000 to 40,000 Da, for example, from 20,000 to 40,000 Da. The size of the polymer may in particular be selected based on the intended use of the product, for example, based on the ability to localize to specific tissues such as tumors, or based on the ability to increase the half-life from the bloodstream (for more information, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, if a product is intended to be removed from the bloodstream for tissue penetration, eg for use in tumor treatment, it would be preferable to use a low molecular weight polymer, eg, about 5000 Da molecular weight. In applications where the product remains in the blood stream, it may be preferable to use a higher molecular weight polymer, for example, in the range of 20,000 Da to 40,000 Da.

Suitable polymers include a polyalkylene polymer such as polyethylene glycol, or in particular methoxy polyethylene glycol or a derivative thereof, and in particular with a molecular weight in the range of from about 15,000 Da to about 40,000 Da.

In one example, antibodies for use in the present invention are attached to fragments of polyethylene glycol (PEG). In one specific example, an antibody is an antibody fragment, and the PEG molecules can be attached through any available side chain or terminal amino acid functional group located in the antibody fragment, such as through any free amino group, imino group, thiol group, hydroxyl group, or carboxyl group. group. Such amino acids in an antibody fragment may be naturally occurring, or they may be engineered in the fragment using recombinant DNA techniques (see, for example, US 5219996; US 5667425; WO98/25971, WO2008/038024). In one example, an antibody molecule of the present invention is a modified Fab fragment, where the modification consists of adding one or more amino acid fragments to the C-terminal end of the heavy chain to allow attachment of an effector molecule. Accordingly, the added amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can be attached. Multiple sites may be used to attach two or more PEG molecules.

Accordingly, the PEG molecules are covalently linked through the thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulfur atom of the cysteine residue located in the fragment. The covalent bond will typically be a disulfide bond, or in particular a sulfur-carbon bond. When a thiol group is used as the point of attachment for suitably activated effector molecules, for example, thiol-selective derivatives such as maleimides and cysteine derivatives can be used. In the preparation of the polymer-modified antibody fragments described above, an activated polymer can be used as a starting material. The activated polymer may be any polymer containing a reactive thiol group such as an α-halocarboxylic acid or an ester, eg iodoacetamide, an imide, eg maleimide, vinyl sulfone or disulfide. Such starting materials are commercially available (eg, Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be prepared from commercially available starting materials using standard chemical techniques. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

In one embodiment, the antibody is a modified Fab fragment, Fab' or diFab fragment that is pegylated, that is, has PEG (polyethylene glycol) covalently attached to it, for example, by using the method disclosed in patent documents EP 0948544 or EP1090037 [ see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, PEG is attached to cysteine at the hinge region. In one example, a PEG-modified Fab fragment has a maleimide group covalently linked to one thiol group in the modified hinge region. The lysine moiety can be covalently bonded to the maleimide group, and a methoxypolyethylene glycol polymer having a molecular weight of about 20,000 Da can be attached to each of the amino groups on the lysine moiety. The total molecular weight of the PEG attached to the Fab fragment may therefore be approximately 40,000 Da.

Specific PEG molecules include 2-[3-(N-maleimido)propionamido]ethylamide N,N'-bismethoxypolyethylene glycol (MW 20000) modified lysine, also known as PEG2MAL40K (available from Nektar, formerly Shearwater).

Alternative manufacturers of PEG linkers include NOF, which sells them under the brand name GL2-400MA3 (where m in the structure shown below is 5) and under the brand name GL2-400MA (where m is 2) and n is approximately 450:

.

.

That is, the molecular weight of each PEG is approximately 20,000 Da.

Thus, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxo-hexyl)amino]propyloxy}hexane (PEG with two branches, - CH ₂ )3NHCO(CH ₂ ) ₅ -MAL, Mw 40000, known as SUNBRIGHT GL2-400MA3.

In addition, alternative PEG effector molecules of the following type

produced by Dr Reddy, NOF and Jenkem.

In one embodiment, the Fab or Fab' of the present invention is conjugated to a PEG molecule.

In one embodiment, an antibody is provided that is pegylated (e.g., with a PEG as described herein), fused via a cysteine amino acid residue at or near amino acid 226 of the chain, e.g., amino acid 226 of the heavy chain (denoted using sequential numbering), for example, at amino acid 224 of SEQ ID NO:26.

In one embodiment, the present invention provides a Fab'PEG molecule comprising one or more PEG polymers, for example 1 or 2 polymers such as a 40 kD polymer or polymers.

The Fab'-PEG molecules of the present invention may be particularly preferred if they have a half-life independent of the Fc moiety. In one embodiment, a Fab' is provided conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule. In one embodiment, a scFv conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule is provided. In one embodiment, the Fab or Fab' of the present invention is conjugated to human serum albumin. In one embodiment, the antibody or fragment is conjugated to a starch molecule, for example, to increase the half-life. Methods for conjugating starch to protein are described in US Pat. No. 8,017,739, the contents of which are incorporated herein by reference.

The present invention also provides an isolated polynucleotide encoding an antibody or antigen-binding fragment of the present invention. An isolated polynucleotide of the present invention may include synthetic DNA, for example, produced by chemical processing, cDNA, genomic DNA, or any combination thereof.

Standard molecular biology techniques can be used to prepare DNA sequences encoding an antibody or antigen-binding fragment thereof of the present invention. The desired DNA sequences can be synthesized in whole or in part using oligonucleotide synthesis techniques. Where appropriate, site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used.

In one embodiment, an isolated polynucleotide of the invention encodes:

a. the variable region of the light chain, where the polynucleotide:

i. at least 90% identical to the sequence of SEQ ID NO: 15 or SEQ ID NO: 19 or SEQ ID NO: 23; or

ii. includes SEQ ID NO: 15 or SEQ ID NO: 19 or SEQ ID NO: 23; or

iii. consists mainly of SEQ ID NO: 15 or SEQ ID NO: 19 or SEQ ID NO: 23;

b. heavy chain variable region, where the polynucleotide:

i. at least 90% identical to the sequence of SEQ ID NO: 27; or

ii. includes SEQ ID NO: 27; or

iii. consists mainly of SEQ ID NO: 27;

c. light chain, where the polynucleotide:

i. at least 90% identical to the sequence of SEQ ID NO: 16 or SEQ ID NO: 20 or SEQ ID NO: 24; or

ii. includes SEQ ID NO: 16 or SEQ ID NO: 20 or SEQ ID NO: 24; or

iii. consists mainly of SEQ ID NO: 16 or SEQ ID NO: 20 or SEQ ID NO: 24;

d. heavy chain, where the polynucleotide:

i. at least 90% identical to the sequence of SEQ ID NO: 28; or

ii. includes SEQ ID NO: 28; or

iii. consists essentially of SEQ ID NO: 28.

The present invention also provides a cloning or expression vector comprising one or more of the polynucleotides described herein. In one example, a cloning or expression vector of the present invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NOs: 15, 16, 19, 20, 23, 24, 27, or 28.

General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art. Detailed information on these methods is provided in “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Further provided is a host cell comprising one or more isolated polynucleotide sequences of the invention, or one or more cloning or expression vectors comprising one or more isolated polynucleotide sequences encoding an antibody of the present invention. Any suitable host cell/vector system can be used to express the polynucleotide sequences encoding the antibody or antigen-binding fragment of the present invention. Bacterial, eg, E. coli, and other microorganism systems may be used, or expression systems based on eukaryotic host cells, eg, mammalian host cells, may also be used. Suitable mammalian host cells include Chinese hamster ovary (CHO) cells, myeloma cells, or hybridoma cells.

Suitable Chinese Hamster Ovary (CHO) cell types for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with selectable a DHFR marker, or CHOK1-SV cells, which can be used with a selectable glutamine synthetase marker. Other cell types useful in antibody expression include lymphocytic cell lines such as NSO myeloma cells and SP2 cells, COS cells. The host cell can be stably transformed or transfected with the isolated polynucleotide sequences or expression vectors of the present invention.

In one embodiment, the host cell of the present invention is a CHO-DG44 cell stably transfected with an expression vector comprising the isolated polynucleotide sequences of the present invention, preferably comprising the isolated polynucleotide sequences corresponding to SEQ ID NOs: 15 and 27, or SEQ ID NOs: 19 and 27 or SEQ ID NOs: 23 and 27 or SEQ ID NOs: 16 and 28 or SEQ ID NOs: 20 and 28 or SEQ ID NOs: 24 and 28.

The present invention also provides a method for producing an antibody or antigen-binding fragment of the present invention, comprising culturing a host cell of the present invention under conditions suitable for producing an antibody or antigen-binding fragment of the invention, and isolating the antibody or antigen-binding fragment thereof.

The antibody, or antigen-binding fragment thereof, may comprise only the heavy or light chain polypeptide, in which case only the heavy or light chain coding sequence is required for transfection of the host cells. For the production of antibodies or antigen-binding fragments thereof, comprising both heavy chains and light chains, the cell line can be transfected with two vectors, the first vector encoding the light polypeptide chain and the second vector encoding the heavy polypeptide chain. Alternatively, only one vector may be used, which includes sequences encoding light and heavy polypeptide chains.

Thus, a method is provided for culturing a host cell and expressing an antibody or fragment thereof, isolating the antibody or fragment and optionally purifying them to obtain an isolated antibody or fragment. In one embodiment, the method further comprises the step of conjugating the effector molecule to the isolated antibody or fragment, eg, conjugating to a PEG polymer, particularly as described herein.

Thus, in one embodiment, a purified anti-alpha-synuclein antibody or fragment thereof is provided, e.g., a humanized antibody or fragment thereof, in particular an antibody or fragment thereof of the invention, in a substantially purified form, in particular free or substantially free of endotoxin. and/or host cell protein or DNA.

Generally, the expression "substantially free of endotoxin" is intended to refer to an endotoxin content of 1 endotoxin unit per 1 milligram of antibody produced or less, such as 0.5 or 0.1 endotoxin unit per 1 milligram of product. Generally, the phrase "substantially free of host cell protein or DNA" is intended to refer to a host cell protein or DNA content of 400 μg per milligram of antibody produced or less, as appropriate, e.g., 100 μg per milligram or less, in particular 20 micrograms in 1 milligram.

Since the antibodies of the present invention are useful in the treatment, diagnosis and/or prevention of a pathological condition such as alpha synucleinopathy, the present invention also provides a pharmaceutical or diagnostic composition comprising an antibody or antigen-binding fragment of the present invention in combination with one or more of a pharmaceutically acceptable carrier, excipient or diluent. In one embodiment, the antibody or antigen-binding fragment of the present invention is the only active ingredient. In another embodiment, the antibody or antigen-binding fragment of the present invention is present in combination with one or more additional active ingredients. Alternatively, pharmaceutical compositions include an antibody or antigen-binding fragment of the present invention, which is the only active ingredient, and it can be administered individually to the patient in combination (for example, simultaneously, sequentially or separately) with other agents, drugs or hormones.

In another embodiment, the pharmaceutical composition comprises an antibody or antigen-binding fragment thereof comprising the light chain variable region of SEQ ID NO: 13 or 17 or 21 and containing the heavy chain variable region of SEQ ID NO: 25, e.g., SEQ ID NO: 13 and SEQ ID NO: 25, or SEQ ID NO: 17 and SEQ ID NO: 25, or SEQ ID NO: 21 and SEQ ID NO: 25.

Pharmaceutical compositions of the invention may be appropriately administered to a patient to determine the required therapeutically effective amount. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent necessary to treat, alleviate, or prevent a particular disease or condition, or to exert a detectable therapeutic or prophylactic effect. For any antibody, a therapeutically effective amount can be assessed initially by conducting studies either in cell culture or in animal models, typically rodents, rabbits, dogs, pigs, or primates. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine appropriate dosages and routes of administration for humans.

The precise therapeutically effective amount in a human will depend on the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), drug response sensitivity, and tolerance/response. for ongoing therapy. This amount can be determined by routine experimentation and is determined by the attending physician. Typically, a therapeutically effective amount will be 0.01 mg/kg to 500 mg/kg, eg 0.1 mg/kg to 200 mg/kg, eg 100 mg/kg. Pharmaceutical compositions are conveniently formulated in single dose dosage forms containing a predetermined amount of the active agent of the invention in a single dose.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, adjuvants such as wetting agents or emulsifiers or buffering agents may be present in such compositions to maintain the desired pH. Such carriers make it possible to prepare pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, pastes and suspensions for ingestion by a patient.

Suitable administration forms include those suitable for parenteral administration, eg by injection or infusion, eg by bolus injection or continuous infusion, intravenous, inhalation or subcutaneous form. When the product is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous medium and may contain adjuvants such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment of the invention may be prepared in a dry form for dissolution in an appropriate sterile liquid prior to use. Solid forms suitable for dissolution or suspension in liquid media prior to injection may also be prepared.

Once prepared, the compositions of the invention may be administered directly to the subject. Accordingly, the invention provides the use of an antibody or antigen-binding fragment of the invention for the manufacture of a medicament.

The subjects to be treated may be animals. Preferably, the pharmaceutical compositions of the present invention are adapted for administration to humans.

In this regard, in another aspect, the present invention provides an antibody or antigen-binding fragment or a pharmaceutical composition comprising the antibody or antigen-binding fragment, for use in therapy, and in particular for use in the treatment of one or more alpha synucleinopathies. In yet another aspect, the present invention provides a method of treating one or more synucleinopathies in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment of the present invention or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for use in therapy is an antibody or antigen-binding fragment thereof, comprising:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a light chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of SEQ ID NO: 8 and wherein the antibody or antigen-binding fragment thereof is for use in therapy. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

In another embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is for use in the treatment of one or more synucleinopathies, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of SEQ ID NO: 8. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha. -synuclein in fibrils.

In another embodiment, a method is provided for treating one or more synucleinopathies in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of SEQ ID NO: 8. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha. -synuclein in fibrils.

The alpha synucleinopathies of the present invention include, but are not limited to, Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD), a variant of Alzheimer's disease with Lewy bodies (LBVAD), mixed type of Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1). Preferably, the alpha synucleinopathy is Parkinson's disease (PD).

In another embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is for use in the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), dementia with Lewy bodies (DLB) , diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBVAD), mixed Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1), preferably, Parkinson's disease (PD), where the antibody or antigen-binding fragment includes:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein optionally the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of the SEQ sequence ID NO: 8, and where the antibody or antigen-binding fragment is intended for use in therapy. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

In another embodiment, a method is provided for the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBVAD) , mixed type of Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1), preferably Parkinson's disease (PD), in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof or a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein optionally the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of the SEQ sequence ID NO: 8, and where the antibody or antigen-binding fragment is intended for use in therapy. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

Alternatively, the invention also provides the use of an antibody or an antigen-binding fragment thereof for the manufacture of a medicament for the treatment of alpha synucleinopathy, wherein the alpha synucleinopathy is preferably Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBVAD), mixed Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with iron accumulation in the brain type 1 ( NBIA-1), more preferably Parkinson's disease (PD), wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

wherein the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, wherein optionally the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of the SEQ sequence ID NO: 8, and where the antibody or antigen-binding fragment is intended for use in therapy. Preferably, the antibody or antigen-binding fragment thereof does not cross-react with beta-synuclein and bind alpha-synuclein with a dissociation constant (K _D ) value at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

In addition, it is part of the present invention to use anti-alpha-synuclein antibodies or antigen-binding fragments thereof as diagnostic tools or in diagnostic studies, for example, for diagnosing alpha-synucleinopathies such as Parkinson's disease (PD) (including idiopathic and hereditary forms of the disease). Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBVAD), mixed Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1).

