EA041378B1 - ANTIBODIES TO ALPHA-SYNUCLEIN - Google Patents

Description

Field of technology to which the invention relates

The invention relates to antibodies to alpha-synuclein and a method for using them to treat synucleinopathies. In particular, the present invention relates to antibodies to human alpha-synuclein and their use to treat Parkinson's disease.

State of the art

Alpha-synuclein is a small, soluble protein of 140 amino acids that exists in many different forms. Alpha-synuclein is primarily found in presynaptic nerve terminals, and although its exact function is unknown, researchers believe it plays a central role in numerous 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 increases in the number of copies of the alpha-synuclein gene cause the development of a familial form of early-onset Parkinson's disease (PD). Interestingly, in families with increased copy numbers of gene loci, the pathogenic effect is clearly dependent on the gene dosage. Gene duplications cause the development of a relatively early form of PD (~47 years) with a normal course of the disease, while gene triplications 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 component of Lewy bodies, the key pathological hallmark of the disease.

Lewy body pathology spreads during the course of the disease and it is proposed that alphasynuclein acts as a misfolded prion-like protein, resulting in the formation of toxic oligomers and aggregates that can spread from affected to unaffected neurons (Olanow C.W. et al., Movement Disorders, Vol. 28, No. 1, 2013). Current treatments are unable to stop the spread of the disease and only help in treating symptoms associated with the progressive loss of motor neuron-dependent activities. In 2014, Tran H.T. et al. (Tran H.T. et al., Cell Reports 7, 2054-2065, 2014) showed that intraperitoneal administration of a monoclonal antibody to misfolded alpha-synuclein to mice that had previously received an intrastriatal injection of preformed alpha-synuclein fibrils resulted in decreased Lewy body pathology, reduced the loss of substantia nigra dopaminergic neurons, and attenuated motor impairment. Therefore, there remains a need for passive immunotherapy that could be therapeutically effective in PD and other alpha-synucleinopathies.

The essence of the invention

The present invention satisfies the above-defined need by providing 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 comprises a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 comprising SEQ ID NO: 2; and iii. CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising:

i. CDR-H1 comprising SEQ ID NO: 4;

ii. CDR-H2 comprising SEQ ID NO: 45; and iii. CDR-H3 comprising SEQ ID NO: 46.

Embodiment 2. The antibody or antigen-binding fragment thereof according to embodiment 1, which binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131; wherein the epitope optionally comprises A124 and G132.

Embodiment 3:3. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody or antigen-binding fragment prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils.

Embodiment 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-3, wherein the antibody or antigen-binding fragment thereof is capable of binding alphasynuclein as a monomer and in fibrils.

Embodiment 5. 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 compared to alpha-synuclein in monomeric form, characterized by a dissociation constant (KD) that is at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

Embodiment 6. The antibody or antigen-binding fragment thereof according to any of the preceding embodiments with a KD value (KD) to alpha-synuclein in fibrils of 300 pM or less.

Embodiment 7. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, which does not bind to beta-synuclein and/or gamma-synuclein.

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

Embodiment 9. The antibody of any of the preceding embodiments, which is a full-length antibody.

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

Embodiment 11. An antigen-binding fragment thereof according to any one of embodiments 1-8, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, scFv, dAb or VHH.

Embodiment 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. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SeQ ID NO: 4; a CDR-H2 comprising SeQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; and

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

Embodiment 13. An antibody or antigen-binding fragment thereof according to any one of claims 111, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; and

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 23; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 25.

Embodiment 14. An antibody or antigen-binding fragment thereof according to any one of claims 1-

11, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SeQ ID NO: 4; a CDR-H2 comprising SeQ ID NO: 8, and a CDRH3 comprising SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 27 or 35; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 29 or 37.

Embodiment 15. An antibody or antigen-binding fragment thereof according to any one of claims 1-

11, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 7, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 23 or 31; or

c. a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 25 or 33.

Embodiment 16. An antibody or antigen-binding fragment thereof according to any one of claims 1-

11, wherein the antibody or antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 7, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SeQ ID NO: 4; a CDR-H2 comprising SeQ ID NO: 8, and a CDRH3 comprising SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 27 or 35; or

c. a light chain comprising SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 29 or 37.

Embodiment 17. An antibody or antigen-binding fragment thereof that:

a. competes for binding of alpha-synuclein with the antibody or antigen-binding fragment thereof of any of the preceding claims; and/or

b. cross-blocks or is cross-blocked with respect to alpha-synuclein binding by the antibody or antigen-binding fragment thereof of any of the preceding claims; and/or

- 2 041378

c. binds to the same epitope of alpha-synuclein as the antibody or antigen-binding fragment thereof of any of the preceding claims; and/or

d. comprises a heavy chain variable region having at least 80% identity or similarity to the sequence set forth in SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 27, or

SEQ ID NO: 35; and/or

e. comprises a light chain variable region having at least 80% identity or similarity to the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 19.

Embodiment 18. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of embodiments 1-16.

Embodiment 19. An isolated polynucleotide according to embodiment 18, encoding:

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

i. is at least 90% identical to SEQ ID NO: 16 or SEQ ID NO: 20; or ii. comprises SEQ ID NO: 16 or 20; or iii. consists essentially of SEQ ID NO: 16 or SEQ ID NO: 20;

b. a heavy chain variable region, where the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or ii. comprises SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or iii. consists essentially of SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36;

c. light chain, where the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 22; or ii. comprises SEQ ID NO: 18 or 22; or iii. consists essentially of SEQ ID NO: 18 or SEQ ID NO: 22;

d. heavy chain, where the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or ii. comprises SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or iii. consists essentially of SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38.

Embodiment 20. A cloning or expression vector comprising one or more polynucleotides according to any one of embodiments 18 or 19.

Embodiment 21. A host cell comprising:

a. one or more polynucleotides of any one of embodiments 18 or 19; or

b. one or more expression vectors according to embodiment 20.

Embodiment 22. A method for producing an antibody or antigen-binding fragment thereof according to any one of embodiments 1-17, comprising culturing a host cell according to embodiment 21 under conditions suitable for producing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

Embodiment 23. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1-17 and one or more pharmaceutically acceptable carriers, excipients or diluents.

Embodiment 24. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-17 or a pharmaceutical composition according to embodiment 23 for use in therapy.

Embodiment 25. The antibody or antigen-binding fragment thereof of any one of embodiments 1-17 or the pharmaceutical composition of embodiment 23 for use in the treatment of one or more synucleinopathies.

Embodiment 26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the synucleinopathies are selected from 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation type 1 (NBIA-1).

Embodiment 27. Use of an antibody or antigen-binding fragment thereof according to embodiment 26, wherein the synucleinopathy is Parkinson's disease.

Embodiment 28. A method of treating synucleinopathy 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 embodiments 1-17 or a pharmaceutical composition according to embodiment 23.

Embodiment 29. The method of embodiment 29, wherein the synucleinopathy is selected

- 3 041378 from 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with brain iron accumulation type 1 (NBIA-1), preferably Parkinson's disease.

Embodiment 30. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-16 for use in diagnosing alpha-synucleinopathies, preferably diagnosing Parkinson's disease.

Brief description of the figures

Fig. 1. (A) SDS-PAGE of alpha-synuclein expression patterns. Alpha-synuclein with His-tag (1) and after His-tag removal with TEV protease (2), size exclusion chromatography on Superdex 75 of human alpha-synuclein treated with TEV protease (3). Protein molecular weight marker SeeBluePlus2 (Invitrogen) (M). (B) SDS-PAGE of human alpha-synuclein purified from Expi293 supernatant as untagged wild-type protein. (4) Protein molecular weight marker SeeBluePlus2 (Invitrogen) (M).

Fig. 2. (A) JC-1 assay analysis of fibrils of monomer without fluorescence and fibrils with maximum fluorescence at 540 nm. (B) Representative example of the random coil spectrum of monomeric human alpha-synuclein (wavelength 1646 cm ^-1 ) and the inner β-sheet formed in fibrils of recombinant human alpha-synuclein (wavelength 1625-1630 cm ^-1 ).

Fig. 3. ELISA binding assay. ELISA binding of rabbit IgG1 6470 to recombinant human alpha-synuclein monomer and fibrils and human alpha-synuclein peptide PVDPDNEAYE.

Fig. 4. (A) Western blot showing binding of rabbit IgG1 6470 to human alpha-synuclein and human beta-synuclein. 1, human alpha-synuclein; 2, human alpha-synuclein (tPeptide); 3, human beta-synuclein (rPeptide); Marker, MagicMark XP. (B) NMR chemical shift changes revealing the predicted epitope of 6470 on human alpha-synuclein.

Fig. 5. Inhibition of IgG 6470 binding to immobilized alpha-synuclein (left columns, monomer and regular fibrils, respectively, of each of the tested peptides).

Fig. 6. Schematic representation of Fab 6470 in complex with peptide 123-132.

Fig. 7. Schematic representation of the contacts of the heavy chain of Fab 6470 with the peptide 123-132. Peptide residues are labeled directly, variable residues of the heavy chain of 6470 are labeled with the vH residue number.

Fig. 8. Schematic representation of the contacts of the light chain of Fab 6470 with the peptide 123-132. Peptide residues are labeled directly, residues of the variable region of the light chain of 6470 are labeled with the vL-residue number.

Fig. 9. Humanization of the light chain. 6470 denotes the variable region sequence of rabbit light chain. 6470gL3 denotes a humanized graft of the variable region of 6470 light chain using the human germline IGKV1-16 as an acceptor scaffold. CDRs are shown in bold/underlined. Donor residues are shown in bold/italic and shaded: Q48 and Q72. The mutation in CDRL1 N33R is shown in bold/underlined and shaded.

Fig. 10. Humanization of the heavy chain. 6470 denotes the variable region sequence of rabbit heavy chain. 6470gH23 and gH36 denote humanized grafts of the variable region of the heavy chain of antibody 6470 using the human germline IGHV3-23 as an acceptor scaffold. CDRs are shown in bold/underlined. Donor residues are shown in bold/italic and shaded: V24, Y47, I48, G49, S73, V78, and R97. The S56N and N102H mutations in CDRH2 and CDRH3, respectively, are shown in bold/underlined and shaded.

Fig. 11. Air-liquid interface stress of 6470 antibodies and mutants in three pre-prepared buffers after 3 and 24 h of shaking.

Fig. 12. Immunohistochemistry. Immunoreactivity in brain sections from patients with (A–E) and without PD (F–H). (A–C) In the temporal cortex of PD patients, antibody 6470 stained neuropil and Lewy body-like structures (white arrows) in the gray matter. (D, E) antibody 6470 stained Lewy body-like structures (white arrows) in the substantia nigra of PD patients. (F, G) In temporal cortex tissues without PD, 6470 also stained neuropil but did not stain Lewy body-like structures. (H) No Lewy body-like structures were observed in the substantia nigra of a non-PD individual; black arrows indicate nonspecific staining. Scale bar: 50 μm.

Fig. 13. Cell-based aggregation assay (HEK cells). The antibodies of the present invention are capable of inhibiting alpha-synuclein fibril-induced aggregation with an _IC50 of less than 5 nM. Error bars represent the standard error of measurement (SEM, N=3, n=9). In the legend, FL at the end of each antibody name stands for full-length.

Fig. 14. Cell-based aggregation assay (primary neurons). Representative antibody for

- 4 041378 the present invention is capable of inhibiting alpha-synuclein aggregation induced by alpha-synuclein fibrils in primary mouse neurons expressing endogenous levels of alpha-synuclein with an IC ₅₀ of less than 4 nM. Error bars represent the standard error of measurement (SEM, N=4, n=18).

Fig. 15. Immunohistochemical staining images of alpha-synuclein pathology (arrows) in different brain regions of wild-type C57B1/6J mice (A) and SNCA-OVX mice (B) injected with murine or human PFF, respectively.

Fig. 16. Quantification of pathological alpha-synuclein in different brain regions of wild-type C57B1/6J mice (A: cerebral cortex; B: striatum; C: amygdala; and D: substantia nigra) injected with murine PFF.

Fig. 17. Pharmacokinetic profiles of antibodies to alpha-synuclein: A. antibody 6470 in wild-type mouse; B. antibody 6470 and reference antibody in cynomolgus macaques.

Detailed description of the invention

The present invention is described below with reference to specific non-limiting aspects and embodiments thereof and with reference to certain drawings and examples.

Technical terms are used according to their common sense, unless otherwise indicated. Where a specific meaning is conveyed by certain terms, the definitions of the terms are given in the context in which they are used.

When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" should be considered as a preferred variant of the term "comprising".

When the singular is used, the plural of that noun is also included unless otherwise noted.

As used in the present description, the terms treatment, treating, etc. refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in the sense of partially or completely preventing the development of a disease or its symptom and/or may be therapeutic in the sense of partially or completely curing the disease and/or an adverse effect associated with this disease. Thus, treatment covers any treatment of a disease in a mammal, especially in a human, and includes:

(a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed as having the disease;

(b) inhibition of the disease, i.e. stopping its progression; and (c) alleviation of the disease, i.e. causing regression of the disease.

A therapeutically effective amount refers to an amount of an alpha-synuclein antibody or antigen-binding fragment thereof that, when administered to a mammal or other subject for the purpose of treating a disease, is sufficient to effect such treatment of the disease. A therapeutically effective amount will vary depending on the alpha-synuclein antibody or antigen-binding fragment thereof, the disease and its severity, and the age, weight, etc., of the subject being treated.

As used herein, the term "isolated" means that the antibody, antigen-binding fragment, or polynucleotide, as the case may be, exists in a physical environment different from that in which it may be found in nature. The present invention relates to an antibody or antigen-binding fragment thereof that binds to alpha-synuclein, wherein the antibody comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46.

In SEQ ID NO: 44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless of this, in SEQ ID NO: 45, Xaa is serine (Ser; S) or asparagine (Asn; N), and in SEQ ID NO: 46, Xaa is asparagine (Asn; N) or histidine (His; H).

In one embodiment, Xaa of SEQ ID NO: 44 and 46 is asparagine and Xaa of SEQ ID NO: 45 is serine.

In one embodiment, an antibody or antigen-binding fragment thereof that binds to alpha-synuclein comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; And

- 5 041378 iii. CDR-L3 comprising SEQ ID NO: 3; and

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6.

Alpha-synuclein (or alpha-syn; α-synuclein; α-syn or any other known synonym) refers to the generic name of this protein and includes, without limitation, alternative splice variants, mutants, and alpha-synuclein from other species (mouse, monkey, etc.). Unless otherwise indicated, when human alpha-synuclein is referred to or when explicitly listed, such alpha-synuclein comprises the sequence set forth in SEQ ID NO: 10 or in Uniprot P37840.

In the context of the present description, the term antibody generally refers to intact (whole) antibodies, i.e. comprising elements of two heavy chains and two light chains. An antibody may also comprise additional binding domains, for example as in the DVD-Ig molecule described in WO 2007/024715 or in the so-called (FabFv) ₂ Fc described in WO 2011/030107. Thus, an antibody as used in the present invention includes bi-, tri- or tetravalent full-length antibodies.

Antigen-binding fragments of antibodies include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., V _H or V _L or V _HH ), scFv, bi-, tri-, or tetravalent antibodies, Bis-scFv, diabodies, tribodies, triabodies, tetrabodies, and epitope-binding fragments of any of the foregoing (see, e.g., 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 producing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first described in WO 2009/040562, and its disulfide-stabilized version, Fab-dsFv, was first described in WO 2010/035012. Other antibody fragments useful in the present invention include the Fab and Fab' fragments described in International Patent Applications WO 2005/003169, WO 2005/003170 and WO 2005/003171. Polyvalent antibodies may have multiple specificities, for example they may be bispecific or they may be monospecific (see, for example, WO 92/22583 and WO 05/113605). One example of the latter is tri-Fab (or TFM), described in WO 92/22583.

An alternative antigen-binding fragment comprises a Fab linked to two scFvs or dsscFvs, wherein each scFv or dsscFv binds to the same or different targets (e.g., one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that increases half-life by binding, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO 2015/197772, which is incorporated herein by reference in its entirety and is specifically referred to in the context of discussing antibody fragments.

A typical Fab' molecule comprises a pair of heavy and light chains, wherein the heavy chain comprises a variable region VH, a constant domain CH1 and a natural or modified hinge region, and the light chain comprises a variable region VL and a constant domain CL. The Fab' dimers of the present invention form F(ab') ₂ , in which dimerization can be accomplished, for example, via the hinge region.

The antibody or antigen-binding fragment thereof of the present invention binds to an epitope of alpha-synuclein.

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

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46, and binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In SEQ ID NO: 44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless of this, in SEQ ID NO: 45, Xaa is serine (Ser; S) or asparagine (Asn; N), and in SEQ ID NO: 46, Xaa is asparagine (Asn; N) or histidine (His; H).

In one embodiment, Xaa in SEQ ID NO: 44 and 46 is asparagine and Xaa in SEQ ID NO: 45 is serine.

In one embodiment, an antibody or antigen-binding fragment thereof that binds to alpha-synuclein comprises:

a. a light chain variable region comprising:

- 6 041378

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6, and binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In the context of the present invention, the term epitope is used interchangeably for both conformational and linear epitopes, where a conformational epitope consists of discontinuous regions of the primary amino acid sequence of an antigen, and a linear epitope is formed by a sequence of continuously successive amino acids.

An epitope can be identified by any suitable epitope mapping method known in the art in combination with any 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 determining the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognized by the antibody. Alpha-synuclein peptides can be produced synthetically or by proteolytic cleavage of the alpha-synuclein protein. Peptides that bind to the antibody can be identified, for example, by mass spectrometric analysis. In another example, NMR spectroscopy or X-ray crystallography can be used to identify an epitope bound by an antibody of the present invention. Typically, when an epitope is determined by X-ray crystallography, amino acid residues of the antigen within 4A of the CDR are considered to be the amino acid residues comprising the epitope. Once identified, the epitope can be used to produce fragments that bind to the antibody of the present invention and, if necessary, can be used as an immunogen to produce additional antibodies that bind to the same epitope.

In one embodiment, the epitope of the antibody or antigen-binding fragment thereof is determined by X-ray crystallography using an alpha-synuclein peptide comprising residues 123-132 of SEQ ID NO: 10.

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

In this particular context, the term prevent (and grammatical variations thereof) is used herein interchangeably with the term inhibit and refers to the effect exerted by the antibody of the present invention on alpha-synuclein aggregation induced by alpha-synuclein fibrils. This effect may be prophylactic in terms of completely or partially preventing aggregation; or completely or partially reducing, i.e. blocking, aggregation that has already begun after further progression, or completely or partially reducing the likelihood of further aggregation occurring; or completely or partially reversing aggregation that has already occurred.

Without being limited by any theory, it is believed that the antibody or antigen-binding fragment thereof of the present invention binds to alpha-synuclein:

i) in monomeric form and prevents the formation of alpha-synuclein oligomers and aggregates; and/or ii) in oligomeric and fibrillar form and prevents the spread of alpha-synuclein from neuron to neuron; and/or iii) in oligomeric and/or fibrillar form and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, preferably endogenous alpha-synuclein aggregation.

In the context of the present invention, the term fibrils, fibrillar form or in fibrils, when used in relation to alpha-synuclein, is intended to refer to non-monomeric forms of alpha-synuclein, including oligomers of alpha-synuclein, which may be forms that are distributed within and between brain structures.

Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

- 7 041378 iv. CDR-H1, containing SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46, prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils. Preferably, the antibody or antigen-binding fragment thereof binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In SEQ ID NO: 44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless of this, in SEQ ID NO: 45, Xaa is serine (Ser; S) or asparagine (Asn; N), and in SEQ ID NO: 46, Xaa is asparagine (Asn; N) or histidine (His; H).

In one embodiment, Xaa in SEQ ID NO: 44 and 46 is asparagine and Xaa in SEQ ID NO: 45 is serine.

In one embodiment, an antibody or antigen-binding fragment thereof that binds to alpha-synuclein comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6, and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils. Preferably, the antibody or antigen-binding fragment thereof binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

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

In one embodiment, an antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) for monomeric alpha-synuclein of less than 15 nM. In another embodiment, an antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) for alpha-synuclein in fibrils of less than 10 nM. In one preferred embodiment, an antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) for alpha-synuclein in fibrils of less than 300 pM.

As used herein, the term KD refers to a dissociation constant that is derived from the ratio of Kd to Ka (i.e., Kd/K _a ), expressed as a molar concentration (M). Kd and Ka refer to the rate of dissociation and the rate of association, respectively, of a particular antigen-antibody (or antigen-binding fragment thereof) interaction. KD values for antibodies can be determined by methods well known in the art. One method for determining the KD of an antibody is to use surface plasmon resonance, such as with the Biacore® system, such as described in the examples herein, using isolated native or recombinant alpha-synuclein, a suitable fusion protein/polypeptide thereof, or fibrils thereof. In one example, affinity is measured using recombinant human alpha-synuclein, as described in the examples herein. To perform the surface plasmon resonance method, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase flowing along a flow cell. If a ligand binds to the immobilized target, the local refractive index of light changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting the change in the intensity of the reflected light. The rates of change of the SPR signal can be analyzed to obtain an apparent constant of the association and dissociation phase ratio of the binding reaction. The ratio of these values yields the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-65(1993)).

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has a higher binding affinity (i.e., a lower KD value) for alpha-synuclein in fibrils compared to alpha-synuclein in monomer form. The term affinity refers to the strength of the interaction between the antibody or antigen-binding fragment thereof with alpha-synuclein.

