RU2787039C2 - Antibodies against alpha synuclein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody or its antigen-binding fragment, which bind to alpha-synuclein, are presented. At the same time, the antibody or the antigen-binding fragment are capable of binding to alpha-synuclein both in the form of monomer and in the form of fibrils and preventing aggregation of alpha-synuclein, induced by alpha-synuclein fibrils. Isolated polynucleotide encoding an antibody or its antigen-binding fragment, cloning and expression vectors, a host cell, a method for the production of an antibody or its antigen-binding fragment, a pharmaceutical composition for the treatment of Parkinson’s disease, and a method for the treatment of Parkinson’s disease are also presented.

EFFECT: inventions can be assigned for use them for the treatment of synucleopathies, including Parkinson’s disease.

27 cl, 17 dwg, 12 tbl, 10 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

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

BACKGROUND OF THE INVENTION

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

In 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 an increase in the number of copies of the alpha-synuclein gene are the cause of the familial form of early-onset Parkinson's disease (PD). Interestingly, in families with an increased number of copies of gene loci, the pathogenic effect is indisputably dependent on the dose of the gene. Gene duplications are associated with the development of a relatively early form of PD (~47 years) with a normal course of the disease, while gene tripplications are associated with a very early age of onset (~33 years) and a very rapid course of the disease. In all forms of Parkinson's disease, alpha-synuclein is the main component of Lewy bodies, a key pathological feature of the disease.

The pathology of Lewy bodies spreads during the course of the disease, and it is suggested that alpha-synuclein acts as a prion-like protein with misassembly leading to the formation of toxic oligomers and aggregates that can spread from affected to unaffected neurons (Olanow CW et al. Movement Disorders, Vol. 28, No.1, 2013). Current therapies are unable to stop the spread of the disease and are only helpful in treating the 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 against misfolded alpha-synuclein to mice that had previously received intrastriatal injection of preformed alpha-synuclein fibrils resulted in a decrease in the severity of pathology Lewy bodies, reduced the loss of dopaminergic neurons in the substantia nigra and attenuated motor impairment. Therefore, there still remains a need for passive immunotherapy that could be therapeutically effective in PD and other alpha synucleinopathies.

SUMMARY OF THE INVENTION

The present invention satisfies the need defined above with anti-alpha-synuclein antibodies according to the following embodiments.

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

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

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.

Embodiment 2: An antibody or antigen-binding fragment thereof according to Embodiment 1 that binds to an alpha-synuclein epitope comprising, 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.

Embodiment 3: 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.

Embodiment 4: An 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 alpha-synuclein 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 monomeric alpha-synuclein, characterized by a dissociation constant (K _D ) of at least 10 times higher for monomeric alpha-synuclein than for alpha-synuclein in fibrils.

Embodiment 6: An antibody or antigen-binding fragment thereof according to any of the preceding embodiments with a (K _D ) value 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 that 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: An antibody according to 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 V _HH .

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 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 CDR-H3 containing 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 containing SEQ ID NO:17; and a heavy chain containing SEQ ID NO:33.

Embodiment 13: 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 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 CDR-H3 containing 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 containing SEQ ID NO:17; and a heavy chain containing 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 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 CDR-H3 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.

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 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 CDR-H3 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.

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 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 CDR-H3 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

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

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

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

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

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

d. contains a heavy chain variable region having at least 80% identity or similarity to the sequence shown in SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:27 or SEQ ID NO:35; and/or

e. contains a light chain variable region having at least 80% identity or similarity to the sequence shown 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: The isolated polynucleotide of Embodiment 18 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.

Embodiment 20: A cloning or expression vector containing one or more of the polynucleotides of any one of Embodiments 18 or 19.

Embodiment 21: A host cell comprising:

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

b. one or more expression vectors of embodiment 20.

Embodiment 22: A method for producing an antibody or antigen-binding fragment thereof according to any of embodiments 1-17, comprising culturing the host cell of 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 the 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: 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 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), Lewy body dementia (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 iron accumulation in the brain type 1 (NBIA-1).

Embodiment 27: Use of the antibody or antigen-binding fragment thereof of 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 from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), body variant Alzheimer's disease Lewy disease (LBVAD), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1), preferably Parkinson's disease.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig.2. (A) Analysis of fibrils by JC-1 analysis of monomer without fluorescence and fibrils with maximum fluorescence at 540 nm. (B) Typical example of the spectrum of random coils of monomeric human alpha-synuclein (wavelength 1646 cm ^-1 ) and internal 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 PVDPDNEAYE peptide.

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 (rPeptide); 3 - human beta-synuclein (rPeptide); Marker, MagicMark XP. (B) NMR chemical shift changes showing the predicted 6470 epitope on human alpha-synuclein.

Fig.5. Inhibition of IgG 6470 binding to immobilized alpha-synuclein (left bars, monomer and correct 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 Fab 6470 heavy chain contacts with peptide 123-132. Peptide residues are labeled directly, heavy chain variable residues 6470 are labeled with vH residue number.

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

Fig.9. Humanization of the light chain. 6470 denotes the rabbit light chain variable region sequence. 6470gL3 denotes a humanized graft of the 6470 light chain variable region using the human germline IGKV1-16 as an acceptor scaffold. CDRs are shown in bold/underlined. Donor balances 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 rabbit heavy chain variable region sequence. 6470gH23 and gH36 are humanized grafts of the 6470 antibody heavy chain variable region using the human IGHV3-23 germline 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. Stress at the air-liquid interface. 6470 antibodies and mutants in three pre-mixed buffers 3 and 24 hours after shaking.

Fig.12. Immunohistochemistry. Immunoreactivity in brain sections of patients with PD (A-E) and without PD (F-H). (A-C) In the temporal cortex of PD patients, antibody 6470 stained neuropils and Lewy body-type structures (white arrows) in gray matter. (D, E) Antibody 6470 stained Lewy body-type structures (white arrows) in the substantia nigra of PD patients. (F, G). In temporal cortical tissues without PD 6470 also stained the neuropil but did not stain structures similar to Lewy bodies. (H) No Lewy body-like structures were observed in the substantia nigra of an individual without PD; black arrows indicate non-specific 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 aggregation induced by alpha-synuclein fibrils with an IC ₅₀ of less than 5 nM. Error bars represent the standard error of the measurement (SEM, N=3, n=9). In the conventions, the FL at the end of each antibody name stands for "full length".

Fig.14. Cell-based aggregation analysis (primary neurons). A representative antibody of 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 the measurement (SEM, N=4, n=18).

Fig.15. Immunohistochemical staining images of alpha-synuclein pathology (arrows) in different brain regions of C57Bl/6J wild-type mice (A) and SNCA-OVX mice (B) injected with murine or human PFF, respectively.

Fig.16. Quantification of pathological alpha-synuclein in various brain regions of C57Bl/6J wild-type mice (A: cerebral cortex; B: striatum; C: tonsil and D: substantia nigra) injected with mouse PFF.

Fig.17. Pharmacokinetic profiles of anti-alpha-synuclein antibodies: A. 6470 antibody in wild-type mouse; B. antibody 6470 and reference antibody in Cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

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

Technical terms are used according to their common sense, unless otherwise indicated. If a particular 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 the preferred version of the term "comprising".

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

As used herein, the terms "treating", "treating", and the like are relate to obtaining the 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 symptom thereof and/or may be therapeutic in the sense of partially or completely curing a disease and/or an adverse effect associated with the disease. Thus, treatment encompasses any treatment of a disease in a mammal, especially a human, and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease but who has not yet been diagnosed as having the disease; (b) inhibition of the disease, ie. stopping its development; and (c) alleviating the disease, ie. causing regression of the disease.

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

In the context of the present description, the term "isolated" means that the antibody, antigennegative fragment or polynucleotide, as the case may be, exists in a physical environment other than that in which it may occur 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 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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless, 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, the antibody or antigen-binding fragment thereof that binds to alpha-synuclein comprises:

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing SEQ ID NO:6.

Alpha-synuclein (or alpha-synuclein; a-synuclein; a-syn or any other known synonym) refers to the common name for this protein and includes, without limitation, alternative splicing variants, mutants, and alpha-synuclein from other species (mice, monkeys, and etc.). Unless otherwise indicated, when referring to human alpha-synuclein or when explicitly stated, such alpha-synuclein contains the sequence shown in SEQ ID NO:10 or Uniprot P37840.

In the context of the present description, the term "antibody", as a rule, refers to intact (whole) antibodies, i. containing elements of two heavy chains and two light chains. The antibody may also contain additional binding domains, such 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 (ie, full length heavy and light chains); Fab, Fab modified, Fab', Fab' modified, F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single domain antibodies (eg 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 above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and manufacturing 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 WO2009/040562 and its disulfide-stabilized version, Fab-dsFv, was first described in WO2010/035012. 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, may be bispecific, or may be monospecific (see, for example, WO 92/22583 and WO 05/113605). One example of the latter is the tri-Fab (or TFM) described in WO92/22583.

An alternative antigen-binding fragment contains a Fab bound 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 the 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 specifically referred to in the context of the discussion of antibody fragments.

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

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

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

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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46

and binds to an alpha-synuclein 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.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless, 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, the antibody, or antigen-binding fragment thereof, that binds to alpha-synuclein comprises:

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing SEQ ID NO:6

and binds to an alpha-synuclein 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.

In the context of the present invention, the term "epitope" is used interchangeably for both conformational and linear epitopes, where the conformational epitope consists of discontinuous stretches of the antigen's primary amino acid sequence and the linear epitope is formed by a sequence of contiguous amino acids.

An epitope can be identified by any suitable epitope mapping method known in the art, in combination with any of the antibodies provided in 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 an antibody containing an epitope sequence recognized by the antibody. Alpha-synuclein peptides can be obtained synthetically or by proteolytic cleavage of the alpha-synuclein protein. Peptides that bind to an antibody can be identified, for example, using mass spectrometric analysis. In another example, NMR spectroscopy or X-ray crystallography can be used to identify an epitope associated with an antibody of the present invention. Generally, when epitope determination is performed by X-ray crystallography, the amino acid residues of the antigen within 4Å of the CDR are considered to be the amino acid residues that make up the epitope. Once identified, an epitope can serve to generate fragments that bind to an antibody of the present invention and, if necessary, it can be used as an immunogen to generate additional antibodies that bind to the same epitope.

In one embodiment, the epitope of an antibody or antigen-binding fragment thereof is determined by X-ray crystallography using an alpha-synuclein peptide containing 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 its grammatical variants) is used interchangeably herein with the term "inhibit" and refers to the effect that an antibody of the present invention has on alpha-synuclein aggregation induced by alpha-synuclein fibrils. This action may be prophylactic in terms of total or partial prevention of aggregation; or total or partial reduction, i. e. blocking, aggregation that has already begun after further progression, or a complete or partial reduction in the likelihood of further aggregation; or full or partial relief of aggregation that has already occurred.

Without wishing to be bound by 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 oligomers and aggregates of alpha-synuclein; 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" as used in relation to alpha-synuclein is intended to refer to non-monomeric forms of alpha-synuclein, including alpha-synuclein oligomers, which may be intra- and between brain structures.

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

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46,

prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils. Preferably, the antibody or antigen-binding fragment thereof binds to an alpha-synuclein 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.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless, 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, the antibody, or antigen-binding fragment thereof, that binds to alpha-synuclein comprises:

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing 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 alpha-synuclein 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.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is capable of binding alpha-synuclein as a monomer and fibrils. In one embodiment, the antibody or antigen-binding fragment thereof has a higher binding affinity for alpha-synuclein in fibrils compared to monomeric alpha-synuclein. This is characterized by a dissociation constant (K _D ) 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 (K _D ) for monomeric alpha-synuclein of less than 15 nM. In another embodiment, the antibody or antigen-binding fragment of the present invention has a dissociation constant (K _D ) for alpha-synuclein in fibrils of less than 10 nM. In one of the preferred embodiments, the antibody or antigen-binding fragment of the present invention has a dissociation constant (K _D ) for alpha-synuclein in fibrils of less than 300 pM.

In the context of the present description, the term "K _D " refers to the dissociation constant, which is obtained from the ratio of K _d to K _a (ie K _d /K _a ), expressed as a molar concentration (M). K _d and K _a refer to the rate of dissociation and the rate of association, respectively, of a particular antigen-antibody (or antigen-binding fragment thereof) interaction. Antibody K _D values can be determined by methods well known in the art. One way to determine the K _D of an antibody is to use surface plasmon resonance, for example, using the Biacore® system, for example, as described in the examples provided herein, using isolated natural or recombinant alpha-synuclein, its suitable fusion protein/polypeptide, or its fibrils. In one example, affinity is measured using recombinant human alpha-synuclein as described in the examples provided herein. To implement the surface plasmon resonance method, target molecules are immobilized on the solid phase and exposed to ligands in the mobile phase flowing along the flow cell. If the ligand binds to the immobilized target, the local refractive index of the light changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting the change in reflected light intensity. To obtain an apparent constant of the ratio of association and dissociation phases in a binding reaction, the rate of change of the SPR signal can be analyzed. The ratio of these values gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)).