Preferably, the diagnosis can be made on biological samples. The term "biological sample" covers a range of types of samples taken from an individual that can be used in diagnostic testing or health monitoring. This definition includes cerebrospinal fluid, blood, such as plasma and serum, and other samples of biological fluids, such as urine and saliva, solid tissue samples, such as biopsy specimens, or tissue cultures or cells derived from them, and their progeny. This definition also includes samples that have been processed in some way after they are received, such as treatment with reagents, solubilization, or enrichment with specific components such as polynucleotides.

Preferably, the diagnostic study can be carried out on biological samples that are outside the human or animal body. Such a diagnostic study is also called an in vitro study. An in vitro diagnostic test may be based on an in vitro assay for detecting alpha-synuclein in a biological sample that has been taken from an individual, comprising the steps of i) contacting the biological sample with an anti-alpha-synuclein antibody of the invention, or an antigen-binding fragment thereof, and ii) detecting binding antibodies against alpha-synuclein or its antigen-binding fragment with alpha-synuclein. By comparing the detectable level of alpha synuclein or the presence of a specific post-translationally modified form of alpha synuclein with a suitable control, one or more alpha synucleinopathies can be identified. Such a detection method can accordingly be used to determine whether a subject has alpha synucleinopathy or is at risk of developing alpha synucleinopathy, including determining the stage (severity) of alpha synucleinopathy.

Therefore, the present invention provides an antibody or antigen-binding fragment thereof for use in the diagnosis of alpha synucleinopathies, preferably in the diagnosis of Parkinson's disease (PD), wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 corresponding to SEQ ID NO: 1; CDR-L2 corresponding to SEQ ID NO: 2 and CDR-L3 corresponding to SEQ ID NO: 3 or SEQ ID NO: 7; and a heavy chain variable region comprising CDR-H1 corresponding to SEQ ID NO: 4; CDR-H2 corresponding to SEQ ID NO: 5 and CDR-H3 corresponding to SEQ ID NO: 6; or

b. a light chain variable region corresponding to SEQ ID NO: 13 or SEQ ID NO: 17 or SEQ ID NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25; or

c. a light chain corresponding to SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 22, and a heavy chain corresponding to SEQ ID NO: 26;

where, optionally, the antibody or antigen-binding fragment binds alpha-synuclein to an epitope comprising at least residues D119, N122 and Y125 of SEQ ID NO: 8.

The sequences included in the present invention are shown in Table 1.

Table 1

Name SEQID NO: SUBSEQUENCE CDR-L1 1 KASQNINKNLD CDR-L2 2 YANNLQT CDR-L3 3 YQYKNGWT CDR-H1 4 GFTFNNAAMY CDR-H2 5 RIRTKPNNYATSYADSVKG CDR-H3 6 DYSRGDR CDR-L3 option 1 7 YQYKNAWT Alpha synuclein
P37840
human 8 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA Rat VL
5811 9 NIQMTQSPPVLSASVGDRVTLSCKASQNINKNLDWYQQKHGEAPKLLMYYANNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYYCYQYKNGWTFGGGTKLELK Rat VH
5811 10 EMQLVESGGGLVQPKESLKISCAASGFTFNNAAMYWVRQAPGKGLEWVARIRTKPNNYATSYADSVKGRFTISRDDSKSMVYLQMDNLKSEDTAMYYCTADYSRGDRWGQGVMVTVSS Rat VL nucleotide eleven aacatccagatgacccagtctcctccagtcctgtctgcatctgtgggagacagagtcactctcagctgcaaagcaagtcagaacattaataagaacttagactggtatcagcaaaagcatggagaagctccaaaactcctgatgtattatgcaaacaatttacaaacgggcatcccatcaaggttcagtggcagtggatctggaacagattacacgctca ccatcagcagcctgcagcctgaagatgttgccacatattactgctatcagtataagaatgggtggacgttcggtggaggcaccaagctggaactgaaa Rat VH
nucleotide 12 Gaaatgcagctggtggagtctggtggaggattggtgcagcctaaggagtcattgaaaatctcatgtgcagcctctggattcaccttcaataatgctgccatgtactgggtccgccaggctccaggaaagggtctggaatgggttgctcgcataagaactaaacctaataattatgcaacatcttatgctgattcagtgaaaggcagatt caccatctccagagatgattcaaaaagcatggtctacctacaaatggataacttgaaaagtgaggacacagccatgtattactgtacagcagattactccagaggtgacaggtggggccaaggagtcatggtcacagtctcgagc 5811 gL5
V-region 13 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCYQYKNGWTFGQGTKVEIK 5811 gL5
light chain 14 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCYQYKNGWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 5811 gL5
V-region
nucleotide 15 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactg attacaccctgaccattagctcccttcaaccggaggacttcgccacctattactgctaccagtacaagaacggctggacttttggacaaggcaccaaggtcgaaatcaag 5811 gL5
light chain
nucleotide 16 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactg attacaccctgaccattagctcccttcaaccgggaggacttcgccacctattactgctaccagtacaagaacggctggacttttggacaaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctaccccg cgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggc gagtgc 5811 gL8
V-region 17 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYKNGWTFGQGTKVEIK 5811 gL8
light chain 18 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYKNGWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 5811 gL8
V-region
nucleotide 19 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagcccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactga tttcaccctgaccattagctcccttcaaccggaggacttcgccacctattactgctaccagtacaagaacggctggacttttggacaaggcaccaaggtcgaaatcaag 5811 gL8
light chain
nucleotide 20 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagcccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactga tttcaccctgaccattagctcccttcaaccgggaggacttcgccacctattactgctaccagtacaagaacggctggacttttggacaaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctaccccc gcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccgggg cgagtgc 5811gL14
V-region 21 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYKNAWTFGQGTKVEIK 5811gL14
light chain 22 DIQMTQSPSSLSASVGDRVTITCKASQNINKNLDWYQQKPGKAPKLLIYYANNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCYQYKNAWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 5811gL14
V-region
nucleotide 23 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagcccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactga tttcaccctgaccattagctcccttcaaccgggaggacttcgccacctattactgctaccagtacaagaacgcttggacttttggacaaggcaccaaggtcgaaatcaag 5811gL14
light chain
nucleotide 24 Gacatccagatgacccagagcccgagctccctgtccgcatcagtgggggatcgcgtgactattacgtgcaaagcctcgcagaacatcaacaagaacctcgactggtatcagcagaagcccaggaaaggcgcctaagctgctgatctactacgccaacaatctccagaccggcgtgccctcgcggttctccggatctgggtccggtactga tttcaccctgaccattagctcccttcaaccgggaggacttcgccacctattactgctaccagtacaagaacgcttggacttttggacaaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctaccc ccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccgg ggcgagtgc 5811gH4
V-region 25 EVQLVESGGGLVKPGGSLRLSCAASGFTFNNAAMYWVRQAPGKGLEWVARIRTKPNNYATSYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTADYSRGDRWGQGTMVTVSS 5811gH4
heavy chain 26 EVQLVESGGGLVKPGGSLRLSCAASGFTFNNAAMYWVRQAPGKGLEWVARIRTKPNNYATSYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTADYSRGDRWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 5811gH4
V-region
nucleotide 27 Gaagtgcagcttgtggagagcggaggtggactcgtgaagcctggcggatctctgcgcctgtcctgcgccgcctcggggttcacctttaacaatgccgcaatgtattgggtcagacaggccccgggaaagggtttggaatgggtggctaggattcggactaagcccaacaactacgcgacctcctacgccgatagcgtga agggcagattcaccatctcccgggacgactcaaagaacacgctgtacctccaaatgaactccctgaaaaccgaggacaccgccgtgtactactgcaccgcggactactccggggcgatcgctggggacaggggactatggtcactgtctcgagt 5811gH4
heavy chain
nucleotide 28 gaagtgcagcttgtggagagcggaggtggactcgtgaagcctggcggatctgcgcctgtcctgcgccgcctcggggttcacctttaacaatgccgcaatgtattgggtcagacaggccccgggaaagggtttggaatgggtggctaggattcggactaagcccaacaactacgcgacctcctacgccgatagcgt gaagggcagattcaccatctcccgggacgactcaaagaacacgctgtacctccaaatgaactccctgaaaaccgaggacaccgccgtgtactactgcaccgcggactactcccggggcgatcgctggggacaggggactatggtcactgtctcgagtgcctccaccaagggcccctccgtgttccctctggccccttgctcccggtccacctccga gtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctaccctgtaacgt ggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccag ttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatctccaaggccaagggccagccccgcgagcccca ggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtccggtggcagga aggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag Human IGKV1-39 JK1 acceptor scaffold 29 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK Human IGHV3-15 JH3 acceptor scaffold thirty EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDAFDVWGQGTMVTVSS Human IGKV1-39 JK1 acceptor scaffold
nucleotide 31 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagattt cactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtaccccttggacgttcggccaagggaccaaggtggaaatcaaa Human IGHV3-15 JH3 acceptor scaffold
nucleotide 32 Gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcagcctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtggggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattca ccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagatgcttttgatgtctggggccaagggacaatggtcaccgtctcttca Rabbit Fc human 68-140 a-syn 33 GAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAVEKTVAPSTTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTI SKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK 5811 rat-mouse chimeric light chain 34 NIQMTQSPPVLSASVGDRVTLSCKASQNINKNLDWYQQKHGEAPKLLMYYANNLQTGIPSRFSGSGSGTDYTLTISSLQPEDVATYYCYQYKNGWTFGGGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHN SYTCEATHKTSTSPIVKSFNRNEC 5811 rat-mouse chimeric heavy chain 35 EMQLVESGGGLVQPKESLKISCAASGFTFNNAAMYWVRQAPGKGLEWVARIRTKPNNYATSYADSVKGRFTISRDDSKSMVYLQMDNLKSEDTAMYYCTADYSRGDRWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDTLSSSSVTVPSSTWPSETVTCN VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPI MDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 5811 rat-mouse chimeric Fab-HIS heavy chain 36 EMQLVESGGGLVQPKESLKISCAASGFTFNNAAMYWVRQAPGKGLEWVARIRTKPNNYATSYADSVKGRFTISRDDSKSMVYLQMDNLKSEDTAMYYCTADYSRGDRWGQGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDTLSSSSVTVPSSTWPSETVTCN VAHPASSTKVDKKIVPRDCHHHHHHHHHH

In the following, the invention will be further described by way of examples with reference to the embodiments illustrated in the accompanying drawings.

EXAMPLES

Example 1 Expression of alpha-synuclein monomer and human fibrils

The gene encoding human alpha synuclein was synthesized and subcloned into the pMH 10His TEV vector (containing the CMV promoter) using standard molecular biology techniques to create a vector engineered to produce alpha synuclein with the 10His-TEV N-terminal tag. The resulting vector was transfected into Expi293F cells using the Expi293TM expression system (Invitrogen) according to the manufacturer's protocols. The alpha-synuclein protein accumulated in the culture medium, from where it was recovered by immobilized metal ion affinity chromatography on a HisTrap excel column (GE Healthcare). The column was washed with 25 mM Tris-HCl, 300 mM NaCl, pH 8.0, and the protein was eluted using a stepwise gradient of 500 mM imidazole in the same buffer. The 10His label was removed using TEV protease. The sample was then concentrated and desalted, and the digested protein was reloaded onto a HisTrap excel column and the digested alpha-synuclein in the eluate was collected. Alpha-synuclein was further purified by gel filtration on a HiLoad 26/600 Superdex 75 column (GE Healthcare) and endotoxin was removed by passage through a Proteus NoEndo cartridge (Generon). The purified alpha-synuclein was confirmed to be monomeric by light scattering SEC MALS (FIG. 1A).

Wild-type human alpha-synuclein (untagged) was also expressed in Expi293F cells. The protein was extracted from the culture medium by anion exchange in a HiTrap Q column (GE Healthcare). The column was washed with 20 mM Tris-HCl, pH 8.0, and the protein was eluted using a sodium chloride gradient to 400 mM. Fractions were concentrated and desalted by passage through a HiPrep 26/10 column (GE Healthcare) and eluted with 20 mM Tris-HCl, pH 8.0. The protein was then purified on a MonoQ 10/100GL column, eluted using a gradient of sodium chloride to 400 mM in 20 mM TrisHCl, pH 8.0, and then subjected to gel filtration on a HiLoad 26/600 Superdex 75 column (GE Healthcare) eluting in PBS pH 7.4 (Figure 1B).

This unlabeled wild-type alpha-synuclein monomer was used to prepare alpha-synuclein. Fibrils were obtained by continuous stirring for 10 days of purified monomer (9-10 mg/ml in PBS pH 7.4) at 1200 rpm, 37° C. in a Vortemp56 shaker incubator (Labnet). Fibril formation was assessed by JC-1 assay (Lee et al., Biochem. J. 2009, 418, 311-323) and Fourier transform solution infrared spectroscopy. The monomer not included in fibrils in solutions was evaluated by ultracentrifugation and passage through the membrane with cutoff of molecules with a molecular weight of 100 kDa, followed by gel electrophoresis. In further studies, only fibrils with a JC-1>15 response, low soluble monomer (<5%) and an FTIR spectrum with a main absorption between 1625 and 1630 cm ^-1 were used (figure 2). The prepared fibrils were stored at -80°C.

Example 2 Immunization and Antibody Isolation

Numerous immunization strategies have been implemented using various animal species and immunogens. Antibody 5811 was obtained from a female Sprague Dawley rat (weighing > 180 g) immunized by subcutaneous injection of 50 μg of unlabeled wild-type alpha-synuclein monomer expressed as described above (SEQ ID NO: 8).

Rats were given 3 booster injections at 21 day intervals using incomplete Freund's adjuvant (IFA), bled for analysis from the tail vein 14 days after immunization. Termination occurred 14 days after the final boost, after which a suspension of individual cells of the spleen, lymph node, bone marrow and peripheral blood mononuclear cells was obtained and frozen in 10% DMSO/FCS at -80°C.