- 8 041378

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention has an IC ₅₀ of blocking alpha-synuclein aggregation induced by alpha-synuclein in fibrils of less than 10 nM, preferably the antibody or antigen-binding fragment thereof of the present invention has an IC ₅₀ of blocking alpha-synuclein aggregation induced by alpha-synuclein in fibrils of less than 5 nM. Examples of cell-based aggregation assays are disclosed in the examples.

As used herein, the term IC ₅₀ refers to the half-maximal inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a specific biological or biochemical function, aggregation according to the present invention, induced by alpha-synuclein, preferably alpha-synuclein in fibrils. IC50 is a quantitative measure that indicates what amount of a particular substance is needed to inhibit half of a given biological process.

In one embodiment, based on in vitro assays, the antibody or antigen-binding fragment thereof of the present invention has an IC ₅₀ for blocking alpha-synuclein aggregation induced by alpha-synuclein in fibrils of less than 10 nM, preferably the antibody or antigen-binding fragment thereof of the present invention has an IC ₅₀ for blocking alpha-synuclein aggregation induced by alpha-synuclein in fibrils of less than 5 nM.

The antibody or antigen-binding fragment thereof of the present invention does not bind to beta-synuclein and/or gamma-synuclein and is specific for alpha-synuclein.

The term "specific" as used herein is intended to refer to an antibody that recognizes only the antigen for which it is specific, or to an antibody that has a significantly higher binding affinity for the antigen for which it is specific (e.g., alpha-synuclein) than the level of binding to antigens for which it is not specific (gamma- and beta-synuclein), such as having at least a 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold higher binding affinity.

The antibodies of the present invention can be produced by any suitable method known in the art. Fusion proteins comprising a polypeptide/alpha-synuclein protein, cells (recombinantly or naturally) expressing the polypeptide can be used to produce antibodies that specifically recognize alpha-synuclein. The polypeptide can be a mature polypeptide or a biologically active fragment or derivative thereof.

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

Polypeptides used to immunize a host may be obtained by methods well known in the art from genetically engineered host cells containing expression systems, or they may be obtained from natural biological sources. In this application, the term polypeptides includes peptides, polypeptides, and proteins. They are used interchangeably unless otherwise indicated. In some cases, an alpha-synuclein polypeptide or fragment thereof may be part of a larger protein, such as a fusion protein, for example, fused to an affinity or similar tag.

Engineered antibodies to alpha-synuclein polypeptide can be prepared in cases where immunization of an animal is desired by administering the polypeptides to an animal, e.g., an animal, preferably a non-human animal, according to well-known and routine protocols (see, e.g., 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 generally the most suitable.

Monoclonal antibodies can be produced by any method known in the art, such as the hybridoma method (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma method, the human B-cell hybridoma method (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan RLiss, Inc., 1985).

Antibodies for use in the invention may also be prepared using techniques for generating antibodies from a single lymphocyte by cloning and expressing immunoglobulin variable region cDNA obtained from individual lymphocytes selected for the production of specific antibodies, for example, by the methods described by Babcook J. et al., 1996, Proc. Natl. Acad. Sci. USA 93 (15): 7843-78481; WO 92/02551; WO 2004/051268 and WO 2004/106377.

Antibody screening can be performed using assays that measure binding to alpha-synuclein and/or assays that measure inhibition of alpha-synuclein fibril formation in the presence of the antibody or antibody fragment.

The antibody or antigen-binding fragment thereof of the present invention comprises complementarity determining regions (CDRs), three from the heavy chain and three from the light chain. Typically,

The CDRs are located in the framework region and together form the variable region. By convention, the CDRs in the variable region of an antibody heavy chain or its antigen-binding fragment are referred to as

CDR-H1, CDR-H2, and CDR-H3, and in the light chain variable regions, CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially from the N-terminus to the C-terminus of each chain.

CDRs are typically numbered according to the system developed by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter Kabat et al. (see above)). This numbering system is used in the present description unless otherwise indicated.

Kabat residue designations do not always correspond directly to the linear amino acid residue numbering. The actual linear amino acid sequence may contain fewer or additional amino acid residues relative to the strict Kabat numbering, corresponding to a truncation of or insertion into a structural component (either a framework region or a complementarity determining region (CDR)) of the core variable domain structure. The correct Kabat residue numbering can be determined for a given antibody by aligning homologous residues in the antibody sequence relative to a standard Kabat numbered sequence.

The CDRs of the variable domain of the heavy chain 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 the Chothia numbering system (Chothia C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)) the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, in the context of the present disclosure, CDR-H1 refers to residues 26-35 according to the combined Kabat numbering system with the Chothia topological definition of the loop.

The CDRs of the light chain variable domain 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 preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3, and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 5, and a CDR-H3 comprising SEQ ID NO: 6.

Alternatively, the antibody or antigen-binding fragment comprises a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1; CDR-L2 comprising SEQ ID NO: 2, and CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; CDR-H2 comprising SEQ ID NO: 8, and CDR-H3 comprising SEQ ID NO: 9.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 7; CDR-L2 comprising SEQ ID NO: 2, and CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; CDR-H2 comprising SEQ ID NO: 5, and CDR-H3 comprising SEQ ID NO: 6.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 7; CDR-L2 comprising SEQ ID NO: 2, and CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; CDR-H2 comprising SEQ ID NO: 8, and CDR-H3 comprising SEQ ID NO: 9.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention may comprise framework regions of the animal in which the antibody was produced. For example, if the antibody was produced in a rabbit, it will comprise the CDRs defined above and framework regions of a rabbit antibody, for example an antibody comprising a light chain variable region according to SEQ ID NO: 11 (the nucleotide sequence of which is shown in SEQ ID NO: 12) and a heavy chain variable region according to SEQ ID NO: 13 (the nucleotide sequence of which is shown in SEQ ID NO: 14).

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

Chimeric antibodies are usually produced by recombinant DNA techniques. The DNA can be modified by replacing the coding sequence of the human L and H chains with the corresponding non-human (e.g., murine) H and L constant regions (Morrison; PNAS 81, 6851 (1984)).

Human antibodies comprise variable regions of heavy or light chains or full-length heavy or light chains that are the product of or are derived from a particular germline sequence if the variable regions or full-length chains of the antibody are obtained from a system that utilizes human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest or screening a phage-displayed library of human immunoglobulin genes with the antigen of interest. A human antibody or fragment thereof that is the product of or is derived from a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest to the sequence (i.e., has the highest % identity) of the human antibody. A human antibody that is a product of or derived from a particular human germline immunoglobulin sequence may differ in amino acid residues from the germline sequence, for example, due to naturally occurring somatic mutations or the deliberate introduction of a site-directed mutation. However, the selected human antibody typically has an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene and that contains amino acid residues that identify the human antibody as being human when compared to the amino acid sequence of the germline immunoglobulin of another species (e.g., the mouse germline sequence). In some cases, a human antibody may have an amino acid sequence that is at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 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 have no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, 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 by several methods known to those skilled in the art. Human antibodies can be produced by the hybridoma technique using human myeloma or mouse-human heteromyeloma cell lines (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 of which utilizes human variable region repertoires (Winter G; (1994) Annu. Rev. Immunol. 12:433-455, Green L.L., (1999) J. Immunol. Methods 231:11-23).

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

Therefore, the antibody or its antigen-binding fragment binds to alpha-synuclein and contains:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46, wherein the antibody or antigen-binding fragment thereof is humanized.

Preferably, the humanized antibody or antigen-binding fragment thereof prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and, more preferably, binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In SEQ ID NO: 44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless of this, in SEQ ID NO: 45, Xaa is serine (Ser; S) or asparagine (Asn; N), and in SEQ ID NO: 46, Xaa is asparagine (Asn; N) or histidine (His; H).

In one embodiment, the humanized antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46,

- 11 041378 and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123,

Y125, E126, M127, P128, S129, E130 and E131, where in SEQ ID NO: 44 Xaa is asparagine (Asn;

N), in SEQ ID NO: 45 Xaa is serine (Ser; S), and in SEQ ID NO: 46 Xaa is asparagine (Asn N).

In a preferred embodiment, the humanized antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6, and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131.

As used herein, the term "humanized antibody" or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the heavy and/or light chain comprises one or more 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 onto the variable region framework of the heavy and/or light chain of an acceptor antibody (e.g., a human antibody). For review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR to the human antibody framework, only one or more specificity-determining residues from any of the CDRs described above are transferred (see, e.g., 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 in this application are transferred to the framework region of the human antibody. In another embodiment, only the specificity-determining residues from each of the CDRs described above in this application are transferred to the framework region of the human antibody.

Any suitable variable region acceptor framework sequence can be used for grafting CDRs, taking into account the class/type of donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.

Accordingly, the humanized antibody of the present invention has a variable domain comprising human acceptor framework regions and one or more CDRs as specifically provided herein. Thus, in one embodiment, there is provided a blocking humanized antibody that binds to alpha-synuclein, preferably human alpha-synuclein, wherein the variable domain comprises acceptor human framework regions and donor non-human CDRs.

Examples of human framework regions that can be used in the present invention include 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 available at http://www.imgt.org/ can be used.

In the 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, comprise composite chains having framework regions derived from different chains.

A suitable framework region for the light chain of the humanized antibody or antigen-binding fragment thereof of the present invention is obtained from the human germline IGKV1-16 JK4 having SEQ ID NO: 39, the nucleotide sequence of which is set forth in SEQ ID NO: 40.

A suitable framework region for the heavy chain of the humanized antibody or antigen-binding fragment thereof of the present invention is obtained from the human germline IGHV3-23 JH4 having the sequence set forth in SEQ ID NO: 41, the nucleotide sequence of which is set forth in SEQ ID NO: 42.

Accordingly, in one embodiment, there is provided a humanized antibody or antigen-binding fragment thereof comprising:

the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 7 for CDR-L1, the sequence set forth in SEQ ID NO: 2 for CDR-L2, and the sequence set forth in SEQ ID NO: 3

- 12 041378 for CDR-L3, wherein the light chain framework region is derived from the human germline IGKV116 JK4; and the sequence set forth in SEQ ID NO: 4 for CDR-H1, the sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 8 for CDR-H2, and the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 9 for CDR-H3, wherein the heavy chain framework region is derived from the human germline IGHV3-23 JH4.

In the humanized antibody or antigen-binding fragment thereof of the present invention, the framework regions may not have exactly the same sequences as the acceptor antibody. For example, unusual residues may be replaced by residues more commonly found in that class or type of acceptor chain. Alternatively, selected residues in the acceptor framework regions may be altered so that they correspond to residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such alterations should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions that may need to be altered is given in WO 91/09967.

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, there is provided a humanized antibody or antigen-binding fragment thereof, wherein at least residues at each of positions 48 and 72 of the light chain variable domain (referring to SEQ ID NO: 15 or 19) are donor residues, see, for example, the sequences SEQ ID NO: 15, 17, 19 and 21. Preferably, residue 48 of the light chain variable domain is glutamine, and/or residue 72 of the light chain variable domain is glutamine.

More preferably, both residues 48 and 72 in the humanized light chain variable region of the humanized antibody or antigen-binding fragment thereof of the present invention are glutamine.

In another embodiment, there is provided a humanized antibody or antigen-binding fragment thereof, wherein at least residues in each of positions 24, 47, 48, 49, 73 and 97 (referring to SEQ ID NO: 31 or 35) or 24, 47, 48, 49, 78 and 97 of the heavy chain variable domain (referring to SEQ ID NO: 23 and 27) are donor residues, see, for example, the sequences SEQ ID NO: 23, 25, 27, 29, 31, 33, 35 and 37.

Preferably, residue 24 of the variable domain of the heavy chain is valine, and/or residue 47 of the variable domain of the heavy chain is tyrosine, and/or residue 48 of the variable domain of the heavy chain is isoleucine, and/or residue 49 of the variable domain of the heavy chain is glycine, and/or residue 97 of the variable domain of the heavy chain is arginine, and/or residue 73 of the variable domain of the heavy chain is serine, and/or residue 78 of the variable domain of the heavy chain is valine.

Preferably, in the humanized heavy chain variable region of the present invention, residue 24 is valine, residue 47 is tyrosine, residue 48 is isoleucine, residue 49 is glycine, residue 73 is serine, and residue 97 is arginine. It is also preferable that, in the humanized heavy chain variable region of the humanized antibody or antigen-binding fragment thereof of the present invention, residue 24 is valine, residue 47 is tyrosine, residue 48 is isoleucine, residue 49 is glycine, residue 78 is valine, and residue 97 is arginine.

In one preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31.

In another embodiment, the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 23; or a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 27 or 35; or a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 23 or 31; or a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 27 or 35.

In one embodiment, the invention provides an antibody or antigen-binding fragment thereof comprising a sequence that is 80% similar to or identical to a sequence disclosed herein, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% similar to or identical to part or all of a relevant sequence, such as a variable domain sequence, a CDR sequence, or a variable domain sequence excluding the CDRs. In one embodiment, the relevant sequence is SEQ ID NO: 15. In one embodiment, the relevant sequence is SEQ ID NO: 23 or SEQ ID NO: 31.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to 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 set forth in SEQ ID NO: 15 or SEQ ID NO: 19 and/or 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 set forth in SEQ ID NO: 31, SEQ ID NO: 23, SEQ ID NO: 27, or SEQ ID NO: 35.

In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to 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 to or identical to the sequence set forth in SEQ ID NO: 15, wherein the antibody or antigen-binding fragment thereof has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 7 for CDR-L1, the sequence set forth in SEQ ID NO: 2 for CDR-L2, and the sequence set forth in SEQ ID NO: 3 for CDR-L3.

In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to 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 to or identical to the sequence set forth in SEQ ID NO: 31, and wherein the antibody or antigen-binding fragment thereof has the sequence set forth in SEQ ID NO: 4 for CDR-H1, the sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 8 for CDR-H2, and the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 9 for CDR-H3.

In the context of the present disclosure, identity indicates that in the aligned sequences, at any particular position, the amino acid residues are identical between these sequences. In the context of the present disclosure, similarity indicates that in the aligned sequences, at any particular position, the amino acid residues are of a similar type between these sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that can often be substituted 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 with basic (alkaline) side chains); aspartate and glutamate (amino acids with acidic side chains);

asparagine and glutamine (amino acids that have amide side chains); and cysteine and methionine (amino acids that have sulfur-containing side chains). Degrees of identity and similarity can be easily calculated (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, an antigen-binding fragment of the present invention can be, but is not limited to, a Fab, a modified Fab, a Fab', a modified Fab', F(ab') ₂ , Fv, single-domain antibodies (e.g., _VH or VL or _VHH ), scFv, dsscFv, bi-, tri- or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the foregoing (see, e.g., 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 producing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments useful in the present invention include the Fab and Fab' fragments described in WO 2005/003169, WO 2005/003170, and WO 2005/003171. Multivalent antibodies may have multiple specificities, such as being bispecific or monospecific (see, e.g., WO 92/22853, WO 05/113605, WO 2009/040562, and WO 2010/035012).

An alternative antigen-binding fragment comprises a Fab linked to two scFvs or dsscFvs, where each scFv or dsscFv binds to the same or different targets (e.g., one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that enhances

- 14 041378 half-life by binding, for example, to albumin). Such antibody fragments are described in International Patent Application Publication No. WO 2015/197772, which is incorporated herein by reference in its entirety, and particularly in the context of the discussion of antibody fragments.

In another embodiment, an antibody or antigen-binding fragment thereof of the present invention is part of an alpha-synuclein-binding fusion protein that comprises, for example, the fused antigen-binding fragments of the present invention, e.g., as a Fab or Fab' fragment, and one or two single-domain antibodies (dAbs) directly or indirectly linked thereto, e.g., as described in wO 2009/040562, WO 2010035012, WO 2011/030107, WO 2011/061492, and WO 2011/086091, each of which is incorporated herein by reference. In one embodiment, the fusion protein comprises two-domain antibodies, e.g., as paired variable heavy (VH) and variable light (VL) domains, optionally linked by a disulfide bond.

In one embodiment, the Fab or Fab' element of the fusion protein has a specificity that is the same or similar to the specificity of a single-domain antibody or antibodies. In one embodiment, the Fab or Fab' has a specificity for a single-domain antibody or antibodies, in other words, the fusion protein is multivalent. In one embodiment, the multivalent fusion protein of the present invention has an albumin binding site, for example, the VH/V _L pair of the protein provides an albumin binding site.

The constant region domains of the antibody molecule of the present invention, if present, may be selected taking into account the intended function of the antibody molecule and, in particular, the effector functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, the constant region domains of human IgG, especially the IgG1 and IgG3 isotypes, may be used if the antibody molecule is intended for therapeutic use and the effector functions of the antibody are required. Alternatively, the IgG2 and IgG4 isotypes may be used if the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required. It is understood that sequence variants of these constant region domains may also be used. For example, IgG4 molecules in which the serine at position 241 is replaced by proline, as described, for example, in Angal et al., may be used. (Angal et al., Molecular Immunology, 1993, 30(1), 105-108), referred to herein as IgG4P. In one embodiment, the antibody is a full-length antibody, preferably selected from IgG1 and IgG4 or IgG4P.

Accordingly, the present invention relates to a full-length humanized antibody that binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46, wherein the humanized antibody prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and preferably binds to an epitope of alpha-synuclein comprising, with reference to SeQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E13, wherein the epitope optionally comprises A124 and G132, wherein the antibody is an IgG4P isoform.

In SEQ ID NO: 44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless of this, in SEQ ID NO: 45, Xaa is serine (Ser; S) or asparagine (Asn; N), and in SEQ ID NO: 46, Xaa is asparagine (Asn; N) or histidine (His; H).

In one preferred embodiment, a full-length humanized antibody that binds to alpha-synuclein comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46, and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132, wherein the antibody is an IgG4P isoform, wherein in SEQ ID NO: 44 Xaa is

- 15 041378 is asparagine (Asn; N), in SEQ ID NO: 45 Xaa is serine (Ser; S) and in SEQ ID NO: 46, Xaa is asparagine (Asn N).

In a most preferred embodiment, a full-length humanized antibody that binds to alpha-synuclein comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6, and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

One skilled in the art will also appreciate that antibodies may undergo a variety of post-translational modifications. The type and extent of these modifications often depend 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 (alkaline) residue (such as lysine or arginine) by carboxypeptidases (as described in Harris R.J. Journal of Chromatography 705:129-134, 1995). Accordingly, the terminal lysine of the heavy chain of an antibody may be absent.

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

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

In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 23 or SEQ ID NO: 31. For example, the antibody can be a full-length IgG4 antibody comprising a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 23 or SEQ ID NO: 31. In another embodiment, the antibody is a full-length IgG4 antibody comprising a light chain set forth in SEQ ID NO: 17 and a heavy chain selected from SEQ ID NO: 25 or SEQ ID NO: 33. In yet another embodiment, the antigen-binding fragment is a Fab' comprising a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 23 or SEQ ID NO: 31.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35.

For example, the antibody is a full-length IgG4 antibody comprising a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35. In another embodiment, the antibody is a full-length IgG4 antibody comprising a light chain set forth in SEQ ID NO: 17 and a heavy chain selected from SEQ ID NO: 29 or SEQ ID NO: 37. In another embodiment, the antigen-binding fragment is a Fab' comprising a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 19 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35. For example, the antibody is a full-length IgG4 antibody comprising a light chain variable region set forth in SEQ ID NO: 19 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35. In another embodiment, the antibody is a full-length IgG4 antibody comprising a light chain set forth in SEQ ID NO: 21 and a heavy chain selected from SEQ ID NO: 29 or SEQ ID NO: 37. In yet another embodiment, the antigen-binding fragment is a Fab' comprising a light chain variable region set forth in SEQ ID NO: 19 and a heavy chain variable region selected from SEQ ID NO: 27 or SEQ ID NO: 35. a heavy chain region selected from SEQ ID NO: 27 or SEQ ID NO: 35.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 19 and a heavy chain variable region selected from SEQ ID NO: 23 or SEQ ID NO: 31. For example, the antibody is a full-length IgG4 antibody comprising a light chain variable region set forth in SEQ ID NO: 19 and a heavy chain variable region selected from SEQ ID NO: 23 or SEQ ID NO: 31. In another embodiment, the antibody is a full-length IgG4 antibody comprising a light chain set forth in SEQ ID NO: 21 and a heavy chain selected from SEQ ID NO: 25 or SEQ ID NO: 33. In yet another embodiment, the antigen-binding fragment is a Fab' comprising a light chain variable region set forth in SEQ ID NO: 25 or SEQ ID NO: 33. NO: 21, and a heavy chain variable region selected from SEQ ID NO: 25 or SEQ ID NO: 33.

In a preferred embodiment, the antibody binds to alpha-synuclein and is a full-length IgG4 antibody comprising a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31. More preferably, the antibody prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and even more preferably, the antibody binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In another preferred embodiment, the antibody binds to alpha-synuclein and is a full-length IgG4 antibody comprising a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33. More preferably, the antibody prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and even more preferably, the antibody binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In addition, the present invention also relates to an antibody or antigen-binding fragment thereof that competes for binding of alpha-synuclein with the antibody or antigen-binding fragment thereof of the present invention.

Therefore, the present invention relates to an antibody or antigen-binding fragment thereof that competes for binding to alpha-synuclein with the antibodies or antigen-binding fragments of the present invention by cross-blocking or becomes blocked by the antibody or antigen-binding fragment thereof of the invention; and, in particular, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 27 or SEQ ID NO: 35, and a light chain variable region comprising SEQ ID NO: 15 or SEQ ID NO: 19.