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

In one embodiment, the antibody or antigen-binding fragment 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. synuclein induced by alpha-synuclein in fibrils, less than 5 nM. Examples of cell-based aggregation assays are disclosed in the examples.

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

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

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

Antibodies of the present invention can be obtained by any suitable method known in this field. To generate antibodies that specifically recognize alpha-synuclein, fusion proteins comprising the alpha-synuclein polypeptide/protein, cells (recombinantly or naturally) expressing the polypeptide can be used. The polypeptide may be a "mature" polypeptide or a biologically active fragment or derivative thereof.

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

The polypeptides used to immunize the 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 noted. In some instances, the alpha-synuclein polypeptide or fragment thereof may be part of a larger protein, such as a fusion protein, eg fused to an affinity or similar tag.

Engineered antibodies to an alpha-synuclein polypeptide can be prepared in those cases where immunization of an animal is desired by administering the polypeptides to an animal, e.g., 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 prepared 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 R Liss, Inc., 1985).

Antibodies for use according to the invention can also be obtained using methods 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, the methods described by Babcook, J. et al., 1996, Proc., Natl. Acad. sci. USA 93(15):7843-7848l; WO92/02551; WO2004/051268 and WO2004/106377.

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

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

CDRs are usually numbered according to the system developed by Kabat et al. This system is outlined in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Depar™ent of Health and Human Services, NIH, USA (hereinafter "Kabat et al. (see above)"). In the present description, this numbering system is used, unless otherwise indicated.

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

The heavy chain variable domain CDRs are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia numbering (Chothia, C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)), the CDR-H1 equivalent loop extends from residue 26 to residue 32. Thus, unless otherwise stated, in the context of the present description "CDR-H1" refers to residues 26-35, according to the combined numbering system according to Kabat with the topological definition of the loop according to Chothia.

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

In one 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 containing a CDR-H1 containing SEQ ID NO:4, a CDR-H2 containing SEQ ID NO:5, and a CDR-H3 containing SEQ ID NO:6.

Alternatively, the antibody or antigen-binding fragment contains a light chain variable region containing a CDR-L1 containing SEQ ID NO:1; CDR-L2 containing SEQ ID NO:2 and 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 CDR-H3 containing SEQ ID NO:9.

In another embodiment, the antibody or antigen-binding fragment thereof contains a light chain variable region containing a CDR-L1 containing SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 CDR-H3 containing SEQ ID NO:6.

In yet another embodiment, the antibody or antigen-binding fragment contains a light chain variable region containing a CDR-L1 containing SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 CDR-H3 containing SEQ ID NO:9.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention may comprise the framework regions of the animal in which the antibody was produced. For example, if an antibody was produced in a rabbit body, it will contain the CDRs as defined above, and rabbit antibody framework regions, for example, an antibody containing 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 using 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 (eg murine) H and L constant regions (Morrison; PNAS 81, 6851 (1984)).

Human antibodies contain heavy or light chain variable regions or full-length heavy or light chains that are "the product of" or "derived from" a particular germline sequence, if the antibody variable regions or full-length chains are derived from a system that uses human germline immunoglobulin genes . Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest, or screening a phage-displayed human immunoglobulin gene library using an antigen of interest. A human antibody or fragment thereof that is a "product" of or "derived from" a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with 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 a human antibody. A human antibody that is "the 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 define the human antibody as being human when compared to the amino acid sequence of the germline immunoglobulin of other species ( e.g. 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 % is 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 show 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 obtained by several methods known to those skilled in the art. Human antibodies can be generated by the hybridoma method 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 using human variable region repertoires (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23) .

In one of the preferred embodiments of the present invention, the antibody or its antigennegative fragment according to the invention is humanized.

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

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46,

where the antibody or its antigennegative fragment 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 alpha-synuclein 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.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless, 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 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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46,

and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and binds to an alpha-synuclein epitope containing, 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 contains:

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing SEQ ID NO:6,

and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and binds to an alpha-synuclein epitope containing, 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 contains one or more CDRs (including, if necessary, one or more modified CDRs) from a donor antibody ( eg, a non-human antibody, such as a mouse or rabbit monoclonal antibody) grafted onto the heavy and/or light chain variable region framework of an acceptor antibody (eg, a human antibody). For a review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more specificity-determining residues from any of those described above in this application are transferred to the human antibody framework. CDR (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity-determining residues from one or more of the CDRs described above in this application are transferred to the framework region of a human antibody. In another embodiment, only the specificity-determining residues from each of the CDRs described above are transferred to the human antibody framework.

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

Accordingly, a humanized antibody of the present invention has a variable domain containing human acceptor framework regions, as well as one or more CDRs provided, in particular, in the present description. 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 are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences available at: http://www.imgt.org/ can be used.

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

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

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

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

- the sequence given in SEQ ID NO:1 or SEQ ID NO:7 for CDR-L1, the sequence given in SEQ ID NO:2 for CDR-L2, and the sequence given in SEQ ID NO:3 for CDR-L3, where the frame region of the light chain is derived from the human germline IGKV1-16 JK4; and

- the sequence given in SEQ ID NO:4 for CDR-H1, the sequence given in SEQ ID NO:5 or SEQ ID NO:8 for CDR-H2, and the sequence given in SEQ ID NO:6 or SEQ ID NO: 9 for CDR-H3 where the heavy chain framework is derived from the human germline IGHV3-23 JH4.

In a humanized antibody or antigen-binding fragment thereof of the present invention, the framework regions may not have exactly the same sequences as those of the acceptor antibody. For example, uncommon 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 can be modified to match residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in acceptor frameworks that may need to be modified is given in WO91/09967.

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

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

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

In another embodiment, a humanized antibody or antigen-binding fragment thereof is provided wherein at least residues at each of positions 24, 47, 48, 49, 73, and 97 (with reference to SEQ ID NOs: 31 or 35) or 24, 47, 48, 49, 78 and 97 of the heavy chain variable domain (with reference to SEQ ID NOS: 23 and 27) are donor residues, see for example SEQ ID NOS: 23, 25, 27, 29, 31, 33, 35 and 37.

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

- the variable region of the light chain, containing SEQ ID NO:15, and the variable region of the heavy chain, containing 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 containing SEQ ID NO:19 and a heavy chain variable region containing SEQ ID NO:27 or 35.

In one embodiment, the invention relates to an antibody or antigen-binding fragment containing a sequence that is 80% similar or identical to the sequence disclosed in the present description, for example, 85%, 90%, 91%, 92%, 93%, 94 %, 95% 96%, 97%, 98% or 99% is similar or identical to part or all of the relevant sequence, for example, a variable domain sequence, a CDR sequence, or a variable domain sequence, excluding the CDR. In one embodiment, the corresponding sequence is SEQ ID NO:15. In one embodiment, the relevant sequence is SEQ ID NO:23 or SEQ ID NO:31.

In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to human light chain alpha-synuclein, wherein the light chain variable domain contains a sequence having at least 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identity or similarity to the sequence shown in SEQ ID NO:15 or SEQ ID NO:19 and/or heavy chain variable domain contains a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99% identity or similarity to the sequence shown in SEQ ID NO:31, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:35.

In one embodiment, the present invention provides 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 or identical to the sequence shown in SEQ ID NO:15, where the antibody or antigennegative fragment has the sequence shown in SEQ ID NO:1 or SEQ ID NO:7 for CDR-L1, the sequence shown in SEQ ID NO:2 for CDR-L2, and the sequence shown in SEQ ID NO:3 for CDR-L3.

In one embodiment, the present invention provides 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 or identical to the sequence shown in SEQ ID NO:31, and where the antibody or antigennegative fragment has the sequence shown in SEQ ID NO:4 for CDR-H1, the sequence shown in SEQ ID NO:5 or SEQ ID NO:8 for CDR-H2, and the sequence shown in SEQ ID NO:6 or SEQ ID NO:9 for CDR-H3.

In the context of the present description, "identity" indicates that in aligned sequences at any particular position, amino acid residues are identical between these sequences. In the context of the present description, "similarity" indicates that in aligned sequences at any particular position there are amino acid residues of a similar type between these sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that can often substitute for each other include, without limitation:

- 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 having acidic side chains);

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

- cysteine and methionine (amino acids with 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, the antigen binding fragment of the present invention may be, without limitation, Fab modified Fab, Fab' 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, tribodies, tetrabodies, and epitope-binding fragments of any of the above (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). Other antibody fragments useful in the present invention include the Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171. Polyvalent antibodies may have multiple specificities, for example, may be bispecific or monospecific (see, for example, WO 92/22853, WO05/113605, WO2009/040562 and WO2010/035012).

An alternative antigen-binding fragment contains a Fab bound 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 the therapeutic target and one scFv or dsscFv that increases the half-life by binding, for example, albumin). Such antibody fragments are described in International Patent Publication No. WO 2015/197772, which is incorporated herein by reference in its entirety and in particular in the context of the discussion of antibody fragments.

In another embodiment, the antibody or antigen-binding fragment of the present invention is part of an alpha-synuclein-binding fusion protein, which contains, for example, fused antigen-binding fragments of the present invention, for example, in the form of a Fab or Fab' fragment, and one or two single-domain antibodies (dAb), directly or indirectly associated with them, for example, as described in documents WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492 and WO2011/086091, each of which is incorporated herein by reference. In one embodiment, the fusion protein comprises dual domain antibodies, eg, as a paired variable heavy (VH) and variable light (VL) domain, optionally linked by a disulfide bond.

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

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

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

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46,

wherein the humanized antibody prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils and preferentially binds to an alpha-synuclein epitope containing, with reference to SEQ ID NO:10, residues E123, Y125, E126, M127, P128, S129, E130 and E13, where the epitope optionally contains A124 and G132, where the antibody is an IgG4P isoform.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Regardless, 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 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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46,

and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and preferentially binds to an alpha-synuclein 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, where the antibody is an isoform of IgG4P, in which 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 the most preferred embodiment, a full-length humanized antibody that binds to alpha-synuclein contains:

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing SEQ ID NO:6,

and prevents alpha-synuclein aggregation induced by alpha-synuclein fibrils, and preferentially binds to an alpha-synuclein 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.

One of skill in the art will also appreciate that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variants of glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic (alkaline) residue (such as lysine or arginine) by carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the heavy chain of the antibody may be absent.

In one embodiment, the C-terminal amino acid 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 may 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 as 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 as set forth in SEQ ID NO:17 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: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 as 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.

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 as 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: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 alpha-synuclein 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.

In another preferred embodiment, the antibody binds to alpha-synuclein and is a full-length IgG4 antibody containing a light chain containing SEQ ID NO:17 and a heavy chain containing 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 alpha-synuclein 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.

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

Therefore, the present invention relates to an antibody or antigen-binding fragment thereof that competes for alpha-synuclein binding to the antibodies or antigen-binding fragments of the present invention by cross-blocking or becomes blocked by the antibody or antigen-binding fragment 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, an antibody or antigen-binding fragment thereof competes for alpha-synuclein binding 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 containing SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:27, or SEQ ID NO:35, and a light chain variable region containing SEQ ID NO:15 or SEQ ID NO:19, and for binding to the alpha epitope synuclein containing, 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 competitive ELISA or BIAcore assays, in which binding of the cross-blocking antibody to human alpha synuclein prevents binding of the antibody of the present invention, or vice versa. In such competition assays, isolated natural or recombinant alpha synuclein or a suitable fusion protein/polypeptide can be used. 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 (eg, fusion of a 6xHis 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 total "observable" charge will depend on the absolute amino acid sequence of the object, the local environment of the charged groups in the 3D 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 available solvent carries no net electrical charge. In one example, an anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention may be engineered to have a suitable isoelectric point. This makes it possible to obtain antibodies and/or fragments with more stable properties, in particular with suitable solubility and/or stability profiles and/or improved purification characteristics.

Thus, in one aspect, the invention relates to a humanized antibody, or antigen-binding fragment thereof, that binds to alpha-synuclein and is designed to have an isoelectric point that is different from that of the originally identified antibody. An antibody can, for example, be constructed by replacing an amino acid residue, such as replacing an acidic amino acid residue with one or more basic (alkaline) amino acid residues. Alternatively, basic amino acid residues may be introduced, or acidic amino acid residues may be removed. Alternatively, if the molecule has an unacceptably high pI value, then, if necessary, acid residues can be introduced to lower the pI. It is important to note that when pI is manipulated, 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 "unmodified" antibody or fragment thereof.