B cell culture

B cell cultures were prepared using a method similar to that described in Tickle et al., 2015. J Biomol Screen: 20 (4), 492-497. Briefly, B cells derived from lymph nodes or splenocytes of immunized animals were cultured at a density of approximately 2000-5000 cells per well in barcoded 96-well tissue cell culture plates with 200 µl/well of RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (Sigma Aldrich), 2% HEPES (Sigma Aldrich), 1% L-glutamine (Gibco BRL), 1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol (Gibco BRL) , 1% supernatant of activated human PBMCs (BSS) and X-ray irradiated EL4 mouse thymoma mutant cells (5 x 10 ⁴ /well) for seven days at 37° C. in 5% CO ₂ atmosphere. Cultures were established using B cells from all immunized animals and a total of approximately 1.7 x 10 ⁹ B cells were selected for testing.

The 5811 antibody of the present invention was obtained from activated B cells isolated from a lymph node, which were cultured at a density of approximately 2000 cells per well. The lymph node was used in addition to splenocytes for antibody detection in order to have an alternative source of B cells from which new antibodies could be selected and identified. Approximately 3.3 x 10 ⁸ cells of full-length monomeric human alpha-synuclein were harvested from immunized rats.

Primary screening

The presence of specific antibodies to human alpha-synuclein in B-cell culture supernatants was determined by homogeneous fluorescent binding assay using SuperAvidin™ microspheres (Bangs Laboratories) coated with biotinylated recombinant full-length human alpha-synuclein monomer as the target antigen source. The recombinant human alpha-synuclein described herein was biotinylated using a 3-fold molar excess of biotin. A small molar excess of biotin was used to exclude complete modification of all seven lysine residues that are in the alpha-synuclein molecule. Alpha-synuclein monomer was incubated overnight at 40° C. with biotin and free biotin was removed the next day using a Zeba™ spin-desalting column. The screening involved transferring 10 µl of supernatant from barcoded 96-well tissue culture plates to black-walled 384-well barcoded assay plates containing biotinylated recombinant human alpha-synuclein monomer immobilized on Superavidin microspheres (10 µl/ well) using an Agilent Bravo Fluid Transfer Device. Binding was detected using the Alexafluor647 conjugate (Jackson) specific for goat Fcγ fragment against rat IgG). Plates were read on a TTP Labtech Mirrorball fluorescent cytometer to identify wells containing IgG specific for alpha-synuclein.

Secondary scan

After primary screening, positive supernatants were pooled on barcoded 96-well reference plates using a pipetting robot to pipette individual Beckman Coulter BiomekNXP wells, and B cells in culture plates were frozen at -80°C. The reference plates were then screened by streptavidin capture ELISA using biotinylated recombinant human alpha synuclein monomer or biotinylated recombinant human alpha synuclein fibrils. This was done to identify wells in which both monomeric and fibrillar recombinant human alpha-synuclein were binding and to exclude any false positive wells showing off-target binding to the Superavidin™ microspheres. Given the insoluble nature of fibrils, traditional ELISA coating protocols, which are used with proteins in solution, were not suitable in this case. It was decided to use a minimal biotinylation protocol to preserve the fibrillar structure and facilitate efficient fibril coating on the streptavidin pre-coated ELISA plate.

All of the biotinylated alpha-synuclein fibrils described herein were generated by combining the biotinylated recombinant alpha-synuclein monomer (as described above) with a 50-fold excess of unlabeled recombinant alpha-synuclein in PBS. Fibril formation was confirmed by JC1 assay (Lee et al., Biochem. J. 2009, 418, 311-323).

Biotinylated monomer or biotinylated fibrils in PBS were captured on 384-well streptavidin-coated Maxisorp plates in carbonate coating buffer (dH ₂ O+0.16% Na ₂ CO ₃ +0.3% NaHCO ₃ .) The plates were blocked with 1% mass/volume PEG/PBS and then incubated with 10 μl/well of B cell culture supernatant (diluted 1:1 with blocking buffer). A secondary HRP-conjugated goat Fcγ fragment against rat IgG, a specific HRP conjugate (Jackson), was added to the plates, followed by visualization of binding with TMB substrate (3,3',5,5'-tetramethylbenzidine, from EMD Millipore; 10 μl/well ). Optical density at 630 nm was measured using a BioTek Synergy 2 microplate reader. Primary binding analysis revealed 83 bindings, and after screening by ELISA, 29 of these bindings were shown to be both monomeric and fibrillar recombinant alpha binding. human synuclein.

B cell supernatants that showed the strongest binding signals in the ELISA to recombinant fibrils were selected for further surface plasmon resonance analysis to identify those cells that had the best dissociation rate in the case of recombinant human alpha-synuclein monomer, recombinant alpha fibrils. human α-synuclein; and fibrils of recombinant mouse alpha-synuclein. Supernatants of 26 different B cells were examined, and dissociation rates (kd) of <1 x 10 ^-5 were obtained in 19 wells for recombinant human alpha-synuclein monomer. Of these, dissociation rates (kd) of less than 1 x 10 ^-5 were obtained in 6 wells for the recombinant mouse alpha-synuclein monomer. All 19 supernatants were selected for variable region isolation.

Isolation of the variable region

In order to enable the isolation of antibody variable region genes from the supernatants of interest, a deconvolution step had to be performed so that the antigen-specific B cells in a given well, which contained a heterogeneous population of B cells, could be identified. This was achieved by using the fluorescence spot method (Clargo et al., 2014. MAbs: 6 (1), 143-159). Briefly, immunoglobulin-secreting B cells from the positive well were mixed with streptavidin microspheres (New England Biolabs) coated with biotinylated recombinant human alpha-synuclein fibrils (prepared using a 1:50 mixture as described above) and with a final dilution of 1: 1200 goat Fc fragment against rat IgG specific for FITC conjugate (Jackson). After incubation under static conditions at 37° C. for 1 hour, antigen-specific B cells could be identified by the presence of a fluorescent halo surrounding these B cells. A number of these individual B cell clones, identified with an Olympus microscope, were then selected using an Eppendorf micromanipulator and placed in a PCR tube.

The genes for the variable region of the antibody were isolated from individual cells by reverse transcription PCR using primers specific for the variable region of the heavy and light chains. Two rounds of PCR were performed with a nested 2° PCR including restriction sites at the 3' and 5' ends, allowing the variable region to be cloned into a mouse IgG Fcγ1 or Fab-10HIS (VH) or mouse kappa (VL) mammalian expression vector. Anti-alpha-synuclein antibody genes were successfully cloned into expression vectors from 9 different supernatants. The heavy and light chain constructs were co-transfected into Expi-293 cells using ExpiFectamine 293 (Invitrogen) and expressed in a 30 ml volume in a 125 ml Erlenmeyer flask chimeric recombinant rat-mouse antibodies 5811 mFab or 5811 mIgG1 (comprising rat variable regions 5811 and mouse constant regions (corresponding to SEQ ID NOS: 34 and 35 for IgG and SEQ ID NOS: 34 and 36 for Fab) After expression for 5-7 days, supernatants were collected and purified by affinity chromatography.

ELISA screening of intermediate supernatants

Purified rat-mouse chimeric antibodies were then subjected to further screening using the ELISA method. Monomer and fibrils of biotinylated recombinant human alpha-synuclein were captured on Maxisorp 384-well plates (ThermoScientific/Nunc) coated with streptavidin in carbonate coating buffer (dH ₂ O+0.16% Na ₂ CO ₃ +0.3% NaHCO ₃ ) . Separate plates were also coated with a layer of biotinylated peptide corresponding to residues 117 to 126 of human alpha synuclein corresponding to SEQ ID NO: 8 (PVDPDNEAYE peptide) to check if the intermediate processes are related to this or another region of the molecule. The plates were blocked with 1% w/v PEG/PBS and then incubated with several dilutions of the purified intermediate supernatant. Secondary HRP-conjugated goat anti-mouse IgG Fc (Stratech Scientific Ltd/Jackson ImmunoResearch) was added to the plates and then visualized for binding to TMB substrate (3,3',5,5'-tetramethylbenzidine, EMD Millipore; 10 µl/ hole). Absorbance was measured at 630 nm using a BioTek Synergy 2 microplate reader. Data for 5811 antibody as IgG Fc-1 (comprising SEQ ID NOS: 34 and 35) and as Fab (comprising SEQ ID NOS: 34 and 36) shown in figures 3A and 3B. As can be seen, the 5811 antibody exhibits binding to both monomeric and fibrillar recombinant human alpha-synuclein and also exhibits binding to the PVDPDNEAYE peptide.

Next, characterization was performed to determine the activity, affinity and avidity, epitope binding and biophysical properties of such antibodies. Unless specifically stated that a humanized version was used, experiments were performed with rat-mouse chimeric antibodies (5811 mFab or 5811 mIgG1).

Example 3 Characterization of Antibodies

Kinetic studies using the Biacore system

The interaction kinetics was studied by the surface plasmon resonance method on a Biacore T200 instrument. Each of three different ligands, comprising recombinant full-length human alpha-synuclein monomer, purified recombinant human alpha-synuclein fibrils, and purified recombinant mouse alpha-synuclein fibrils prepared as described in the present invention, was immobilized on three different flow cells on the surface of a CM5 chip with using an amine coupling reaction. Three ligands were prepared in 10 mM NaAc, pH 3.5 and immobilized on separate surfaces of the flow cell to achieve an immobilization level of about 30 response units (RU) for alpha-synuclein monomer, about 40 RU for human alpha-synuclein fibrils, and about 300 RU for mouse alpha-synuclein fibrils, respectively, at a flow rate of 10 µl/min. HBS-EP+ buffer (GE Health Bio-Sciences AB) was used as a running buffer for both ligand immobilization and kinetic studies. Then, the binding of monoclonal antibody 5811 mIgG1 against alpha-synuclein and monoclonal antibody 5811 mFab against three ligands was measured. Monoclonal antibodies mIgG1 or mFab were injected at 7 different concentrations from 800 nM to 0.195 nM into 3 flow cells with a contact time of 3 minutes and a dissociation time of 30 minutes at a flow rate of 100 μl/min. The surface was regenerated with one injection of 50 mM HCl for 90 seconds at a flow rate of 10 μl/min and another injection of 50 mM HCl for 60 seconds at a flow rate of 10 μl/min. Data were analyzed using Biacore T200 software (version 3.0) using a bivalent analyte model with assumed no total contribution (RI=0) and global Rmax for the mIgG1 format, and a 1:1 model with flexible total contribution (local RI) and global Rmax.

The kinetic values for both mIgG1 and mFab binding to immobilized targets are shown in Table 2. The mIgG1 format showed an apparent selective affinity for human alpha synuclein fibrils over affinity for human alpha synuclein monomer, as the dissociation constant KD is greater than 10 times lower for human fibrils.

table 2

Sample Human monomer human fibril mouse fibril ka1
(1/ms) kd1
(1/s) KD1
(nM) ka1
(1/ms) kd1
(1/s) KD1
(nM) ka1
(1/ms) kd1
(1/s) KD1
(nM) 5811
mFab 1.26E+06 3.69E-04 0.29 1.04E+06 6.28E-04 0.60 3.12E+04 1.34E-03 42.92 5811
mIgG1 4.21E+05 2.25E-04 0.53 1.05E+06 2.87E-05 0.03 6.94E+05 8.27E-02 119.07

Binding to synuclein

The binding of anti-human alpha-synuclein antibodies to human beta-synuclein was studied by Western blotting using rPeptide beta-synuclein. 1 μg of synuclein was run on a 4-12% Bis/Tris gel and blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in PBS with 3% BSA and 0.1% Tween20. The 5811 mIgG1 antibody was added to the blocked blot and incubated for 1 hour at room temperature, washed with PBS, 0.1% Tween20 and incubated for 1 hour with secondary antibody-HRP conjugate (anti-rabbit H+L HRP conjugate, Bethyl, A120 - 101P). The blot was washed thoroughly in PBS with 0.1% Tween 20, PBS and water. Chemiluminescence was measured after the addition of ECL western blotting substrate (Pierce). As shown in Figure 4(A), lane 3, the 5811 mIgG1 antibody does not bind to human beta-synuclein.

Epitope mapping

NMR

Human alpha synuclein was cloned into the expression vector pET28a so that the protein was expressed without any labels. The construct was transformed into E. coli BL21 (DE3) cells (Stratagene) and the cells were grown in defined media with C ¹³ labeled DL-glucose and N ¹⁵ labeled ammonium sulfate in the presence and absence of deuterium oxide (D ₂ O). Expression was induced at OD 600 nm=1 with 300 mM IPTG and the culture was incubated at 30° C. for 4 hours. Cells were pelleted and lysed by three freeze-thaw cycles in 100 ml lysis buffer (20 mM Tris/HCl, pH 8.0, 25 units of benzonase (Merck Millipore), a complete protease inhibitor cocktail without EDTA (2 tablets, Roche) and 10 mg lysozyme (Sigma)). The lysate was clarified by centrifugation at 18,000 rpm and the clarified lysate was passed through a 0.22 μm filter (Stericup, Millipore). The sterile lysate was loaded onto a MonoQ 10/100GL column (GE Healthcare) equilibrated with 20 mM Tris/HCl pH 8.0, 5CV and the protein was eluted with a gradient to 500 mM NaCl in the same buffer. Further purification of the purest fractions was repeated on a MonoQ 10/100GL column after a 5-fold dilution in 20 mM Tris/HCl, pH 8.0. The purest fractions were pooled, concentrated with a 10 kDa MWCO centrifuge (Centriprep, Millipore), purified by gel permeation chromatography on a HiLoad 26/600 Superdex 75 column (GE Healthcare) and eluted in 25 mM sodium phosphate buffer, 100 mM NaCl ( pH 6.4). Fractions from a Superdex 75 column were pooled and sodium azide (final concentration 0.02%) and AEBSF (final concentration 10 μM) were added. The final protein concentration was approximately 5 mg/ml.

Rat-mouse antibody Fab 5811 was expressed in CHO SXE as His-tagged forms and purified from the supernatant by His-tagged protein affinity chromatography, binding the protein to HisTrap Excel (GE Healthcare) from the supernatant and eluting with 250 mM imidazole in PBS. The eluted pool was loaded onto a HiTrap GammaBind Plus Sepharose (GE Healthcare) column, washed with PBS, and the protein was eluted with 0.1 M glycine-HCl, pH 2.6, and the pH was adjusted to 6 with 0.75 M sodium phosphate, pH 9 The Fab-His protein eluting buffer was changed to NMR buffer (25 mM sodium phosphate, pH 6.4, 100 mM NaCl) on a HiPrep 26/10 desalting column. Fab-His protein fractions were concentrated and protease inhibitors AEBSF (final concentration 10 μM) and sodium azide (final concentration 0.02%) were added, then sterilized on a Millex GV filter with a pore size of 0.22 μm.

Binding of the 5811 antibody having VL corresponding to SEQ ID No: 9 and VH corresponding to SEQ ID No: 10 to the epitope was determined by heteronuclear magnetic resonance (NMR) spectroscopy using the Fab fragment of the antibody.