In another embodiment, the antibody or antigen-binding fragment thereof competes for binding to alpha-synuclein to the same epitope as an antibody or antigen-binding fragment thereof of the present invention, and in particular competes with an antibody or antigen-binding fragment thereof with a heavy chain variable region comprising SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 27 or SEQ ID NO: 35 and a light chain variable region comprising SEQ ID NO: 15 or SEQ ID NO: 19, and for binding to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, at least residues M127, P128, S129, E130 and E131, preferably residues E123, Y125, E126, M127, P128, S129, E130 and E131.

In one embodiment, such an antibody or antigen-binding fragment thereof competes with the antibodies or fragments thereof of the present invention and has a heavy chain variable region having at least 80% identity or similarity to the sequence according to SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 27 or SEQ ID NO: 35; and/or has a light chain variable region having at least 80% identity or similarity to the sequence according to SEQ ID NO: 15 or SEQ ID NO: 19.

Competing antibodies can be identified by any suitable method known in the art, such as by ELISA or BIAcore competition assays in which binding of a cross-blocking antibody to human alpha-synuclein prevents binding of an antibody of the invention, or vice versa. Isolated natural or recombinant alpha-synuclein or a suitable fusion protein/polypeptide can be used in such competition assays. In one example, competition is measured using recombinant human alpha-synuclein (SEQ ID NO: 10). In one example, recombinant human alpha-synuclein tagged at the N-terminus or C-terminus (e.g., fusion of a 6χHis tag to a TEV recognition site) is used in accordance with the examples provided herein. In another example, competition is measured using recombinant human alpha-synuclein fibrils.

In one embodiment, the competing antibodies are fully human or humanized. In one embodiment, the competing antibodies have an affinity for human alpha-synuclein of 100 μM or less, preferably 50 μM or less.

Biological molecules such as antibodies or fragments contain acidic and/or basic (alkaline) functional groups, giving the molecule a net positive or negative charge. The amount of net charge observed will depend on the absolute amino acid

- 17 041 378 sequence of the object, the local environment of the charged groups in the three-dimensional structure and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or its surface in an accessible solvent does not carry a net electrical charge. In one example, the alpha-synuclein antibody or antigen-binding fragment thereof of the present invention can be designed so that they have a suitable isoelectric point. This can produce antibodies and/or fragments with more robust properties, in particular with suitable solubility and/or stability profiles and/or improved purification characteristics.

Thus, in one aspect, the invention provides a humanized antibody or antigen-binding fragment thereof that binds to alpha-synuclein and that is engineered to have an isoelectric point that differs from that of the originally identified antibody. The antibody can, for example, be engineered by substituting an amino acid residue, such as substituting an acidic amino acid residue with one or more basic (alkaline) amino acid residues. Alternatively, basic amino acid residues can be introduced, or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI, acidic residues can be introduced to lower the pI, if desired. It is important to note that when manipulating the pI, care must be taken to maintain 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 "modified" antibody or fragment thereof.

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

It is understood that the affinity of the antibodies of the present invention can be altered by any suitable method known in the art. Accordingly, the present invention also relates to variants of the antibody molecules of the present invention that have improved affinity for alpha-synuclein, in particular human alpha-synuclein. Such variants can be generated by several affinity maturation protocols, including CDR mutation (Yang et al., J. Mol. Biol., 254, 392–403, 1995), strand shuffling (Marks et al., Bio/Technology, 10, 779–783, 1992), 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 sexual mimic PCR (Crameri et al., Nature, 391, 288–291, 1998). A discussion of these affinity maturation methods can be found in Vaughan et al. (see above).

In the context of the present invention, affinity maturation was performed according to IOTA (WO 2014198951).

If desired, the antibody or antigen-binding fragment thereof of the present invention may be conjugated to one or more effector molecules. It is understood that an effector molecule may comprise one effector molecule or two or more such molecules linked to form a single fragment that can be attached to the antibody or antigen-binding fragment thereof of the present invention. If it is desired to obtain an antibody fragment linked to an effector molecule, it can be obtained by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule either directly or through a linking agent. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2 ^nd 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 procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 03/031581. Alternatively, where the effector molecule is a protein or polypeptide, binding may be achieved using recombinant DNA procedures, such as those described in WO 86/01533 and EP 0392745.

In the context of the present description, the term effector molecule includes, for example, antitumor agents, drugs, toxins, biologically active proteins, such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides, in particular radioactive iodine, radioisotopes, chelating metals, nanoparticles and reporter groups, such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is harmful (e.g. kills) to a cell. Examples include combrestatins, dolastatins, epothilones, staurosporine, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin di- hydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologues.

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

Other effector molecules may include chelating radionuclides such as 111In and 90Y, Lu177, bismuth213, californium252, iridium192, and tungsten188/rhenium188; 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 exotoxin or diphtheria toxin, a protein such as insulin, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or an antiangiogenic agent such as angiostatin or endostatin, or a biological response modifier such as a 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 another growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful, for example, for diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents. 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, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; Suitable luminescent materials include luminol; Suitable bioluminescent materials include luciferase, luciferin, and aequorin; and Suitable radioactive nuclides include 125I, 131I, 111In, and 99Tc.

In another example, the effector molecule may increase the half-life of the antibody in vivo and/or reduce the immunogenicity of the antibody and/or improve the 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 the compounds described in WO 05/117984.

When the effector molecule is a polymer, it may typically be a synthetic or naturally occurring polymer, such as an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

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

Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or their derivatives.

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

In the context of the present description, the term derivatives includes reactive derivatives, for example, thiol-selective reactive groups such as maleimides, etc. The reactive group may be linked to the polymer directly or through a linker segment. It is understood that the remainder of such a group in some cases forms part of the product as a linking group between the antibody fragment and the polymer.

The polymer size can be varied as required, but is typically in the range of average molecular weight from 500 to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da.

- 19 041378

The size of the polymer may be selected in particular with regard to the intended use of the product, such as the ability to localize to specific tissues such as tumors, or the ability to increase the half-life in the bloodstream (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, when the product is intended to be cleared from the bloodstream and penetrate into tissues, such as for use in the treatment of a tumor, it may be preferable to use a polymer with a small molecular weight, such as one with a molecular weight of about 5,000 Da. For applications in which the product remains in the bloodstream, it may be preferable to use a polymer with a higher molecular weight, such as one with a molecular weight in the range of 20,000 to 40,000 Da.

Suitable polymers include a polyalkylene polymer such as poly(ethylene glycol) or, especially, methoxypoly(ethylene glycol) or a derivative thereof, and especially with a molecular weight in the range of from about 15,000 Da to about 40,000 Da.

In one example, an antibody or antigen-binding fragment thereof of the present invention is attached to poly(ethylene glycol) (PEG) moieties. In one specific embodiment, the antigen-binding fragment of the present invention and the PEG molecules may be attached via any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may be naturally occurring in the antibody fragment or may be introduced into the fragment using recombinant DNA techniques (see, e.g., US 5,219,996; US 5,667,425; WO 98/25971, WO 2008/038024). In one example, an antibody molecule of the present invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Accordingly, the additional amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can be attached. Several sites can be used to attach two or more PEG molecules.

Accordingly, the PEG molecules are covalently linked via a 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 a sulfur atom of a cysteine residue located on this fragment. The covalent bond is typically a disulfide bond or, in particular, a carbon disulfide bond. When a thiol group is used as an attachment point, suitably activated effector molecules can be used, for example, thiol-selective derivatives such as maleimides and cysteine derivatives can be used. The activated polymer can be used as a starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol reactive group such as a t-halocarboxylic acid or ester such as iodoacetamide, an imide such as maleimide, a vinyl sulfone, or a disulfide. Such starting materials are commercially available (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials by conventional chemical procedures. Particular 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, a Fab' fragment or a diFab that is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently bound thereto, for example according to the method disclosed in EP 0948544 or EP 1090037 [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 D.C. 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 a cysteine in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently linked to one thiol group in the modified hinge region. A lysine residue can be covalently linked to the maleimide group, and a methoxypoly(ethylene glycol) polymer of about 20,000 Da can be attached to each of the amino groups on the lysine residue. Thus, the total molecular weight of PEG attached to the Fab fragment can be about 40,000 Da.

Specific PEG molecules include 2-[3-(I-maleimido)propionamido]ethylamide N,Nbis(methoxypoly(ethylene glycol) MW 20,000) modified with lysine, also known as PEG2MAL40K (available from Nektar, formerly Shearwater).

Alternative sources of PEG linkers include NOF, which supplies GL2-4OOMA3 (where m in the structure below is 5) and GL2-400MA (where m is 2) and n is approximately 450

- 20 041378

In other words, each PEG has a size of approximately 20,000 Da.

Thus, in one embodiment, the PEG is 2,3bis(methylpolyoxyethyleneoxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propyloxy}hexane (di-PEG, _-CH2 )3NHCO( _CH2 )5-MAL, MW 40,000, known as SUNBRIGHT GL2-400MA3.

Other alternative PEG effector molecules are of the following type:

Available from 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 PEG as disclosed herein) linked via a cysteine amino acid residue at or near amino acid 226 of the chain, such as amino acid 226 of the heavy chain (sequential numbering), such as amino acid 223 of SEQ ID NO: 33.

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

The Fab'-PEG molecules of the present invention may be particularly preferred because they have a half-life independent of the Fc portion. In one embodiment, a Fab' is conjugated to a polymer, such as a PEG molecule, a starch molecule, or an albumin molecule. In one embodiment, an scFv is conjugated to a polymer, such as a PEG molecule, a starch molecule, or an albumin molecule. In one embodiment, a 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 half-life. Methods for conjugating starch to a protein are described in U.S. Patent No. 801,739, incorporated herein by reference.

The present invention also relates to an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof of the present invention. An isolated polynucleotide of the present invention may comprise synthetic DNA, such as that obtained by chemical treatment, cDNA, genomic DNA, or any combination thereof.

Standard molecular biology techniques can be used to obtain DNA sequences encoding the 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. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used if necessary.

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

a. a light chain variable region in which the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 16 or SEQ ID NO: 20; or ii. comprises SEQ ID NO: 16 or 20; or iii. consists essentially of SEQ ID NO: 16 or SEQ ID NO: 20;

b. a heavy chain variable region in which the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or ii. comprises SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or iii. consists essentially of SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36;

c. a light chain in which the polynucleotide:

- 21 041378

i. is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 22; or ii. comprises SEQ ID NO: 18 or 22; or iii. consists essentially of SEQ ID NO: 18 or SEQ ID NO: 22;

d. a heavy chain in which the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or ii. comprises SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or iii. consists essentially of SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38;

e. a light chain variable region in which the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 12; or ii. comprises SEQ ID NO: 12; or iii. consists essentially of SEQ ID NO: 12;

f. a variable region of the heavy chain in which the polynucleotide:

i. is at least 90% identical to SEQ ID NO: 14; or ii. comprises SEQ ID NO: 14; or iii. consists essentially of SEQ ID NO: 14.

In one embodiment, the present invention relates to an isolated polynucleotide encoding a heavy chain of a Fab' fragment of an antibody or an IgG1 or IgG4 antibody of the present invention, which comprises a sequence as set forth in SEQ ID NO: 24, 28, 32 or 36. Also provided is an isolated polynucleotide encoding a light chain of a Fab' fragment of an antibody or an IgG1 or IgG4 antibody of the present invention, which comprises a sequence as set forth in SEQ ID NO: 16 or 20.

In another embodiment, the present invention relates to an isolated polynucleotide encoding a heavy chain and a light chain of an IgG4(P) antibody of the present invention, wherein the polynucleotide encoding the heavy chain comprises a sequence as set forth in SEQ ID NO: 26, 30, 34, or 38, and the polynucleotide encoding the light chain comprises a sequence as set forth in SEQ ID NO: 18 or 22.

The present invention also relates to a cloning or expression vector comprising one or more polynucleotides disclosed 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 NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.

General methods by which vectors may be constructed, transfection methods, and culturing methods are well known to those skilled in the art. Reference is made in this regard to Current Protocols in Molecular Biology, 1999, F.M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Also 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 thereof of the present invention. Bacterial, such as E. coli, and other microbial systems can be used, or eukaryotic, such as mammalian, host cell expression systems can be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

Suitable types of Chinese hamster ovary cells (CHO cells) used in the present invention may include CHO and CHO-Ki cells, including dhfr-CHO cells such as CHODG44 cells and CHO-DXB11 cells, which can also be used with a DHFR-selectable marker, or CHOK1-SV cells, which can be used with a glutamine synthetase-selectable marker. Other cell types used for antibody expression include lymphocyte 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 expression vectors comprising the isolated polynucleotide sequences of the present invention, preferably comprising the isolated polynucleotide sequences of SEQ ID NOs: 18 and 26 or SEQ ID NOs: 18 and 34 or SEQ ID NOs: 18 and 30 or SEQ ID NOs: 18 and 38.

The present invention also relates to a method for producing an antibody or antigen-binding fragment thereof according to the present invention, comprising culturing a host cell according to the present invention under conditions suitable for producing the antibody or antigen-binding fragment thereof.

- 22 041378 fragment according to the invention, and isolation of the antibody or its antigen-binding fragment.

An antibody or antigen-binding fragment thereof may comprise only a heavy chain or a light chain polypeptide, in which case only the coding sequence for the heavy chain or light chain polypeptide need be used to transfect host cells. To produce antibodies or antigen-binding fragments thereof containing both heavy and light chains, a cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used that includes sequences encoding both light chain and heavy chain polypeptides.

Thus, a method is provided for culturing a host cell and expressing an antibody or a fragment thereof, isolating the latter and, optionally, purifying it to obtain an isolated antibody or fragment. In one embodiment, the method further comprises a step of conjugating an effector molecule to the isolated antibody or fragment, for example conjugation to a PEG polymer, in particular as disclosed in the present description.

Thus, in one embodiment, a purified alphasynuclein antibody or fragment thereof is provided, such as a humanized antibody or fragment thereof, in particular an antibody or fragment thereof according to the invention, which is substantially purified, in particular free or substantially free of endotoxin and/or host cell protein or DNA.

Substantially endotoxin free generally refers to an endotoxin content of 1 IU per mg of antibody product or less, such as 0.5 or 0.1 IU per mg of product.

Substantially free of host cell protein or DNA refers to a host cell protein and/or DNA content of 400 μg per mg of antibody product or less, such as 100 μg per mg or less, in particular 20 μg per mg, as appropriate.

Since the antibodies of the present invention are useful for the treatment, diagnosis and/or prevention of a pathological condition such as alpha-synucleinopathy, the present invention also relates to a pharmaceutical or diagnostic composition comprising an antibody or antigen-binding fragment thereof of the present invention in combination with one or more of a pharmaceutically acceptable carrier, excipient or diluent.

Preferably, the pharmaceutical or diagnostic composition comprises a humanized antibody that binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46.

More preferably, the pharmaceutical or diagnostic composition comprises a humanized antibody that binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is the only active ingredient. In another embodiment, the antibody or antigen-binding fragment thereof of the present invention is in combination with one or more additional active ingredients. Alternatively, the pharmaceutical compositions comprise an antibody or antigen-binding fragment thereof of the present invention that is the only active ingredient and that can be administered to a patient as a single agent in combination (e.g., 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: 15 or 19 and comprising the heavy chain variable region of SEQ ID NO: 23, 27, 31 or 35, for example, SEQ ID NO: 15 and SEQ ID NO: 23 or SEQ ID NO: 15 and SEQ ID NO: 31.

Preferably, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to alpha-synuclein and comprises the light chain variable region of SEQ ID NO: 15 and the heavy chain variable region of SEQ ID NO: 31.

- 23 041378

The pharmaceutical compositions of the invention can be suitably administered to a patient to determine the necessary therapeutically effective amount. As used herein, the term therapeutically effective amount refers to the amount of a therapeutic agent required to treat, improve or prevent a target disease or condition or to exhibit a detectable therapeutic or prophylactic effect. For any antibody, the therapeutically effective amount can be initially estimated using either cell culture assays or animal models, typically rodents, rabbits, dogs, pigs or primates. The animal model can also be used to determine a suitable concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans.

The precise therapeutically effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, drug combination(s), allergic reaction and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the skill of the physician. Typically, a therapeutically effective amount is from 0.01 to 500 mg/kg, such as from 0.1 to 200 mg/kg, such as 100 mg/kg. The pharmaceutical compositions can be presented in convenient unit dosage forms containing a predetermined amount of the active agent of the invention per dose.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in such compositions. Such carriers allow obtaining pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, gruels and suspensions for patient administration.

Suitable forms for administration include those suitable for parenteral administration, such as by injection or infusion, such as bolus injection or continuous infusion, intravenous, inhalation or subcutaneous administration. If the product is intended for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain agents used to prepare the appropriate dosage form, such as suspending, preserving, stabilizing and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof of the invention may be in dry form, which is reconstituted with an appropriate sterile liquid before use. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared.

Once the dosage form is prepared, the compositions of the invention can be administered directly to a subject. Accordingly, the present invention relates to the use of an antibody or antigen-binding fragment thereof 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.

Therefore, in another aspect, the present invention relates to an antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for use in therapy, wherein the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46.

Preferably, the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and more preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In a 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 that binds to alpha-synuclein and comprises:

i. CDR-L1 comprising SEQ ID NO: 1;

ii. CDR-L2 containing SEQ ID NO: 2; And

- 24 041378 iii. CDR-L3 comprising SEQ ID NO: 3; and

b. variable region of the heavy chain, containing:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 5; and vi. CDR-H3 comprising SEQ ID NO: 6.

Preferably, the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and more preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In particular, use in therapy includes use in the treatment of one or more alphasynucleinopathies.

In another aspect, the present invention relates to 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 thereof of the present invention or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

i. CDR-L1 comprising SEQ ID NO: 44;

ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And

b. a heavy chain variable region comprising:

iv. CDR-H1 comprising SEQ ID NO: 4;

v. CDR-H2 comprising SEQ ID NO: 45; and vi. CDR-H3 comprising SEQ ID NO: 46.

Preferably, the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and more preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In one preferred embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is intended for use in the treatment of one or more alpha-synucleinopathies, wherein the antibody or antigen-binding fragment thereof binds to alpha synuclein and comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

Preferably, the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and, more preferably, binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and G132.

In another preferred embodiment, the present invention relates to a method of treating one or more alpha-synucleinopathies in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

Preferably, the antibody or antigen-binding fragment thereof is humanized and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and more preferably binds to an epitope of alpha-synuclein comprising, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, wherein the epitope optionally comprises A124 and

- 25 041378

G132.

Alternatively, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is intended for use in the therapy or treatment of one or more alpha-synucleinopathies and is an antibody or antigen-binding fragment thereof comprising:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-L2 comprising SEQ ID NO: 2 and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5 or SEQ ID NO: 8 and a CDR-H3 comprising SEQ ID NO: 6 or SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 15 or 19 and a heavy chain variable region comprising SEQ ID NO: 23 or SEQ ID NO: 27 or SEQ ID NO: 31 or SEQ ID NO: 35; or

c. a light chain comprising SEQ ID NO: 17 or SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 25 or SEQ ID NO: 29 or SEQ ID NO: 33 or SEQ ID NO: 37.

In another embodiment of the present invention, a method of treating one or more alphasynucleinopathies in a patient comprises administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-L2 comprising SEQ ID NO: 2 and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5 or SEQ ID NO: 8 and a CDR-H3 comprising SEQ ID NO: 6 or SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 15 or 19 and a heavy chain variable region comprising SEQ ID NO: 23 or SEQ ID NO: 27 or SEQ ID NO: 31 or SEQ ID NO: 35; or

c. a light chain comprising SEQ ID NO: 17 or SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 25 or SEQ ID NO: 29 or SEQ ID NO: 33 or SEQ ID NO: 37.

Alpha-synucleinopathies according to 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), Lewy body variant Alzheimer's disease (LBVAD), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation 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 intended 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation type 1 (NBIA-1), preferably Parkinson's disease (PD), and is an antibody or antigen-binding fragment thereof comprising:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SeQ ID NO: 4; a CDR-H2 comprising SeQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

In another embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is intended 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and neurodegeneration with brain iron accumulation type 1 (NBIA-1), preferably Parkinson's disease (PD), and is an antibody or antigen-binding fragment thereof comprising:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-L2 comprising SEQ ID NO: 2 and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5 or SEQ ID NO: 8 and a CDR-H3 comprising SEQ ID NO: 6 or SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 15 or 19 and a variable region

- 26 041378 heavy chain comprising SEQ ID NO: 23 or SEQ ID NO: 27 or SEQ ID NO: 31 or SEQ ID NO: 35;

or

c. a light chain comprising SEQ ID NO: 17 or SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 25 or SEQ ID NO: 29 or SEQ ID NO: 33 or SEQ ID NO: 37.