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

It is understood that the affinity of the antibodies of the present invention may be altered by any suitable method known in the art. Therefore, 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), the use of E. coli mutator strains (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexually mimic PCR (Crameri et al., Nature, 391 , 288-291, 1998). A discussion of these affinity maturation methods can be found in Vaughan et al. (see above).

Within the scope of the present invention, affinity maturation was performed according to IOTA (WO2014198951).

If necessary, the antibody or antigennegative fragment 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 associated to form a single fragment that can be attached to an antibody or antigen-binding fragment of the present invention. If it is desired to obtain an antibody fragment bound to an effector molecule, it can be prepared using standard chemical or recombinant DNA procedures in which the antibody fragment is bound to the effector molecule, either directly or through a binding agent. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., Eds., 1987, pp. 623-53; Thorpe et al., 1982 , Immunol Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO 03/031581. Alternatively, when the effector molecule is a protein or polypeptide, binding can be achieved using recombinant DNA procedures, such as those described in WO 86/01533 and EP0392745.

As used herein, the term effector molecule includes, for example, antitumor agents, drugs, toxins, biologically active proteins, such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids, and fragments thereof, such as DNA, RNA. and their fragments, radionuclides, in particular radioactive iodine, radioisotopes, chelate 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 detrimental (eg, kills) to a cell. Examples include combrestatins, dolastatins, epothilones, staurosporine, maytansinoids, spongistatins, rizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daubnorubicin dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs or homologues thereof.

Effector molecules also include, without limitation, 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-dichloroplatinum(II) diamine (DDP), cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin)) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamycins or duocarmycins) and antimitotic agents (eg vincristine and vinblastine).

Other effector molecules may include chelated 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, without limitation, 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, protein such as insulin, tumor necrosis factor, interferon α, interferon β, nerve growth factor , platelet growth factor or tissue plasminogen activator, thrombotic agent or anti-angiogenic agent such as angiostatin or endostatin, or biological response modifier such as lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful, for example, for diagnosis. 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 US Pat. 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, an effector molecule can increase the in vivo half-life of an antibody and/or decrease the immunogenicity of an antibody and/or improve delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO 05/117984.

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

Specific optional substituents that may be present in the aforementioned 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 derivatives thereof.

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

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

The size of the polymer may vary as desired, but is typically in the average molecular weight range of 500 Da to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The size of the polymer, in particular, may be selected based on the intended use of the product, such as the ability to localize to certain tissues, such as tumors, or the ability to increase the half-life from the circulation (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, when the product is intended for clearance from the circulation and penetration into tissues, such as for use in tumor treatment, it may be preferable to use a polymer with a low molecular weight, for example, with a molecular weight of about 5000 Da. For applications in which the product remains in the bloodstream, it may be preferable to use a polymer with a higher molecular weight, for example, a molecular weight in the range of 20,000 Da 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 ranging 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) fragments. In one specific embodiment, the antigen-binding fragment of the present invention and the PEG molecules can be attached through any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, for example, any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the antibody fragment, or may be inserted into the fragment using recombinant DNA techniques (see, for example, US 5219,996; US 5667425; WO98/25971, WO2008/038024). In one example, an antibody molecule of the present invention is a modified Fab fragment, where 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, 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 through the thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment can be covalently linked to the sulfur atom of the cysteine residue located on this fragment. The covalent bond is usually a disulfide bond, or in particular a carbon disulfide bond. When a thiol group is used as the point of attachment, appropriately 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 an ester, eg iodoacetamide, an imide, eg maleimide, vinyl sulfone or disulfide. Such starting materials are commercially available (eg, from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly known as Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly known as Shearwater).

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

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

Alternative sources of PEG linkers include NOF supplying GL2-400MA3 (where m is 5 in the structure below) and GL2-400MA (where m is 2) and n is approximately 450:

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

Thus, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propyloxy}hexane (di-branched PEG, -CH2) 3NHCO (CH2)5-MAL, MM 40000, 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 a PEG disclosed herein) attached via a cysteine amino acid residue at or near amino acid 226 of the chain, e.g., amino acid 226 of the heavy chain (sequential numbering), e.g., amino acid 223 in SEQ ID NO:33.

In one embodiment, the present invention relates to a Fab'-PEG molecule containing one or more PEG polymers, for example 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 moiety. 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 conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule is provided. In one embodiment, the Fab or Fab' of the present invention is conjugated to human serum albumin. In one embodiment, the antibody or fragment is conjugated to a starch molecule, for example to increase the half-life. Methods for conjugating starch to protein are described in US Pat. No. 801,739, incorporated herein by reference.

The present invention also relates to an isolated polynucleotide encoding an antibody or antigen-binding fragment of the present invention. The isolated polynucleotide of the present invention may contain synthetic DNA, for example, obtained by chemical processing, using cDNA, genomic DNA, or any combination thereof.

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

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. contains SEQ ID NO:16 or 20; or

iii. essentially consists 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. 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. a light chain in which 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. 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. 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;

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

i. is at least 90% identical to SEQ ID NO:12; or

ii. contains SEQ ID NO:12; or

iii. essentially consists of SEQ ID NO:12;

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

i. is at least 90% identical to SEQ ID NO:14; or

ii. contains SEQ ID NO:14; or

iii. essentially consists of SEQ ID NO:14.

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

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

The present invention also relates to a cloning or expression vector containing one or more of the polynucleotides disclosed in the present description. In one example, the cloning or expression vector of the present invention contains one or more isolated polynucleotides containing a sequence selected from SEQ ID NOS: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.

The general methods by which vectors, transfection methods and culture methods can be constructed are well known to those skilled in the art. Reference is made in this connection 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 containing one or more isolated polynucleotide sequences of the invention or one or more cloning or expression vectors containing one or more isolated polynucleotide sequences encoding an antibody of the present invention. Any suitable host/vector system can be used to express polynucleotide sequences encoding an antibody or antigen-binding fragment thereof of the present invention. Bacterial, eg E. coli , and other microbial systems may be used, or eukaryotic, eg mammalian host cell expression systems may be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

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

In one embodiment, the host cell of the present invention is a CHO-DG44 cell stably transfected with expression vectors containing the isolated polynucleotide sequences of the present invention, preferably containing the isolated polynucleotide sequences of SEQ ID NO:18 and 26 or SEQ ID NO: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 of the present invention, comprising culturing a host cell of the present invention under conditions suitable for obtaining an antibody or antigen-binding fragment of the invention, and isolating the antibody or antigen-binding fragment thereof.

An antibody, or antigen-binding fragment thereof, may contain only a heavy or light chain polypeptide, in which case only the coding sequence for the heavy chain or light chain polypeptide needs to be used for transfection of host cells. To produce antibodies or antigen-binding fragments 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 light chain and heavy chain polypeptides.

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

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

Substantially free of endotoxin 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, depending on situations.

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

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

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing SEQ ID NO:46.

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

a. a light chain variable region containing:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing SEQ ID NO:6.

In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is the sole active ingredient. In another embodiment, the antibody or antigen-binding fragment of the present invention is in combination with one or more additional active ingredients. Alternatively, pharmaceutical compositions contain an antibody or antigen-binding fragment of the present invention, which is the only active ingredient and which can be administered to a patient as a single agent in combination (for example, simultaneously, sequentially or separately) with other agents, drugs or hormones.

In another embodiment, the pharmaceutical composition comprises an antibody, or an 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, e.g., 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.

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

The exact therapeutically effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), allergic response, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the skill of the physician to determine. Typically, a therapeutically effective amount is 0.01 to 500 mg/kg, eg 0.1 to 200 mg/kg, eg 100 mg/kg. Pharmaceutical compositions may 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, adjuvants such as wetting or emulsifying agents or pH buffering agents may be present in such compositions. Such carriers make it possible to prepare pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by a patient.

Suitable administration forms include those suitable for parenteral administration, eg by injection or infusion, eg bolus injection or continuous infusion, for 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 formulate the appropriate dosage form, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment of the invention may be in a dry form which is reconstituted with an appropriate sterile liquid prior to use. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared.

Once in dosage form, the compositions of the invention may be administered directly to a subject. Accordingly, the present invention relates to the use of an antibody or antigen-binding fragment of the invention for the manufacture of a medicament.

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 or a pharmaceutical composition containing the antibody or antigen-binding fragment, for use in therapy, where the antibody or antigen-binding fragment binds to alpha-synuclein and 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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing 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 alpha-synuclein 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.

In a preferred embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing 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 contains:

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

ii. CDR-L2 containing SEQ ID NO:2, and

iii. CDR-L3 containing SEQ ID NO:3; and

b. heavy chain variable region containing:

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

v. CDR-H2 containing SEQ ID NO:5, and

vi. CDR-H3 containing 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 alpha-synuclein 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.

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

In yet 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 of the present invention or a pharmaceutical composition containing the antibody or antigen-binding fragment, where the antibody or antigen-binding fragment binds with alpha-synuclein and contains:

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:

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

v. CDR-H2 containing SEQ ID NO:45, and

vi. CDR-H3 containing 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 alpha-synuclein 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.

In one preferred embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is for use in the treatment of one or more alpha synucleinopathies, wherein the antibody or antigen-binding fragment thereof binds to alpha synuclein and 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 CDR-H3 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.

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 alpha-synuclein 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.

In another preferred embodiment, the present invention provides a method for 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 the antigen-binding fragment binds to alpha-synuclein and 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 CDR-H3 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.

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 alpha-synuclein 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.

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

a. a light chain variable region containing a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 or SEQ ID NO:8 and CDR-H3 containing 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 containing SEQ ID NO:17 or SEQ ID NO:21, and a heavy chain containing 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 alpha synucleinopathies 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 contains:

a. a light chain variable region containing a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 or SEQ ID NO:8 and CDR-H3 containing 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 containing SEQ ID NO:17 or SEQ ID NO:21, and a heavy chain containing SEQ ID NO:25 or SEQ ID NO:29 or SEQ ID NO:33 or SEQ ID NO:37.

Alpha synucleinopathies of the present invention include, without limitation, 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). Preferably, the alpha synucleinopathy is Parkinson's disease (PD).

In another embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is for use in the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy bodies (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), preferably Parkinson's disease (PD ), and is an antibody or antigen-binding fragment containing:

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 CDR-H3 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.

In another embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is for use in the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy bodies (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), preferably Parkinson's disease (PD ), and is an antibody or antigen-binding fragment containing:

a. a light chain variable region containing a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 or SEQ ID NO:8 and CDR-H3 containing 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 containing SEQ ID NO:17 or SEQ ID NO:21, and a heavy chain containing SEQ ID NO:25 or SEQ ID NO:29 or SEQ ID NO:33 or SEQ ID NO:37.

In another embodiment, a method is provided for the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), Lewy body variant Alzheimer's disease (LBVAD), combination disease 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 an antigen-binding fragment thereof, or a pharmaceutical composition containing an antibody or antigen-binding fragment, 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 CDR-H3 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.

In another embodiment, a method of treating 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), combination disease Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and brain iron storage 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 an antigen-binding fragment thereof, or a pharmaceutical composition containing an antibody or antigen-binding fragment, where the antibody or antigen-binding fragment contains:

a. a light chain variable region containing a CDR-L1 containing SEQ ID NO:1 or SEQ ID NO:7; CDR-L2 containing SEQ ID NO:2 and 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 or SEQ ID NO:8 and CDR-H3 containing 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 containing SEQ ID NO:17 or SEQ ID NO:21, and a heavy chain containing 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), 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 brain iron accumulation type 1 (NBIA-1), more preferably Parkinson's disease (PD), 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 CDR-H3 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.

It is also part of the present invention to use anti-alpha-synuclein antibodies or antigen-binding fragments for use as diagnostic active agents or in diagnostic assays, e.g. for diagnosing alpha-synucleinopathies such as 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).

Diagnosis can preferably be made on biological samples. "Biological sample" covers many types of samples obtained from an individual, and can be used in diagnostic analysis or for monitoring. The definition includes cerebrospinal fluid, 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 sample or tissue culture or cells derived from it, and their progeny. The definition also includes samples that have been manipulated in any manner after they have been obtained, such as treatment with reagents, solubilization, or enrichment for certain components such as polynucleotides.

Diagnostic testing can preferably be performed on biological samples that are not in contact with the human or animal body. Such diagnostic testing is also referred to as 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 the biological sample with an anti-alpha-synuclein antibody or antigen-binding fragment disclosed herein; and ii) detecting binding of the anti-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 is suffering from or at risk of developing alpha synucleinopathy, including determining the stage (severity) of alpha synucleinopathy.