Basic assignments of the NMR spectrum of α-synuclein

Samples for NMR analysis were typically 350 µl with a protein concentration of 360 µM ¹³ C/ ¹⁵ N labeled or 430 µM ² H/ ¹³ C/ ¹⁵ N labeled human alpha-synuclein in 5-inch Shigemi ampoules. mm. The buffer was 100 mM NaCl, 25 mM sodium phosphate, pH 6.4, 10 μM AEBSF, 0.02% NaN ₃ . All experiments were recorded at 20°C on a Bruker AVIII spectrometer with a frequency of 600 MHz or a Bruker AVII spectrometer with a frequency of 800 MHz equipped with cryogenically cooled probes. Sequential relationships between the basic NMR signals of residues in the protein, HN(i)-N(i)-N(i±1), were established using the 3D (H)N(CA)NNH experiment (Weisemann et al., 1993 3D Triple -resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins. J. Biomol. NMR 3), recorded with a spectral width of 28, 28 and 10 ppm and a signal recording time of 117 (F1) 117 ( F2) and 140 (F3) ms when measured at ¹⁵ N, ¹⁵ N and ¹ H, respectively, with 8 scans per increment and a relaxation delay of 1.5 seconds. A heterogeneous sample was used with a sample density of 10% (4000 out of 40000 hypercomplex points), giving a total data collection time of 2.75 days. Sequential relationships were confirmed and residue types were identified using TROSY-HNCA experiments (Grzesiek and Bax, 1992 Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J. Magn. Reson. 96, 432-440; Salzmann et.al ., 1998. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins (Proc. Natl. Acad. Sci. USA. 95, 13585-90) and TROSY-HNCACB (Wittekind and Mueller, 1993 HNCACB, a High-Sensitivity 3D NMR Experiment to Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta-Carbon Resonances in Proteins J. Magn Reson Ser B 101, 201-205 Salzmann et al., 1999 TROSY -type Triple Resonance Experiments for Sequential NMR Assignment of Large Proteins, J. Am. Chem. Soc. 121, 844-848). The TROSY-HNCA experiment was recorded with spectral widths of 23, 28, 10 ppm and signal acquisition times of 12.1 (F1), 21.7 (F2), and 100 (F3) ms measured at ¹³ C, ¹⁵ N, and ¹ H, respectively. (8 scans per increment, relaxation delay 1.5 seconds, total acquisition time 1 day), while the TROSY-HNCACB experiment was recorded with a spectral width of 56, 28 and 10 ppm and a signal acquisition time of 8.2 (F1), 21.7 (F2) and 100 (F3) ms measured at ¹³ C, ¹⁵ N and ¹ H, respectively (8 scans per increment, 1.5 s relaxation delay, 1.7 days total acquisition time). Base carbonyl assignments were obtained from the TROSY-HNCO spectrum (Grzesiek and Bax, 1992 Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J. Magn. Reson. 96, 432-440; Salzmann et.al., 1998. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. USA. 95, 13585-90), recorded with a spectral width of 10, 29, 10 ppm and a signal acquisition time of 80 (F1 ), 21.7 (F2) and 150 (F3) ms when measured at ¹³ C, ¹⁵ N and ¹ H, respectively (8 scans per increment, relaxation delay 1.5 s). A heterogeneous sample was used with a sample density of 15% (1208 out of 8050 hypercomplex points), giving a total data collection time of 19 hours. NMR spectra were processed using the NMRPipe system (Delaglio et al., 1995 NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-93) with linear prediction used to increase the effective signal acquisition time on nitrogen up to a single increase. Non-uniformly sampled data were transformed using the Harvard iterative method based on soft thresholding (Hyberts et al., 2012 Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. J Biomol NMR 52, 315-27 ) with data transformation to the next Fourier number, increasing the time of indirect data collection up to 60%. Data analysis was performed using the Sparky method (Goddard and Kneller, DG SPARKY 3. In., University of California, San Francisco), which resulted in amide proton and nitrogen resonance assignments of 133 residues corresponding to 99% of the residues, excluding proline and N residues. -terminal methionine. The only other alpha-synuclein residue that was not assigned was aspartic acid at position 2

Binding Site Mapping of the 5811Fab Antibody Fragment

5811 binding site mapping was performed using a 150 μM sample of ² H/ ¹³ C/ ¹⁵ N labeled human α-synuclein containing a 10% molar excess of unlabeled Fab 5811. Samples were prepared in the same buffer as described above for baseline assignment of α-synuclein . Chemical shift changes at ¹ H, ¹⁵ N and ¹³ C were determined by comparing the TROSY-HNCO spectrum (Grzesiek and Bax, 1992 Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J. Magn. Reson. 96, 432-440 Salzmann et.al., 1998. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. USA. 95, 13585-90) registered for the alpha-synuclein/ Fab, with an equivalent control spectrum recorded for free alpha-synuclein. The control TROSY-HNCO experiment with free alpha-synuclein was recorded with a spectral width of 10, 28 and 10 ppm and a signal acquisition time of 80 (F1), 21.7 (F2) and 150 (F3) ms when measured at ¹³ C, ¹⁵ N and ¹ H, respectively (16 scans per increment, relaxation delay 1.4 seconds). A non-homogeneous sample (NUS) was used with a sample density of 25% (2013 of 8050 hypercomplex points), giving a total data collection time of 2.5 days. The TROSY-HNCO experiment for the α-synuclein/Fab complex was recorded with a spectral width of 10, 28 and 10 ppm and a signal acquisition time of 80 (F1), 21.7 (F2) and 80 (F3) ms when measured at ¹³ C, ¹⁵ N and ¹ H, respectively (32 scans per increment, relaxation delay 1.5 seconds). A non-homogeneous sample (NUS) was used with a sample density of 25% (1119 out of 4477 hypercomplex points), giving a total data collection time of 2.8 days. NMR spectra were processed using the NMRPipe system (Delaglio et al., 1995 NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-93) with NUS data transformation performed using mddnmr multidimensional decomposition (Orekhov and Jaravine, 2011. Analysis of non-uniformly sampled spectra with Multi-Dimensional Decomposition. Prog. Nucl. Magn. Reson. Spectrosc., 59, p 271-292). In the process of data conversion, the effective signal acquisition time when measured on nitrogen was increased up to a single increase.

Chemical shift changes were analyzed using the minimum shift technique (Williamson et al., 1997 Mapping the binding site for matrix metalloproteinase on the N-terminal domain of the tissue inhibitor of metalloproteinases-2 by NMR chemical shift perturbation. Biochemistry 36, 13882-9) , in much the same way as previously described (Veverka et al., 2008 Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR. Oncogene 27, 585-95), except that the equation used to calculate the combined chemical shift change (Δδ) was modified to include the carbonyl chemical shift, resulting in the following equation:

where Δδ _HN , Δδ _N and Δδ _C are changes in chemical shifts ¹ H, ¹⁵ N and ¹³ C, respectively. αN and αC correspond to the scaling factors of 0.2 and 0.35, respectively, used for the chemical shift ranges for the amide, nitrogen, and carbonyl protons.

To identify Fab binding sites (epitopes) on alpha synuclein, a combined minimum shift histogram versus protein sequence was used to identify regions of alpha synuclein containing significantly perturbed signals. If the size of the combined chemical shift change for individual amino acids exceeded the threshold value of the average combined chemical shift change for all amino acids plus one standard deviation from this average, then these residues were selected for further evaluation as possible contact residues in the Fab binding site.

Significantly perturbed residuals were identified as residuals whose minimum shift was at least greater than the mean plus one standard deviation of all calculated shifts. Four different thresholds were used to identify Fab-associated residues. Residues that participate in the binding site are estimated with increasing accuracy as: residues whose minimum shift exceeds the mean plus one standard deviation of all calculated shifts (component > 0.025574); residuals whose minimum shift exceeds the mean plus two standard deviations of all calculated shifts (component > 0.042552); residuals whose minimum shift exceeds the mean plus three standard deviations of all calculated shifts (component > 0.059530); residuals whose minimum shift exceeds the mean plus four standard deviations of all calculated shifts (amount > 0.076508). In this assay, proline residues cannot be identified as they do not contain an amide proton.

Therefore, the alpha-synuclein epitope for 5811 Fab is determined with increasing accuracy as the mean plus one standard deviation for all calculated shifts: E114, D115, V118, D119, D121, N122, E123, A124, Y125, E126, M127, S129, Q134 , D135 and Y136; mean plus two standard deviations of all calculated shifts: V118, D119, D121, N122, Y125, M127, D135 and Y136; mean plus three standard deviations of all calculated shifts: V118, D119, D121, N122, M127, D135 and Y136; there were no residuals for which the deviation of shifts exceeded the mean plus four standard deviations of all calculated shifts

Using the amino acid numbering used in the National Center for Biotechnology Information (NCBI) reference sequence NP_000336.1, 5811 Fab was found to bind at least the following alpha-synuclein residues (mean+3 SD) V118, D119, D121, N122 , Y125, M127, D135 and Y136. The antibody can also bind all of the following residues (mean+1 SD) E114, D115, V118, D119, D121, N122, E123, A124, Y125, E126, M127, S129, Q134, D135 and Y136.

As shown in Figure 4B, the NMR chemical shift changed for human alpha-synuclein dCN after binding to 5811 mFab. The predicted 5811 mFab epitope appears to include residues at amino acids 114 and 136 of human alpha synuclein (SEQ ID NO: 8)

Peptide mapping

Further characterization of the epitope bound by the 5811 mFab antibody was performed using short (usually 9-mer or 10-mer) peptides representing and covering the C-terminal region of human alpha synuclein. They were used in a competitive surface plasmon resonance assay to test their ability to inhibit antibody binding to monomeric alpha-synuclein or to preformed alpha-synuclein fibrils immobilized on the Biacore chip. The peptide showing the maximum level of inhibition was then selected for co-crystallization studies with the antibody to confirm the exact epitope.

Peptides were purchased from Peptide Protein Research Ltd., Bishop's Waltham, U.K. and synthesized by solid phase peptide synthesis using the Fmoc group according to the method of Atherton and Sheppard (see Atherton, E.; Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press). The N and C ends of the peptides were blocked with acetyl and amide groups, respectively, except when the peptides represented the N-terminus and C-terminus of α-synuclein, where the amino groups and carboxyl groups, respectively, remained free. Peptide stock solutions were prepared in DMSO with a content of 10 mM. A complete list of peptides is shown in Table 3.

Table 3

Peptide identification number Subsequence AS104-113 EEGAPQEGIL AS109-118 QEGILEDMPV AS111-120 GILEDMPVDP AS113-122 LEDMPVDPDN AS115-124 DMPVDPDNEA AS117-126 PVDPDNEAYE AS119-128 DPDNEAYEMP AS121-130 DNEAYEMPSE AS123-132 EAYEMPSEEG AS125-134 YEMPSEEGYQ AS127-136 MPSEEGYQDY

Recombinant human alpha-synuclein monomer and preformed alpha-synuclein fibrils were immobilized on a CM5 chip using a Biacore 3000 instrument (GE Healthcare). After activating the surface of carboxymethyl dextran by injecting 100 µl of a freshly prepared 1:1 (v/v) mixture of 50 mM N-hydroxysuccimide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a flow rate of 10 µl/min HBS-EP (GE Healthcare ) as running buffer, the reaction was initiated by passing 100 μl of monomer and fibrils at 5 μM in 10 mM acetate, pH 5.0, through separate flow cells. The reference flow cell was activated in the same manner, and then all surfaces of the flow cell were deactivated by pulse injection of 50 μl of 1 M ethanolamine ⋅ HCl pH 8.5.

Peptide solutions were prepared in running buffer at 100 μM, and a blank peptide sample was prepared by diluting DMSO 1 to 100 in running buffer. A solution of 5811 mFab was prepared at 50.5 nM in running buffer prior to pre-incubation with 198 μl with either 2 μl of blank peptide probe or diluted peptide to give a final mixture of 50 nM Fab and 1 μM peptide or control. Sensograms were recorded for each sample by injecting 30 μl of the mixture at a rate of 10 μl/min and registering the reporting point 5 seconds before the end of the injection. The chip was regenerated at the end of each cycle with two injections of 10 μl of 40 mm HCl and one injection of 5 mm NaOH. Control cycles were alternated with peptide cycles.

The degree of inhibition of each peptide was calculated as the percentage change in response units measured at the reporting point compared to the mean of adjacent control cycles.

The level of inhibition of each alpha-synuclein peptide is shown in Figure 5. Significant inhibition of 5811 mFab against either alpha-synuclein monomer or fibrils was observed only for the C-terminal peptides AS113-122, AS115-124, AS117-126 and AS119-128, where levels of inhibition of either form of α-synuclein were very similar. The highest level of inhibition was observed for the AS117-126 peptide at 81% and 90% for monomer and fibril binding, respectively. The common residues for the four above peptides are residues 119 to 122, which respectively form part of the epitope. Since the level of inhibition in the case of peptides AS113-122 and AS119-128 falls by only 2 to 5%, residues 115 to 118 and 123 to 124 should also form part of the epitope.

The result of this study suggested that the epitope of antibody 5811 includes residues 115 to 124 (DMPVDPDNEA).

Alanine scan

Single-point mutants of human alpha-synuclein amino acid (His-labeled) were obtained and expressed in the Expi293 system (Thermo Fisher Scientific) according to the manufacturer's instructions. Alanine residues were placed at positions 118 to 128, with the exception of residue 124, which was already alanine and was replaced with serine (Table 4). Mutant proteins were purified from supernatants obtained after centrifugation (4200 rpm, 2 hours), then subjected to sterilizing filtration (Stericup, Millipore). Supernatants were loaded onto a HisTrap Excel column (GE Healthcare) pre-equilibrated and washed with 25 mM sodium phosphate and 500 mM NaCl. Bound protein was eluted with an imidazole gradient up to 500 mM in the same buffer. Fractions with protein of interest were identified by NuPage gel electrophoresis, pooled, concentrated using Centriprep 10 kDa MWCO (Millipore) and buffer exchanged on a PD-10 column in PBS. Protein fractions were concentrated using an Ultracel 3KDa MWCO concentrating centrifuge (Millipore). The concentrate was passed through a sterilizing filter with a pore size of 0.22 μm (Millex GV, Millipore) and stored at -20°C. All mutants were expressed in the same amount as wild-type human alpha-synuclein (Figure 6A).

Table 4

Mutant alpha synuclein V118A D119A P120A D121A N122A E123A A124S Y125A E126A M127A P128A

Human alpha-synuclein mutants were assayed on a 4-12% Bis/Tris NuPage gel using 1 microgram per protein per strip and blotted onto a PVDF membrane (iBlot mini stack, Thermo Fisher Scientific). The blot was blocked in blocking buffer (3% bovine serum albumin, 0.1% Tween 20 in phosphate buffered saline, PBS) and incubated with either 5811 mFab or 5811 mIgG1 antibody. After incubation for 1 hour at room temperature, the blot was washed with 0.1% Tween in PBS. The blot was incubated for 1 hour with an anti-mouse IgG Fc HRP secondary identification antibody conjugate (AB5879, Abcam) in blocking buffer, washed, and chemiluminescence determined after the addition of ECL Western Blotting Substrate (Pierce).