In another embodiment, there is provided a method of treating 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with brain iron accumulation 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 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 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SeQ ID NO: 4; a CDR-H2 comprising SeQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

In another embodiment, a method of treating 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA), and brain iron accumulation neurodegeneration type 1 (NBIA-1), preferably Parkinson's disease (PD), in a patient, comprises administering to said patient a therapeutically effective embodiment 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 comprising SEQ ID NO: 1 or SEQ ID NO: 7; a CDR-L2 comprising SEQ ID NO: 2 and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5 or SEQ ID NO: 8 and a CDR-H3 comprising SEQ ID NO: 6 or SEQ ID NO: 9; or

b. a light chain variable region comprising SEQ ID NO: 15 or 19 and a heavy chain variable region comprising SEQ ID NO: 23 or SEQ ID NO: 27 or SEQ ID NO: 31 or SEQ ID NO: 35; or

c. a light chain comprising SEQ ID NO: 17 or SEQ ID NO: 21 and a heavy chain comprising SEQ ID NO: 25 or SEQ ID NO: 29 or SEQ ID NO: 33 or SEQ ID NO: 37.

Alternatively, the invention also relates to 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), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD), variant Alzheimer's disease with Lewy bodies (LBVAD), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with brain iron accumulation type 1 (NBIA-1), more preferably Parkinson's disease (PD), wherein the antibody or an antigen-binding fragment thereof comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

Also part of the present invention is the use of alpha-synuclein antibodies or antigen-binding fragments for use as diagnostically active agents or in diagnostic assays, for example for the diagnosis of alpha-synucleinopathies such as 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with brain iron accumulation type 1 (NBIA-1).

The diagnosis can preferably be made on biological samples. A biological sample covers a variety of sample types obtained from an individual and can be used in diagnostic testing or for monitoring. The definition covers cerebrospinal fluid,

- 27 041378 blood, such as plasma and serum, and other liquid samples of biological origin, such as urine and saliva, solid tissue samples, such as a biopsy or tissue culture sample or cells derived therefrom, and their progeny. The definition also includes samples that have been manipulated in any way after their collection, such as treatment with reagents, solubilization or enrichment for specific components, such as polynucleotides.

Diagnostic testing may preferably be performed on biological samples that are not in contact with the human or animal body. Such diagnostic testing is also called in vitro testing. In vitro diagnostic testing may be based on an in vitro method for detecting alpha-synuclein in a biological sample that has been obtained from an individual, comprising the steps of:

i) contacting a biological sample with an alpha-synuclein antibody or antigen-binding fragment thereof disclosed herein; and ii) detecting binding of the alpha-synuclein antibody or antigen-binding fragment thereof to alpha-synuclein.

By comparing the detected level of alpha-synuclein or the presence of a particular post-translationally modified form of alpha-synuclein with a suitable control, one or more alpha-synucleinopathies can be identified. Thus, such a detection method can be used to determine whether a subject suffers from or is at risk of developing alpha-synucleinopathies, including determining the stage (severity) of alpha-synucleinopathies.

Therefore, the present invention relates to an antibody or antigen-binding fragment thereof for use in the diagnosis of alpha-synucleinopathies, preferably for the diagnosis of or in the presence of Parkinson's disease, wherein the antibody or antigen-binding fragment thereof binds to alpha-synuclein and comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 44, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 45, and a CDRH3 comprising SEQ ID NO: 46.

Preferably, the antibody or antigen-binding fragment thereof for use in diagnosing alpha-synucleinopathies, preferably in diagnosing or in the presence of Parkinson's disease, binds to alpha-synuclein and comprises:

a. a light chain variable region comprising a CDR-L1 comprising SEQ ID NO: 1, a CDR-L2 comprising SEQ ID NO: 2, and a CDR-L3 comprising SEQ ID NO: 3; and a heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO: 4; a CDR-H2 comprising SEQ ID NO: 5, and a CDRH3 comprising SEQ ID NO: 6; or

b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or

c. a light chain comprising SEQ ID NO: 17 and a heavy chain comprising SEQ ID NO: 33.

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

Table 1

Name SEQ ID NO: Subsequence CDR-L1 1 QASQSVYKNNYLA CDR-L2 2 GASTLAS CDR-L3 3 AGYKGGRNDGFA CDR-H1 4 GIDLSSHDMY CDR-H2 5 AIYASGSTYYASWAKG CDR-H3 6 IHYGNSGGL CDR-L1 N33R 7 QASQSVYKNRYLA CDR-H2 S56N 8 AIYASGNTYYASWAKG CDR-H3 N102H 9 IHYGHSGGL Human alphasynuclein P37840 10 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVL YVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTA VAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILED MPVDPDNEAYEMPSEEGYQDYEPEA rabbit VL AND AIVMTQTPSSKSVAVGDTVTINCQASQSVYKNNYLAWFQQ KPGQPPKQLIYGASTLASGVPSRFKGSGSGTQFTLTISDVVC

- 28 041378

DDAATYYCAGYKGGRNDGFAFGGGTEVVVK N.p. rabbit VL 12 Gccatcgtgatgacccagactccatcttccaagtctgtcgctgtggggagacacagtcaccatc aattgccaggccagtcagagtgtttataagaacaactacttagcctggtttcaacagaaaccag ggcagcctcccaaacaactgatctatggtgcgtccactctggcatctggggtcccatcgcggt tcaaaggcagtggatctgggacacagttcactctcaccatcagcgatgtggtgtgtgacgatg ctgccacttactactgtgcaggatataaaggtggtcgtaatgatggttttgctttcggcggaggg accgaggtggtggtcaaa rabbit VH 13 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAP GKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTS LTTEDTATYFCARIHYGNSGGLWGQGTLVTVSS N.p. rabbit VH 14 Cagtcggtggagggagtccggggggtcgcctggtcacgcctgggacacccctgacactcacc tgcacagtctctggaatcgacctcagtagccacgacatgtattgggtccgccaggctccagg gaaggggctggaatacattggagccatttatgctagtggtagcacatactacgcgagctggg cgaaaggccgattcaccatctctccaagacctcgaccacggtggatctgaaaatgaccagtctg acaaccgaggacacggccacctatttctgtgccagaattcattatggtaatagtggtgggttgt ggggccaaggcaccctggtcaccgtctcgagt 6470 gL3 VL 15 DIQMTQSPSSLSSASVGDRVTITCQASQSVYKNNYLAWFQQ KPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQP EDFATYYCAGYKGGRNDGFAFGGGTKVEIK 6470 gL3 N.p. VL 16 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccat tacgtgccaagccagccagtccgtgtacaagaacaactacctggcctggttccagcaaaagc ccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgag ccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccga ggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttg gtggaggcaccaaggtcgaaatcaag Light chain 6470 gL3 17 DIQMTQSPSSLSASVGDRVTITCQASQSVYKNNYLAWFQQ KPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQP EDFATYYCAGYKGGRNDGFAFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLS SP VTKSFNRGEC N.p. light chain 6470 gL3 18 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccat tacgtgccaagccagccagtccgtgtacaagaacaactacctggcctggttccagcaaaagc ccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgag ccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccga ggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttg gtggaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttccca ccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttcta cccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactccca ggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccc tgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcc tgtccagccccgtgaccaagtccttcaaccggggcgagtgc 6470 gL3 VLN33R 19 DIQMTQSPSSLSSASVGDRVTITCQASQSVYKNRYLAWFQQ KPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQP EDFATYYCAGYKGGRNDGFAFGGGTKVEIK 6470 gL3 N.p. N33RVL 20 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccat tacgtgccaagccagccagtccgtgtacaagaaccgttacctggcctggttccagcaaaagc ccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgag ccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccga ggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttg gtggaggcaccaaggtcgaaatcaag 6470 gL3 21 DIQMTQSPSSLSSASVGDRVTITCQASQSVYKNRYLAWFQQ

- 29 041378

Light chain N33R KPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQP EDFATYYCAGYKGGRNDGFAFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLS SP VTKSFNRGEC 6470 gL3 N.p. light chain N33R 22 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccat tacgtgccaagccagccagtccgtgtacaagaaccgttacctggcctggttccagcaaaagc ccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgag ccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccga ggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttg gtggaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttccca ccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttcta cccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactccca ggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccc tgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcc tgtccagccccgtgaccaagtccttcaaccggggcgagtgc 6470 gH23 VH 23 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGSTYYASWAKGRFTISRDNSKNTVYLQ MNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSS 6470 gH23 N.p. VH 24 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctct cttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccg ggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgg gcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaac tctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtgg gttgtggggtcaggtactctggttaccgtctcgagc 6470 gH23 Heavy Chain 25 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGSTYYASWAKGRFTISRDNSKNTVYLQ MNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 6470 gH23 Heavy chain N.p. 26 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctc ttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgg gtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctggg cgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaact ctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtggg ttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttcc ctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaagg actacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcac accttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccct cctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaa ggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaat ttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccg gacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttc aattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacag ttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacgg

- 30 041378

caaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatc tccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaa gagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacatt gccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtg ctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcag gaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaa gtccctgtccctgagcctgggcaag 6470 gH23 VH S56N N102H 27 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGNTYYASWAKGRFTISRDNSKNTVYL QMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSS 6470 gH23 H.n. VH S56N N102H 28 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctct cttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccg ggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgg gcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaac tctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgg gttgtggggtcagggtactctggttaccgtctcgagc 6470 gH23 Heavy Chain S56NN102H 29 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGNTYYASWAKGRFTISRDNSKNTVYL QMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 6470 gH23 Heavy chain N.p. S56N N102H 30 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctc ttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgg gtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctggg cgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaact ctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgg gttgtggggtcaggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttc cctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaag gactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgca caccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccc tcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacacca aggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctga atttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcc cggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagt tcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaac agttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaac ggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagacc atctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccag gaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccga cattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccct gtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtgg caggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacaccca gaagtccctgtccctgagcctgggcaag 6470 gH36 VH 31 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGSTYYASWAKGRFTISRDSSKNTLYLQ

- 31 041378

MNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSS 6470 gH36 N.p. VH 32 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctct cttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccg ggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgg gcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaa ctctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtg ggttgtggggtcagggtactctggttaccgtctcgagc 6470 gH36 Heavy Chain 33 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGSTYYASWAKGRFTISRDSSKNTLYLQ MNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 6470 gH36 Heavy chain n.p. 34 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctc ttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgg gtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctggg cgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaact ctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtggg ttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttcc ctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaagg actacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcac accttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccct cctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaa ggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaat ttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccg gacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttc aattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacag ttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacgg caaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatc tccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaa gagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacatt gccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtg ctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcag gaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaa gtccctgtccctgagcctgggcaag 6470 gH36 VH S56N N102H 35 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGNTYYASWAKGRFTISRDSSKNTLYLQ MNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSS 6470 gH36 N.p. VH S56N N102H 36 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctct cttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccg ggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgg gcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaa ctctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtg ggttgtggggtcagggtactctggttaccgtctcgagc 6470 gH36 Heavy Chain 37 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQA PGKGLEYIGAIYASGNTYYASWAKGRFTISRDSSKNTLYLQ

- 32 041378

S56NN102H MNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 6470 gH36 Heavy chain N.p. S56N N102H 38 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctc ttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgg gtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctggg cgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaact ctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgg gttgtggggtcaggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttc cctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaag gactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgca caccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccc tcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacacca aggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctga atttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcc cggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagt tcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaac agttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaac ggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagacc atctccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccag gaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccga cattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccct gtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtgg caggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacaccca gaagtccctgtccctgagcctgggcaag Acceptance. frame person IGKV1-16 JK4 39 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPG KAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYNSYPLTFGGGTKVEIK N.p. acceptance, frame person IGKV1-16 JK4 40 Gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatc acttgtcgggcgagtcagggcattagcaattatttagcctggtttcagcagaaaccaggggaaa gcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcg gcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaa cttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtggagat caaa Acceptance. frame man. IGHV3-23 JH4 41 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYAMSWVRQA PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYC AKYFDYWGQGTL VTVS S N.p. acceptance, frame person IGHV3-23 JH4 42 Gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactct cctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccag ggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagac tccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatg aacagcctgagagccgaggacacggccgtatattactgtgcgaaatactttgactactgggg ccaaggaaccctggtcaccgtctcctca

- 33 041378

Person 68-140 asyn in rabbit Fc 43 GAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEG APQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAVEKTVA PSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVV VDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVS TLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLE PKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGK AEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCS VMHEALHNHYTQKSISRSPGK CDR-L1 xzz 44 QASQSVYKNXYLA (X = N or R) CDR-H2 X56 45 AIYASGXTYYASWAKG (X = SorN) CDR-H3 X102 46 IHYGXSGGL (X = N or Η) Rabbit Light Chain 6470 47 AIVMTQTPSSKSVAVGDTVTINCQASQSVYKNNYLAWFQQ KPGQPPKQLIYGASTLASGVPSRFKGSGSGTQFTLTISDVVC DDAATYYCAGYKGGRNDGFAFGGGTEVVVKRTPVAPTVL IFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTT GIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQ GTTSVVQSFNRGDC Rabbit Heavy Chain 6470 48 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAP GKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTS LTTEDTATYFCARIHYGNSGGLWGQGTLVTVSSGQPKAPS VFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTN GVRTFPS VRQ SSGL YSLS S VVS VTS S SQP VTCNVAHP ATNT KVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISR TPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQ FNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTIS KARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDIS VEWEKNGK AEDNYKTTP AVLD SDGS YFL YSKL S VPT SEW QRGDVFTCSVMHEALHNHYTQKSISRSPGK Heavy Chain Fab Rabbit 6470 49 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAP GKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTS LTTEDTATYFCARIHYGNSGGLWGQGTLVTVSSGQPKAPS VFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTN GVRTFPS VRQ SSGL YSLS S VVS VTS S SQP VTCNVAHP ATNT KVDKTVAPSTCSKPHHHHHHHHHH

The invention is further described below by way of examples with reference to embodiments shown in the accompanying drawings.

Examples

Example 1. Expression of human alpha-synuclein monomer and 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 generate a vector engineered to produce alpha-synuclein with an N-terminal 10His-TEV tag. The resulting vector was transfected into Expi293F cells using the Expi293TM Expression System (Invitrogen) according to the manufacturer's protocols. Alpha-synuclein protein was accumulated in the culture medium and then recovered by 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 with a step gradient of 500 mM imidazole in the same buffer. The 10His tag was removed using TEV protease. The sample was then concentrated and desalted before reloading the digested protein onto a HisTrap excel column and collecting the digested alpha-synuclein in the flow-through. Alpha-synuclein was further purified by gel filtration on a HiLoad 26/600 Superdex 75 column (GE Healthcare) and endotoxin was removed by passing through a Proteus NoEndo cartridge (Generon). SEC MALS confirmed that the purified alpha-synuclein was monomeric (Fig. 1A).

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

This monomer of (untagged) wild-type alpha-synuclein was used to prepare fibril

- 34 041378 rills of alpha-synuclein were prepared by stirring purified recombinant alpha-synuclein monomer (9-10 mg/ml in PBS pH 7.4) at 1200 rpm at 37°C with shaking in a Vortemp56 incubator (Labnet) continuously for 10 days. Fibril formation was assessed by the JC-1 assay (Lee et al., Biochem. J. 2009, 418, 311-323) and Fourier transform infrared spectroscopy of the solution. Unincorporated monomer in fibril solutions was assessed by ultracentrifugation and passage through a 100 kDa cutoff membrane followed by gel electrophoresis. Only fibrils with a JC-1 response >15, low amounts of soluble monomer (<5%) and an FTIR spectrum with a major absorption between 1625 and 1630 cm ^-1 (Fig. 2) were used in further studies. The prepared fibrils were stored at -80 °C.

Example 2. Immunization and antibody production.

Several immunization strategies were performed using different substances and immunogens. Antibody 6470 was obtained from a female New Zealand White rabbit (>2 kg) that was immunized subcutaneously with a rabbit Fc fusion protein containing human alpha-synuclein 68140 residues fused to rabbit Fc (SEQ ID NO: 43).

A fusion protein consisting of alpha-synuclein (68-140) and rabbit Fc was expressed for immunization in Expi293F cells using the Expi293TM expression system (Invitrogen) according to the manufacturer's protocol. The protein was purified from the supernatant by affinity chromatography on a MabSelectSure column (GE Healthcare). The column was equilibrated with 50 mM glycine/sodium glycinate buffer, pH 8.8, and eluted with a gradient of 0.1 M citric acid, pH 2.0, in the same buffer. Protein fractions were neutralized with 2 M Tris HCl, pH 8.5, concentrated, and further purified by gel filtration on a HiLoad 26/600 Superdex 200 column (GE Healthcare) equilibrated and eluted in PBS, pH 7.4. Rabbits received a primary immunization containing 500 μg of fusion protein emulsified in an equal volume of complete Freund's adjuvant (CFA). Rabbits received two booster injections 21 days apart using incomplete Freund's adjuvant (IFA) and were bled from the ear 14 days after immunization. Bleeding was stopped 14 days after the last booster injection of single-cell suspensions of spleen, bone marrow, and peripheral blood mononuclear cells prepared and frozen in 10% dimethyl sulfoxide (DMSO) in fetal bovine serum (FCS) at -80°C.

B-cell culture.

B cell cultures were generated as described by Tickle et al., 2015. J. Biomol. Screen: 20(4), 492-497. Briefly, B cells obtained from lymph nodes or splenocytes of immunized animals were cultured at a density of approximately 2,000-5,000 cells/well in barcoded 96-well tissue 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% activated human PBMC supernatant (BSS), and X-ray-irradiated mutant murine thymoma EL4 cells (5 x 10 ⁴ /well) for seven days at 37°C in an atmosphere of 5% _CO2 . Cultures were established using B cells obtained from all immunized animals, and a total of approximately 1.7x109 B cells were collected.

6470, an antibody of the invention, was prepared from activated lymph node-derived B cells that were cultured at a density of approximately 5,000 cells per well. The lymph node was used in addition to splenocytes, which have proven to be an alternative source for obtaining B cell samples and identifying novel antibodies. Antibodies with related sequences were identified from lymph node-derived B cells, but not from the spleen. Approximately 9.6 x 10 ⁷ cells were collected from a rabbit immunized with human alpha synuclein C-terminal protein.

Primary screening.

The presence of antibodies specific for human alpha-synuclein in B-cell culture supernatants was determined by a homogeneous fluorescence binding assay using Superavidin™ beads (Bangs Laboratories) coated with biotinylated recombinant full-length human alpha-synuclein as the source of target antigen. Recombinant human alpha-synuclein, as disclosed herein, was biotinylated with a 3-fold molar excess of biotin. A low molar excess of biotin was used to avoid complete modification of all seven lysine residues that are present in the alpha-synuclein molecule. Alpha-synuclein monomer was incubated overnight at 40°C with biotin, and free biotin was removed the following day using a Zeba™ spin desalting column. Screening involved transferring 10 μl of supernatant from barcoded 96-well tissue culture plates to barcoded black-walled 384-well assay plates containing biotinylated recombinant human alpha-synuclein monomer immobilized on superavidin beads (10 μl/well) using an Agilent Bravo liquid handler. Binding was detected using goat anti-rabbit IgG Fcy-specific Alexafluor647 conjugate (Jackson). Plates were read on a

- 35 041378 Labtech Mirrorball TTR device for identification of wells containing alpha-synuclein-specific IgG.

Secondary screening.

Following primary screening, positive supernatants were pooled into barcoded 96-well master plates using a Beckman Coulter BiomekNXP robotic arm, with B cells in cell culture plates frozen at -80°C. Master plates were then screened in a streptavidin capture ELISA assay using biotinylated rhα-synuclein monomer or biotinylated rhα-synuclein fibrils to identify wells that demonstrated binding to both monomeric and fibrillar rhα-synuclein and to exclude any false-positive wells demonstrating off-target binding to Superavidin™ beads. Given the insoluble nature of the fibrils, conventional ELISA coating protocols used with proteins in solution were not suitable. It was decided to use a minimal biotinylation protocol to preserve the fibrillar structure and to facilitate efficient fibril coating of the ELISA plate, which was pre-coated with streptavidin.

A total pool of biotinylated alpha-synuclein fibrils was prepared as described herein by combining 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 streptavidin-coated 384-well Maxisorp plates in carbonate coating buffer ( _dH2O + 0.16% _Na2CO3 + 0.3% _NaHCO3 ). Plates were blocked with 1 w/v% PEG/PBS and then incubated with 10 _μl /well of B cell culture supernatant (diluted 1:1 in blocking buffer). Secondary HRP-conjugated goat anti-rabbit IgG Fc antibodies (Stratech Scientific Ltd/Jackson ImmunoResearch) were added to the plates, followed by visualization of binding to TMB substrate (3,3',5,5'tetramethylbenzidine, EMD Millipore; 10 μl/well). Optical density was measured at 630 nM using a BioTek Synergy 2 microplate reader. Primary binding assay identified 640 target events, and 491 of these were shown to bind to both monomeric and fibrillar recombinant human alpha-synuclein after ELISA screening.

B cell supernatants showing the strongest ELISA binding signals to recombinant fibrils were selected for further analysis by surface plasmon resonance to identify those showing the best off-rates from recombinant human alpha-synuclein monomer, recombinant human alpha-synuclein fibrils, and recombinant mouse alpha-synuclein fibrils. Supernatants from 80 different B cells were tested, nine wells showed off-rates (kd) from recombinant human fibrils <1x10’5. Of these, seven had off-rates (kd) from recombinant mouse fibrils less than 1x10’5, and two gave off-rates (kd) from recombinant human monomer less than 1x10’5. All nine supernatants were selected for variable region extraction.

Extraction of variable region.