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

a. a light chain variable region containing a CDR-L1 containing SEQ ID NO:44, a CDR-L2 containing SEQ ID NO:2, and a CDR-L3 containing SEQ ID NO:3; and a heavy chain variable region containing a CDR-H1 containing SEQ ID NO:4, a CDR-H2 containing SEQ ID NO:45 and a CDR-H3 containing SEQ ID NO:46.

Preferably, the antibody or antigen-binding fragment thereof for use in the diagnosis of alpha synucleinopathies, preferably in the diagnosis of or in the presence of Parkinson's disease, binds to alpha synuclein and 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 CDR-H3 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.

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

Table 1 Name SEQID NO: Sequence CDR-L1 1 QASQSVYKNNYLA CDR-L2 2 GASTLAS CDR-L3 3 AGYKGGRNDGFA CDR-H1 four GIDLSSHDMY CDR-H2 five AIYASGSTYYASWAKG CDR-H3 6 IHYGNSGGL CDR-L1N33R 7 QASQSVYKNRYLA CDR-H2 S56N 8 AIYASGNTYYASWAKG CDR-H3 N102H nine IHYGHSGGL Human alpha synuclein
P37840 10 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA rabbit VL eleven AIVMTQTPSSKSVAVGDTVTINCQASQSVYKNNYLAWFQQKPGQPPKQLIYGASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCAGYKGGRNDGFAFGGGTEVVVK N.p. rabbit VL 12 Gccatcgtgatgacccagactccatcttccaagtctgtcgctgtgggagacacagtcaccatcaattgccaggccagtcagagtgtttataagaacaactacttagcctggtttcaacagaaaccagggcagcctcccaaacaactgatctatggtgcgtccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgatgtggtgtgtgacgatgctgccacttactactgtgcaggatataaaggtggtcgtaatgatggttttgctttcggcggagggaccgaggtggtggtcaaa rabbit vh 13 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARIHYGNSGGLWGQGTLVTVSS N.p. rabbit vh fourteen Cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggaatcgacctcagtagccacgacatgtattgggtccgccaggctccagggaaggggctggaatacattggagccatttatgctagtggtagcacatactacgcgagctgggcgaaaggccgattcaccatctccaagacctcgaccacggtggatctgaaaatgaccagtctgacaaccgaggacacggccacctatttctgtgccagaattcattatggtaatagtggtgggttgtggggccaaggcaccctggtcaccgtctcgagt 6470gL3
VL 15 DIQMTQSPSSLSASVGDRVTITCQASQSVYKNNYLAWFQQKPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCAGYKGGRNDGFAFGGGTKVEIK 6470gL3
N.p. VL sixteen Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccattacgtgccaagccagccagtccgtgtacaagaacaactacctggcctggttccagcaaaagcccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgagccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccgaggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttggtggaggcaccaaggtcgaaatcaag Light chain 6470 gL3 17 DIQMTQSPSSLSASVGDRVTITCQASQSVYKNNYLAWFQQKPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCAGYKGGRNDGFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESTKVTEQDSKDSTYVSLSSTLNKSSKADEQ N.p. light chain 6470 gL3 eighteen Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccattacgtgccaagccagccagtccgtgtacaagaacaactacctggcctggttccagcaaaagcccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgagccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccgaggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttggtggaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 6470gL3
VL-N33R nineteen DIQMTQSPSSLSASVGDRVTITCQASQSVYKNRYLAWFQQKPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCAGYKGGRNDGFAFGGGTKVEIK 6470gL3
N.p. N33R VL 20 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccattacgtgccaagccagccagtccgtgtacaagaaccgttacctggcctggttccagcaaaagcccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgagccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccgaggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttggtggaggcaccaaggtcgaaatcaag 6470gL3
Light chain N33R 21 DIQMTQSPSSLSASVGDRVTITCQASQSVYKNRYLAWFQQKPGKAPKQLIYGASTLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCAGYKGGRNDGFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESTKVTEQDSKDSTYVSLSSTLKSSKADYEK 6470gL3
N.p. light chain N33R 22 Gacattcagatgacccagtccccttcatcactgtccgcgagcgtgggcgacagagtgaccattacgtgccaagccagccagtccgtgtacaagaaccgttacctggcctggttccagcaaaagcccgggaaggcgccaaaacagcttatctacggtgcatccactctcgcctcgggagtgccgagccgcttctcgggatctgggtccggaactcagttcaccctgactatctcgtccctgcaacccgaggatttcgccacctactactgcgccggctataagggaggacggaacgacggcttcgcttttggtggaggcaccaaggtcgaaatcaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc 6470gH23
vh 23 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSS 6470gH23
N.p. vh 24 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtgggttgtggggtcagggtactctggttaccgtctcgagc 6470gH23
heavy chain 25 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 6470gH23
N.p. heavy chain 26 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtgggttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatct ccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 6470gH23
VH S56N N102H 27 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGNTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSS 6470gH23
N.p. VH S56N N102H 28 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgggttgtggggtcagggtactctggttaccgtctcgagc 6470gH23
Heavy chain S56N N102H 29 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGNTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 6470gH23
N.p. heavy chain S56N N102H thirty gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgacaactctaaaaacaccgtgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgggttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatct ccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 6470gH36
vh 31 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSS 6470gH36
N.p. vh 32 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtgggttgtggggtcagggtactctggttaccgtctcgagc 6470gH36
heavy chain 33 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARIHYGNSGGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 6470gH36
N.p. heavy chain 34 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtagcacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtaatagtggtgggttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatct ccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag 6470gH36
VH S56N N102H 35 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGNTYYASWAKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSS 6470gH36
N.p. VH S56N N102H 36 Gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgggttgtggggtcagggtactctggttaccgtctcgagc 6470gH36
Heavy chain S56N N102H 37 EVQLLESGGGLVQPGGSLRLSCAVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGNTYYASWAKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARIHYGHSGGLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 6470gH36
N.p. heavy chain S56N N102H 38 gaggttcagctgctggagtctggaggcgggcttgtccagcctggagggagcctgcgtctctcttgtgcagtaagcggcatcgacctgtccagccacgacatgtattgggtacgtcaggcaccgggtaaaggtctggaatacatcggcgccatttatgctagtggtaatacatactacgcgagctgggcgaaaggccgtttcaccatctcccgtgactccagcaaaaacaccctgtacctgcagatgaactctctgcgtgcggaagacactgcggtttactattgcgcgcgtattcattatggtcacagtggtgggttgtggggtcagggtactctggttaccgtctcgagcgcttctacaaagggcccctccgtgttccctctggccccttgctcccggtccacctccgagtctaccgccgctctgggctgcctggtcaaggactacttccccgagcccgtgacagtgtcctggaactctggcgccctgacctccggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtcgtgaccgtgccctcctccagcctgggcaccaagacctacacctgtaacgtggaccacaagccctccaacaccaaggtggacaagcgggtggaatctaagtacggccctccctgccccccctgccctgcccctgaatttctgggcggaccttccgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaatgccaagaccaagcccagagaggaacagttcaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccctccagcatcgaaaagaccatct ccaaggccaagggccagccccgcgagccccaggtgtacaccctgccccctagccaggaagagatgaccaagaaccaggtgtccctgacctgtctggtcaagggcttctacccctccgacattgccgtggaatgggagtccaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctccttcttcctgtactctcggctgaccgtggacaagtcccggtggcaggaaggcaacgtcttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagcctgggcaag Acceptance. person frame IGKV1-16JK4 39 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK N.p. acceptance. frame pers. IGKV1-16JK4 40 Gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggcattagcaattatttagcctggtttcagcagaaaccagggaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtggagatcaaa Acceptance. person frame IGHV3-23JH4 41 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYFDYWGQGTLVTVSS N.p. acceptance. frame pers. IGHV3-23JH4 42 Gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaatactttgactactggggccaaggaaccctggtcaccgtctcctca Pers. 68-140 a-syn in rabbit Fc 43 GAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEAVEKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVY™GPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK CDR-L1
X33 44 QASQSVYKNXYLA
(X = N or R) CDR-H2
X56 45 AIYASGXTYYASWAKG
(X=S or N) CDR-H3
X102 46 IHYGXSGGL
(X = N or H) Rabbit light chain 6470 47 AIVMTQTPSSKSVAVGDTVTINCQASQSVYKNNYLAWFQQKPGQPPKQLIYGASTLASGVPSRFKGSGSGTQFTLTTISDVVCDDAATYYCAGYKGGRNDGFAFGGGTEVVVKRTPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQNSHTCVKEY Rabbit heavy chain 6470 48 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARIHYGNSGGLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVY™GPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK Heavy chain Fab rabbit 6470 49 QSVEESGGRLVTPGTPLTLTCTVSGIDLSSHDMYWVRQAPGKGLEYIGAIYASGSTYYASWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARIHYGNSGGLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVVTSSSQPVTCNVAHPATNTKVDKTVHTCH

The invention is further described below by way of examples with reference to the 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) by standard molecular biology techniques to create a vector designed to produce alpha synuclein with the 10His-TEV N-terminal tag. The resulting vector was transfected into Expi293F cells using the Expi293™ expression system (Invitrogen) according to the manufacturer's protocols. The alpha-synuclein protein was accumulated in the culture medium, from where it was then recovered by immobilized metal ion affinity chromatography on a HisTrap excel column (GE Healthcare). The column was washed with 25 mM Tris-HCl, 300 mM NaCl, pH 8.0 and the protein was eluted with a stepwise gradient of 500 mM imidazole in the same buffer. The 10His label was removed with TEV protease. The sample was then concentrated and desalted before re-introducing the digested protein to the HisTrap excel column and collecting the digested alpha-synuclein on stream. 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 is monomeric (FIG. 1A).

Wild type (untagged) human alpha synuclein was also expressed in Expi293F cells. The protein was recovered 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, eluted with a gradient of sodium chloride 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 wild-type (unlabeled) alpha-synuclein monomer was used to prepare alpha-synuclein fibrils obtained by mixing purified recombinant alpha-synuclein monomer (9-10 mg/ml in PBS pH 7.4) at 1200 rpm at 37°C. C by shaking the incubator Vortemp56 (Labnet) continuously for 10 days. Fibril formation was assessed by JC-1 assay (Lee et al., Biochem. J. 2009, 418, 311-323) and Fourier transform solution infrared spectroscopy. The unincorporated monomer in fibril solutions was evaluated by ultracentrifugation and passage through a membrane with a cut-off particle mass of 100 kDa, followed by gel electrophoresis. In further studies, only fibrils with a JC-1 response >15, low soluble monomer (<5%), and an FTIR spectrum with a main absorbance between 1625 and 1630 cm ^-1 were used (FIG. 2). The prepared fibrils were stored at -80°C.

Example 2 Immunization and Antibody Isolation

Several immunization strategies were performed using various substances and immunogens. Antibody 6470 was obtained from a female New Zealand white rabbit (>2 kg), which was subcutaneously immunized with a rabbit Fc fusion protein containing human alpha-synuclein residues 68-140 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 Expi293™ 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 pH 8.8 buffer and eluted with a gradient of 0.1 M citric acid pH 2.0 in the same buffer. Protein fractions were neutralized with 2M 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 comprising 500 μg of fusion protein emulsified in an equal volume of complete Freund's adjuvant (CFA). Rabbits were given 2 booster injections 21 days apart using Incomplete Freund's Adjuvant (IFA) and blood was taken from the ear 14 days after immunization. Blood sampling 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 prepared in a manner similar to that described by Tickle et al., 2015. J Biomol Screen: 20(4), 492-497. Briefly, B cells derived from lymph nodes or splenocytes of immunized animals were cultured at a density of approximately 2000-5000 cells per well in 96-well barcoded 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% supernatant of activated human PBMCs (BSS), and X-rayed EL4 mouse thymoma mutant cells (5×10 ⁴ /well) for seven days at 37° C. in 5% CO ₂ atmosphere. Cultures were created using B cells obtained from all immunized animals and a total of approximately 1.7×10 ⁹ B cells were selected.

6470, an antibody of the present invention, was obtained 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 proved to be an alternative source for obtaining B cell samples and identifying new antibodies. Antibodies with related sequences were identified from B cells derived from the lymph node, but not from the spleen. Approximately 9.6×10 ⁷ cells were harvested from a rabbit immunized with C-terminal human alpha-synuclein protein.