As shown in Figures 6B and 6C, 3 mutants corresponding to human alpha synuclein D119A, N122A and Y125A were not recognized by the 5811 mFab and 5811 mIgG1 antibodies. This analysis suggests that these amino acids in human alpha synuclein may be important for the binding of antibodies 5811 mFab and 5811 mIgG1 to human alpha synuclein.

Example 4 Humanization of an Antibody

Rat antibody 5811 was humanized by grafting hypervariable regions (CDRs) from a rat V region onto a human germline antibody V region framework. To restore antibody activity, a number of framework residues from the rat V region were also maintained in a humanized sequence. These residues were selected using the protocol set forth in Adair et al., WO91/09967. Sequence alignments of rat (donor) antibody V region sequences with human germline (acceptor) V region sequences are shown in Figures 7 and 8, along with engineered humanized sequences. Hypervariable regions (CDRs) grafted from the donor to the acceptor sequence are defined by the Kabat numbering system (Kabat et al., 1987), except for CDR-H1, which uses the combined Chothia/Kabat numbering system (see patent document Adair et al. ., 1991 Humanized antibodies. WO91/09967).

Genes encoding a range of heavy and light chain V region sequence variants were designed and constructed using DNA2.0 Inc. automated synthesis techniques. Other heavy and light chain V region variants have been generated by modifying the VH and VK genes by oligonucleotide directed mutagenesis, including, in some cases, mutations in the CDRs. For transient expression in mammalian cells, the humanized light chain V region genes were cloned into the pMhCK UCB light chain expression vector, which contains DNA encoding the human kappa chain constant region (Km3 allotype). The humanized heavy chain V region genes were cloned into the human UCB gamma-4 heavy chain expression vector pMh-4PFL, which contains the DNA encoding the human gamma-4 heavy chain constant region with the S241P hinge-stabilizing mutation (Angal et al., Mol. Immunol 1993, 30 (1): 105-8). Chimeric rat-human antibody 5811 (comprising SEQ ID NOS: 9 and 10) was also prepared in a similar manner. Co-transfection of the resulting heavy and light chain vectors into suspension cultures of Expi293TM cells was performed using the ExpiFectamineTM 293 transfection reagent (A14525, ThermoFisher Scientific) and resulted in the expression of humanized, recombinant antibodies in either human IgG4P format or Fab-HIS format.

The human V region of IGKV1-39 plus the J region of JK1 (IMGT, http://www.imgt.org/) was chosen as an acceptor for the light chain hypervariable regions (CDRs) of antibody 5811. Light chain framework residues in gL5 grafts, gL8 and gL14 were all derived from the human germline gene except for residue 71 (corresponding to SEQ ID No: 13), where a donor tyrosine residue (Y71) was retained. Residue 94 in CDRL3 of the gL14 graft was mutated from a glycine (G) residue to an alanine (A) residue, thus modifying a potential asparagine deamidation site.

The human IGHV3-15 V region plus JH3 J region (IMGT, http://www.imgt.org/) was chosen as the heavy chain hypervariable region (CDR) acceptor of antibody 5811. Heavy chain scaffold residues in the gH4 graft all occurred from the human germline gene, except for residues 49 and 100 (corresponding to SEQ ID No: 25), where the donor residues alanine (A49) and alanine (A100), respectively, were retained.

Humanized antibody chain variants and combinations thereof were expressed and evaluated for their potency relative to the parent antibody, their biophysical properties and suitability for further processing.

As shown in Table 5, all inoculations retained the same or similar affinity as the parent rat-human antibody for alpha-synuclein in fibrils.

Table 5

Antibody 5811
option Donor Light Chain Residues Heavy chain donor residues ka1
(1/ms) kd1
(1/s) Affinity (KD)
pM Chimeric Rat-Human 5811 - - 1.56E+06 2.42E-05 15.6 5811gL5gH4 Y71 A49, A94 2.74E+06 4.10E-05 15.0 5811gL8gH4 - A49, A94 3.26E+06 5.17E-05 15.9 5811gL14gH4 A100 A49, A94 1.10E+06 2.04E-05 18.5

Example 5 Immunohistochemical Study

Immunohistochemical studies were performed by Asterand Bioscience (Royston, United Kingdoms). Cryosections (10 µm) were first subjected to antigen unmasking using a Dako PT Link instrument and EnVision FLEX Target Retrieval Solutions (pH 6) at 97° C. for 20 minutes with automatic heating and cooling. All subsequent stages of incubation were carried out at room temperature. Cryosections were air dried for 30 minutes, fixed in 4% paraformaldehyde prepared in 1X PBS for 10 minutes, washed in Dako EnVision™ FLEX Wash Buffer (Dako) and then loaded into a Dako Autostainer Plus automatic staining system. Endogenous peroxidase activity was blocked by incubating sections with a Dako peroxidase block (Dako) for 5 minutes. Sections were then washed twice with 1X PBS and incubated with Dako CSA II protein block (Dako) for 10 minutes. The protein block solution was removed with a stream of air and the sections were incubated for 30 minutes with rat-mouse IgG1 antibody 5811 (containing SEQ ID NOS: 34 and 35) diluted (0.05 μg/ml) in Dako antibody diluent (Dako). After incubation, sections were washed twice with 1X PBS, then incubated with Dako Flex polymer-HRP anti-mouse substrate (Dako) for 20 minutes, washed twice, and then incubated with diaminobenzidine substrate (Dako) for 10 minutes. The chromogenic reaction was stopped by rinsing the slides with distilled water. After chromogenesis, sections were removed from the Dako Autostainer Plus and manually counterstained with hematoxylin, dehydrated with sequential ethanol, clarified in three xylene changes, and embedded with a layer of DPX embedding medium (Sigma-Aldrich). Digital images of stained sections were obtained using the Aperio ScanScope AT Turbo system (Leica Biosystems). The 5811 mIgG1 antibody was tested on brain sections obtained from five different pS129 alpha synuclein positive and three different pS129 alpha synuclein negative donors (1 slice/donor). Antibody 5811 mIgG1 methylated neuropil and random Lewy body-like structures in the temporal cortex and substantia nigra of patients with Parkinson's disease (Figure 9A-E). In brain tissues in the absence of Parkinson's disease, the 5811 mIgG1 antibody labeled the neuropil in the temporal cortex, but no Lewy body-like structures were observed in the temporal cortex and substantia nigra (FIG. 9F-H). These observations suggest that the 5811 mIgG1 antibody binds to normal alpha-synuclein in the neuropil of the brain tissues of patients with and without Parkinson's disease, and, at the same time, it binds to pathological alpha-synuclein, which is present only in Lewy bodies. in patients with Parkinson's disease.

Example 6 Characterization of Humanized Antibodies

Three Ab5811 humanized IgG4P antibodies (5811gL5gH4; 5811gL8gH4 5811gL14gH4; sequences are shown in Table 1) were tested to evaluate their biochemical and biophysical characteristics, including thermal stability (T _m ), experimental isoelectric point (pI), hydrophobicity, solubility (precipitation method by PEG), resistance to aggregation at the air/liquid interface, and chemical stability in relation to the propensity to deamidate Asn93 at CDR-L3 (5811gL14gH4 has an N(93)A motif, and both 5811gL8gH4 and gL5gH4 have an N(93)G motif ).

Thermal stability measurements (T _m )

The melting point (Tm) or temperature at the key point of protein unfolding was determined using Thermofluor assay. In this method, SYPRO orange fluorescent dye was used to monitor the process of protein unfolding by binding to hydrophobic regions that become exposed with increasing temperature.

The reaction mixture contained 5 µl of 30x SYPRO® Orange dye (Invitrogen™) diluted with PBS from a 5000X stock solution and 45 µl of test sample at a concentration of 0.12 mg/ml (in PBS pH 7.4). In four replicates, approximately 10 μl of the mixture was dispensed into an optical 384-well PCR plate and the reaction was run on a 7900HT real-time fast PCR system (Applied BiosystemsTM). The heating device of the PCR system was set to a temperature range of 20°C to 99°C with a temperature rise rate of 1.1°C/min. Changes in fluorescence in the wells were monitored using a semiconductor light detector. The increase in intensity was plotted and the slope of the tangent to the inflection curve(s) was used to calculate the Tm value as described below. The Tm value for each antibody molecule was obtained in both PBS pH 7.4 and 50 mM sodium acetate/125 mM sodium chloride pH 5.0, which are common pretreatment buffers.

Thermal stability for all three humanized antibodies is shown in Table 6. In PBS pH 7.4, one transition was observed and was attributed to both CH2 and Fab domain deployment. In 50 mM sodium acetate/125 mM sodium chloride pH 5.0, two transitions were observed. The lower Tm value was attributed to the unfolding of the CH2 domain, and the second transition was attributed to the Fab domain. This analysis showed that the 5811gL8gH4 and 5811gL5gH4 antibodies have comparable thermal stability to the 5811gL14gH4 antibody, which is only slightly less thermally stable.

Table 6

Antibody Average
_Tm value (1)°C standard error Average
_Tm value (2)°C standard error PBS
pH 7.4 5811gL14gH4 65.3 0 ND ND 5811gL8gH4 67.6 0 ND ND 5811gL5gH4 67.6 0 ND ND Acetate
pH 5 5811gL14gH4 56.3 0.1 66.2 0 5811gL8gH4 56.3 0 69 0 5811gL5gH4 56.4 0 69.1 0.1

Experimental determination of the isoelectric point (pI)

Experimental pI values for 5811gL14gH4, 5811gL8gH4, and 5811gL5gH4 were obtained using the cIEF iCE3™ complete capillary imaging system (Protein Simple). Samples were prepared by mixing the following components: 30 µl of sample (from a stock solution of 1 mg/ml in HPLC grade water), 35 µl of 1% methylcellulose solution (Protein Simple), 4 µl of pH 3-10 ampholytes (Pharmalyte) , 0.5 µl 4.65 and 0.5 µl 9.77 synthetic pI markers (ProteinSimple), 12.5 µl 8M urea solution (Sigma-Aldrich®). HPLC grade water was used to bring the final volume to 100 µl. Before analysis, the mixture was briefly shaken to ensure complete mixing and centrifuged at 10,000 rpm for 3 minutes to remove air bubbles. Samples were focused for 1 minute at 1.5 kV followed by 5 minutes at 3 kV and images of the A280 capillary were acquired using ProteinSimple software. The resulting electropherograms were first analyzed using the iCE3 software and pI values were assigned (linear relationship between pI markers). The calibrated electropherograms were then integrated using Empower® software (Waters).

The experimental value of pI for all molecules was in the range of 8.80-9.23, mainly at 9.09. There was no difference in the distribution of acidic/basic species, which was typical for IgG4P molecules. The experimental pI values were high and therefore could have a positive effect on the antibody production process.

Hydrophobic Interaction Chromatography (HIC)

Humanized antibodies 5811gL14gH4, 5811gL8gH4, and 5811gL5gH4 were evaluated by hydrophobic interaction chromatography (HIC) for their hydrophobicity. In HIC analysis, antibodies bind to the hydrophobic stationary phase in the presence of high concentrations of polar salts and desorb into the mobile phase as the salt concentration decreases. Longer retention time corresponds to higher hydrophobicity.

Samples of antibodies with a concentration of 2 mg/ml were diluted 1:2 with 1.6 M ammonium sulfate and PBS (pH 7.4). 5 μg (10 μl) of sample was injected into a Dionex ProPac™ HIC-10 column (100 mm x 4.6 mm) connected in series to an Agilent 1200 binary HPLC system with a fluorescent detector. Separation was controlled by intrinsic fluorescence (excitation and emission wavelengths, 280 nm and 340 nm, respectively).

Using buffer A (0.8 M ammonium sulfate 100 mM phosphate pH7.4) and buffer B (100 mM phosphate pH 7.4), the sample was analyzed using gradient elution as follows, (i) exposure for 2 minutes at 0 % B, (ii) linear gradient from 0 to 100% B over 30 minutes (0.8 ml/minute) (iii) column washes with 100% B for 2 minutes and reequilibrated at 0% B in for 10 minutes and then the introduction of the next sample. The column temperature was maintained at 20°C. Low and high hydrophobicity standards were also analyzed plus a control sample in the same run sequence to normalize retention times. The retention time (RT) of the sample was normalized to low and high hydrophobicity standards using the following equation:

[(Sample (RT) - low hydrophobicity (RT) reference / High hydrophobicity (RT) reference - low hydrophobicity (RT) reference] x 100

All three antibodies 5811gL14gH4, 5811gL8gH4 and 5811gL5gH4 had similar normalized retention times and similar low hydrophobicity, i.e., eluted from the column in less than 5 minutes (see Table 7). Low hydrophobicity has a positive effect on stability (i.e., reduces aggregation) during production.

Table 7

Antibody (main peak) Retention time (min) Normalized retention time (min) 5811gL14gH4 4.7 2.9 5811gL8gH4 4.7 2.9 5811gL5gH4 4.6 2.0

Solubility measurement by polyethylene glycol (PEG) precipitation.

The colloidal stability of the humanized antibodies 5811gL14gH4, 5811gL8gH4 and 5811gL5gH4 was analyzed using the polyethylene glycol (PEG) precipitation method. PEG was used to quantify protein solubility by increasing the PEG concentration (w/v) and measuring the amount of protein remaining in solution. This analysis served to simulate the effect of high concentration on solubility without the use of traditional methods of measuring concentrations.

PEG-induced precipitation of humanized antibodies 5811gL14gH4, 5811gL8gH4 and 5811gL5gH4 was studied in the presence of 7-18% PEG-3350 in PBS pH 7.4 and 50 mM sodium acetate/125 mM sodium chloride pH 5.0. Antibody samples were buffer exchanged using dialysis and adjusted to 2 mg/mL. To minimize non-equilibrium precipitation, 2x protein and 2x PEG solutions were mixed in sample preparation at a 1:1 volume ratio. After mixing, the samples were incubated at 37°C for 30 minutes to re-dissolve non-equilibrium aggregates. After incubation overnight at 20°C, the samples were centrifuged for 60 minutes (4000 g). Aliquots of the supernatant were transferred to half the volume of 96-well optical plates and absorbance measured at 280 nm using a BMG Labtech FLUOstar® Omega LVIS A280 plate reader. The concentrations were plotted against the percentage of PEG, and the calculated midpoint (LogEC ₅₀ ) (obtained from fitting a non-linear regression curve, with variable slope) was determined as a measure of the relative colloidal solubility of the samples. In this assay, a higher LogEC ₅₀ value corresponds to a higher colloidal stability.

As shown in Table 8, no differences were found in colloidal stability between the three different antibodies. Higher colloidal stability was observed in acetate buffer pH 5 as illustrated by the higher logEC ₅₀ value.