To extract the antibody variable region genes from the selected supernatants of interest, a deconvolution step was necessary to allow identification of antigen-specific B cells in a given well, which contained a heterogeneous population of B cells. This was achieved using the fluorescent focus assay (Clargo et al., 2014. MAbs: 6(1), 143159). Briefly, immunoglobulin-secreting B cells from a positive well were mixed with streptavidin beads (New England Biolabs) coated with biotinylated recombinant human alpha-synuclein fibrils (prepared using a 1:50 mixture as described above) and goat anti-rabbit Fcy-specific FITC conjugate (Jackson) at a final dilution of 1:1200. After static incubation at 37°C for 1 h, antigen-specific B cells could be identified by the presence of a fluorescent halo surrounding the B cell. Several of these individual B cell clones, identified using the Olympus microscope, were then picked using the Eppendorf micromanipulator and placed in a PCR tube.

The antibody variable region genes were isolated from single cells by reverse transcription (RT) PCR using primers specific for the variable region of the heavy and light chains. Two rounds of PCR were performed by nested 2° PCR incorporating restriction sites at the 3' and 5' ends to allow cloning of the variable region into a mammalian expression vector expressing rabbit IgG (VH) or rabbit IgG kappa chain (VL). Anti-alphasynuclein antibody genes from 5 different supernatants were successfully cloned into the expression vectors. The heavy and light chain constructs were co-transfected into Expi-293 cells using ExpiFectamine 293 (Invitrogen) to express the recombinant antibody in a 125 ml 30 ml Erlenmeyer flask. After 5-7 days of expression, supernatants were collected and purified by affinity chromatography.

- 36 041378

ELISA screening of transient-containing supernatants.

Purified antibodies were then further screened by ELISA. Biotinylated recombinant human alpha-synuclein monomers and fibrils were captured on streptavidin-coated 384-well Maxisorp plates (ThermoScientific/Nunc) in carbonate coating buffer (dH2O + 0.16% _Na2CO3 + 0.3% _NaHCO3 ). Individual plates were also coated with a biotinylated peptide corresponding to residues 117-126 of human alpha-synuclein given in SEQ ID NO: 10 (peptide PVDPDNEAYE) to test whether the transients were bound to this or another region of the molecule. Plates were blocked with 1 w/v% PEG/PBS and then incubated with several dilutions of the cleared transient supernatant. Secondary HRP-conjugated goat anti-rabbit IgG Fc antibodies (Stratech Scientific Ltd/Jackson ImmunoResearch) were added to the plates followed by visualization of binding to TMB substrate (3,3',5,5'-tetramethylbenzidine, EMD Millipore; 10 μl/well). The optical density was measured at 630 nM using a BioTek Synergy 2 microplate reader. The data for 6470 are shown in Fig. 3. As can be seen, 6470 shows binding to both monomeric and fibrillar recombinant human alphasynuclein but no binding to the 117-126 peptide.

The antibodies (IgG) were then tested using a cell-based aggregation assay as described further in Example 7. The binding kinetics of all antibodies showing activity in the cell-based assay were subsequently determined using surface plasmon resonance. The antibodies were tested in IgG and Fab format to determine avidity (bivalent binding) and affinity (monovalent binding), respectively.

Example 3. Characteristics of antibodies.

Kinetics according to Biacore.

The kinetics of interactions were determined by surface plasmon resonance on a Biacore T200 instrument. Each of three different ligands, including recombinant full-length human alpha-synuclein monomer, purified recombinant human alpha-synuclein fibrils, and purified recombinant mouse alpha-synuclein fibrils, prepared as described herein, were immobilized in three different flow cells on the surface of the CM5 chip by amine immobilization. The three ligands were prepared in 10 mM NaAc, pH 3.5, and immobilized on the surface of individual flow cells to achieve immobilization levels of approximately 30 response units (RU) for alpha-synuclein monomer, approximately 40 RU for human alpha-synuclein fibrils, and approximately 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 the running buffer for both ligand immobilization and kinetic analysis. Binding of monoclonal rabbit IgG1 6470 (containing SEQ ID NOs: 47 and 48) and monoclonal rabbit Fab 6470 (containing SEQ ID NOs: 47 and 49) to the three ligands was then measured. Monoclonal IgG or Fab antibodies were injected at 7 different concentrations from 800 to 0.195 nM, with each concentration in 3 flow wells, with a contact time of 3 min and a dissociation time of 30 min at a flow rate of 100 μl/min. To regenerate the surface, one injection of 50 mM HCl was performed for 90 s at 10 μl/min and another injection of 50 mM HCl for 60 s at 10 μl/min. Data were analyzed with Biacore T200 software (version 3.0) using a bivalent analyte model with an assumed no overall contribution (RI=0) and a global R _max for the IgG format, and a 1:1 model with a flexible overall contribution (local RI) and a global R _max .

The kinetic values for binding of both IgG and Fab to the immobilized targets are shown in Table 2. The IgG format showed a clear selective affinity for human alpha-synuclein fibrils compared to the affinity for human alpha-synuclein monomer, since the dissociation constant K _D for human fibrils was more than 10-fold lower.

Table 2

sample Human monomer Human fibrils mouse fibrils kal (1/ms) kdl (1/s) KD1 (nM) kal (1/ms) kdl (1/s) KD1 (nM) kal (1/ms) kdl (1/s) KD1 (nM) 6470 Rabbit Fab 1.80E+06 2,67E- 02 14.79 1.83E+06 2,25E- 02 12.35 1.38E+06 2,43E- 02 17.65 6470 rabbit IgGl 4.75E+06 1,42E- 02 2.98 1.76E+06 4.78E- 04 0.27 8.89E+05 3.07E- 04 0.34

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Binding to beta-synuclein.

Binding of anti-human alpha-synuclein antibodies to human beta-synuclein was tested by Western blot analysis using beta-synuclein rPeptide. One microgram of synuclein was run on 4-12% Bis/Tris gel and blotted onto PVDF membrane. The membrane was blocked in PBS with 3% BSA and 0.1% Tween 20. Rabbit IgG1 6470 was added to the blocked blots and incubated for 1 h at room temperature, washed with PBS, 0.1% Tween 20 and incubated for 1 h with secondary antibody-HRP conjugate (anti-rabbit H+L conjugate with HRP, Bethyl, A120101P). The blot was washed extensively in PBS with 0.1% Tween 20, PBS and water. Chemiluminescence was measured after addition of ECL Western blot substrate (Pierce). As shown in Fig. 4(A), rabbit IgG1 6470 (lane 3) 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 tags. The construct was transformed into E. coli BL21 (DE3) cells (Stratagene), and the cells were grown in defined medium with ^13C -labeled DL-glucose and ^15K -labeled ammonium sulfate in the presence and absence of deuterium oxide ( _D2O ). Expression was induced at _OD600hm = 1 with 300 mM IPTG and the culture was incubated at 30°C for 4 h. 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), 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 cleared 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 the MonoQ 10/100GL column after 5-fold dilution in 20 mM Tris/HCl, pH 8.0. The purest fractions were pooled, concentrated using a 10 kDa MWCO centrifugal concentrator (Centriprep, Millipore), purified by size exclusion 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 the 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.

Rabbit Fab 6470 (containing VL of SEQ ID NO: 11 and VH of SEQ ID NO: 13, and also containing SEQ ID NOs: 47 and 49) was expressed in CHO SXE as His-tagged entities and purified from the supernatant by His-tag affinity chromatography by coupling the protein to HisTrap Excel (GE Healthcare) from the supernatant and eluting it with 250 mM imidazole in PBS. The elution pool was loaded onto a HiTrap GammaBind Plus Sepharose column (GE Healthcare), the column was 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 eluted FabHis protein was buffer exchanged with NMR buffer (25 mM sodium phosphate, pH 6.4, 100 mM NaCl) on a HiPrep 26/10 desalting column. Before sterilization on a 0.22 μm Millex GV Fab-His filter, the protein fractions were concentrated, and the protease inhibitors AEBSF (final concentration 10 μM) and sodium azide (final concentration 0.02%) were added. For crystallography, concentrated Fab-His 6470 was purified by preparative size exclusion chromatography on a HiLoad 26/600 Superdex 75 column (GE Healthcare) and eluted with 25 mM sodium phosphate, pH 6.4, 100 mM NaCl. The purity of the final pools was checked by UPLC-SEC to be >99%. The final pools were passed through a 0.22 mm Millex GV filter for sterilization.

Determination of the α-synuclein backbone.

NMR samples were typically 350 μl in volume with a protein concentration of 360 μM ^13C / ^15N -labeled or 430 μM ^2H / ^13C / ^15N -labeled human α-synuclein in 5 mm Shigemi tubes. Buffer conditions were 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 either a Bruker AVIII 600 MHz or Bruker AVII 800 MHz spectrometer equipped with cryogenically cooled probes. Sequential assignments of NMR signals of residues in the protein backbone, H _N (i)-N(i)-N(i±1), were performed in a 3D (H)N(CA)NNH experiment (Weisemann et al., 1993 3D Triple-resonance NMR techniques for the sequential assignment of NH and ¹⁵ N resonances in ¹⁵ N- and ¹³ C-labelled proteins. J. Biomol. NMR 3), recorded with spectral widths of 28, 28, and 10 ppm and scan times of 117 (F1), 117 (F2), and 140 (F3) ms in measurements on ¹⁵ N, ¹⁵ N, and 1H nuclei, respectively, with 8 scans per increment and a relaxation delay of 1.5 s. A heterogeneous sample with a sampling density of 10% (4000 of 40,000 hypercomplex points) was used, resulting in a total scanning time of 2.75 days. Sequential relationships were confirmed and residue types were identified using TROSY-HNCA (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

- 38 041378 with the Alpha- and Beta-Carbon Resonances in Proteins. J. Magn. Reason. 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). In the TROSY-HNCA experiment, spectra with a width of 23, 28, 10 ppm and a scan time of 12.1 (F1), 21.7 (F2) and 100 (F3) ms were recorded in measurements on ¹³ C, ¹⁵ N and ¹ H nuclei, respectively (8 scans per increment, relaxation delay 1.5 s, total scanning time 1 day), while in the TROSY-HNCACB experiment spectra with a width of 56, 28 and 10 ppm and a scan time of 8.2 (F1) were recorded, 21.7 (F2) and 100 (F3) ms in measurements on ^13C , ^15N and 1H nuclei, respectively (8 scans per increment, relaxation delay 1.5 s, total scan time 1.7 days). The arrangement of carbonyl groups in the backbone was 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, 432440; 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), which was recorded with spectral widths of 10, 29, 10 ppm and a scan time of 80 (F1) , 21.7 (F2) and 150 (F3) ms in measurements on ^13C , ^15N and 1H nuclei, respectively (8 scans per increment and a relaxation delay of 1.5 s). A non-uniform sample with a sampling density of 15% (1208 of 8050 hypercomplex points) was used, resulting in a total scan time of 19 h. NMR spectra were processed using the NMRPipe software (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 scan time one-fold over nitrogen nuclei. The heterogeneous sample data were reconstructed using the Harvard iterative method with a soft threshold criterion (Hyberts et al., 2012), where the data was reconstructed to the next Fourier number, resulting in a 60% increase in indirect scan time. Data analysis was performed using the Sparky method (Goddard and Kneller, DG SPARKY 3. In., University of California, San Francisco), resulting in the determination of amide proton and nitrogen resonances of 133 residues, corresponding to 99% of the residues (excluding proline and N-terminal methionine residues) .

Mapping of the Fab 6470 binding site was accomplished using 150 μM ^2H / ^13C / ^15N -labeled human alpha-synuclein sample containing a 10% molar excess of unlabeled Fab 6470. Samples were prepared in the same buffer as described above for alpha-synuclein scaffold determination. Chemical shift changes in the 1H, ^15N , and ^13C spectra were determined by comparing the spectrum obtained in the TROSY-HNCO experiment (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) obtained for the alpha-synuclein/Fab complex with an equivalent control spectrum obtained for free alpha-synuclein. The control spectrum in the TROSY-HNCO experiment with free alpha-synuclein was acquired with spectral widths of 10, 28, and 10 ppm and scan times of 80 (F1), 22 (F2), and 150 (F3) ms in measurements on ^13C , ^15N , and 1H nuclei, respectively (16 scans per increment, 1.5 s relaxation delay). A non-uniform sample with a sampling density of 25% (2013 of 8050 hypercomplex points) was used, resulting in a total scan time of 2.7 days. The TROSY-HNCO experiment for the alpha-synuclein/Fab complex was performed using spectral widths of 10, 28, and 10 ppm and scan times of 80 (F1), 21.7 (F2), and 80 (F3) ms in ^13C , ^15N , and 1H measurements, respectively (32 scans per increment, 1.5 s relaxation delay). Non-uniform sampling with a sampling density of 25% was used (1119 of 4477 hypercomplex points), resulting in a total scan time of 2.8 days. NMR spectra were processed with the NMRPipe program (Delaglio et al., 1995 NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-93) with reconstruction of the NUS data performed using mddnmr. Analysis of non-uniformly sampled spectra with Multi-Dimensional Decomposition. Prog. Nucl. Magn. Reson. Spectrosc, 59, p 271-292). The effective scanning time for nitrogen nuclei was increased by 1-fold during data recovery.

Chemical shift changes were analyzed using the minimal shift approach (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), essentially as described previously (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 for modifying the equation used to calculate the combined chemical shift change (Δδ) to include the chemical shift of the carbonyl atom, resulting in the following equation:

^(ΔδΗΝ) ² + (ΔδΝαΝ) ² + (ДбСаС) ^{2 Лб =} $ Ϊ i ~ where Δδ _H N, Δδ\ and Δδ _C are the differences in the chemical shifts of 1H, ¹⁵ N and ¹³ C, respectively. aN and aC correspond to the scaling factors of 0.2 and 0.35, respectively, used to account for the differences in the chemical shift ranges of the amide proton, nitrogen and carbonyl atom.

Histochemicals were used to identify the binding site (epitopes) of Fab on alpha-synuclein.

- 39,041,378 grams of combined minimal shift as a function of protein sequence to identify regions of alpha-synuclein containing highly distorted signals. If the combined chemical shift change size for individual amino acids exceeded a threshold of the mean combined chemical shift change for all amino acids plus one standard deviation from that mean, these residues were selected for further evaluation as potential contact residues in the Fab binding site.

Highly distorted residues were identified as residues with a minimum shift greater than at least the mean calculated for all shifts plus one standard deviation. Four different cutoff values were used to identify residues bound to Fab. Residues involved in the binding site were scored with increasing levels of stringency as: residues with a minimum shift greater than the mean calculated for all shifts plus one standard deviation (score >0.018925); residues with a minimum shift greater than the mean calculated for all shifts plus two standard deviations (score >0.032049); residues with a minimum shift greater than the mean calculated for all shifts plus three standard deviations (score >0.045174); residues with a minimum shift greater than the mean calculated for all shifts plus four standard deviations (score >0.058299). Proline residues could not be identified in this analysis because they do not contain an amide proton.

Therefore, the epitope for Fab 6470 is defined with increasing levels of stringency as the mean value calculated for all shifts plus one standard deviation: D121, N122, E123, A124, Y125, E126, M127, S129, E130, Y133, Q134, D135, and Y136; the mean value calculated for all shifts plus two standard deviations: E123, A124, Y125, E126, M127, S129, E130, D135, and Y136; the mean value calculated for all shifts plus three standard deviations: Y125, M127, S129, and D135; the mean value calculated for all shifts plus four standard deviations: M127, S129, and D135.

As shown in Fig. 4B, antibody 6470 was found to bind to at least the following residues (mean +3SD) Y125, M127, S129, and D135 and further also binds to all of the following residues (mean +1SD) D121, N122, E123, A124, E126, E130, Y133, Q134, and Y136 of human alpha-synuclein (SEQ ID NO: 10) by NMR studies.

Peptide mapping.

Further characterization of the 6470-bound epitope was accomplished using short (usually 9- or 10-mer) peptides representative of and spanning the C-terminal region of human alpha-synuclein. These were used in a competitive surface plasmon resonance assay to test whether any of them were able to inhibit antibody binding to monomeric alpha-synuclein or preformed alpha-synuclein fibrils immobilized on a Biacore chip. The peptide showing the highest 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 Fmoc solid-phase peptide synthesis according to the method of Atherton and Sheppard (Ref: Atherton, E.; Sheppard R.C. (1989). Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press). The N- and C-terminal ends of the peptides were capped with acetyl and amide groups, respectively, except in the case of the peptides representing the N-terminus and C-terminus of α-synuclein, in which case the amino and carboxyl groups, respectively, were left free. Peptide stock solutions were prepared in DMSO at 10 mM. The complete list of peptides is given in Table 3.

Table 3

Peptide ID 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

- 40 041378 alpha-synuclein fibrils were immobilized on the CM5 chip using a Biacore 3000 instrument (GE Healthcare). After activation of the carboxymethyldextran surface by injection of 100 μl of a fresh 1:1 (v/v) mixture of 50 mM N-hydroxysuccinide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a flow rate of 10 μl/min with HBS-EP (GE Healthcare) as the running buffer, binding was achieved by injecting 100 μl of monomer and fibrils at a concentration of 5 μM in 10 mM acetate, pH 5.0, through separate flow cells. A control flow cell was activated in the same manner, and then all flow cell surfaces were deactivated with a 50 µl flush of 1 M ethanolamine HCl, pH 8.5.

Peptide solutions were prepared in running buffer at 100 μM and a control without peptide was prepared as a 1:100 dilution using DMSO in the running buffer. A solution of 6470 rabbit Fab (containing SEQ ID NOs: 47 and 49) was prepared in 50.5 nM running buffer before pre-incubation of 198 μL with 2 μL of blank control or diluted peptide to yield a final mixture of 50 nM Fab and 1 μM peptide or control. Sensorgrams were recorded for each sample by injecting 30 μL of the mixture at 10 μL/min and recording the signal at the time point 5 s before the end of the injection. At the end of each cycle, the chip was reconstituted with two 10 μL injections 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 signal recording point compared to the average of adjacent control cycles.

The inhibition levels of each alpha-synuclein peptide are shown in Fig. 5. Significant inhibition of Fab 6470 binding to either alpha-synuclein monomer or fibrils was observed for only three peptides: AS121-130, AS123-132, and AS125-134, with the highest inhibition levels observed for AS123-132, which were 37 and 54% for antibody binding to monomer and fibrils, respectively. Slightly lower inhibition levels were obtained for the AS125-134 peptide, 34 and 52%, respectively, indicating that the major component of the epitope contains residues 125-132. The inhibition levels provided by the AS121-130 peptide were lower, 20 and 27%, respectively, suggesting that the common residues of all three peptides are 125-130, which contribute the major portion to the epitope.

Since the epitope of antibody 6470 appears to contain at least the YEMPSEEG sequence, the AS123-132 peptide was examined in co-crystallization studies with Fab 6470.

X-ray crystallography.

To prepare the complexes, 1 ml of purified rabbit Fab 6470 at a concentration of approximately 10 mg/ml was mixed with alpha-synuclein peptide 123-132 (EAYEMPSEEG) at a Fab:peptide molar ratio of 1:2 and incubated for 1 h at room temperature. Conditions suitable for crystal growth were determined by sessile drop vapor diffusion using commercially available crystallization screens (Qiagen). Hanging drop vapor diffusion was used to obtain diffraction-grade crystals.

To prepare the Fab 6470-peptide 123-132 complex, 1 μl of the protein solution was mixed with 1 μl of a reservoir solution containing 1.6 M ammonium sulfate and 0.1 M Hepes buffer, pH 7.5. The crystals were collected and snap frozen in liquid nitrogen after being rapidly passed through a cryoprotectant solution containing 1.6 M ammonium sulfate, 0.1 M Hepes buffer, pH 7.5, and 20% glycerol. The crystals were collected and snap frozen in liquid nitrogen after being rapidly passed through a cryoprotectant solution containing 0.2 M ammonium sulfate and 35 v/v % polyethyleneglycol 8000.

Single crystal diffraction data of Fab 6470-peptide 123-132 were obtained at a resolution of 2.9 Å at beamline i04-1 on the Diamond Synchrotron, Didcot, Oxfordshire, UK, and processed using Mosflm, Aimless and Truncate. The structure of the complex was solved by molecular replacement using Phaser software, using the in-house Fab coordinates as a search model.

Refinement and model building rounds were performed using CNS (BBrunger et al., (2007) Nature Protocols 2, 2728–2733) and COOT (Emsley et al., (2004) Acta crystallographica. Section D, Biological crystallography 60, 2126–2132) until all refinement statistics converged for both models. Model geometry was verified using Molprobity software43. Molecular visualizations were generated using Pymo144. The epitope information described below was obtained by examining atoms at 4Å resolution at the Fab/peptide interface. Data collection and refinement statistics are summarized in Table 4A and Table 4B.

Table 4A

Structure Fab VR6470-peptide 123-132 Space group P312 1 Cell size

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a, b, c (A) 111.78, 111.78, 71.93 α, β, Y, (°) 90.00, 90.00, 120.00 Resolution (A) 30.00-2.90 (3.08-2.90) Rmerge 0.07 (0.36) I/σΙ 17.0(5.1) Completeness (%) 99.9 (100) Redundancy 9.8(10.2)

Table 4B

Clarification Fab VR6470-peptide 123-132 Resolution (A) 30.00-2.90 No. of reflection 11762 ^R work ^/R free 0.2587/0.3192 No. atoms Protein 3259 Water 0 Ligand 30 B-factors Peptide 81.8 Fab 67.4 Standard deviations Lengths of connection (A) 0,005 Connection angles (°) 1,125

Values in brackets refer to the shell obtained at higher resolution.