Primary screening

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

Secondary Screening

After primary screening, positive supernatants were pooled in 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 using biotinylated recombinant human alpha-synuclein monomer or biotinylated recombinant human alpha-synuclein fibrils to identify wells that showed binding to both monomeric and fibrillar recombinant human alpha-synuclein, and to exclude any false positive wells showing off-target binding to the Superavidin™ spheres. Given the insoluble nature of fibrils, conventional ELISA coating protocols that are 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 an ELISA plate that had been pre-coated with streptavidin.

A total biotinylated alpha-synuclein fibril pool was generated 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 384-well streptavidin-coated Maxisorp plates in carbonate coating buffer (dH ₂ O + 0.16% Na ₂ CO ₃ + 0.3% NaHCO ₃ .) The plates were blocked with 1% wt. /about. PEG/PBS and then incubated with 10 μl/well of B cell culture supernatant (diluted 1:1 with 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 substrate ™B (3,3',5,5'-tetramethylbenzidine, from EMD Millipore; 10 µl/well) . Optical density was measured at 630 nM using a BioTek Synergy 2 microplate reader. Primary binding analysis revealed 640 events of interest, and after ELISA screening, 491 of them were shown to bind to both monomeric and fibrillar recombinant human alpha-synuclein.

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 rate of dissociation from recombinant human alpha-synuclein monomer, recombinant human alpha-synuclein fibrils and fibrils. recombinant mouse alpha-synuclein. Supernatants from 80 different B cells were tested, nine wells showed dissociation rate (kd) values from recombinant human fibrils <1×10 ^-5 . Of these, seven had dissociation rate (kd) values from recombinant mouse fibrils less than ^{1x10 -5} and two gave dissociation rates (kd) from recombinant human monomer less than ^{1x10 -5} . All nine supernatants were selected for variable region extraction.

Variable Region Extraction

In order to extract antibody variable region genes from selected supernatants of interest, a deconvolution step had to be performed to ensure 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 technique (Clargo et al., 2014. MAbs: 6(1), 143-159). Briefly, positive well immunoglobulin-secreting B cells were mixed with streptavidin spheres (New England Biolabs) coated with biotinylated recombinant human alpha-synuclein fibrils (prepared with a 1:50 mixture as described above) and goat anti-rabbit conjugate. Fcγ-specific FITC (Jackson) with a final dilution of 1:1200. After static incubation at 37° C. for 1 hour, 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 an Olympus microscope, were then selected using an Eppendorf micromanipulator and placed in a PCR tube.

The antibody variable region genes were isolated from individual cells by reverse transcription (RT) PCR using heavy and light chain variable region primers. Two rounds of PCR were performed using nested 2° PCR, including restriction sites at the 3' and 5' ends, allowing the variable region to be cloned into a mammalian expression vector expressing rabbit IgG (VH) or kappa chain (VL) of rabbit IgG. Anti-alpha-synuclein antibody genes from 5 different supernatants were successfully cloned into 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.

ELISA screening of supernatants containing transients

The purified antibodies were then subjected to further screening by ELISA. Biotinylated monomers and fibrils of recombinant human alpha-synuclein were captured on 384-well streptavidin-coated Maxisorp plates (ThermoScientific/Nunc) in carbonate coating buffer (dH ₂ O + 0.16% Na ₂ CO ₃ + 0.3% NaHCO ₃ ) . Separate plates were also coated with a biotinylated peptide corresponding to residues 117-126 of human alpha synuclein shown in SEQ ID NO:10 (PVDPDNEAYE peptide) to check if transients are associated with this or another region of the molecule. Tablets blocked 1% wt./about. PEG/PBS and then incubated with several dilutions of the purified temporary supernatant. Secondary HRP-conjugated goat anti-rabbit IgG Fc antibodies (Stratech Scientific Ltd/Jackson ImmunoResearch) were added to plates followed by visualization of substrate binding ™B (3,3',5,5'-tetramethylbenzidine, from EMD Millipore; 10 µl/well) . Absorbance was measured at 630 nM with a BioTek Synergy 2 microplate reader. Data for 6470 is shown in FIG. 3. As can be seen, 6470 shows binding to both monomeric and fibrillar recombinant human alpha-synuclein, but does not bind to peptide 117-126.

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

Example 3 Characterization of Antibodies

Kinetics by Biacore

The interaction kinetics was determined by the surface plasmon resonance method 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 a CM5 chip by amino group immobilization . The three ligands were prepared in 10 mM NaAc, pH 3.5 and immobilized on the surface of individual flow cells until an immobilization level of about 30 response units (RU) for alpha-synuclein monomer, about 40 RU for human alpha-synuclein fibrils, and about 300 RU was achieved. 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 running buffer for both ligand immobilization and kinetic analysis. The binding of monoclonal rabbit IgG1 6470 (containing SEQ ID NO:47 and 48) and monoclonal rabbit Fab 6470 (containing SEQ ID NO:47 and 49) to the three ligands was then measured. Monoclonal antibodies IgG or Fab were injected at 7 different concentrations from 800 nM to 0.195 nM, with each concentration in 3 flow cells, with a contact time of 3 minutes and a dissociation time of 30 minutes at a flow rate of 100 μl/min. For surface regeneration, one injection of 50 mM HCl for 90 s at a rate of 10 μl/min and another injection of 50 mM HCl for 60 s at a rate of 10 μl/min were administered. Data were analyzed using Biacore T200 software (version 3.0) using a bivalent analyte model with assumed no total contribution (RI = 0) and global Rmax for the IgG format, and using a 1:1 model with flexible total contribution (local RI value ) and the global value of Rmax.

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

table 2 sample Human monomer human fibrils mouse fibrils ka1
(1/ms) kd1
(1/s) KD1
(nM) ka1
(1/ms) kd1
(1/s) KD1
(nM) ka1
(1/ms) kd1
(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 IgG1 4.75E+06 1.42E-02 2.98 1.76E+06 4.78E-04 0.27 8.89E+05 3.07E-04 0.34

Binding to beta-synuclein

The binding of anti-human alpha-synuclein antibodies to human beta-synuclein was tested by Western blot analysis using rPeptide beta-synuclein. One microgram of synuclein was run on a 4-12% bis/tris gel and blotted onto a PVDF membrane. The membrane was blocked in PBS with 3% BSA and 0.1% Tween20. Rabbit IgG1 6470 was added to the blocked blots and incubated for 1 hour at room temperature, washed with PBS, 0.1% Tween20 and incubated for 1 hour with secondary antibody-HRP conjugate (anti-rabbit H+L conjugate with HRP, Bethyl, A120 -101P). The blot was washed thoroughly in PBS with 0.1% Tween20, PBS and water. Chemiluminescence was measured after the addition of ECL western blotting substrate (Pierce). As shown in Figure 4(A), rabbit IgG1 6470 (lane 3) does not bind to human beta-synuclein.

Epitope mapping

NMR

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

Rabbit Fab 6470 (containing VL of SEQ ID NO:11 and VH of SEQ ID NO:13, and also containing SEQ ID NO:47 and 49) was expressed in CHO SXE as His-tagged objects and purified from the supernatant with 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 Fab-His protein was buffer exchanged with NMR buffer (25 mM sodium phosphate, pH 6.4, 100 mM NaCl) on a HiPrep 26/10 desalting column. Prior to sterilization on a 0.22 µm Millex GV Fab-His filter, protein fractions were concentrated and 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 final pools were tested for >99% purity by UPLC-SEC. The final pools were passed through a 0.22 mm Millex GV filter for sterilization.

Determination of the α-synuclein backbone

NMR samples typically had a volume of 350 μl with a protein concentration of 360 μM ¹³ C/ ¹⁵ N-labeled or 430 μM ² H/ ¹³ C/ ¹⁵ N-labeled human α-synuclein in 5 mm Shigemi tubes. Buffer conditions were as follows: 100 mM NaCl, 25 mM sodium phosphate, pH 6.4, 10 μM AEBSF, 0.02% NaN ₃ . All experiments were recorded at 20° C. either on a 600 MHz Bruker AVIII spectrometer or on an 800 MHz Bruker AVIII spectrometer equipped with cryogenically cooled probes. Sequential relationships between 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 15N resonances in 15N- and 13C-labelled proteins J. Biomol NMR 3) were recorded with a spectral width of 28, 28 and 10 ppm and a scan time of 117 (F1) 117 (F2) and 140 (F3) ms in measurements on ¹⁵ N, ¹⁵ N and ¹ H nuclei, respectively, with 8 scans per increment and a relaxation delay of 1.5 s. A heterogeneous sample was used with a sample density of 10% (4000 out of 40000 hypercomplex points), giving a total scan time of 2.75 days. Sequential relationships were confirmed and residue types were identified using TROSY-HNCA experiments (Grzesiek and Bax, 1992 Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J. Magn. Reson. 96, 432-440; Salzmann et al. , 1998. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins (Proc. Natl. Acad. Sci. USA. 95, 13585-90) and TROSY-HNCACB (Wittekind and Mueller, 1993 HNCACB, a High -Sensitivity 3D NMR Experiment to Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta-Carbon Resonances in Proteins, J. Magn. Reson. Ser. B 101, 201-205; Salzmann et al., 1999. TROSY-type Triple Resonance Experiments for Sequential NMR Assignment of Large Proteins, J. Am. Chem. Soc. 121, 844-848). 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 scan time 1 day), while in the TROSY-HNCACB experiment, spectra were recorded with a width of 56, 28 and 10 ppm and a scan time of 8.2 (F1), 21.7 (F2) and 100 (F3) ms in measurements on ¹³ C, ¹⁵ N and ¹ H nuclei, respectively (8 scans per increment, relaxation delay 1.5 s, total scan time 1.7 days). The location 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, 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), which was recorded with a spectral width of 10, 29, 10 ppm and a scan time of 80 ( F1), 21.7 (F2) and 150 (F3) ms in measurements on ¹³ C, ¹⁵ N and ¹ H nuclei, respectively (8 scans per increment and a relaxation delay of 1.5 s). A heterogeneous sample was used with a sample density of 15% (1208 out of 8050 hypercomplex points), giving a total scan time of 19 hours. 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 once by nitrogen nuclei. Heterogeneous sample data was reconstructed using the Harvard soft threshold iterative method (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 by the Sparky method (Goddard and Kneller, DG SPARKY 3. In University of California, San Francisco), resulting in amide proton and nitrogen resonances of 133 residues, corresponding to 99% residues (excluding proline and N-terminal methionine residues) .

Fab 6470 binding site mapping was performed using a 150 μM sample of ² H/ ¹³ C/ ¹⁵ N-labeled human alpha-synuclein containing a 10% molar excess of unlabeled Fab 6470. Samples were prepared in the same buffer as described above for alpha scaffold determination. -synuclein. Changes in the chemical shift in the spectra ^{of 1} H, ¹⁵ N and ¹³ C 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 complex alpha-synuclein/Fab, with an equivalent control spectrum obtained for free alpha-synuclein. The control spectrum in the TROSY-HNCO experiment with free alpha-synuclein was obtained with a spectral width of 10, 28 and 10 ppm and a scan time of 80 (F1), 22 (F2) and 150 (F3) ms in measurements on the nuclei ¹³ C, ¹⁵ N and ¹ H, respectively (16 scans per increment, relaxation delay 1.5 s). A heterogeneous sample was used with a sample density of 25% (2013 out of 8050 hypercomplex points), giving 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 measurements on ¹³ C, ¹⁵ N and ¹ H, respectively (32 scans per increment, relaxation delay 1.5 s). A heterogeneous sample was used with a sample density of 25% (1119 out of 4477 hypercomplex points), giving a total scan time of 2.8 days. NMR spectra were processed using 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 scan time for nitrogen nuclei was increased by 1-fold during data recovery.

Chemical shift changes were analyzed using a minimum 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 take into account the chemical shift of the carbonyl atom, resulting in the following equation:

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

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

Highly skewed residuals were identified as residuals with a minimum shift greater than at least the average calculated for all shifts plus one standard deviation. Four different thresholds were used to identify Fab-associated residues. Residues that are involved in the binding site were evaluated with increasing levels of rigor as: residues whose minimum shift exceeds the average calculated for all shifts, plus one standard deviation (constituting >0.018925); residuals whose minimum shift exceeds the mean calculated for all shifts plus two standard deviations (>0.032049); residuals whose minimum shift is greater than the mean calculated for all shifts plus three standard deviations (>0.045174); residuals whose minimum shift is greater than the mean calculated for all shifts plus four standard deviations (>0.058299). In this assay, proline residues cannot be identified as they do not contain an amide proton.