Table 8

50 mM NaOAc/125 mM NaCl pH 5 PBS pH 7.4 Best fit values 5811gL14gH4 5811gL8gH4 5811gL5gH4 5811gL14gH4 5811gL8gH4 5811gL5gH4 Log EC ₅₀ 15.1 14.9 15.3 11.9 11.7 11.9 Slope angle -0.91 -0.77 -0.66 -0.84 -0.62 -0.69 R ² standard error 0.987 0.989 0.965 0.994 0.985 0.99 Sigmoidal dose-response form (variable slope)

Effect of Stress at the Air-Liquid Interface (Aggregation Analysis)

This assay is used to simulate the stress that antibodies may be subjected to during their production (eg, ultrafiltration). Proteins, such as antibodies, tend to unfold when exposed to air-liquid interface conditions, where hydrophobic surfaces are due to the hydrophobic environment (air) and hydrophilic surfaces are due to the hydrophilic environment (water). Agitation of protein solutions results in the formation of an extensive air-liquid interface, which can initiate aggregation.

Humanized antibody samples 5811gL14gH4, 5811gL8gH4 and 5811gL5gH4 in PBS pH 7.4 and 50 mM sodium acetate/125 mM sodium chloride pH 5.0 were stressed by vigorous agitation using an Eppendorf Thermomixer ComfortTM. Before mixing, the concentration was adjusted to 1 mg/ml using the appropriate extinction coefficients (1.41 absorbance at 280 nm, 1 mg/ml, path length 1 cm) and absorbance at 280 nm, 340 nm and 595 nm obtained using Varian Cary® 50-Bio spectrophotometer to establish the origin of the read time. An aliquot of each sample was placed in 1.5 ml conical Eppendorf tubes with caps (4 x 250 μl) and subjected to severe conditions for a aggregation resistance test by vigorous agitation at 1400 rpm at 25° C. for 24 hours. Time-dependent aggregation (haze) was monitored by measuring samples 24 hours after stirring at 595 nm using a Varian Cary™ 50-Bio spectrophotometer. For each sample, graphical dependences of the average absorption values on time were plotted.

The 5811gL5gH4 antibody molecule was characterized by a higher resistance to aggregation compared to the 5811gL14gH4 and 5811gL8gH4 antibodies. All antibodies were more resistant to aggregation in PBS pH 7.4.

Chemical stability - study of the effect of stress on the deamidation reaction

An accelerated study of the impact of stress on all three antibodies 5811gL5gH4, 5811gL8gH4 and 5811gL14gH4 was performed to determine the predisposition to deamidation of one identified as a potential site: Asn(93) in the CDR3 light chain, where the 5811gL14gH4 antibody has the Asn(93)Ala motif and both antibodies 5811gL 8gH4 and gL5gH4 have the Asn(93)Gly motif.

Three antibodies were exposed to conditions known to promote deamidation of Asn(N) residues (50 mM Tris/125 mM sodium chloride, pH 8.0/37° C.). In addition, samples were also prepared in 50 mM sodium acetate/125 mM sodium chloride pH 5.0 as a control condition to evaluate basal deamidation prior to stress exposure. The final concentration of the sample in each of the buffers was adjusted to ~5 mg/ml, and then the sample was divided into two aliquots, where one was stored at 4°C and one at 37°C for up to 5 weeks. An aliquot was removed immediately (T0) and after 2 weeks and 5 weeks and stored at -20°C.

Basal deamidation at the Asn(93) residue was measured on unstressed samples (50 mM sodium acetate/125 mM sodium chloride, pH 5/4°C) and the propensity of Asn(93) to deamidate was determined using samples exposed to stress 2 weeks/pH 8/37°C, generating a tryptic peptide containing the appropriate sequence. Briefly, 80 μg of each sample was reconstituted with DTT and denatured with guanidine hydrochloride at 37°C. The samples were then subjected to alkylation with iodoacetamide at room temperature, and then buffer exchange was performed with 7.5 mM Tris/1.5 mM CaCl ₂ , pH 7.9 (ZebaTM 7 kDa MWCO centrifuge columns, Thermo Fisher) and incubated for approximately 3 hours. with trypsin (1:23 by weight) at 37°C. Proteolysis was stopped by adding trifluoroacetic acid to 0.1% by volume, and the samples were stored at -20°C. Upon thawing, the samples were centrifuged to remove the precipitate.

The resulting peptides were separated and analyzed on a Waters BEH C18 column connected to a Thermo Fusion™ mass spectrometer operating in positive ion chemical ionization mode using a data-dependent orbital ion trap-orbital ion trap fragmentation technique with collision-induced dissociation (CID ). LC-MS and MS2 data were analyzed using Thermo Xcalibur™ and Pepfinder™ software. The corresponding peptide (LC N93-K102) was analyzed to evaluate the percentage of chemical modification.

Mass spectrometric peptide mapping showed that the basal level of Asn(93) deamidation was lower for the 5811gL14gH4 antibody compared to the 5811gL8gH4 and 5811gL5gH4 antibodies. This difference between molecules was not unexpected, as it was shown that, despite the difficulty in predicting the presence of deamidation propensity, Asn (N) residues followed by Gly (G) residues have a higher deamidation propensity than residues followed by followed by the bulkier residue Ala (A) (Robinson, NE et al., Proc. Natl. Acad. Sci. USA 2001, 98, 4367-4372).

For all three antibodies, a low degree of deamidation to Asn(93) was observed, namely 1.2% - 1.4% per week. The light chain mass difference of the N93-K102 peptide in the case of antibodies 5811gL5gH4 and 5811gL8gH4 included modification -17 Da (probably a succinimide intermediate) and +1 Da (completely deamidated product on Asn(93)), while in the case of antibody 5811gL14gH4the, succinimide did not found (table 9).

Table 9

Percent(%) Deamidation of Asn(93) Succinimide formation Deamidation plus succinimide Basal 2 weeks Basal 2 weeks Growth of total chemical modification per week 5811gL14gH4 1.2 4.1 Not detected Not detected 1.4 5811gL8gH4 4.4 7.1 4.6 4.3 1.2 5811gL5gH4 5.6 8.6 4.8 4.3 1.3

The overall degree of deamidation was low for all antibodies, although higher heterogeneity was observed for the 5811gL5gH4 and 5811gL8gH4 antibodies.

Example 7 Cell Aggregation Study

HEK Freestyle 293F cells (suspension cell culture) were prepared in Freestyle 293 expression medium (Invitrogen TM) at a density of 0.7 x 106 cells/ml and cultured to a density of 300 x 106 cells/ml. Transfection was performed according to the manufacturer's instructions, and, briefly, 600 μg of the expression vector pcDNA3.1(+) containing the alpha-synuclein gene was mixed in 20 ml of OptiMEM medium, while 293-fectin was diluted in OptiMEM medium (Invitrogen TM) and incubated for 5 minutes at room temperature. Added diluted DNA, and incubated at room temperature for 20 minutes, and then added dropwise to the cells (20 ml per flask). Cells were incubated for 24 hours at 37°C, 125 rpm, 8% CO ₂ . Cells were either used immediately or frozen at 5 million cells/ml in FBS+10% DMSO.

If the cells were pre-frozen, the cryovials were thawed and the cells were resuspended in Freestyle 293 medium, centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in Freestyle 293 medium (Life TechnologiesTM) + Pen/Strep (InvitrogenTM) at density 2 × 10 ⁶ cells/ml. In a 384-well plate (GrainerTM) was added 20 μl of suspension cell culture (total about 40,000 cells/well). 150 nM human alpha synuclein fibrils (prepared as described in the invention in Example 1) were added to each well, and then antibodies 5811gL5gH4 IgG4P, 5811 gL8gH4 IgG4P and 5811 gL14gH4 IgG4P (sequences are shown in Table 1) were added for testing in PBS (at various concentrations). The plates were incubated at 37°C, 5% CO2, 95% humidity in a cell culture incubator for 2 days.

At the end of the second day, medium was removed from all wells and the plate was washed, leaving 20 µl in each well. Approximately 50 μl PBS was added to each well and the plates were centrifuged at 500 g for 5 minutes. The supernatant was aspirated from all wells using a plate washer, leaving 20 µl of medium in each well. Versene (LonzaTM) (50 μl/well) was added and the plates were centrifuged at 500 g for 5 minutes, the supernatant was aspirated leaving only 20 μl of medium in each well. 20 μl of 8% formaldehyde (16% solution in water, Life TechnologiesTM) + 2% Triton X-100 (VWRTM) in PBS was added to each well. The plates were incubated at room temperature for 15 minutes and then 50 μl of FACS buffer consisting of HBSS (VWRTM without calcium and magnesium) + 2% FBS + 2 mM EDTA (Life TechnologiesTM) was added. The plates were centrifuged at 2000 g for 1 minute and the supernatant was aspirated leaving only 20 µl of medium in each well. An additional 20 μl of FACS buffer with anti-pSer129 alpha-synuclein antibody (AbCamTM) diluted 1:300 was added to each well. The plates were incubated for 1 hour at room temperature, and then 50 μl of FACS buffer was added to each well, and re-centrifuged at 2000 g for 1 minute. The supernatant was removed, then a 1:500 diluted Alexafluor647-conjugated anti-rabbit secondary antibody (Life TechnologiesTM) and DAPI (Life TechnologiesTM) was added to each well. The plates were incubated for 1 hour at room temperature in the dark, and then 50 μl of FACS buffer was added and the plates were centrifuged at 2000 g for 1 minute. After washing, additional FACS buffer was added and the plates were prepared for reading on a flow cytometer (BD FACS Canto II).

Flow cytometry (FACS) data was analyzed using FlowJo software. First, live individual cells were selected over time using forward and side scatter. Then, DAPI+ cells were time-selected and their number was used as a measure of the number of live nucleated single cells. Finally, 129-alpha-synuclein-positive (pSer129+) cells were selected over time. The percentage of pSer129+ cells relative to all DAPI+ cells was used as a measure of aggregation. Data are normalized to wells treated with fibrils only and without antibodies and are expressed as a percentage. The results are shown in Figure 10, which illustrate the ability of test antibodies to inhibit aggregation induced by alpha-synuclein fibrils in cells expressing alpha-synuclein. These data confirm that the antibodies of the present invention are capable of blocking aggregation induced by alpha-synuclein fibrils with an IC ₅₀ value of approximately 5 nM or less.

Example 8 Primary Neuron Aggregation Study

Hippocampi from E17 mouse embryos were dissected in dissection buffer (Hanks Balanced Salt Solution (HBSS) without calcium and without magnesium, 0.6% D-(+)-glucose, 20 mM Hepes). The dissection buffer was then removed and replaced with a dissociation solution (HBSS without calcium and without magnesium, 0.6% D-(+)-glucose, 20 mM HEPES, 40 units of papain, 1 mg/ml deoxyribonuclease, 1 mM L-cysteine , 0.5 mm EDTA). After 30 min incubation at 37°C, the dissociation buffer was removed and the hippocampus was washed 3 times with seed medium (Neurobasal™ medium, 2% B27 supplement, 1 mM GlutaMAX, 2.5% FBS, 50 U/mL penicillin-streptomycin) . Tissue clots were triturated with a 1 ml pipette to obtain a single cell suspension. Cells were diluted to the appropriate concentration in seeding medium. Approximately 15,000 cells were seeded in each well of a 384-well PDL-coated plate. The cells were then kept in a cell culture incubator at 37° C., 5% CO ₂ , 95% humidity.

The next day, 80% of the medium was changed to seeding medium without FBS [Neurobasal™ medium supplemented with 2% B27, 1 mM GlutaMAX, 50 U/ml penicillin-streptomycin). Seven days after seeding, the medium was removed, leaving 20 µl in each well. 100 nM human alpha synuclein fibrils (prepared as described in the invention in Example 1) were added to each well, then 5811gL5gH4 IgG4P, 5811 gL8gH4 IgG4P, and 5811 gL14gH4 IgG4P antibodies (sequences shown in Table 1) were added for testing in PBS (at various concentrations). The plate was incubated at 37° C., 5% CO ₂ , 95% humidity in a cell culture incubator for an additional 7 days. Fourteen days after seeding, the medium was aspirated from all wells, leaving 20 μl in the wells. Each well was washed with 80 μl of Dulbecco's sodium phosphate buffer (DPBS). DPBS was removed and cells were incubated in 40 μl of fixing buffer (DPBS with 4% paraformaldehyde) per well for 15 minutes. Then the fixing buffer was removed and the cells were washed again with 80 μl of DPBS. DPBS was removed and replaced with 40 μl per well of permeabilization buffer (DPBS with 0.1% Triton X-100). After 10 minutes, the permeabilization buffer was removed and the cells were incubated for 1 hour in 40 μl per well of blocking buffer (PBS with 1% BSA and 0.1% Triton X-100). The blocking buffer was then removed and replaced with 40 µl per well of the primary antibody solution (blocking buffer with 0.3% rabbit antibody against phosphoserine 129-alpha-synuclein (AbCamTM ab51253). The antibody solution was incubated on the cells for 1 hour, then washed three times ( 90 µl/each, PBS) After the last wash, PBS was removed and replaced with 40 µl secondary antibody solution (0.1% AlexaFluor647-conjugated anti-rabbit antibody in PBS with 0.2% AlexaFluor488-conjugated anti-beta-III- tubulin).The secondary antibody solution was incubated on the cells for 1 hour, then removed and replaced with 40 µl PBS containing 0.3% CellMask BlueTM. well, and sealed the plate with a transparent polymer film.

Plates were visualized using an Arrayscan plate imaging device (ThermoFisher ScientificTM). The images were analyzed using the HCS ScanTM software from the same manufacturer. The density of neurons was controlled by the beta-III-tubulin signal. Sparse visual fields or visual fields with a damaged layer of neuronal cells, characterized by a significant decrease in the beta-III-tubulin signal surface, were to be excluded. Finally, the pSer129 alpha-synuclein signal surface in the field of view was used to quantify abnormal alpha-synuclein aggregation.

Phosphorylation of alpha-synuclein at the S129 site is believed to play an important role in the control of normal alpha-synuclein functions, as well as the regulation of its aggregation, LBs formation, and neurotoxicity. Under normal conditions, only a small proportion of alpha-synuclein is constitutively phosphorylated at the S129 site in the brain (Fujiwara H, et al. (2002) Nat Cell Biol, 4, 160-164), whereas in the brains of patients suffering from synucleinopathy, dramatic accumulation of pS129 (Kahle PJ, et al. (2000) Ann N Y Acad Sci, 920, 33-41); Okochi M, et al. (2000) J Biol Chem, 275, 390-397); Anderson JP, et al. (2006) J Biol Chem, 281, 29739-29752).