R _sy m=Σ|(I-<I>)|/Σ(I), where I is the observed integrated intensity, <I> is the average integrated intensity obtained from several measurements, and the summation is performed over all observed reflections.

^R work= ^Σ||F obs ^|- k ^|F calc ^||/Σ|F obs ^|, where F _obs and F _calc are the observed and calculated structural factors, respectively.

R _free is calculated as R _work using 5% of the reflectance data selected at random and not included in the refinement calculations.

The major contact sites between the heavy and light chain residues and the peptide are shown in Table 5.

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Table 5

Chain Residue (SEQ ID NOT3) Atom Chain Amino acid peptide Target atom of the peptide Distance (A) N 96 (HIS) NE2 [N] A 125(TYR) CD1 [C] 3.93 N 96 (HIS) CD2 [C] A 125(TYR) CD1 [C] 3.93 A 125(TYR) CE1 [C] 3.69 N 99(ASN) SV [C] A 125(TYR) CD2 [C] 3.34 N 99(ASN) N[N] A 125(TYR) CD2 [C] 3.57 N 99(ASN) SV [C] A 125(TYR) CE2 [C] 3.85 N 96 (HIS) CA [C] A 125(TYR) CE2 [C] 3.85 N 96 (HIS) SV [C] A 125(TYR) CE2 [C] 3.93 N 97(TYR) N[N] A 125(TYR) CE2 [C] 3.81 N 99(ASN) CA [C] A 125(TYR) CE2 [C] 3.99 N 98(GLY) N[N] A 125(TYR) CE2 [C] 3.42 N 98(GLY) CA [C] A 125(TYR) CE2 [C] 3.91 N 98(GLY) C[C] A 125(TYR) CE2 [C] 4 N 99(ASN) N[N] A 125(TYR) CE2 [C] 3.12 N 96 (HIS) CA [C] A 125(TYR) CZ [C] 3.5 N 96 (HIS) SV [C] A 125(TYR) CZ [C] 3.96 N 96 (HIS) With [C] A 125(TYR) CZ [C] 3.79 N 97(TYR) N[N] A 125(TYR) CZ [C] 3.32 N 98(GLY) N[N] A 125(TYR) CZ [C] 3.72 N 96 (HIS) CA [C] A 125(TYR) HE [0] 3.19 N 97(TYR) N[N] A 125(TYR) HE [0] 2.32 N 97(TYR) With [C] A 125(TYR) HE [0] 3.64 N 98(GLY) N[N] A 125(TYR) HE [0] 3.1 n 97(TYR) CG [C] A 125(TYR) HE [0] 3.51 n 97(TYR) CE1 [C] A 125(TYR) HE [0] 3.86 n 97(TYR) CA [C] A 125(TYR) HE [0] 3.19 n 97(TYR) SV [C] A 125(TYR) HE [0] 3.38 n 97(TYR) CD1 [C] A 125(TYR) HE [0] 2.84 n 32( ASP) OD1 [O] A 126(GLU) CA [C] 3.34 A 126(GLU) SV [S] 3.78 n 52(ALA) SV [C] A 126(GLU) CG [C] 3.92 n 32( ASP) OD1 [0] A 126(GLU) CG [C] 3.16 A 126(GLU) CD [C] 3.47 n 52(ALA) N[N] A 126(GLU) 0E2 [0] 3.82 n 52(ALA) SV [C] A 126(GLU) 0E2 [0] 3.92 n 32( ASP) 0D1 [0] A 126(GLU) 0E2 [0] 2.99 n 51 (TYR) SV [C] A 126(GLU) 0E2 [0] 3.93 n 32( ASP) CG [C] A 126(GLU) 0E2 [0] 3.89 n 51 (TYR) CD2 C] A 126(GLU) 0E2 [0] 3.7 n 32( ASP) 0D1 [0] A 126(GLU) With [With] 3.2 n 32( ASP) CG [C] A 126(GLU) With [With] 3.72 n 32( ASP) 0D2 [0] A 126(GLU) With [With] 3.79 n 32( ASP) 0D1 [0] A 126(GLU) o [0] 2,4 n 32( ASP) CG [C] A 126(GLU) 0 [0] 2.8 n 32( ASP) 0D2 [0] A 126(GLU) 0[0] 2.73 n 51 (TYR) CD2 [C] A 126(GLU) o [0] 3.87 n 51 (TYR) CE2 [C] A 126(GLU) 0[0] 3.96 A 127(MET) SV [S] 3.99 A 127(MET) With [With] 3.85 n 51 (TYR) CD2 [C] A 127(MET) o [0] 3.1 n 51 (TYR) CE2 [C] A 127(MET) o [0] 3.03

-43041378

N 57(TYR) OH [0] A 129(SER) SV [C] 3.05 N 57(TYR) CZ [C] A 129(SER) SV [C] 3.96 N 57(TYR) OH [0] A 129(SER) OG [0] 2.78 N 57(TYR) CE2 [C] A 129(SER) OG [0] 3.79 N 57(TYR) CZ [C] A 129(SER) OG [0] 3.7 Chain Residue (SEQ ID NO:11) Atom Chain Amino acid peptide Target atom of the peptide Distance (Angstrom) L 34(TYR) OH [0] A 123 (GLU) CD [C] 3.63 A 123 (GLU) 0E1 [0] 3.46 L 34(TYR) CE1 [C] A 123 (GLU) 0E2 [0] 3.68 L 34(TYR) CZ [C] A 123 (GLU) 0E2 [0] 3.74 L 34(TYR) OH [0] A 123 (GLU) 0E2 [0] 3.01 A 125(TYR) CE2 [C] 3.89 L 34(TYR) CE2 [C] A 125(TYR) CE2 [C] 3.85 A 125(TYR) CZ [C] 3.59 L 34(TYR) CD2 [C] A 125(TYR) HE [0] 3.66 L 34(TYR) CE2 [C] A 125(TYR) HE [0] 3.39 L 93 (TYR) CE2 [C] A 127(MET) SV [S] 3.75 L 93 (TYR) CZ [C] A 127(MET) SV [S] 3.62 L 93 (TYR) OH [0] A 127(MET) SV [S] 3.05 L 93 (TYR) CE2 [C] A 127(MET) CG [C] 3,4 L 93 (TYR) CZ [C] A 127(MET) CG [C] 3.69 L 93 (TYR) OH [0] A 127(MET) CG [C] 3.67 L 34(TYR) CE2 [C] A 127(MET) CG [C] 3.95 L 34(TYR) CZ [C] A 127(MET) SD [S] 3.49 L 34(TYR) OH [0] A 127(MET) SD [S] 3.16 L 34(TYR) CE2 [C] A 127(MET) SD [S] 3.05 L 34(TYR) CZ [C] A 127(MET) CE [C] 3.72 L 34(TYR) OH [0] A 127(MET) CE [C] 3.75 L 93 (TYR) CE2 [C] A 127(MET) CE [C] 3.22 L 30(TYR) CG [C] A 127(MET) CE [C] 3.79 L 30(TYR) CD2 [C] A 127(MET) CE [C] 3.35 L 30(TYR) CE2 [C] A 127(MET) CE [C] 3.33 L 34(TYR) CE2 [C] A 127(MET) CE [C] 3.33 L 30(TYR) CZ [C] A 127(MET) CE [C] 3.75 L 30(TYR) OH [0] A 128(PRO) CD [C] 3.86 A 128(PRO) CG [C] 3.43 L 30(TYR) CZ [C] A 128(PRO) CG [C] 3.98 L 93 (TYR) OH [0] A 128(PRO) 0 [0] 3.45 L 96(GLY) N [N] A 128(PRO) 0 [0] 3.41 L 96(GLY) CA [C] A 129(SER) CA [C] 3.88 L 96(GLY) 0 [0] A 129(SER) CA [C] 3,4 L 96(GLY) N [N] A 129(SER) CA [C] 3.99 L 96(GLY) 0 [0] A 129(SER) SV [S] 3.56

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A 129(SER) With [C] 3.52 L 96(GLY) CA [C] A 130(GLU) N[N] 3.99 L 96(GLY) With [C] A 130(GLU) N[N] 3.6 L 96(GLY) 0 [O] A 130(GLU) N[N] 2.73 L 96(GLY) N[N] A 130(GLU) N[N] 3.78 L 96(GLY) 0 [O] A 130(GLU) CA [C] 3.71 A 130(GLU) SV [C] 3.81 L 97(ARG) CD [C] A 130(GLU) SV [C] 3.78 A 130(GLU) CG [C] 3.93 A 130(GLU) CD [C] 3.72 L 97(ARG) CG [C] A 130(GLU) OE1 [O] 3.85 L 94(LYS) CG [C] A 130(GLU) OE1 [O] 3.77 L 97(ARG) CD [C] A 130(GLU) OE1 [O] 2.77 L 97(ARG) NE [N] A 130(GLU) OE1 [O] 3.75 L 94(LYS) CG [C] A 130(GLU) OE2 [O] 3.69 L 94(LYS) 0 [O] A 130(GLU) OE2 [0] 3.63 L 94(LYS) CD [C] A 130(GLU) OE2 [0] 3.77 L 94(LYS) CE [C] A 130(GLU) OE2 [0] 3.26 L 97(ARG) NE [N] A 131(GLU) With [C] 3.94 L 97(ARG) CD [C] A 131(GLU) 0 [0] 3.75 L 97(ARG) NE [N] A 131(GLU) 0 [0] 2.94 L 97(ARG) CZ [C] A 131(GLU) 0 [0] 3.79 L 97(ARG) NH2 [N] A 131(GLU) 0 [0] 2.79

Thus, the epitope contains residues E123, Y125, E126, M127, P128, S129, E130 and E131. Figure 6 shows Fab 6470 in complex with peptide 123-132, and Figures 7 and 8 show contacts between the heavy chain and light chain of Fabs 64-132 and 6470, respectively.

Example 4. Antibody humanization and affinity maturation.

Rabbit antibody 6470 was humanized by grafting CDRs from rabbit V regions onto the human germline antibody V region framework. To restore antibody activity, several residues of the rabbit V region framework were also retained in the humanized sequence. These residues were selected using the protocol described by Adair et al. (1991) (WO 91/09967). The alignment of rabbit antibody V region (donor) sequences with human germline (acceptor) V region sequences is shown in Figs. 9 and 10, along with the generated humanized sequences. The CDRs grafted from the donor to the acceptor sequence are defined according to Kabat (Kabat et al., 1987), except for CDR-H1, where the combined Chothia/Kabat definition is used (see Adair et al., WO 91/09967).

Genes encoding several heavy and light chain V region sequence variants were designed and constructed using the DNA2.0 Inc. automated synthesis approach. Other heavy and light chain V region variants were generated by modifying the VH and VK genes using 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 UCB light chain expression vector pMhCK, which contains DNA encoding the human kappa chain constant region (Km3 allotype). The humanized heavy chain V region genes were cloned into the UCB human gamma-4 heavy chain expression vector pMhY4PFL, which contains DNA encoding the human gamma-4 heavy chain constant region with the S241P mutation stabilizing the hinge region (Angal et al., Mol. Immunol. 1993, 30(1): 105-8). Chimeric 6470 containing rabbit V regions (SEQ ID NOs: 11 and 13) and human constant regions was also prepared in a similar manner and used as a reference antibody. Cotransfection of the resulting heavy and light chain vectors into Expi293TM suspension cells resulted in expression of humanized recombinant antibodies in the human IgG4P format.

The human V region of IGKV1-16 plus the J region of JK4 (IMGT, http://www.imgt.org/) were selected as an acceptor for the light chain CDRs of antibody 6470. All framework residues of the light chain in the gL3 graft were of human germline origin except residues 48 and 72 (referring to SEQ ID NO: 15), which retained the donor glutamine (Q48) and glutamine (Q72) residues, respectively. Retention of residues Q48 and Q72 was important to maintain the full activity of the humanized antibody (Fig. 9 and Table 6) for binding human alpha-synuclein fibrils.

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Table 6

Antibody variants Light chain Heavy chain Affinity for human fibrils (KD) Donor remains Donor remains rM Chimeric 6470 - - 99 6470gL3gH23 Q48, Q72 V24, Y47,148, G49, V78, R97 148 6470gL3gH36 Q48, Q72 V24, Y47,148, G49, S73, R97 166 6470gL6gH23 Q72 V24, Y47,148, G49, V78, R97 547 6470gL6gH36 Q72 V24, Y47,148, G49, S73, R97 377 6470gL8gH23 Q48 V24, Y47,148, G49, V78, R97 246 6470gL8gH36 Q48 V24, Y47,148, G49, S73, R97 198 6470gL3gH25 Q48, Q72 V24,148, G49, K71, S73, V78, R97 76000 6470gL3gH26 Q48, Q72 V24, Y47, G49, K71, S73, V78, R97 300 6470gL3gH27 Q48, Q72 V24, Y47,148, K71, S73, V78, R97 22300 6470gL3gH35 Q48, Q72 V24, Y47,148, G49, K71, S73, V78 5282 6470gL3gH46 Q48, Q72 Y47,148, G49, V78, R97 316 6470gL3gH50 Q48, Q72 Y47,148, G49, S73, R97 580

The human V region of IGHV3-23 plus the J region of JH4 (IMGT, http://www.imgt.org/) was chosen as the heavy chain CDR acceptor of the 6470 antibody. As in many rabbit antibodies, the VH gene of the 6470 antibody is shorter than that of the chosen human acceptor. When aligned with the human acceptor sequence, framework 1 of the 6470 VH region lacks the N-terminal residue that is retained in the humanized antibody (Fig. 10). In the framework 3 region of rabbit 6470 VH, two residues (75 and 76) are also missing in the loop between the D and E strands of the beta sheet: in humanized grafts, the gap is filled by the corresponding residues (lysine 75, K75; asparagine 76, N76) from the selected human acceptor sequence (Fig. 10). All heavy chain framework residues in the gH23 and gH36 grafts are derived from the human germline gene except for one or more residues from the group consisting of residues 24, 47, 48, 49, 73, 78, and 97 (referring to SEQ ID NOs: 23 and 31), which retain the donor residues valine (V24), tyrosine (Y47), isoleucine (148), glycine (G49), serine (S73), valine (V78), and arginine (R97), respectively. Conservation of residues V24, Y47,148, G49, and R97 was important to ensure full activity of the humanized antibody to bind to human alpha-synuclein fibrils.

In addition, humanized VH genes were cloned into the pMhFablOHIS human Fab-HIS UCB expression vector, which contains DNA encoding the human gamma-1 CH1 hinge domain with a C-terminal ten-histidine tag. The histidine tag facilitates purification of expressed Fabs by affinity chromatography. Cotransfection of the resulting heavy and light chain vectors into Expi293™ suspension cells resulted in expression of humanized recombinant antibodies in Fab-HIS formats.

Affinity maturation was performed according to the UTA methods described in WO2014198951. The interface between the rabbit 6470 Fab and the EAYEMPSEEG peptide of alpha-synuclein (123132) in a complex determined by X-ray crystallography was analyzed to identify mutations that could potentially improve the affinity of the rabbit 6470 Fab for the alpha-synuclein protein. UTA is a statistically potential tool for determining the probability of occurrence of a given type of contact atom in a protein interface or binding site.

To assess the effect of these mutations on the activity of the antibodies to bind human alpha-synuclein monomer or fibrils, the mutations were first examined on the rabbit 6470 Fab (Table 7A). The kinetics of the interaction were determined by surface plasmon resonance on a Biacore T200 instrument as described in Example 3. Residue 33 in CDRL1 (referring to SEQ ID NO: 11) was mutated from asparagine (N) to arginine (R) or lysine (K): mutation of residue 33 to arginine resulted in increased affinity for alpha-synuclein (Table 7A). Residue 55 in CDRH2 was mutated from serine (S) to asparagine (N) and residue 99 in CDRH3 was mutated from asparagine (N) to lysine (K) or glutamine (Q) or histidine (H) or tryptophan (W) (referring to SEQ ID NO: 13), mutation of residue 55 to asparagine and residue 99 to histidine resulted in increased affinity for alpha-synuclein (Table 7A). Mutation of asparagine in CDRH3 (N99H) also removes a potential deamidation site.

-46 041378

Table 7A

Mutation Chain Monomer Fibril kal (1/mc) kdl (1/c) KD1 (nM) kal (1/mc) kdl (1/c) KD1 (nM) 6470 Rabbit Fab 7.23E+06 l,24E-01 17.2 4.58E+06 4,76E-02 10.3 Y30W L 2.99E+06 l,51E-01 50.4 4.75E+06 8,95E-02 18.8 N33R L l,92E+07 7,99E-02 7.9 9.98E+06 2,89E-02 2.9 N33K L 2.84E+06 4,33E-02 15.2 6.93E+06 4,98E-02 7.1 H31R H 2.65E+05 4,40E-01 1658,4 5.98E+06 3,73E-01 62.3 H31K H 6D7E+06 9,38E-01 151.9 9.06E+06 4,23E-01 46.7 H31Q H 8.00E+06 l,85E-01 23.1 7.83E+06 9,72E-02 12.4 S53N H 4.56E+04 5D7E-02 1132.6 5.75E+06 2,86E-01 49.6 S55N H l,39E+07 l,91E-02 1,9 9.20E+06 l,30E-02 1,4 N99R H l,22E+07 l,70E-01 16.9 l,89E+07 l,54E-01 15.3 N99K H 7.89E+06 l,89E-01 23.9 l,58E+07 2,07E-01 20.7 N99Q H 3.75E+05 4,93E-01 1314,1 l,03E+07 4,25E-01 42.4 N99H H l,33E+07 7,45E-03 0,7 6.20E+06 4,01E-03 0,4 N99W H 9.22E+06 5,66E-01 61.3 5.27E+06 l,34E-01 25.3

Finally, newly identified mutations in the previously prepared full-length humanized antibodies (Table 6) were also tested and their selectivity for human fibrils was verified (Table 7B). The interaction kinetics were determined by surface plasmon resonance on a Biacore T200 instrument as described in Example 3. As shown in Table 7B, mutations at position 33 in the light chain (referring to SEQ ID NO: 19) and 56 and 102 in the heavy chain (referring to SEQ ID NOs: 27 and 35) result in increased affinity for human fibrils, which is an advantageous characteristic of antibodies that need to cross the blood-brain barrier to bind to their target. When antibodies are administered systemically, a large amount of the administered antibody may be lost because antibodies have limited systems to cross complex physiological barriers.

Table 7B

Humanized antibodies Human monomer Human fibril kal (1/ms) kdl (1/c) KD1 (nM) kal (1/ms) kdl (1/c) KD1 (nM) VR6470 gL3; gH23 l,20E+06 0,02416 20,15 8.55E+05 l,42E-04 0.166 VR6470 gL3; gH36 1D5E+06 0,01742 15,10 l,07E+06 ZD7E-04 0.298 VR6470 gL3; gH23S56N-N102H 9.66E+05 0,00445 4.62 l,04E+06 7,08E-05 0,068 VR6470 gL3; gH36S56N-N102H 1D9E+06 0,00488 4.10 l,25E+06 7,44E-05 0,059 VR6470 gL3-N33R; gH23-S56N-N102H 3.48E+06 0,00594 1.71 2.07E+06 1D6E-04 0,056 VR6470 gL3-N33R; gH36-S56N-N102H 4.97E+06 0,00648 1.31 2.39E+06 l,26E-04 0,053

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

Example 5. Characterization of humanized antibodies.

Biophysical characterization was performed for six humanized 64G IgG4P antibodies (Table 8, sequences are given in Table 1).

Table 8 ____________Description____________ _____________gL3gH23_____________ _____________gL3gH36_____________ gL3-N33RgH23-S56N-N102H gL3-N33RgH36-S56N-N102H _______gL3gH23-S56N-N102H_______ gL3gH36-S56N-N102H

- 47 041378

All antibodies were screened for thermal stability ( _Tm ), experimental pI, hydrophobicity, solubility (PEG precipitation assay) and aggregation stability at the air/liquid interface to determine whether mutations had any impact, particularly on affinity, stability and developability.

The screening process also included an assessment of chemical stability (deamidation, tendency to aspartic acid isomerization), since the antibodies contain:

1) Asn(102)S motif (deamidation) in CDR3 of the heavy chain of gL3gH23 and gL3gH36 only;

2) the Asn(98)Asp(99) motif (deamidation) in CDR3 of the light chain of all antibodies;

3) the Asn(32)Asn(33) motif (deamidation) in CDR1 of the light chain of all mutants except N33;

4) the Asp(99)G motif (Asp isomerization) in CDR3 of the light chain of all antibodies. Chemical instability in these regions may lead to product heterogeneity and immunogenicity.

Measurement of thermal stability (Tm).

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

The reaction mixture contained 5 μl of 30x SYPRO® Orange Dye (Invitrogen™) diluted in PBS from a 5000x stock solution and 45 μl of sample at 0.12 mg/ml (in PBS pH 7.4). Approximately 10 μl of the mixture was dispensed into a 384-well optical assay plate in quadruplicate and run on a 7900HT Fast PCR System (Applied Biosystems™). The PCR system heater was set to a temperature of 20 to 99°C with an increase rate of 1.1°C/min. Changes in fluorescence in the wells were monitored using a charge-coupled device. The intensity increase data were plotted and the inflection point of the slope(s) was used to calculate _Tm as described below.