Therefore, the epitope for Fab 6470 is defined with increasing levels of stringency as the mean calculated for all shifts plus one standard deviation: D121, N122, E123, A124, Y125, E126, M127, S129, E130, Y133, Q134, D135, and Y136 ; mean value calculated for all shifts plus two standard deviations: E123, A124, Y125, E126, M127, S129, E130, D135 and Y136; mean value calculated for all shifts plus three standard deviations: Y125, M127, S129 and D135; the average 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 (average +3SD) Y125, M127, S129, and D135 by NMR studies and, in addition, also binds to all of the following residues ( mean +1SD) D121, N122, E123, A124, E126, E130, Y133, Q134 and Y136 human alpha synuclein (SEQ ID NO:10).

Peptide mapping

Further characterization of the epitope associated with 6470 was performed using short (usually 9-mer or 10-mer) peptides characteristic of and spanning the C-terminal region of human alpha-synuclein. They were used in a competitive surface plasmon resonance assay to test if any of them were able to inhibit antibody binding to monomeric alpha-synuclein or preformed alpha-synuclein fibrils immobilized on the Biacore chip. The peptide showing the maximum level of inhibition was then selected for co-crystallization study with the antibody to confirm the exact epitope.

Peptides were purchased from Peptide Protein Research Ltd., Bishop's Waltham, U.K., and synthesized using 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-peptide ends were capped with acetyl and amide groups, respectively, except for the case when the peptides were the N-terminus and C-terminus of α-synuclein, while the amino and carboxyl groups, respectively, remained free. Peptide stock solutions were prepared in DMSO at 10 mM. A complete list of peptides is shown in Table 3.

Table 3 Peptide ID Sequence AS104-113 EEGAPQEGIL AS109-118 QEGILEDMPV AS111-120 GILEDMPVDP AS113-122 LEDMPVDPDN AS115-124 DMPVDPDNEA AS117-126 PVDPDNEAYE AS119-128 DPDNEAYEMP AS121-130 DNEAYEMPSE AS123-132 EAYEMPSEEG AS125-134 YEMPSEEGYQ AS127-136 MPSEEGYQDY

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

Peptide solutions were prepared in running buffer at 100 μM and control without peptide was prepared as a 1:100 dilution using DMSO in running buffer. A solution of 6470 rabbit Fab (comprising SEQ ID NOS: 47 and 49) was prepared in 50.5 nM run buffer prior to pre-incubation of 198 µl with 2 µl blank control or diluted peptide to produce 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 a rate of 10 μl/min, and a signal was recorded at the time point 5 seconds before the end of the injection. At the end of each cycle, the chip was regenerated 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 level of inhibition of each alpha-synuclein peptide is 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, of which AS123-132 had the highest levels of inhibition, which amounted to 37 % and 54% for antibody binding to monomer and fibrils, respectively. Slightly lower levels of inhibition were obtained for the AS125-134 peptide, 34% and 52%, respectively, indicating that the main component of the epitope contains residues 125-132. The levels of inhibition provided by the AS121-130 peptide were lower, 20% and 27%, respectively, suggesting that the total residues of all three peptides are 125-130, which are the main contributors to the epitope.

Since the epitope of the 6470 antibody appears to contain at least the YEMPSEEG sequence, the AS123-132 peptide was studied in cocrystallization 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 123-132 peptide (EAYEMPSEEG) in a 1:2 molar ratio of Fab:peptide and incubated for 1 hour at room temperature. Conditions suitable for crystal growth were determined by the sessile drop vapor diffusion method using commercially available crystallization screens (Qiagen). To obtain crystals of diffraction quality, the hanging drop vapor diffusion method was used.

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 flash frozen in liquid nitrogen after a quick run 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 flash frozen in liquid nitrogen after a quick run through a cryoprotectant solution containing 0.2 M ammonium sulfate and 35% (v/v) polyethylene glycol 8000.

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

Refinement and model building cycles 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 converge for both models. The model geometry was validated using the Molprobity43 software. Molecular imaging was obtained using Pymol44. The epitope information described below was obtained by viewing the atoms at 4Å resolution on the Fab/peptide interface. Data collection and refinement statistics are shown in Table 4A and Table 4B.

Table 4A Structure Fab VR6470-peptide 123-132 space group P 3 ₁ 2 1 Cell size a, b, c (Å) 111.78, 111.78, 71.93 α, β, γ (°) 90.00, 90.00, 120.00 Resolution (Å) 30.00-2.90 (3.08-2.90) R _merge 0.07 (0.36) I/σI 17.0 (5.1) Completeness (%) 99.9 (100) Redundancy 9.8(10.2)

Table 4B

Clarification Fab VR6470-peptide 123-132 Resolution (Å) 30.00-2.90 no. reflections 11762 R _work / R _free 0.2587/0.3192 no. atoms Protein 3259 Water 0 ligand thirty B -factors Peptide 81.8 fab 67.4 Standard deviations Bond lengths (Å) 0.005 Communication angles (°) 1.125

Values in brackets refer to the shell obtained at a higher resolution. Rsym = Σ⎪(I - <I>)⎪/Σ(I), where I is the observed integrated intensity, <I> is the average integrated intensity obtained from several measurements, and summation is made over all observed reflections. Rwork = Σ⎪⎪Fobs⎪ - k⎪Fcalc⎪⎪/Σ⎪Fobs⎪, where Fobs and Fcalc are the observed and calculated structural factors, respectively. Rfree is calculated as Rwork using 5% randomly selected reflectance data and not taken into account in refinement calculations.

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

Table 5 Chain Remainder Atom Chain Peptide Amino Acid Target peptide atom Distance (Å) (SEQ ID NO:13) H 96 (HIS) NE2[N] A 125(TYR) CD1[C] 3.93 H 96 (HIS) CD2[C] A 125(TYR) CD1[C] 3.93 A 125(TYR) CE1[C] 3.69 H 99(ASN) CB[C] A 125(TYR) CD2[C] 3.34 H 99(ASN) N[N] A 125(TYR) CD2[C] 3.57 H 99(ASN) CB[C] A 125(TYR) CE2[C] 3.85 H 96 (HIS) CA[C] A 125(TYR) CE2[C] 3.85 H 96 (HIS) CB[C] A 125(TYR) CE2[C] 3.93 H 97(TYR) N[N] A 125(TYR) CE2[C] 3.81 H 99(ASN) CA[C] A 125(TYR) CE2[C] 3.99 H 98(GLY) N[N] A 125(TYR) CE2[C] 3.42 H 98(GLY) CA[C] A 125(TYR) CE2[C] 3.91 H 98(GLY) C[C] A 125(TYR) CE2[C] four H 99(ASN) N[N] A 125(TYR) CE2[C] 3.12 H 96 (HIS) CA[C] A 125(TYR) CZ[C] 3.5 H 96 (HIS) CB[C] A 125(TYR) CZ[C] 3.96 H 96 (HIS) C[C] A 125(TYR) CZ[C] 3.79 H 97(TYR) N[N] A 125(TYR) CZ[C] 3.32 H 98(GLY) N[N] A 125(TYR) CZ[C] 3.72 H 96 (HIS) CA[C] A 125(TYR) OH[O] 3.19 H 97(TYR) N[N] A 125(TYR) OH[O] 2.32 H 97(TYR) C[C] A 125(TYR) OH[O] 3.64 H 98(GLY) N[N] A 125(TYR) OH[O] 3.1 H 97(TYR) CG[C] A 125(TYR) OH[O] 3.51 H 97(TYR) CE1[C] A 125(TYR) OH[O] 3.86 H 97(TYR) CA[C] A 125(TYR) OH[O] 3.19 H 97(TYR) CB[C] A 125(TYR) OH[O] 3.38 H 97(TYR) CD1[C] A 125(TYR) OH[O] 2.84 H 32(ASP) OD1[O] A 126(GLU) CA[C] 3.34 A 126(GLU) CB[C] 3.78 H 52(ALA) CB[C] A 126(GLU) CG[C] 3.92 H 32(ASP) OD1[O] A 126(GLU) CG[C] 3.16 A 126(GLU) CD[C] 3.47 H 52(ALA) N[N] A 126(GLU) OE2[O] 3.82 H 52(ALA) CB[C] A 126(GLU) OE2[O] 3.92 H 32(ASP) OD1[O] A 126(GLU) OE2[O] 2.99 H 51(TYR) CB[C] A 126(GLU) OE2[O] 3.93 H 32(ASP) CG[C] A 126(GLU) OE2[O] 3.89 H 51(TYR) CD2 C] A 126(GLU) OE2[O] 3.7 H 32(ASP) OD1[O] A 126(GLU) C[C] 3.2 H 32(ASP) CG[C] A 126(GLU) C[C] 3.72 H 32(ASP) OD2[O] A 126(GLU) C[C] 3.79 H 32(ASP) OD1[O] A 126(GLU) O[O] 2.4 H 32(ASP) CG[C] A 126(GLU) O[O] 2.8 H 32(ASP) OD2[O] A 126(GLU) O[O] 2.73 H 51(TYR) CD2[C] A 126(GLU) O[O] 3.87 H 51(TYR) CE2[C] A 126(GLU) O[O] 3.96 A 127(MET) CB[C] 3.99 A 127(MET) C[C] 3.85 H 51(TYR) CD2[C] A 127(MET) O[O] 3.1 H 51(TYR) CE2[C] A 127(MET) O[O] 3.03 H 57(TYR) OH[O] A 129(SER) CB[C] 3.05 H 57(TYR) CZ[C] A 129(SER) CB[C] 3.96 H 57(TYR) OH[O] A 129(SER) OG[O] 2.78 H 57(TYR) CE2[C] A 129(SER) OG[O] 3.79 H 57(TYR) CZ[C] A 129(SER) OG[O] 3.7 Chain Remainder Atom Chain Peptide Amino Acid Target peptide atom Distance (Angstrom) (SEQ ID NO:11) L 34(TYR) OH[O] A 123(GLU) CD[C] 3.63 A 123(GLU) OE1[O] 3.46 L 34(TYR) CE1[C] A 123(GLU) OE2[O] 3.68 L 34(TYR) CZ[C] A 123(GLU) OE2[O] 3.74 L 34(TYR) OH[O] A 123(GLU) OE2[O] 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) OH[O] 3.66 L 34(TYR) CE2[C] A 125(TYR) OH[O] 3.39 L 93(TYR) CE2[C] A 127(MET) CB[C] 3.75 L 93(TYR) CZ[C] A 127(MET) CB[C] 3.62 L 93(TYR) OH[O] A 127(MET) CB[C] 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[O] 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[O] 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[O] 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[O] 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[O] A 128(PRO) O[O] 3.45 L 96(GLY) N[N] A 128(PRO) O[O] 3.41 L 96(GLY) CA[C] A 129(SER) CA[C] 3.88 L 96(GLY) O[O] A 129(SER) CA[C] 3.4 L 96(GLY) N[N] A 129(SER) CA[C] 3.99 L 96(GLY) O[O] A 129(SER) CB[C] 3.56 A 129(SER) C[C] 3.52 L 96(GLY) CA[C] A 130(GLU) N[N] 3.99 L 96(GLY) C[C] A 130(GLU) N[N] 3.6 L 96(GLY) O[O] A 130(GLU) N[N] 2.73 L 96(GLY) N[N] A 130(GLU) N[N] 3.78 L 96(GLY) O[O] A 130(GLU) CA[C] 3.71 A 130(GLU) CB[C] 3.81 L 97(ARG) CD[C] A 130(GLU) CB[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) O[O] A 130(GLU) OE2[O] 3.63 L 94(LYS) CD[C] A 130(GLU) OE2[O] 3.77 L 94(LYS) CE[C] A 130(GLU) OE2[O] 3.26 L 97(ARG) NE[N] A 131(GLU) C[C] 3.94 L 97(ARG) CD[C] A 131(GLU) O[O] 3.75 L 97(ARG) NE[N] A 131(GLU) O[O] 2.94 L 97(ARG) CZ[C] A 131(GLU) O[O] 3.79 L 97(ARG) NH2 [N] A 131(GLU) O[O] 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 Fab 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 a human germline antibody V region scaffold. To restore antibody activity, a few scaffold residues from rabbit V regions were also retained in the humanized sequence. These residues were selected using the protocol described by Adair et al. (1991) (WO91/09967). Sequence alignments of rabbit antibody V regions (donor) with human germline (acceptor) V region sequences are shown in FIG. 9 and 10 along with the generated humanized sequences. CDRs grafted from donor to acceptor sequence are defined by Kabat (Kabat et al., 1987), with the exception of CDR-H1, where a combined Chothia/Kabat definition is used (see Adair et al., WO91/09967).