Data were normalized to wells treated with fibrils only and without antibodies and expressed as a percentage. As shown in Figure 11, all three antibodies inhibited alpha-synuclein aggregation induced by alpha-synuclein fibrils on mouse primary neurons expressing endogenous levels of alpha-synuclein. These data confirm that the 5811gL5gH4 IgG4P, 5811gL8gH4 IgG4P, and 5811gL14gH4 IgG4P antibodies were able to block alpha-synuclein fibril-induced aggregation on mouse primary neurons with an IC ₅₀ value below 5 nM.

Example 9 In Vivo Evaluation of VR5811 Efficacy

The 5811gL5gH4 IgG4P antibody (comprising SEQ ID NO: 14 and SEQ ID NO: 26 and hereinafter referred to simply as VR5811) was tested in an α-synuclein knockout mouse transgenic model expressing human alpha-synuclein (hereinafter referred to as SNCA-OVX; Charles River, France).

SNCA-OVX mice were injected with the VR5811 antibody and mouse preformed fibrils (PFFs) (prepared as described in the invention in Example 1). Negative control antibody and placebo were also administered simultaneously with an anti-alpha-synuclein reference antibody (C-term Ab reference antibody) that binds alpha-synuclein at the last nine C-terminal residues. This reference antibody (which has different hypervariable regions (CDRs) from the antibodies of the present invention) exhibited binding characteristics that were comparable to those of the antibodies of the present invention. The reference antibody C-term Ab has the same affinity for alpha-synuclein as the antibodies of the present invention and similar biophysical properties. It was also effective in preventing alpha-synuclein aggregation in cell assays (Table 10).

Table 10

Antibody Human fibill _IC50 ka1 (1/ms) kd1 (1/s) KD1 (nM) (nM) VR5811 2.74E+06 4.10E-05 0.015 Less than 5 Reference antibody
C-term Ab 1.08E+06 2.20E-05 0.02 Less than 5

Antibodies were preincubated with PFF for 30 minutes on a shaker at room temperature, then injected directly into the brain of animals. Antibody/PFF mixtures were prepared in PBS at a ratio of 1 μg PFF/10 μg antibody. PBS at pH 7.4 was used as the placebo solution. The antibody was administered 24 hours prior to the combined intracerebral administration.

The antibodies were then intraperitoneally administered to mice at a dose of 30 mg/kg. The second IP injection was given 7 days after the first injection, and then the same treatment regimen was followed (one IP injection per week at a dose of 30 mg/kg for an injection volume of 10 ml/kg) for 11 weeks, a total of 12 injections, for mice of the strain SNCA-OVX. Mice were randomly assigned to drug treatment groups, and the experimenters were in the dark about the drugs used.

Animal experiments were carried out in accordance with the guidelines of European Directive 2010/63/EU and Belgian law. The Animal Research Ethics Committee of UCB Biopharma SPRL (LA1220040 and LA2220363) approved the experimental protocol (ASYN-IC-PARKINSON-MO). The mice weighed 25 to 30 g and were 17 weeks old at the time of surgery. Mice were kept in cages (4 mice per cage, Macrolon type 2). They were kept on a 12:12 light/dark cycle with lights on at 06:00. The temperature was maintained at 20-21° C. and the humidity was approximately 40%. Prior to allocation to test groups, all animals had ad libitum access to standard kibble and water. Before and after surgery, mice were provided with supplemental food and more comfortable living conditions (Enviro-dri, Pharma Serv). Animal health was monitored daily by animal care staff. Every effort has been made to minimize animal suffering. The killings were carried out under anesthesia.

Surgery was performed under general anesthesia using a mixture of 50 mg/kg ketamine (Nimatek, Eurovet Animal Health B.V.) and 0.5 mg/kg medetomidine (Domitor, Orion Corporation) administered intraperitoneally. In addition, 2.5 mg/kg of atipamezole (Antisedan, Orion Corporation) was administered to facilitate awakening from anesthesia. Recombinant purified fibrils (PFF) were thawed and sonicated at room temperature (Qsonica 500-20 kHz; power 65%, 30 pulses at 1 second on, 1 second off for one minute). PFF was then premixed with antibodies for 30 minutes and shaken at room temperature for 30 minutes prior to injection into the brain. The solution (2 μl) was infused at a rate of 0.2 μl/min and the needle was left in place for an additional 2.5 minutes before being slowly withdrawn. Injection was performed from the same side into the right striatum at the following coordinates: AP = + 0.20 mm, ML = -2.00 mm, DV = -3.20 mm.

Following anesthesia, mice were perfused by transcardial perfusion of ice-cold 0.9% PBS containing 10 U/ml heparin for 9 minutes at a flow rate of 6 ml/min through the left ventricle. The right atrium was excised to provide outflow. Animals were then perfused by perfusion of ice-cold 4% paraformaldehyde in PBS for 15 minutes at a flow rate of 6 ml/min. The brains were fixed overnight in PBS containing 4% paraformaldehyde at 4°C (day 0). The next morning (day+1), the 4% paraformaldehyde was discarded and the brains were washed in cold PBS and incubated overnight. The next day (day +2), the brains were washed in PBS for at least 1 hour and transferred to PBS containing 15% sucrose and stored at 4°C until transport.

Brain sections were made at Neuroscience Associates (TN, USA). First, brains were treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freezing artifacts and embedded in a gelatin matrix using MultiBrain® technology. After curing, the blocks were flash-frozen by immersion in isopentane cooled to -70°C with crushed dry ice and mounted on the freezing table of an AO860 sliding microtome. MultiBrain® blocks were sectioned in the coronal plane with a thickness of 40 µm. All sections were collected sequentially in 24 containers per block, which were filled with Antigen Preserve solution (49% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone). Unstained immediately sections were stored at -20°C.

Free-floating sections were stained immunochemically using an antibody against pSer129alpha-synuclein (mouse alpha-synuclein (pSer129), biotin - (Wako - 010-26481)) diluted 1:30,000. All blocking serum incubation solutions, in this case and elsewhere, used Tris buffered saline (TBS) with Triton X-100 as medium; all rinses were performed with TBS. Endogenous peroxidase activity was blocked by treatment with 0.9% hydrogen peroxide and non-specific binding was blocked with 1.26% whole normal serum. After rinsing, sections were stained with primary antibody overnight at room temperature. The permeabilization carrier solution contained 0.3% Triton X-100. After rinsing, sections were incubated with avidin-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) for one hour at room temperature. After rinsing, sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to create a visually visible reaction product mounted on gelatinized (replaceable) glass slides, air dried, lightly stained with thionine, dehydrated in alcohols, clarified in xylene and coated with Permount environment layer.

Quantification of the pSer129 alpha-synuclein signal in the field of view of the pSer129 alpha-synuclein signal was used to quantify abnormal alpha-synuclein aggregation in the ipsilateral striatum, cortex, basolateral tonsil, and substantia nigra. Regions of Interest (ROI) boundaries were manually determined and automatic quantification of the pSer129 alpha-synuclein signal in various brain regions was performed using VisioPharm 6 software (VisioPharm). To quantify the pSer129 alpha-synuclein signal, a linear Bayesian algorithm was used to obtain the signal area (marker area in µm ² ). The area of the marker count reflects the pathology of pSer129 alpha-synuclein, which covers different areas of the brain. All quantitative estimates were blinded until the end of the statistical analysis.

Data analysis was performed by % marker area (ie, by the ratio between the pSer129 signal area in µm ² and the area of interest in µm ² ). Percentage (%) of marker area was reassessed for several brain slices located rostro-caudally (striatum: 13-14 slices from bregma +1.1 to -0.94; cortex: 13-14 slices from +1.1 to -0 .94; basolateral tonsil: 6-10 sections from -0.58 to -2.06; substantia nigra: 6-8 sections from -2.54 to -3.88), and separately for each test object, the area under pharmacokinetic curve (AUC).

For statistical analysis, one-way analysis of variance (ANOVA) was used. The ANOVA method was followed by multiple pairwise comparisons between means without adjustment for multiplicity (** for p<0.01 and * for p<0.05). The data were converted to logarithmic form to satisfy the criteria of normality and homoscedasticity. Plots represent the geometric means of the untransformed data.

As shown in Figure 12, the VR5811 antibody markedly reduced alpha-synuclein pathology (i.e., pSer129 alpha-synuclein signal) in various brain regions, including the striatum, cerebral cortex, tonsils, and substantia nigra, 3 months after injection of fibrils (PFF) into mice. SNCA-OVX lines.

Antibody for negative control and antibody for comparison C-term. had no effect in reducing alpha-synuclein pathology (alpha-synuclein pSer129 signal) compared to placebo-treated mice injected with the same human fibrils (PFF).

The figure 13 shows the quantitative determination of alpha-synuclein, phosphorylated at Ser129 in each of the analyzed regions of the brain of SNCA-OVX mice. The results confirm that VR5811 significantly (p < 0.05) reduces alpha-synuclein pathology in various brain areas, including the ipsilateral striatum, cerebral cortex, and substantia nigra, compared with three control groups (i.e., placebo, 101.4 and antibody for comparison C-term Ab).

As a result, when tested in SNCA-OVX mice, the 5811 group showed a significant decrease in pSer129 α-synuclein levels in three different structures, two of which were distal from the injection site (cortex and substantia nigra) .

This confirms that the antibodies having the distinctive structural features of the present invention are able to prevent Ser129-phosphorylated alpha-synuclein in vivo. In addition, the results show that not all antibodies that bind alpha-synuclein at the C-terminal region are effective in vivo: a reference antibody that binds to the C-terminus of alpha-synuclein with high affinity and effectively prevents alpha-synuclein aggregation in cellular assays failed to prevent Ser129 phosphorylation in vivo.

Therefore, the antibody or antigen-binding fragment thereof of the present invention can be used to treat alpha synucleinopathies characterized by an increase in Ser129 phosphorylation, including Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy bodies (DLBD), Lewy body variant Alzheimer's disease (LBVAD), mixed type of Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation type 1 (NBIA-1).

Example 10 Pharmacokinetics of Antibody 5811 in Mouse

Male C57/Bl6 mice (n=3 per drug) were intravenously injected with the 5811gL14gH4 IgG4P antibody (5811) as a single dose of 2 mg/kg.

Blood samples were taken (at 0.083, 1, 4, 8, 24, 72, 120, 168, and 336 hours post-injection) from the tail vein of mice, and kept at room temperature for blood clotting. Serum was isolated by centrifugation and then frozen until analysis. The 5811 antibody was quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Serum samples for the study were thawed and quantified by a calibration curve prepared using the 5811 antibody injected at various concentrations into mouse control serum. Before introducing samples into the LC-MS/MS system, serum was denatured, reduced and alkylated using acetonitrile (VWR, UK), TCEP-Tris(2-carboxyethyl)phosphine hydrochloride (Sigma, UK) and iodoacetamide (Sigma, UK), respectively . The alkylated samples were then dissolved in 100 mM ammonium bicarbonate buffer (Sigma, UK) and digested overnight using trypsin enzyme (Promega, UK) at 37°C. Digestion was stopped by adding formic acid to the samples to lower the pH and then desalted using a Waters HLB SPE plate. The resulting eluent was concentrated using a vacuum evaporator. After the samples were completely dried, they were dissolved in a mixture of 95/5: water/acetonitrile containing 0.1% formic acid and injected into the LC-MS/MS system. LC-MS/MS analysis was performed on a Schimadzu High Performance Liquid Chromatography (HPLC) system connected to an AB Sciex QTrap 6500 Triple Quadrupole Mass Spectrometer. Aeris C18 peptide 100 × 2.1 mm, 2.6 µm) in which the temperature was maintained at 50°C. A linear gradient of 5-70% acetonitrile in 0.1% formic acid was applied over 6 minutes and then increased to 95% acetonitrile in 0.1% formic acid over 0.8 minutes at a flow rate of 0.6 ml/min. The mass spectrometer was configured to monitor multiple reactions to detect multiple transitions of the 5811 peptides with a dwell time of 50 milliseconds per transition. Data analysis was performed using the Analyst 1.6 software version.

These data show that the 5811 antibody has very good pharmacokinetics (Table 11 and Figure 14) in mice, as evidenced by the low clearance values measured. They appear to exceed the typical range referred to for human IgG preparations dosed to mice (3-16 ml/day/kg; Deng et al 2011 mabs 3: 1 61-66).

Table 11

Antibody Clearance (standard deviation) ml/day/kg Mouse 5811 3.1 (1.0)

--->

SEQUENCE LIST

<110> YUSB Biopharma SRL

<120> Antibodies

<130> PF0129-WO

<150>GB1720970.1

<151> 2017-12-15

<160> 47

<170>PatentIn version 3.5

<210> 1

<211> 11

<212> Protein

<213> Rattus norvegicus

<400> 1

Lys Ala Ser Gln Asn Ile Asn Lys Asn Leu Asp

1 5 10

<210> 2

<211> 7

<212> Protein

<213> Rattus norvegicus

<400> 2

Tyr Ala Asn Asn Leu Gln Thr

5

<210> 3

<211> 8

<212> Protein

<213> Rattus norvegicus

<400> 3

Tyr Gln Tyr Lys Asn Gly Trp Thr

15

<210> 4

<211> 10

<212> Protein

<213> Rattus norvegicus

<400> 4

Gly Phe Thr Phe Asn Asn Ala Ala Met Tyr

1 5 10

<210> 5

<211> 19

<212> Protein

<213> Rattus norvegicus

<400> 5

Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210> 6

<211> 7

<212> Protein

<213> Rattus norvegicus

<400> 6

Asp Tyr Ser Arg Gly Asp Arg

5

<210> 7

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-L3 Option 1

<400> 7

Tyr Gln Tyr Lys Asn Ala Trp Thr

15

<210> 8

<211> 140

<212> Protein

<213> Homo sapiens

<400> 8

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

1 5 10 15

Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys

20 25 30

Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val

35 40 45

Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr

50 55 60

Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys

65 70 75 80

Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys

85 90 95

Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile

100 105 110

Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

115 120 125

Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

130 135 140

<210> 9

<211> 106

<212> Protein

<213> Rattus norvegicus

<400> 9

Asn Ile Gln Met Thr Gln Ser Pro Pro Val Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys His Gly Glu Ala Pro Lys Leu Leu Met

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 10

<211> 118

<212> Protein

<213> Rattus norvegicus

<400> 10

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Ala

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met

65 70 75 80

Val Tyr Leu Gln Met Asp Asn Leu Lys Ser Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Thr Ala Asp Tyr Ser Arg Gly Asp Arg Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser

115

<210> 11

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Rat VL nucleotides

<400> 11

aacatccaga tgacccagtc tcctccagtc ctgtctgcat ctgtgggaga cagagtcact 60

ctcagctgca aagcaagtca gaacattaat aagaacttag actggtatca gcaaaagcat 120

ggagaagctc caaaactcct gatgtattat gcaaacaatt tacaaacgggg catcccatca 180

aggttcagtg gcagtggatc tggaacagat tacacgctca ccatcagcag cctgcagcct 240

gaagatgttg ccacatatta ctgctatcag tataagaatg ggtggacgtt cggtggaggc 300

accaagctgg aactgaaa 318

<210> 12

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Rat VH nucleotides

<400> 12

gaaatgcagc tggtggagtc tggtggagga ttggtgcagc ctaaggagtc attgaaaatc 60

tcatgtgcag cctctggatt caccttcaat aatgctgcca tgtactggggt ccgccaggct 120

cggaaagg gtctggaatg ggttgctcgc ataagaacta aacctaataa ttatgcaaca 180

tcttatgctg attcagtgaa aggcagattc accatctcca gagatgattc aaaaagcatg 240

gtctacctac aaatggataa cttgaaaagt gaggacacag ccatgtatta ctgtacagca 300

gattactcca gaggtgacag gtggggccaa ggagtcatgg tcacagtctc gagc 354

<210> 13

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> V-region gL5 5811

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 14

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> light chain gL5 5811

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 15

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides of the V region gL5 5811

<400> 15

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat tacaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg gctggacttt tggacaaggc 300

accaaggtcg aaatcaag 318

<210> 16

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> light chain nucleotides gL5 5811

<400> 16

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat tacaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg gctggacttt tggacaaggc 300

accaaggtcg aaatcaagcg tacggtggcc gctccctccg tgttcatctt cccaccctcc 360

gacgagcagc tgaagtccgg caccgcctcc gtcgtgtgcc tgctgaacaa cttctacccc 420

cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggaa 480

tccgtcaccg agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg 540

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 600

tccagccccg tgaccaagtc cttcaaccgg ggcgagtgc 639

<210> 17

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> V-region gL8 5811

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> light chain gL8 5811

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 19

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> V region nucleotides 5811

<400> 19

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat ttcaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg gctggacttt tggacaaggc 300

accaaggtcg aaatcaag 318

<210> 20

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> light chain nucleotides gL8 5811

<400> 20

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat ttcaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg gctggacttt tggacaaggc 300

accaaggtcg aaatcaagcg tacggtggcc gctccctccg tgttcatctt cccaccctcc 360

gacgagcagc tgaagtccgg caccgcctcc gtcgtgtgcc tgctgaacaa cttctacccc 420

cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggaa 480

tccgtcaccg agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg 540

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 600

tccagccccg tgaccaagtc cttcaaccgg ggcgagtgc 639

<210> 21

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> V-region gL14 5811

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Ala Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> light chain gL14 5811

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Ala Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 23

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides of the V region gL14 5811

<400> 23

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat ttcaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg cttggacttt tggacaaggc 300

accaaggtcg aaatcaag 318

<210> 24

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> light chain nucleotides gL14 5811

<400> 24

gacatccaga tgacccagag cccgagctcc ctgtccgcat cagtggggga tcgcgtgact 60

attacgtgca aagcctcgca gaacatcaac aagaacctcg actggtatca gcagaagcca 120

ggaaaggcgc ctaagctgct gatctactac gccaacaatc tccagaccgg cgtgccctcg 180

cggttctccg gatctgggtc cggtactgat ttcaccctga ccattagctc ccttcaaccg 240

gaggacttcg ccacctatta ctgctaccag tacaagaacg cttggacttt tggacaaggc 300

accaaggtcg aaatcaagcg tacggtggcc gctccctccg tgttcatctt cccaccctcc 360

gacgagcagc tgaagtccgg caccgcctcc gtcgtgtgcc tgctgaacaa cttctacccc 420

cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agtccggcaa ctcccaggaa 480

tccgtcaccg agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg 540

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 600

tccagccccg tgaccaagtc cttcaaccgg ggcgagtgc 639

<210> 25

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> V-region gH4 5811

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Ala

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Ala Asp Tyr Ser Arg Gly Asp Arg Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser

115

<210> 26

<211> 445

<212> Protein

<213> Artificial sequence

<220>

<223> heavy chain gH4 5811

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Ala

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Ala Asp Tyr Ser Arg Gly Asp Arg Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 27

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> V region nuclotides gH4 5811

<400> 27

gaagtgcagc ttgtggagag cggaggtgga ctcgtgaagc ctggcggatc tctgcgcctg

tcctgcgccg cctcggggtt cacctttaac aatgccgcaa tgtattggggt cagacaggcc 120

ccgggaaagg gtttggaatg ggtggctagg attcggacta agcccaacaa ctacgcgacc 180

tcctacgccg atagcgtgaa gggcagattc accatctccc gggacgactc aaagaacacg 240

ctgtacctcc aaatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcaccgcg 300

gactactccc ggggcgatcg ctggggacag gggactatgg tcactgtctc gagt 354

<210> 28

<211> 1335

<212> DNA

<213> Artificial sequence

<220>

<223> heavy chain nucleotides gH4 5811

<400> 28

gaagtgcagc ttgtggagag cggaggtgga ctcgtgaagc ctggcggatc tctgcgcctg

tcctgcgccg cctcggggtt cacctttaac aatgccgcaa tgtattggggt cagacaggcc 120

ccgggaaagg gtttggaatg ggtggctagg attcggacta agcccaacaa ctacgcgacc 180

tcctacgccg atagcgtgaa gggcagattc accatctccc gggacgactc aaagaacacg 240

ctgtacctcc aaatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcaccgcg 300

gactactccc ggggcgatcg ctggggacag gggactatgg tcactgtctc gagtgcctcc 360

accaagggcc ccctccgtgtt ccctctggcc ccttgctccc ggtccacctc cgagtctacc 420

gccgctctgg gctgcctggt caaggactac ttccccgagc ccgtgacagt gtcctggaac 480

tctggcgcc tgacctccgg cgtgcacacc ttccctgccg tgctgcagtc ctccggcctg 540

tactccctgt cctccgtcgt gaccgtgccc tcctccagcc tgggcaccaa gacctacacc 600

tgtaacgtgg accacaagcc ctccaacacc aaggtggaca agcgggtgga atctaagtac 660

ggccctccct gccccccctg ccctgcccct gaatttctgg gcggaccttc cgtgttcctg 720

ttccccccaa agcccaagga caccctgatg atctcccggga cccccgaagt gacctgcgtg 780

840

gaagtgcaca atgccaagac caagcccaga gaggaacagt tcaactccac ctaccgggtg 900

gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 960

gtgtccaaca agggcctgcc ctccagcatc gaaaagacca tctccaaggc caagggccag 1020

ccccgcgagc cccaggtgta caccctgccc cctagccagg aagagatgac caagaaccag 1080

gtgtccctga cctgtctggt caagggcttc tacccctccg acattgccgt ggaatggggag 1140

tccaacggcc agcccgagaa caactacaag accaccccccc ctgtgctgga cagcgacggc 1200

tccttcttcc tgtactctcg gctgaccgtg gacaagtccc ggtggcagga aggcaacgtc 1260

ttctcctgct ccgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgtcc 1320

ctgagcctgg gcaag 1335

<210> 29

<211> 107

<212> Protein

<213> Homo sapiens

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 116

<212> Protein

<213> Homo sapiens

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 31

<211> 321

<212> DNA

<213> Homo sapiens

<400> 31

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccttggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 32

<211> 348

<212> DNA

<213> Homo sapiens

<400> 32

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggttggccgt attaaaagca aaactgatgg tgggacaaca 180

gactacgctg cacccgtgaa aggcagattc accatctcaa gagatgattc aaaaaacacg 240

ctgtatctgc aaatgaacag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaca 300

gatgcttttg atgtctgggg ccaagggaca atggtcaccg tctcttca 348

<210> 33

<211> 306

<212> Protein

<213> Artificial sequence

<220>

<223> a-syn rabbit Fc human 68-140

<400> 33

Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys Thr Val Glu

1 5 10 15

Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys Lys Asp Gln

20 25 30

Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu Glu Asp

35 40 45

Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu

50 55 60

Gly Tyr Gln Asp Tyr Glu Pro Glu Ala Val Glu Lys Thr Val Ala Pro

65 70 75 80

Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly

85 90 95

Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

100 105 110

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp

115 120 125

Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Val Gln Val Arg

130 135 140

Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg

145 150 155 160

Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys

165 170 175

Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu

180 185 190

Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr

195 200 205

Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu

210 215 220

Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp

225 230 235 240

Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val

245 250 255

Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro

260 265 270

Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His

275 280 285

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro

290 295 300

Gly Lys

305

<210> 34

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> rat-mouse chimeric light chain 5811

<400> 34

Asn Ile Gln Met Thr Gln Ser Pro Pro Val Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gln Asn Ile Asn Lys Asn

20 25 30

Leu Asp Trp Tyr Gln Gln Lys His Gly Glu Ala Pro Lys Leu Leu Met

35 40 45

Tyr Tyr Ala Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Tyr Gln Tyr Lys Asn Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys

210

<210> 35

<211> 442

<212> Protein

<213> Artificial sequence

<220>

<223> rat-mouse chimeric heavy chain 5811

<400> 35

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Ala

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met

65 70 75 80

Val Tyr Leu Gln Met Asp Asn Leu Lys Ser Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Thr Ala Asp Tyr Ser Arg Gly Asp Arg Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser Ala Lys Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 36

<211> 230

<212> Protein

<213> Artificial sequence

<220>

<223> Fab-His rat-mouse chimeric heavy chain 5811

<400> 36

Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Ala

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Thr Lys Pro Asn Asn Tyr Ala Thr Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met

65 70 75 80

Val Tyr Leu Gln Met Asp Asn Leu Lys Ser Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Thr Ala Asp Tyr Ser Arg Gly Asp Arg Trp Gly Gln Gly Val

100 105 110

Met Val Thr Val Ser Ser Ala Lys Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys His His His His

210 215 220

His His His His His His His

225 230

<210> 37

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 37

Glu Glu Gly Ala Pro Gln Glu Gly Ile Leu

1 5 10

<210> 38

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 38

Gln Glu Gly Ile Leu Glu Asp Met Pro Val

1 5 10

<210> 39

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 39

Gly Ile Leu Glu Asp Met Pro Val Asp Pro

1 5 10

<210> 40

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 40

Leu Glu Asp Met Pro Val Asp Pro Asp Asn

1 5 10

<210> 41

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 41

Asp Met Pro Val Asp Pro Asp Asn Glu Ala

1 5 10

<210> 42

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 42

Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu

1 5 10

<210> 43

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 43

Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro

1 5 10

<210> 44

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 44

Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu

1 5 10

<210> 45

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 45

Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly

1 5 10

<210> 46

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 46

Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln

1 5 10

<210> 47

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> C-terminal fragment of alpha-synclein

<400> 47

Met Pro Ser Glu Glu Gly Tyr Gln Asp Tyr

1 5 10

<---

Claims (60)

1. An antibody or antigen-binding fragment thereof that binds to alpha-synuclein, wherein the antibody or antigen-binding fragment thereof contains: a. a light chain variable region containing CDR-L1 of SEQ ID NO:1; CDR-L2 with SEQ ID NO:2 and CDR-L3 with SEQ ID NO:3; And b. a heavy chain variable region comprising CDR-H1 of SEQ ID NO:4; CDR-H2 of SEQ ID NO:5 and CDR-H3 of SEQ ID NO:6. 2. An antibody or antigen-binding fragment thereof that binds to alpha-synuclein, wherein the antibody or antigen-binding fragment thereof contains: a. a light chain variable region containing CDR-L1 of SEQ ID NO:1; CDR-L2 with SEQ ID NO:2 and CDR-L3 with SEQ ID NO:7; And b. a heavy chain variable region comprising CDR-H1 of SEQ ID NO:4; CDR-H2 of SEQ ID NO:5 and CDR-H3 of SEQ ID NO:6. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds two or more alpha-synuclein amino acid residues between positions 113 and 129 of SEQ ID NO:8, where the antibody or antigennegative fragment binds at least amino acid residues D119, N122 and Y125 in the sequence of SEQ ID NO:8. 4. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils. 5. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is capable of binding alpha-synuclein in monomer form and in fibril form. 6. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has a higher binding affinity for alpha-synuclein in fibril form compared to alpha-synuclein in monomer form, characterized by a dissociation constant (K _D ) that is at least 10 times higher in relation to monomeric alpha-synuclein than in relation to alpha-synuclein in the form of fibrils. 7. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has a (K _D ) value for alpha-synuclein in fibrils of 60 pM or less. 8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a chimeric, humanized, or human antibody. 9. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a full length antibody. 10. An antibody or antigen-binding fragment thereof according to claim 9, wherein the full length antibody is selected from IgG1, IgG4, or IgG4P. 11. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ , scFv, dAb or V _HH . 12. 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. the light chain variable region of SEQ ID NO:13, and heavy chain variable region of SEQ ID NO:25; or b. the light chain variable region of SEQ ID NO:17, and heavy chain variable region of SEQ ID NO:25; or c. the light chain variable region of SEQ ID NO:21, and heavy chain variable region of SEQ ID NO:25. 13. The antibody or antigen-binding fragment according to any one of claims 1-11, where the antibody or antigen-binding fragment contains: a. a light chain of SEQ ID NO:14, and the heavy chain of SEQ ID NO:26; or b. a light chain of SEQ ID NO:18, and the heavy chain of SEQ ID NO:26; or c. a light chain of SEQ ID NO:22, and the heavy chain of SEQ ID NO:26. 14. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-13. 15. The isolated polynucleotide according to claim 14, where the polynucleotide encodes: a. the variable region of the light chain, where the polynucleotide: i. at least 90% identical to the sequence of SEQ ID NO:15, 19 or 23; or ii. contains SEQ ID NO:15 or 19 or 23; or iii. consists essentially of SEQ ID NO:15, 19 or 23; or b. heavy chain variable region, where the polynucleotide: i. at least 90% identical to the sequence of SEQ ID NO:27; or ii. contains SEQ ID NO:27; or iii. consists mainly of SEQ ID NO:27; or c. light chain, where the polynucleotide: i. at least 90% identical to the sequence of SEQ ID NO:16, 20 or 24; or ii. contains SEQ ID NO:16, 20 or 24; or iii. consists essentially of SEQ ID NO:16, 20 or 24; d. heavy chain, where the polynucleotide: i. at least 90% identical to the sequence of SEQ ID NO:28; or ii. contains SEQ ID NO:28; or iii. consists mainly of SEQ ID NO:28. 16. A cloning vector containing one or more polynucleotides according to any one of claims 14, 15. 17. An expression vector containing one or more polynucleotides according to any one of claims 14, 15. 18. A host cell for producing an antibody according to any one of claims 1-13, containing: a. one or more polynucleotides according to any one of claims 14 or 15 or b. one or more expression vectors according to claim 16 or 17. 19. A method for producing an antibody or antigen-binding fragment according to any one of claims 1-13, including culturing the host cell according to claim 18 under suitable conditions to produce an antibody or antigen-binding fragment thereof, and isolation of the antibody or its antigen-binding fragment. 20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for use in therapy. 21. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for use in the therapy of Parkinson's disease (PD). 22. A method of treating Parkinson's disease (PD) in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-13. 23. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for use in the diagnosis of Parkinson's disease.

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