All antibodies had two conformational transitions during globule unfolding. The first one can be attributed to the Tm of the CH2 domain. The second one can be attributed to the average Tm of the unfolding Fab domain and the CH3 domain, according to the literature (Garber E., Demarest S.J. Biochem. Biophys. Res. Commun. 2007 Apr 13; 355(3):751-7). The results are summarized in Table 9.

Table 9

Description T _m domain Fab SD T _m domain CH ₂ SD gL3gH23 73.1 0,6 64.8 0,2 gL3gH36 73.5 0,3 64.7 o,o gL3gH23-S56N-N102H 72.8 0,2 64.8 0,3 gL3gH36-S56N-N102H 73.4 0,2 65.1 0,3 gL3-N33R-gH23-S56N-N102H 73.9 0,5 65.2 0,1 gL3-N33R-gH36-S56N-N102H 73.3 0,2 64.7 0,3

Thermal stability is within the normal range (Heads et al., Relative stabilities of IgG1 and IgG4 Fab domains: influence of the light-heavy interchain disulfide bond architecture. Protein Sci. 2012 Sep; 21(9):1315-22) expected for IgG4 molecules.

Experimental value of pI.

The experimental pI value of the 6470 antibody was obtained using the cIEF iCE3™ system with full capillary imaging (ProteinSimple).

Samples were prepared by mixing 30 µl of sample (from a 1 mg/mL stock solution in HPLC grade water), 35 µl of 1% methylcellulose solution (Protein Simple), 4 µl (pH 3-10) ampholytes (Pharmalyte), 0.5 µl of pI 4.65 and 0.5 µl of pI 9.77 synthetic markers (ProteinSimple), and 12.5 µl of 8 M urea solution (Sigma-Aldrich®). HPLC grade water was used to bring the final volume to 100 µl. The mixture was vortexed briefly to ensure complete mixing and centrifuged at 10,000 rpm for 3 min to remove air bubbles prior to analysis. Samples were focused for 1 min at 1.5 kV, then 5 min at 3 kV, and A280 capillary images were acquired using ProteinSimple software. The resulting electropherograms were first analyzed using iCE3 software and pI values (linear relationship between pI markers) were determined. The calibrated electropherograms were then integrated using Empower® software (Waters).

The experimental pI values for all 6470 antibodies were in the range of 8.4-9.2. There was a slight difference between the molecules in the ratio of charged particles, but for the IgG4P molecule this is not unexpected. All pIs were high and can therefore be used for

- 48 041378 production of antibodies.

Hydrophobic interaction chromatography (HIC).

Hydrophobic interaction chromatography (HIC) separates molecules in order of increasing hydrophobicity. Molecules bind to a 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 times correspond to greater hydrophobicity.

Samples at 2 mg/mL were diluted 1:2 with 1.6 M ammonium sulfate and PBS (pH 7.4). 5 μg (5 μL) of sample were injected onto a Dionex ProPacTM HIC-10 column (100 mm x 4.6 mm) connected in series to an Agilent 1200 HPLC binary pump with a fluorescence detector. Separation was monitored by intrinsic fluorescence (excitation and emission wavelengths, 280 and 340 nm, respectively).

Using buffer A (0.8 M 100 mM ammonium phosphate, pH 7.4) and buffer B (100 mM phosphate, pH 7.4), the sample was analyzed by gradient elution as follows:

(i) holding for 2 min at 0% B;

(ii) linear gradient from 0 to 100% B in 30 min (0.8 ml/min);

(iii) washing the column with 100% B for 2 min and re-equilibrating in 0% B for 10 min before the next sample injection.

The column temperature was maintained at 20°C.

Standards exhibiting low and high hydrophobicity plus a control were also analyzed in the same sequence of runs to normalize retention times (Table 11). The retention time (RT) of the sample was normalized to the low and high hydrophobicity standards using the following equation:

[(Sample(RT)-Low Standard(RT)/High Standard(RT)-Low Standard(RT)]x100.

__________________________________________________________ Table 10

Time Normalized time Antibody (Main peak) holdings (min) holding (min) gL3gH23 9 3.8 gL3gH36 8,8 3.1 gL3gH23-S56N-N102H 8.9 3.5 gL3gH36-S56N-N102H 8,8 3.1 gL3-N33RgH23-S56N-N102H 8,8 3.1 gL3-N33RgH36-S56N-N102H 8,8 3.1

All 6470 antibodies and mutants showed similar normalized retention times and similar low hydrophobicity. Commercially available therapeutic antibodies tend to exhibit low hydrophobicity (Jain et al., Biophysical properties of the clinical-stage antibody landscape, Proc. Natl. Acad. Sci. USA. 2017 Jan 31; 114(5):944–949). Low hydrophobicity promotes stability (i.e., reduces aggregation) during manufacturing.

Solubility measurement using polyethylene glycol (PEG) precipitation analysis.

Colloidal solution stability was analyzed using a polyethyleneglycol (PEG) precipitation assay. PEG was used to quantify protein solubility by increasing the PEG concentration (w/v) and measuring the amount of protein remaining in solution. This assay served to simulate the effect of high solubility without the use of traditional concentration methods. PEG-induced precipitation of antibody 6470 was studied in the presence of 7-18% PEG-3350 in PBS, pH 7.4, 50 mM sodium acetate/125 mM sodium chloride, pH 5.0 (acetate, pH 5), and 20 mM L-histidine, 140 mM NaCl, pH 6.0. Samples were buffer exchanged where necessary by dialysis and the concentration was adjusted to 2 mg/mL. To minimize nonequilibrium precipitation, sample preparation consisted of mixing 2x protein and 2x PEG solutions in a 1:1 volume ratio. After mixing, samples were incubated at 37°C for 30 min to redissolve nonequilibrium aggregates. After overnight incubation at 20°C, samples were centrifuged for 60 min (4000 g). Aliquots of the supernatant were transferred to half the volume of 96-well optical assay plates and absorbance was measured at 280 nm using a BMG Labtech FLUOstar® Omega LVIS A280 plate reader. Concentration data were plotted versus PEG% and the calculated midpoint (LogEC ₅₀ ), obtained by nonlinear curve fitting with varying slopes, was obtained as a measure of the relative solubility of the colloidal sample solution. In this assay, a higher LogEC ₅₀ value corresponds to a higher stability of the colloidal solution.

The results (not shown) showed that with increasing buffer pH, the stability of the colloidal solution decreased for all 6470 antibodies. In addition, the following trend was observed: from more to

- 49 041378 less soluble: gL3gH23 and gL3gH36>gL3gH23-S56N-N102H and gL3gH36-S56N-N102H>gL3N33RgH23-S56N-N102H and gL3-N33RgH36-S56N-N102H.

Therefore, the mutations introduced for affinity maturation reduced the stability of the colloidal solution of antibody molecules. No differences were observed between gL3gH23 and gL3gH36 transplants.

Effect of stress at the air-liquid interface (aggregation analysis).

Proteins tend to unfold when exposed to an air-liquid interface, where hydrophobic surfaces are exposed to a hydrophobic environment (air) and hydrophilic surfaces are exposed to a hydrophilic environment (water). Mixing protein solutions provides a large air-liquid interface that can promote aggregation. This assay serves to simulate the stresses the molecule is subjected to during manufacturing processes (e.g., ultrafiltration) and to provide stringent conditions to attempt to distinguish different antibody molecules.

Samples in PBS pH 7.4, 50 mM sodium acetate/125 mM sodium chloride pH 5.0 (acetate pH 5) and 20 mM L-histidine, 140 mM NaCl, pH 6.0 were vortexed on an Eppendorf Thermomixer Comfort™. Buffers were chosen as common pre-made buffers. Before vortexing, the concentration was adjusted to 1 mg/mL using the appropriate extinction coefficients (1.35 Ab 280 nm, 1 mg/mL, 1 cm pathlength) and absorbances at 280, 340 and 595 nm obtained on a Varian Cary® 50-Bio spectrophotometer to establish the zero reading point. Each sample was sub-aliquoted into 1.5 ml Eppendorf® capped conical tubes (4 x 250 µl) and subjected to stringent stability testing by shaking at 1400 rpm at 25°C for up to 24 h. Time-dependent aggregation (turbidity) was monitored by measuring samples at 3 and 24 h after shaking at 595 nm using a Varian CaryTM 50-Bio spectrophotometer. The average absorbance values for each sample were plotted against time.

The results obtained are shown in Fig. 11. No differences were observed between the 6470 antibodies in any of the three buffers after 24 h, but a slight difference was found in the aggregation propensity after 3 h of shaking, which appeared to be buffer dependent.

Investigation of the effect of extreme conditions on Asp deamidation/isomerization.

An extreme condition study was performed using 6470 gL3gH23 and gL3gH36 antibodies to determine the deamidation/Asp isomerization susceptibility of four identified candidate sequences: Asn(102)S (deamidation motif) in CDR3 of the heavy chain; Asn(98)Asp(99) (deamidation motif) in CDR3 of the light chain; Asn(32)Asn(33) (deamidation motif) in CDR1 of the light chain; and Asp(99)G (Asp isomerization motif) in CDR3 of the light chain. The propensity/rate of deamidation and Asp isomerization cannot be predicted because it depends on the primary sequence and three-dimensional structure, as well as on solution properties (R.C. Stephenson and S. Clarke (1989); K. Diepold et al. (2012); Jasmin F. Sydow et al. (2014); N.E. Robinson et al. (2004).

Baseline levels of deamidation (non-stressed samples) were also obtained; low levels indicate low susceptibility, but levels may vary between batches/manufacturing conditions.

The buffer in the two 6470 antibodies was exchanged with buffers (i) known to promote deamidation of Asn(N) residues (Tris pH 8/37°C) and (ii) known to promote Asp isomerization (acetate, pH 5/37°C). The final sample concentration in each buffer was adjusted to ~6.5 mg/mL and then divided into two aliquots, where one was stored at 4°C and one at 37°C for up to 4 weeks. The aliquot was removed immediately (TO) and after 2 and 4 weeks and stored at -20°C.

Two-week old samples were thawed and analyzed by tryptic digestion/mass spectroscopy (MS) to analyze chemical modification as follows. Stressed protein samples were reduced with TCEP and alkylated with chloroacetic acid in TrisHCL buffer, pH 8.0, containing 0.1% w/v Rapigest detergent. Trypsin (1:25 w/w) was added and samples were digested overnight at room temperature. Proteolysis was stopped by adding formic acid to 1% v/v and samples were diluted to 0.5 mg/mL before centrifugation to remove precipitated Rapigest™. The resulting peptides were separated and analyzed on a Waters BEH C18 column coupled to a Thermo Fusion™ mass spectrometer, which utilizes the data-dependent +ve-ion orbitrap-orbitrap method with collision-induced dissociation (CID) fragmentation. LC-MS data were analyzed using Thermo Xcalibur™ and PepfinderTM software.

Size exclusion HPLC and SDS PAGE were also performed to monitor aggregation/degradation.

Peptide mapping/mass spectrometry results showed that the basal level of Asn deamidation at all three CDR sites is <1.5%, and that deamidation maximally increases to ~6% after 2 weeks at pH 8.0 and 37°C for the Asn(102)S site in heavy chain CDR3.

Asp(99) modification (succinimide formation) in light chain CDR3 was ~25% after 2 weeks at pH 5.0 and 37°C.

The effect of chemical modification (deamidation at Asn(102) in CDR3 of the heavy chain) was assessed.

- 50 041378 and formation of a succinimide intermediate at Asp(99) in CDR3 of the light chain) on the affinity/avidity of recombinant full-length human alpha-synuclein monomer and purified recombinant human alpha-synuclein fibrils. The fully deamidated product (Asn(102)Asp) and stress-stressed material (pH 5/2 week/37°C) were used in the study.

Example 6. Immunohistochemistry.

Immunohistochemistry was performed on an Asterand Bioscience system (Royston, United Kingdoms). Cryosections (10 μm) were first subjected to antigen retrieval with Dako PT Link Target Retrieval Solutions and EnVision FLEX Target Retrieval Solutions (pH 6) at 97°C for 20 min with automated heating and cooling. All subsequent incubation steps were performed at room temperature. Cryosections were air dried for 30 min, fixed in 4% paraformaldehyde prepared in 1X PBS for 10 min, washed in Dako EnVision™ FLEX Wash Buffer (Dako), and then loaded into a Dako Autostainer Plus. Endogenous peroxidase activity was blocked by incubating sections with Dako Peroxidase Block (Dako) for 5 min. Sections were then washed twice with 1X PBS before incubation with Dako CSA II Protein Block (Dako) for 10 min. The protein block solution was removed by air flushing and sections were incubated for 30 min with rabbit IgG1 6470 (comprising SEQ ID NOs: 47 and 48) diluted (0.05 μg/mL) in Dako Antibody Diluent (Dako). Following incubation, sections were washed twice with 1X PBS, then incubated with Dako Flex Anti-Rabbit Polymer-HRP Substrate (Dako) for 20 min, washed twice and then incubated with Diaminobenzidine Substrate (Dako) for 10 min. The chromogenic reaction was stopped by rinsing the slides with distilled water. Following chromogenesis, sections were removed from the Dako Autostainer Plus and manually stained with hematoxylin, dehydrated with increasing concentrations of ethanol, cleared with three xylene washes, and mounted with DPX mounting medium (Sigma-Aldrich). Digital images of stained sections were acquired with an Aperio ScanScope AT Turbo (Leica Biosystems). Antibody 6470 was tested on brain sections from five different p8129-a-synuclein-positive and three different p8129-a-synuclein-negative donors (1 section/donor). Antibody 6470 stained neuropils and Lewy body-like structures in the temporal cortex and substantia nigra of PD patients (Figs. 12A–E). In non-PD brain tissues, antibody 6470 stained neuropils in the temporal cortex, but no Lewy body-like structures were observed in the cortex or substantia nigra (Fig. 12F-H). These observations suggest that antibody 6470 binds to normal α-synuclein in the neuropil of brain tissues from PD and non-PD patients, whereas it binds to abnormal α-synuclein present in Lewy bodies only in PD patients.

Example 7. Cell-based aggregation assay.

HEK Freestyle 293F cells (suspension cells) were seeded at a density of 0.7x106 cells/mL in Freestyle 293 Expression Medium (Invitrogen™) and grown to a density of 300x106 cells/mL. Transfection was performed according to the manufacturer's instructions and quickly mixed with 600 μg of pcDNA3.1(+) containing the alpha-synuclein gene in 20 mL of OptiMEM medium, and 293Fectin was diluted in OptiMEM medium (Invitrogen™) and incubated for 5 min at room temperature.

Diluted DNA was added and incubated at room temperature for 20 min and then added dropwise to the cells (20 ml per flask). Cells were incubated for 24 h at 37°C, 125 rpm, 8% CO ₂ . Cells were either used immediately or frozen at a density of 5 million cells/ml in FBS + 10% DMSO.

If the cells were previously frozen, the cryovials were thawed and the cells were resuspended in Freestyle 293 medium, centrifuged at 500g for 5 min, the supernatant was discarded and the pellet was resuspended in Freestyle 293 medium (Life Technologies™) containing Pen/Strep (Invitrogen™) at a density of 2x106 cells/mL. 20 µL of the cell suspension (a total of approximately 40,000 cells/well) were added to a 384-well plate (Grainer™). To each well, 150 nM human alphasynuclein fibrils (prepared as disclosed herein in Example 1) were added, followed by antibodies (6470 gL3gH23; 6470 gL3gH36; 6470 gL3N33gH23 S56NN102; 6470 gL3N33gH36 S56N N102; 6470 gL3gH23 S56N N102; 6470 gL3gH36 S56N N102; all as full-length IgG4P, sequences are given in Table 1) in PBS for testing (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 day 2, the medium was removed from all wells and the plate was washed, leaving 20 μl per well. Approximately 50 μl of PBS was added to each well and the plates were centrifuged at 500 g for 5 min. The supernatant was aspirated from all wells using a plate washer, leaving 20 μl of medium in each well. Versene (Lonza™) (50 μl/well) was added and the plates were centrifuged at 500 g for 5 min and the supernatant was aspirated, leaving only 20 μl of medium per well. 20 μl of 8% formaldehyde (16% aqueous solution, Life Technologies™) + 2% Triton X-100 (VWRTM) in PBS was added to each well. The plates were incubated at room temperature for 15 min, after which 50 µl of FACS buffer consisting of HBSS (VWR™ calcium-free and

- 51 041378 magnesium) + 2% FBS + 2 mM EDTA (Life Technologies™). The plates were centrifuged at 2000 g for 1 min and the supernatant was aspirated, leaving only 20 µl of medium in each well. An additional 20 µl of FACS buffer with the alpha-synuclein antibody pSer129 (AbCam™) diluted 1:300 was added to each well. The plates were incubated for 1 h at room temperature and then 50 µl of FACS buffer was added to each well before centrifugation again at 2000 g for 1 min. The supernatant was removed before adding Alexafluor647-conjugated secondary anti-rabbit antibody (Life Technologies™) diluted 1:500 and DAPI (Life Technologies™) to each well. The plates were incubated for 1 h at room temperature in the dark, after which 50 μl of FACS buffer was added and the plates were centrifuged at 2000 g for 1 min. After washing, more FACS buffer was added and the plates were ready for loading onto a flow cytometer (BD FACS Canto II) for reading.

FACS data were analyzed using FlowJo software. First, live single cells were gated using forward and side scatter. Second, DAPI+ events were gated and their number was used as a measure of live, nucleated single cells. Finally, phosphoserine 129-labeled alpha-synuclein-positive (pSer129+) cells were gated. The percentage of pSer129+ cells relative to all DAPI+ cells was used as a measure of aggregation. Data were normalized to wells treated with fibrils only and without antibodies and expressed as percentages.

The results are shown in Fig. 13, which shows the ability of the tested antibodies to inhibit alpha-synuclein fibril-induced aggregation on alpha-synuclein expressing cells. These data confirm that the antibodies of the present invention are capable of blocking alpha-synuclein fibril-induced aggregation with an IC ₅₀ of less than 5 nM.

Error bars represent standard error of measurement (SEM, N=3, n=9).

Example 8. Analysis of primary neuron aggregation.

Hippocampi from E17 mouse embryos were dissected in dissection buffer (calcium-free and magnesium-free HBSS, 0.6% D-(+)-glucose, 20 mM Hepes). The dissection buffer was then removed and replaced with dissociation solution (calcium-free and magnesium-free HBSS, 0.6% D-(+)-glucose, 20 mM HEPES, 40 U/m papain, 1 mg/mL DNase, 1 mM L-cysteine, 0.5 mM EDTA). After 30 min of incubation at 37°C, the dissociation buffer was removed and the hippocampi were washed 3 times with seeding medium (Neurobasal™ medium supplemented with 2% B27, 1 mM GlutaMAX, 2.5% FBS, 50 U/ml penicillin-streptomycin). The tissue clumps were broken up using a 1 ml pipette to obtain single cell suspensions. Cells were diluted to the appropriate concentration in seeding medium. Approximately 15,000 cells were seeded into each well of a 384-well PDL-coated plate. The cells were then stored in a cell culture incubator at 37°C, 5% CO ₂ , 95% humidity.

The following day, 80% of the medium was replaced with FBS-free plating medium [Neurobasal™ medium, 2% B27 supplement, 1 mM GlutaMAX, 50 U/ml penicillin-streptomycin]. Seven days after plating, the medium was removed, leaving 20 μl in each well. 100 nM human alpha-synuclein fibrils (prepared as described herein in Example 1) were added to each well, followed by antibody 6470 (6470gL3gH36 hIgG4P; VR6470 in Fig. 14, containing SEQ ID NO: 17 and SEQ ID NO: 33) in PBS for testing (at various concentrations). The plate was incubated at 37°C, 5% _CO2 , 95% humidity in a cell culture incubator for an additional 7 days. Fourteen days after plating, the medium was aspirated from all wells, leaving 20 μl per well. Each well was washed with 80 μl of Dulbecco's phosphate-buffered saline (DPBS). DPBS was removed and cells were incubated in 40 μl of fixation buffer (DPBS with 4% paraformaldehyde) per well for 15 min. Fixation buffer was then removed and cells were washed again with 80 μl of DPBS. DPBS was removed and replaced with 40 μl of permeabilization buffer (DPBS with 0.1% Triton X-100) per well. After 10 min, the permeabilization buffer was removed and the cells were incubated for 1 h in 40 µl blocking buffer (PBS with 1% BSA and 0.1% Triton X-100) per well. The blocking buffer was then removed and replaced with 40 µl/well of primary antibody (blocking buffer with 0.3% phosphoserine 129-labeled rabbit anti-alpha-synuclein antibody (AbCam™ ab51253). The antibody solution was incubated on the cells for 1 h and then washed three times (90 µl/each, PBS). After the last wash, the PBS was removed and replaced with 40 µl of secondary antibody (0.1% AlexaFluor647-conjugated anti-rabbit antibody in PBS with 0.2% AlexaFluor488-conjugated anti-beta-III-tubulin antibody). The secondary antibody solution was incubated in the wells for 1 h, then removed and replaced with 40 µl PBS containing 0.3% CellMask Blue™. After 5 min incubation, wells were washed 3 times with 80 µl PBS, then filled with 50 µl PBS per well before the plate was sealed with clear plastic film.

Plates were processed on an Arrayscan plate imager (ThermoFisher Scientific™). Images were analyzed using the HCS Scan™ software from the same manufacturer. Neuronal density was monitored using the beta-III tubulin signal. Areas of sparse cell arrangement or areas with damaged neuronal cell layer, defined by a significant decrease in the beta-III tubulin signal surface, were excluded. Finally, the pSer129 alpha-synuclein signal surface per zone was used to quantify pathological alpha-synuclein aggregation.