Genes encoding multiple variants of heavy and light chain V region sequences were designed and constructed using the DNA2.0 Inc. automated synthesis approach. Other heavy and light chain V region variants have been generated by modifying the VH and VK genes by oligonucleotide directed mutagenesis, including, in some cases, mutations in the CDR. For transient expression in mammalian cells, the genes for the humanized light chain V region were cloned into the pMhCK light chain expression vector UCB, which contains DNA encoding the human kappa chain constant region (Km3 allotype). The genes for the humanized heavy chain V region were cloned into the human gamma-4 heavy chain expression vector UCB pMhγ4PFL, which contains the DNA encoding the human gamma-4 heavy chain constant region with the hinge stabilizing mutation S241P (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. Co-transfection of the resulting heavy and light chain vectors into Expi293™ suspension cells resulted in the expression of humanized recombinant antibodies in human IgG4P format.

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

Table 6 Antibodies
options light chain heavy chain Affinity for human fibrils (KD) Donor balances Donor balances pM Chimeric 6470 - - 99 6470gL3gH23 Q48, Q72 V24, Y47, I48, G49, V78, R97 148 6470gL3gH36 Q48, Q72 V24, Y47, I48, G49, S73, R97 166 6470gL6gH23 Q72 V24, Y47, I48, G49, V78, R97 547 6470gL6gH36 Q72 V24, Y47, I48, G49, S73, R97 377 6470gL8gH23 Q48 V24, Y47, I48, G49, V78, R97 246 6470gL8gH36 Q48 V24, Y47, I48, G49, S73, R97 198 6470gL3gH25 Q48, Q72 V24, I48, G49, K71, S73, V78, R97 76000 6470gL3gH26 Q48, Q72 V24, Y47, G49, K71, S73, V78, R97 300 6470gL3gH27 Q48, Q72 V24, Y47, I48, K71, S73, V78, R97 22300 6470gL3gH35 Q48, Q72 V24, Y47, I48, G49, K71, S73, V78 5282 6470gL3gH46 Q48, Q72 Y47, I48, G49, V78, R97 316 6470gL3gH50 Q48, Q72 Y47, I48, 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 antibody 6470. As with many rabbit antibodies, the VH gene of antibody 6470 is shorter than the selected 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). Rabbit 6470 VH region framework 3 also lacks two residues (75 and 76) in the loop between strands D and E of the beta sheet: in humanized grafts, the gap is filled with the appropriate residues (lysine 75, K75; asparagine 76, N76) from the selected human acceptor sequence (Fig. 10). All residues of the heavy chain framework region in the gH23 and gH36 transplants 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 (with reference to SEQ ID NO: 23 and 31), which retains donor residues of valine (V24), tyrosine (Y47), isoleucine (I48), glycine (G49), serine (S73), valine (V78), and arginine (R97), respectively. Retention of residues V24, Y47, I48, G49, and R97 was important to ensure that the humanized antibody was fully active to bind to human alpha-synuclein fibrils.

In addition, the humanized VH genes were cloned into the pMhFab10HIS human Fab-HIS UCB expression vector, which contains DNA encoding the human gamma-1 CH1 hinge region domain with a C-terminal tag of ten histidine residues. The histidine tag facilitates the purification of expressed Fabs by affinity chromatography. Co-transfection of the resulting heavy and light chain vectors into Expi293™ suspension cells gave expression of humanized recombinant antibodies in Fab-HIS formats.

Affinity maturation was performed according to the IOTA methods described in WO 2014198951. The interface between the rabbit Fab 6470 and the EAYEMPSEEG alpha-synuclein (123-132) peptide in the X-ray crystallography-determined complex was analyzed to identify mutations that could potentially improve Fab affinity. rabbit 6470 to the alpha-synuclein protein. IOTA is a statistically potent tool for determining the probability of occurrence of a given type of contact atom at a protein interface or binding site.

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

Table 7A Mutation Chain Monomer fibril ka1 (1/ms) kd1 (1/s) KD1 (nM) ka1 (1/ms) kd1 (1/s) KD1 (nM) 6470 rabbit fab - 7.23E+06 1.24E-01 17.2 4.58E+06 4.76E-02 10.3 Y30W L 2.99E+06 1.51E-01 50.4 4.75E+06 8.95E-02 18.8 N33R L 1.92E+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 6.17E+06 9.38E-01 151.9 9.06E+06 4.23E-01 46.7 H31Q H 8.00E+06 1.85E-01 23.1 7.83E+06 9.72E-02 12.4 S53N H 4.56E+04 5.17E-02 1132.6 5.75E+06 2.86E-01 49.6 S55N H 1.39E+07 1.91E-02 1.9 9.20E+06 1.30E-02 1.4 N99R H 1.22E+07 1.70E-01 16.9 1.89E+07 1.54E-01 15.3 N99K H 7.89E+06 1.89E-01 23.9 1.58E+07 2.07E-01 20.7 N99Q H 3.75E+05 4.93E-01 1314.1 1.03E+07 4.25E-01 42.4 N99H H 1.33E+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 1.34E-01 25.3

Finally, newly identified mutations in previously obtained full-length humanized antibodies were also tested (Table 6) and their selectivity for human fibrils was verified (Table 7B). 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 (with reference to SEQ ID NO:19) and 56 and 102 in the heavy chain (with referring to SEQ ID NOs: 27 and 35) lead to an increase in 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 systematically administered, a large amount of administered antibody can be lost because antibodies have limited systems to overcome complex physiological barriers.

Table 7B Humanized antibodies Human monomer human fibril ka1 (1/ms) kd1 (1/s) KD1 (nM) ka1 (1/ms) kd1 (1/s) KD1 (nM) VR6470gL3; gH23 1.20E+06 0.02416 20.15 8.55E+05 1.42E-04 0.166 VR6470gL3; gH36 1.15E+06 0.01742 15.10 1.07E+06 3.17E-04 0.298 VR6470gL3; gH23-S56N-N102H 9.66E+05 0.00445 4.62 1.04E+06 7.08E-05 0.068 VR6470gL3; gH36-S56N-N102H 1.19E+06 0.00488 4.10 1.25E+06 7.44E-05 0.059 VR6470 gL3-N33R; gH23-S56N-N102H 3.48E+06 0.00594 1.71 2.07E+06 1.16E-04 0.056 VR6470 gL3-N33R; gH36-S56N-N102H 4.97E+06 0.00648 1.31 2.39E+06 1.26E-04 0.053

Variants of the humanized antibody chains and combinations thereof were expressed and evaluated for their potency relative to the parent antibody, their biophysical properties, and suitability for subsequent processing.

Example 5 Characterization of Humanized Antibodies

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

Table 8 Description gL3gH23 gL3gH36 gL3-N33RgH23-S56N-N102H gL3-N33RgH36-S56N-N102H gL3gH23-S56N-N102H gL3gH36-S56N-N102H

All antibodies were screened for thermal stability (™), experimental pI, hydrophobicity, solubility (PEG precipitation assay), and air/liquid aggregation stability to determine if mutations had any effect, in particular on affinity, stability and ability to develop.

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

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

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

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

4. Asp(99)G motif (Asp isomerization) in the light chain CDR3 of all antibodies.

Chemical instability at these sites can lead to heterogeneity and immunogenicity of the product.

Thermal stability measurement (Tm)

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

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

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

Table 9 Description ™ Domain Fab SD ™ domain CH ₂ SD gL3gH23 73.1 0.6 64.8 0.2 gL3gH36 73.5 0.3 64.7 0.0 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 pI value

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

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

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

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic Interaction Chromatography (HIC) separates molecules in order of increasing hydrophobicity. Molecules bind to the hydrophobic stationary phase in the presence of high concentrations of polar salts and desorb into the mobile phase as the salt concentration decreases. Longer retention time corresponds to 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 was injected into a Dionex ProPac™ HIC-10 column (100 mm x 4.6 mm) connected in series to an Agilent 1200 binary HPLC pump with a fluorescence detector. Separation was controlled by intrinsic fluorescence (excitation and emission wavelengths, 280 nm 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) exposure for 2 minutes at 0% B, (ii) linear gradient from 0 to 100% B over 30 minutes (0.8 ml/min) (iii) column wash with 100% B for 2 minutes and re-equilibrate in 0% B for 10 minutes before next sample injection. The column temperature was maintained at 20°C.

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

[(Sample (RT) - Low Standard (RT)/High Standard (RT) - Low Standard (RT)] x 100.

Table 10 Antibody (Main Peak) Retention time (min) Normalized retention time (min) gL3gH23 nine 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 the same normalized retention times and the same 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 U S A. 2017 Jan 31;114(5):944-949). The low hydrophobicity promotes stability (i.e. reduces aggregation) during production.

Solubility measurement by polyethylene glycol (PEG) precipitation assay.

The stability of the colloidal solution was analyzed using a polyethylene glycol (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 mimic the effect of high solubility without the use of traditional concentration methods. PEG-induced precipitation of 6470 antibodies was examined 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 replaced with buffer where necessary by dialysis and the concentration was adjusted to 2 mg/mL. To minimize non-equilibrium precipitation, sample preparation consisted of mixing solutions of 2x protein and 2x PEG in a 1:1 volume ratio. After mixing, the samples were incubated at 37°C for 30 minutes to re-dissolve non-equilibrium aggregates. After incubation overnight at 20°C, the samples were centrifuged for 60 minutes (4000 g). Aliquots of the supernatant were transferred to half volume 96-well optical measurement plates and absorbance at 280 nm was measured using a BMG Labtech FLUOstar® Omega LVIS A280 plate reader. Concentration data was plotted against PEG%, and the calculated mean point (LogEC50) obtained by non-linear curve fitting with varying slope was obtained as a measure of the relative solubility of the colloidal solution of the samples. In this assay, a higher LogEC50 value corresponds to a higher stability of the colloidal solution.

The results (not shown) showed that with increasing pH of the buffer, the stability of the colloidal solution decreased for all 6470 antibodies. In addition, the following trend was observed: from more to less soluble: gL3gH23 and gL3gH36 > gL3gH23-S56N-N102H and gL3gH36-S56N-N102H > gL3-N33RgH23-S56N-N102H and gL3-N33RgH36-S56N-N102H.

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

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

Proteins tend to unfold upon contact with an air-liquid interface, where hydrophobic surfaces are represented by a hydrophobic medium (air) and hydrophilic surfaces by a hydrophilic medium (water). Stirring of protein solutions provides a large air-liquid interface that can stimulate aggregation. This assay serves to simulate the stresses a molecule is subjected to during manufacture (eg, ultrafiltration) and to provide stringent conditions to try to distinguish between 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 selected as conventional pre-made buffers. Before shaking, the concentration was adjusted to 1 mg/mL using the appropriate extinction coefficients (1.35 Ab 280 nm, 1 mg/mL, path length 1 cm) and absorbance at 280 nm, 340 nm, and 595 nm obtained with a Varian spectrophotometer. Cary® 50-Bio for establishing a zero reading point. Each sample was sub-aliquoted into 1.5 ml Eppendorf® capped conical tubes (4×250 µl) and subjected to stringent conditions to test stability by shaking at 1400 rpm at 25° C. for up to 24 hours. Time-dependent aggregation (turbidity) was monitored by measuring samples at 3 hours and 24 hours after shaking at 595 nm using a Varian Cary™ 50-Bio spectrophotometer. The average absorbance values for each sample were plotted against time.

The results obtained are shown in Fig. 11. No difference was observed between 6470 antibodies in any of the three buffers at 24 hours, however, a slight difference was found in the tendency to aggregate at 3 hours after shaking, which appeared to be buffer dependent.

Investigation of the impact of extreme conditions on the deamidation/isomerization of Asp

An extreme test was performed using antibodies 6470 gL3gH23 and gL3gH36 to determine the propensity for deamidation/Asp isomerization of four potential sequences identified: Asn(102)S (deamidation motif) in heavy chain CDR3; Asn(98)Asp(99) (deamidation motif) in light chain CDR3; Asn(32)Asn(33) (deamidation motif) in light chain CDR1 and Asp(99)G (Asp isomerization motif) in light chain CDR3. 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 the properties of the solution (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 were also obtained (unstressed samples) - low levels indicate low susceptibility, but levels may vary between batches/manufacturing conditions.

Buffer in two 6470 antibodies was replaced 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 concentration of the sample in each of the buffers 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. An aliquot was removed immediately (T0) and after 2 weeks and 4 weeks and stored at -20°C.

Two week old samples were thawed and analyzed by tryptic digestion/mass spectroscopy (MS) for chemical modification analysis as follows. Samples of stressed proteins were restored with TCEP and alkylated with chloroacetic acid in Tris-HCL 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% by volume and samples were diluted to 0.5 mg/mL prior to 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 using a +ve-ion orbitrap-orbitrap data-dependent 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 baseline Asn deamidation at all three CDR sites is <1.5%, and that deamidation maximizes to ~6% after 2 weeks at pH 8.0 and 37°C for the Asn site. (102)S in the CDR3 heavy chain.