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

Data were normalized to wells treated with fibrils alone and without antibodies and expressed as percentages. As shown in Fig. 14, 6470gL3gH36 (designated VR6470) inhibited alpha-synuclein fibril-induced aggregation in mouse primary neurons expressing endogenous levels of alpha-synuclein. Error bars represent the standard error of measurement (SEM, N=4, n=18). These data confirm that 6470gL3gH36 is able to block alpha-synuclein fibril-induced aggregation in mouse primary neurons with an _IC50 lower than 4 nM.

Example 9. Evaluation of the efficacy of VR6470 in vivo.

An antibody containing the variable regions of SEQ ID NOs: 17 and 33 and the constant region of mouse IgG1 (called VR6470 in this example) was tested in wild-type male C57B1/6J mice (Janvier, France) and in a transgenic α-synuclein knockout mouse model expressing human alpha-synuclein (hereinafter referred to as SNCA-OVX; Charles River, France).

C57B1/6J and SNCA-OVX mice were injected with the antibody and mouse preformed fibrils (PFF) (prepared as disclosed herein in Example 1). A negative control antibody (101.4) and vehicle were also injected together with a reference antibody to alpha-synuclein (C-terminal reference Ab), which binds to the last nine C-terminal residues of alpha-synuclein. This reference antibody (which has CDRs different from the antibodies of the invention) showed comparable binding characteristics to the antibodies of the invention. The reference antibody has a higher affinity for alpha-synuclein than the antibodies of the invention and similar biophysical properties. It is also equally effective in preventing alpha-synuclein aggregation in the HEK cell-based assays according to Example 8 (Table 11).

Table 11

Antibody Monomer Fibrils IC50 kal (1/ms) kdl (1/s) KD1 (nM) kal (1/ms) kdl (1/s) KD1 (nM) (nM) VR6470 1.15E+06 0,01742 15,10 1.07E+06 ZD7E-04 0.298 less than 5 C-term AЬ comparison 4.76E+05 1,03E-02 21.64 1.08E+06 2,20E-05 0,02 less than 5

Antibodies were pre-incubated with PFF for 30 min on a shaker at room temperature before direct injection into the brain of animals. Antibody/PFF mixtures were prepared in PBS at a ratio of 1 μg PFF/10 μg antibody. PBS (pH 7.4) was used as a carrier solution. One injection was administered 24 h before the combined intracerebral administration.

The antibodies were then administered intraperitoneally to mice at a dose of 30 mg/kg. A second intraperitoneal injection was administered 7 days after the first, and then the same treatment regimen was used (one intraperitoneal injection per week at a dose of 30 mg/kg with an injection volume of 10 ml/kg) for 4 weeks for a total of 4 injections in male wild-type C57B1/6J mice and for 11 weeks for a total of 12 injections in SNCA-OVX mice. In both experiments, mice were randomly assigned to drug treatment groups and the experiment was performed blinded to treatment.

Animal experiments were performed in accordance with the guidelines of the European Directive 2010/63/EU and Belgian law. The Ethical Committee for Animal Experimentation of UCB Biopharma SPRL (LA1220040 and LA2220363) approved the experimental protocol (ASYN-ICPARKINSON-MO). At the time of surgery, the mice weighed between 25 and 30 g and were 17 weeks old. Mice were housed in cages (4 mice per cage, Macrolon type 2). They were maintained under a 12:12 light/night cycle and lights were on at 06:00. The temperature was maintained at 20-21°C and the humidity was approximately 40%. All animals had free access to standard food pellets and water before allocation to the experimental groups. Enriched chow and proper housing conditions (Enviro-dri, Pharma Serv) were provided before and after surgery. Animal health was monitored daily by animal care staff. Every effort was made to minimize animal suffering. Animals were euthanized under anesthesia.

- 53 041378

The 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 atipamezole (Antisedan, Orion Corporation) was given to maintain the animals awake. Purified recombinant PFFs were thawed and sonicated at room temperature (Qsonica 500 - 20 kHz; power 65%, 30 pulses of 1 sec (ON) with 1 sec rest (OFF) for one minute). The PFFs were then premixed with antibodies for 30 min and shaken at room temperature for 30 min before injection into the brain. The solution (2 μl) was injected at a rate of 0.2 μl/min, and the needle was left in place for another 2.5 min before being slowly retracted. Injections were made unilaterally into the right striatum at the following coordinates: AP=+0.20 mm, ML=-2.00 mm, DV=-3.20 mm.

After anesthesia, mice were perfused transcardially with ice-cold 0.9% PBS containing 10 U/ml heparin for 9 min at a flow rate of 6 ml/min through the left ventricle. The right atrium was incised as an outflow route. Animals were then perfused with ice-cold 4% paraformaldehyde in PBS for 15 min at a flow rate of 6 ml/min. Brains were fixed overnight in PBS containing 4% paraformaldehyde at 4°C (day 0). The following morning (day +1), 4% paraformaldehyde was removed and brains were washed with cold PBS and incubated overnight. The following day (day +2), brains were washed with PBS for a minimum of 1 h and transferred to PBS containing 15% sucrose and stored at 4°C until shipment.

Brain sections were performed at Neuroscience Associates (TN, USA). Brains were first treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freezing artifacts and embedded in a gelatin matrix according to the MultiBrain® method. After solidification, blocks were rapidly frozen by immersion in isopentane cooled to -70°C with crushed dry ice and mounted in the freezing stage of an AO860 sled microtome. Blocks were sectioned at 40 μm thickness using MultiBrain® in the coronal plane. 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 sections were immediately stored at -20°C.

Free-floating sections were immunostained with pSer129 alpha-synuclein antibody (mouse anti-alpha-synuclein (pSer129), biotin (Wako -010-26481)) diluted 1:30,000. All blocking serum incubations were followed by Tris-buffered saline (TBS) with Triton X-100 as carrier; all washes were performed with TBS. Endogenous peroxidase activity was blocked by treatment with 0.9% hydrogen peroxide, and nonspecific binding was blocked with 1.26% whole normal serum. After washing, sections were stained with primary antibody overnight at room temperature. The carrier solution contained 0.3% Triton X-100 for permeabilization. After washing, sections were incubated with avidin-biotin HRP complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) for one hour at room temperature. After washing, sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to create a visible reaction product mounted on gelatinized (subbed) glass slides that were air-dried, lightly stained with thionine, dehydrated in alcohols, cleared in xylene, and coated with Permount.

Fluorescent immunohistochemistry for p62/SQSTM1 (p62 is known to co-aggregate in human Lewy bodies) and Amytracker (for protein aggregates in general) was performed on free-floating brain sections. VR6470 was shown to reduce the amount of Amytracker-stained aggregated proteins and to co-localize with pS129. This indicates that the VR6470 antibody not only reduces the amount of phosphosynuclein but also reduces the amount of synuclein aggregates (data not shown).

The amount of pSer129 alpha-synuclein signal per field of view of pSer129 alpha-synuclein signal was used to quantify the pathological aggregation of alpha-synuclein in the ipsilateral striatum, cortex, basolateral complexes of the amygdala, and substantia nigra. Regions of interest (ROIs) were manually delineated, and automated quantification of pSer129 alpha-synuclein signal in different brain regions was performed using VisioPharm 6 software (VisioPharm). A linear Bayesian algorithm was used to quantify the pSer129 alpha-synuclein signal, which provides a signal area value (label area in μm ² ). The label area reflects the amount of pSer129 alpha-synuclein pathology that covers different brain regions. All quantifications were performed in a blinded manner until the end of statistical analysis.

Data analysis was performed for % label area (i.e., the ratio between the pSer129 signal area in ^μm2 and the area of the region of interest in ^μm2 ). The % label area was estimated repeatedly for several brain sections located rostro-caudally (striatum: 13-14 sections from Bregma +1.1 to -0.94; cortex: 13-14 sections from +1.1 to -0.94; basolateral amygdala: 6-10 sections from -0.58 to -2.06; substantia nigra: 6-8 sections from -2.54 to -3.88), and AUC was calculated separately for each subject.

One-way ANOVA was used for statistical analysis. ANOVA was accompanied by

- 54 041378 multiple pairwise comparisons between means without any consideration of multiple comparisons (** p<0.01 and * p<0.05). Data were log-transformed to meet the criteria for normality and homoscedasticity. Graphs represent geometric means of untransformed data.

As shown in Figs. 15A and 15B, antibody 6470 (comprising the variable regions of SEQ ID NOs: 17 and 33 and the constant region of mouse IgG1) significantly reduced alpha-synuclein pathology (i.e., pSer129 alpha-synuclein signal) compared to three controls in four different ipsilateral brain regions, including the striatum, cerebral cortex, amygdala, and substantia nigra, one month after administration of mouse PFFs to male wild-type C57B1/6J mice (Fig. 15A) and three months after administration of human PFFs to male SNCA-OVX mice (Fig. 15B).

Figure 16 shows the quantification of alpha-synuclein phosphorylated at Ser129 (AUC% of marker area) in the ipsilateral cortex, striatum, amygdala, and substantia nigra of wild-type C57B1/6J mice, respectively. The negative control antibody and the C-terminal comparison antibody did not reduce alpha-synuclein pathology compared to the vehicle group. In contrast, the 6470 antibody significantly reduced the level of pathology (i.e., pSer129alpha-synuclein signal) in the cortex, striatum, amygdala, and substantia nigra (p < 0.01) compared to the three control groups of C57B1/6J mice injected with mouse PFFs. Among the wild-type C57B1/6J mice tested, the 6470-treated group showed significant reductions in pSer129 alpha-synuclein levels in four different structures, three of which were regions distal to the injection site (cortex, substantia nigra, and amygdala).

In SNCA OVX mice injected with human PFFs, the 6470 antibody significantly reduced the level of pathology in the cortex and striatum compared to mice that received vehicle, a negative control antibody (101.4), and a C-terminal reference antibody. In SNCA-OVX 6470 mice, significant reductions in pSer129 were observed in at least two different brain structures (cortex and striatum), with at least one (the cerebral cortex) located distal to the injection site.

These results confirm that antibodies containing the structural features of the present invention are capable of preventing the in vivo appearance of alpha-synuclein phosphorylated at Ser129.

In addition, the results showed that not all antibodies that bind to alpha-synuclein in the C-terminal region are effective in vivo. A comparison antibody that binds to the C-terminus of alpha-synuclein itself with high affinity and that effectively prevents alpha-synuclein aggregation in cellular assays failed to prevent Ser129 phosphorylation in vivo.

Therefore, the antibody of the present invention can be used to treat alphasynucleinopathies, such as those characterized by increased phosphorylation of Ser129, including 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), combined 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 6470 in mice.

Male C57/B16 mice (n=3 per drug) were intravenously administered a single 2 mg/kg dose of the 6470gL3gH36 IgG4P antibody (containing SEQ ID NOs: 17 and 33; referred to as 6470 in Fig. 17 and hereinafter).

Blood samples were collected (0.083, 1, 4, 8, 24, 72, 120, 168, and 336 h after injection) from the tail vein and allowed to coagulate at room temperature. Serum was isolated after centrifugation, frozen, and stored until analysis. 6470 was quantified by LC-MS/MS. Serum samples obtained during the study were thawed and quantified using a calibration curve generated using 6470 or reference antibody added at different concentrations to control mouse serum. Prior to injection of samples into the LC-MS/MS system, serum was denatured, reduced and alkylated with acetonitrile (VWR, UK), TCEP-Tris (2-carboxyethyl) phosphine hydrochloride (Sigma, UK) and iodoacetamide (Sigma, UK), respectively. The alkylated samples were then reduced in 100 mM ammonium bicarbonate buffer (Sigma, UK) and digested overnight with trypsin (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 evaporated using a vacuum evaporator. After the samples were completely dried, they were reconstituted with 95/5 water/acetonitrile containing 0.1% formic acid and injected into the LC-MS/MS system. LC-MS/MS analysis was performed using a Prominence HPLC system (Shimadzu) coupled to a Triple Quad 6500 mass spectrometer. The digested sample was injected via an autosampler onto a reversed-phase high-performance liquid chromatography column (Phenomenex Aeris C18 peptide column 100 x 2.1 mm, 2.6 μm), which was maintained at 50 °C. A linear gradient of 5-70% acetonitrile in 0.1% formic acid was applied for 6 min, then increased to 95% acetonitrile in 0.1% formic acid.

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Claims (31)

acid for 0.8 min at a flow rate of 0.6 ml/min. The mass spectrometer was set to perform a multiple reaction monitoring assay to detect multiple transitions of 6470 or 5811 peptides with a dwell time of 50 ms per transition. Data analysis was performed using Analyst 1.6 software. These data demonstrate that the 6470 antibody showed very good pharmacokinetic properties in mice (Table 12 and FIG. 17A), as reflected by the low clearance values measured. They appear to exceed the typical ranges reported for human IgG preparations administered to mice (3-16 ml/day/kg; Deng et al., 2011, mabs 3:161-66). The pharmacokinetic properties of the 6470 antibody were also studied in Cynomolgus monkeys and compared with the prior art antibody. Male Cynomolgus monkeys (n=3 or n=6 per preparation) were intravenously injected with a single dose of 2 or 3 mg/kg of the 6470gL3gH36 IgG4P antibody (6470) and another reference antibody to alpha-synuclein (IgG1 antibody to alpha-synuclein that binds to alpha -synuclein at amino acids 118-126; WO 2013/063516). Blood samples were taken at various time points (0.083, 1, 3, 6, 24, 48, 96, 168, 240, 336, 504, 576, 672 hours after injection) and left to coagulate at room temperature. Serum was isolated after centrifugation, which was then frozen and stored until analysis. Samples were thawed and analyzed by LC/ESI MS/MS. For 6470, the method described in this example above was used, with quantification performed by obtaining a standard curve in Cynomolgus serum. For the reference antibody, equine myoglobin was used as an internal standard and quantification was performed by comparing the signals with an internal standard signal. To do this, the samples were mixed with an internal standard. The samples were then denatured, alkylated and further subjected to overnight enzymatic digestion (trypsin). After digestion, the samples were diluted and the signature peptides of all analytes were subjected to LCMS/MS analysis. Samples were prepared only once and administered twice (once for each method). Concentration versus time profiles were analyzed using a Pharsight Phoenix 6 using non-compartmental analysis to determine pharmacokinetic parameters for clearance and half-life in each individual animal. Mean and standard deviation values are given for each molecule. As shown in FIG. 17B and in table. 12, in Cynomolgus monkeys, antibody 6470 also exhibits excellent pharmacokinetic properties, with a low clearance value. As in mice, its pharmacokinetic profile appears to exceed the typical range reported for human IgG preparations administered to Cynomolgus monkeys (5-12 ml/day/kg; Deng et al., 2011, mabs 3:161-66) . The rapid clearance of reference antibodies observed in Cynomolgus is consistent with published human data (JAMA Neurology 2018, 75, 10:1206-14). The 6470 antibody is superior to the reference antibody, which exhibits unsatisfactory, atypical pharmacokinetics and parameters in both exposure and clearance. Table 12 Antibody Clearance (SD) ml/day/kg mouse Cynomolgus 6470 3.1 (0.7) 4.7 (0.8) Reference antibody 23.4 (9.8) CLAIM 1. An antibody or antigen-binding fragment thereof that binds to alpha-synuclein, where the antibody or antigen-binding fragment contains: a. a light chain variable region containing: i. CDR-L1 containing SEQ ID NO: 44; ii. CDR-L2 containing SEQ ID NO: 2; and iii. CDR-L3 containing SEQ ID NO: 3; And b. heavy chain variable region containing: i. CDR-H1 containing SEQ ID NO: 4; ii. CDR-H2 containing SEQ ID NO: 45; and iii. CDR-H3 containing SEQ ID NO: 46. 2. An antibody or antigen-binding fragment according to claim 1 that binds to an alphasynuclein epitope containing, with reference to SEQ ID NO: 10, residues E123, Y125, E126, M127, P128, S129, E130 and E131, where the epitope optionally contains A124 and G132. 3. An antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody or antigen-binding fragment prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils. - 56 041378 4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is capable of binding alpha-synuclein both as a monomer and in fibrils. 5. An antibody or antigen-binding fragment thereof according to any of the preceding claims, which has a higher binding affinity for alpha-synuclein in fibrils compared to monomeric alpha-synuclein, characterized by a dissociation constant (KD) at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils. 6. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which has a (K _D ) to alpha-synuclein in fibrils of 300 pM or less. 7. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which does not bind to beta-synuclein and/or gamma-synuclein. 8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a chimeric or humanized 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 according to any one of the preceding claims, wherein the full length antibody is selected from IgG1, IgG4, or IgGP. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ , scFv or dAb. 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. a light chain variable region containing a CDR-L1 containing SEQ ID NO: 1, a CDR-L2 containing SEQ ID NO: 2, and a CDR-L3 containing SEQ ID NO: 3; and a heavy chain variable region containing CDR-H1 containing SEQ ID NO: 4; CDR-H2 containing SEQ ID NO: 5 and CDRH3 containing SEQ ID NO: 6; or b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 31; or c. a light chain containing SEQ ID NO: 17 and a heavy chain containing SEQ ID NO: 33. 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 variable region containing a CDR-L1 containing SEQ ID NO: 1, a CDR-L2 containing SEQ ID NO: 2, and a CDR-L3 containing SEQ ID NO: 3; and a heavy chain variable region containing CDR-H1 containing SEQ ID NO: 4; CDR-H2 containing SEQ ID NO: 5 and CDRH3 containing SEQ ID NO: 6; or b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 23; or c. a light chain containing SEQ ID NO: 17 and a heavy chain containing SEQ ID NO: 25. 14. 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 variable region containing a CDR-L1 containing SEQ ID NO: 1, a CDR-L2 containing SEQ ID NO: 2, and a CDR-L3 containing SEQ ID NO: 3; and a heavy chain variable region containing CDR-H1 containing SeQ ID NO: 4; CDR-H2 containing SeQ ID NO: 8 and CDRH3 containing SEQ ID NO: 9; or b. a light chain variable region comprising SEQ ID NO: 15 and a heavy chain variable region comprising SEQ ID NO: 27 or 35; or c. a light chain containing SEQ ID NO: 17 and a heavy chain containing SEQ ID NO: 29 or 37. 15. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises: a. a light chain variable region containing a CDR-L1 containing SEQ ID NO: 7, a CDR-L2 containing SEQ ID NO: 2, and a CDR-L3 containing SEQ ID NO: 3; and a heavy chain variable region containing CDR-H1 containing SEQ ID NO: 4; CDR-H2 containing SEQ ID NO: 5 and CDRH3 containing SEQ ID NO: 6; or b. a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 23 or 31; or c. a light chain containing SEQ ID NO: 21 and a heavy chain containing SEQ ID NO: 25 or 33. 16. 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 variable region containing a CDR-L1 containing SEQ ID NO: 7, a CDR-L2 containing SEQ ID NO: 2, and a CDR-L3 containing SEQ ID NO: 3; and a heavy chain variable region containing CDR-H1 containing SEQ ID NO: 4; CDR-H2 containing SEQ ID NO: 8 and CDRH3 containing SEQ ID NO: 9; or b. a light chain variable region comprising SEQ ID NO: 19 and a heavy chain variable region comprising SEQ ID NO: 27 or 35; or - 57 041378 c. a light chain containing SEQ ID NO: 21 and a heavy chain containing SEQ ID NO: 29 or 37. 17. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-16. 18. The isolated polynucleotide according to claim 17, encoding: a. the variable region of the light chain, where the polynucleotide: i. is at least 90% identical to SEQ ID NO: 16 or SEQ ID NO: 20; or ii. contains SEQ ID NO: 16 or 20; or iii. essentially consists of SEQ ID NO: 16 or SEQ ID NO: 20; b. heavy chain variable region, where the polynucleotide: i. is at least 90% identical to SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or ii. contains SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; or iii. essentially consists of SEQ ID NO: 24 or SEQ ID NO: 28 or SEQ ID NO: 32 or SEQ ID NO: 36; c. light chain, where the polynucleotide: i. is at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 22; or ii. contains SEQ ID NO: 18 or 22; or iii. essentially consists of SEQ ID NO: 18 or SEQ ID NO: 22; d. heavy chain, where the polynucleotide: i. is at least 90% identical to SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or ii. contains SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38; or iii. essentially consists of SEQ ID NO: 26 or SEQ ID NO: 30 or SEQ ID NO: 34 or SEQ ID NO: 38. 19. A cloning vector containing one or more polynucleotides according to any one of claims 17 or 18. 20. An expression vector containing one or more polynucleotides according to any one of claims 17 or 18. 21. Host cell containing: a. one or more polynucleotides according to any one of claims 17 or 18; or b. one or more expression vectors according to claim 20. 22. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, comprising culturing the host cell according to claim 21 under suitable conditions to produce the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof. 23. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and one or more pharmaceutically acceptable carriers, excipients or diluents. 24. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 23 in therapy. 25. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 23 for the treatment of one or more synucleinopathies. 26. Use according to claim 25, 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1). 27. Use according to claim 26, wherein the synucleinopathy is Parkinson's disease. 28. A method of treating synucleinopathy 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 to 16 or a pharmaceutical composition according to claim 23. 29. The method of claim 28, 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), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1). 30. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 for the diagnosis of alpha synucleinopathy. 31. Use according to claim 30, wherein the alpha synucleinopathy is Parkinson's disease. -