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

Example 6: Immunohistochemistry

Immunohistochemistry was performed on an Asterand Bioscience device (Royston, United Kingdoms). Cryosections (10 μm) were first exposed to antigen demasking with Dako PT Link and EnVision FLEX Target Retrieval Solutions (pH 6) at 97° C. for 20 minutes with automatic heating and cooling. All subsequent stages of incubation were carried out at room temperature. Cryosections were air dried for 30 minutes, fixed in 4% paraformaldehyde prepared in 1X PBS for 10 minutes, washed in Dako EnVision™ FLEX wash buffer (Dako) and then loaded into a Dako Autostainer Plus. Endogenous peroxidase activity was blocked by incubating sections with a Dako peroxidase block (Dako) for 5 minutes. Sections were then washed twice with 1X PBS before incubation with Dako CSA II protein block (Dako) for 10 minutes. The protein block solution was removed with an air jet and the sections were incubated for 30 minutes with rabbit IgG1 6470 (comprising SEQ ID NOS: 47 and 48) diluted (0.05 μg/ml) in Dako antibody diluent (Dako). After incubation, sections were washed twice with 1X PBS, then incubated with anti-rabbit Dako Flex polymer-HRP substrate (Dako) for 20 minutes, washed twice and then incubated with diaminobenzidine substrate (Dako) for 10 minutes. The chromogenic reaction was stopped by washing the glass slides with distilled water. After chromogenesis, sections were removed from the Dako Autostainer Plus and manually stained with hematoxylin, dehydrated with increasing concentrations of ethanol, cleaned with three xylene washes, and covered with DPX embedding medium (Sigma-Aldrich). Digital images of stained sections were obtained using the Aperio ScanScope AT Turbo system (Leica Biosystems). Antibody 6470 was tested on brain sections obtained from five different pS129-α-synuclein-positive and three different pS129-α-synuclein-negative donors (1 slice/donor). Antibody 6470 stained neuropils and Lewy body-like structures in the temporal cortex and substantia nigra of PD patients (Fig. 12A-E). In brain tissue without PD, 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 both PD and non-PD patients, while it binds to abnormal α-synuclein present in Lewy bodies only in PD patients.

Example 7 Cell Based Aggregation Assay

Freestyle 293F HEK cells (suspension cells) were seeded at a density of 0.7×10 ⁶ cells/ml in Freestyle 293 expression medium (Invitrogen™) and cultured to a density of 300×10 ⁶ 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, while diluting 293Fectin in OptiMEM medium (Invitrogen™) and incubating for 5 minutes at room temperature. The diluted DNA was added and incubated at room temperature for 20 minutes and then added dropwise to the cells (20 ml per flask). Cells were incubated for 24 hours at 37°C, 125 rpm, 8% CO ₂ . Cells were either used immediately or frozen at a density of 5 million cells/ml in FBS + 10% DMSO.

If the cells were pre-frozen, the cryovials were thawed and the cells were resuspended in Freestyle 293 medium, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in Freestyle 293 medium (Life Technologies™) containing Pen/Strep (Invitrogen™) with a density of 2×10 ⁶ cells/ml. 20 μl of cell suspension was added to a 384-well plate (Grainer™) (about 40,000 cells/well in total). 150 nM human alpha-synuclein fibrils (prepared as described herein in Example 1) were added to each well, followed by antibodies (6470 gL3gH23; 6470 gL3gH36; 6470 gL3N33gH23 S56N N102; 6470 gL3N33gH36 S56N N102; 6470 gL3gH36 S56N N102; gL3gH36 S56N N102, all as full length IgG4P, sequences shown in Table 1) in PBS for testing (at different concentrations). The plates were incubated at 37°C, 5% CO2, 95% humidity in a cell culture incubator for 2 days.

At the end of the second day, 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 minutes. 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 minutes, 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 (VWR™) in PBS was added to each well. The plates were incubated at room temperature for 15 minutes and then 50 μl of FACS buffer consisting of HBSS (VWR™ without calcium and magnesium) + 2% FBS + 2 mM EDTA (Life Technologies™) was added. The plates were centrifuged at 2000 g for 1 minute and the supernatant was aspirated leaving only 20 μl of medium in each well. An additional 20 μl of FACS buffer with anti-alpha-synuclein antibody pSer129 (AbCam™) diluted 1:300 was added to each well. The plates were incubated for 1 hour at room temperature, and then 50 μl of FACS buffer was added to each well before centrifuging again at 2000 g for 1 minute. The supernatant was removed before addition of secondary Alexafluor647-conjugated anti-rabbit antibody (Life Technologies™) diluted 1:500 and DAPI (Life Technologies™) to each well. The plates were incubated for 1 hour at room temperature in the dark, and then 50 μl of FACS buffer was added and the plates were centrifuged at 2000 g for 1 minute. After washing, more FACS buffer was added and the plates were ready to be placed on a flow cytometer (BD FACS Canto II) for reading.

FACS data was analyzed using FlowJo software. First, living single cells were subjected to gating using forward and side scatter. Second, DAPI+ events were gated and their number was used as a measure of the number of live, nucleated single cells. Finally, phosphoserine-labeled 129 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 a percentage.

The results are shown in FIG. 13, which shows the ability of test antibodies to inhibit alpha-synuclein fibril-induced aggregation on cells expressing alpha-synuclein. 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 the standard error of the measurement (SEM, N=3, n=9).

Example 8: Primary Neuron Aggregation Analysis

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

The next day, 80% of the medium was replaced with seeding medium without FBS [Neurobasal™ medium supplemented with 2% B27, 1 mM GlutaMAX, 50 U/ml penicillin-streptomycin). Seven days after seeding, the medium was removed, leaving 20 μl in each well. 100 nM human alpha-synuclein fibrils (prepared as described 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% CO ₂ , 95% humidity in a cell culture incubator for an additional 7 days. Fourteen days after seeding, the medium was aspirated from all wells, leaving 20 μl 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 fixation buffer (DPBS with 4% paraformaldehyde) per well for 15 minutes. The fixation buffer was then removed and the cells were washed again with 80 μl DPBS. DPBS was removed and replaced with 40 μl permeabilization buffer (DPBS with 0.1% Triton X-100) per well. After 10 minutes, the permeabilization buffer was removed and the cells were incubated for 1 hour in 40 μl of blocking buffer (PBS with 1% BSA and 0.1% Triton X-100) per well. The blocking buffer was then removed and replaced with a 40 µl/well primary antibody solution (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 hours, then washed three times (90 µl/each, PBS) After the last wash, PBS was removed and replaced with 40 µl of secondary antibody solution (0.1% anti-rabbit antibody conjugated to AlexaFluor647 in PBS with 0.2% anti- beta-III-tubulin conjugated with AlexaFluor488) The secondary antibody solution was incubated in the wells for 1 hour, then removed and replaced with 40 µl of PBS containing 0.3% CellMask Blue™ After 5 minutes of incubation, the wells were washed 3 times with 80 µl of PBS , then filled with 50 μl of PBS per well before the plate was sealed with clear plastic film.

Plates were processed on an Arrayscan plate visualizer (ThermoFisher Scientific™). Images were analyzed using HCS Scan™ software from the same manufacturer. The density of neurons was controlled using the beta-III-tubulin signal. Zones of sparse arrangement of cells or zones with damage with a layer of damaged neuronal cells, determined by a significant decrease in the surface with a beta-III-tubulin signal, were excluded. Finally, a surface with pSer129 alpha-synuclein signal per zone was used to quantify abnormal 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), while significant accumulation of pS129 is observed in the brains of patients suffering from synucleinopathies (Kahle PJ, et al (2000) Ann NY Acad Sci, 920, 33-41); Okochi M, et al. (2000) J Biol Chem, 275, 390-397); Anderson JP, et al. (2006) J Biol Chem, 281, 29739-29752).

Data were normalized to wells treated with only fibrils without antibodies and expressed as a percentage. As shown in FIG. 14, 6470gL3gH36 (designated VR6470) inhibits alpha-synuclein aggregation induced by alpha-synuclein fibrils on mouse primary neurons expressing endogenous levels of alpha-synuclein. Error bars represent the standard error of the measurement (SEM, N=4, n=18). These data confirm that 6470gL3gH36 is able to block alpha-synuclein fibril-induced aggregation in primary mouse neurons with an IC ₅₀ lower than 4 nM.

Example 9 Evaluation of the effectiveness of VR6470 in vivo

An antibody containing the variable region of SEQ ID NO:17 and SEQ ID NO:33 and the mouse IgG1 constant region (named VR6470 in this example) was tested in wild-type C57Bl/6J male mice (Janvier, France) and in an α knockout transgenic model mouse α-synuclein expressing human alpha-synuclein (hereinafter referred to as SNCA-OVX; Charles River, France).

C57Bl/6J and SNCA-OVX mice were injected with the indicated antibody and preformed mouse fibrils (PFF) (obtained as disclosed herein in Example 1). A negative control antibody (101.4) and vehicle were also injected together with an alpha-synuclein reference antibody (C-terminal reference Ab) that binds to the last nine C-terminal residues of alpha-synuclein. This reference antibody (which has different CDRs from the antibodies of the present invention) showed comparable binding characteristics to the antibodies of the present invention. The reference antibody has a higher affinity for alpha-synuclein than the antibodies of the present invention and similar biophysical properties. It is also equally effective in preventing alpha-synuclein aggregation in HEK cell assays according to Example 8 (Table 11).

Table 11 Antibody Monomer fibrils _IC50 ka1 (1/ms) kd1 (1/s) KD1 (nM) ka1 (1/ms) kd1 (1/s) KD1 (nM) (nM) VR6470 1.15E+06 0.01742 15.10 1.07E+06 3.17E-04 0.298 less than 5 C-term Ab 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 minutes on a shaker at room temperature before direct injection into the brains 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 the carrier solution. One injection was administered 24 hours prior to the combined intracerebral administration.

The antibodies were then administered to mice intraperitoneally at a dose of 30 mg/kg. The second intraperitoneal injection was administered 7 days after the first, and then the same treatment regimen (one intraperitoneal injection per week at a dose of 30 mg/kg with an injection volume of 10 ml/kg) was used for 4 weeks, receiving a total of 4 injections C57Bl/6J wild-type male mice, and for 11 weeks, receiving a total of 12 injections of SNCA-OVX mice. In both experiments, mice were randomly assigned to drug treatment groups and the experiment was blinded to treatment.

Animal experiments were performed in accordance with the guidelines of 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-IC-PARKINSON-MO). At the time of surgery, the mice weighed 25 to 30 g and were 17 weeks old. Mice were kept in cages (4 mice per cage, Macrolon type 2). They were kept on a 12:12 day/night cycle and the lights were turned 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 distribution to the experimental groups. Enriched food and proper housing conditions were provided before and after surgery (Enviro-dri, Pharma Serv). Animal health was monitored daily by animal care staff. Every effort has been made to minimize animal suffering. Animals were sacrificed under anesthesia.

The operation was performed under general anesthesia using a mixture of 50 mg/kg ketamine (Nimatek, Eurovet Animal Health B.V.) and 0.5 mg/kg medetomidine (Domitor, Orion Corporation) administered intraperitoneally. In addition, 2.5 mg/kg of atipamezole (Antisedan, Orion Corporation) was given to keep 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 break (OFF) for one minute). PFF was then premixed with antibodies for 30 minutes and shaken at room temperature for 30 minutes prior to brain injection. The solution (2 μl) was injected at a rate of 0.2 μl/min and the needle was left in place for an additional 2.5 minutes before it was slowly retracted. Injections were made unilaterally into the right striatum in the following coordinates: AP = +0.20 mm, ML = -2.00 mm, DV = -3.20 mm.

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

Brain sections were performed at Neuroscience Associates (TN, USA). First, the brain was treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent the formation of artifacts during freezing and placed in a gelatin matrix according to the MultiBrain® method. After curing, the blocks were flash frozen by immersion in isopentane cooled to -70° C. with crushed dry ice and placed in the freeze stage of an AO860 sledge microtome. Sections of blocks 40 µm thick were performed using the 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 anti-alpha synuclein antibody pSer129 (mouse anti-alpha synuclein (pSer129), biotin - (Wako - 010-26481)) diluted 1:30,000. All incubation solutions from blocking serum were further used with Tris buffer solution (TBS) with Triton X-100 as vehicle; all washes were performed with TBS. Endogenous peroxidase activity was blocked by treatment with hydrogen peroxide 0.9%, and non-specific binding was blocked by 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, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to create a visible reaction product mounted on gelled (sub-poor) glass slides, air dried, lightly stained with thionine, dehydrated in alcohols, purified in xylene and coated with Permount.

Fluorescent immunohistochemistry for p62/SQS™1 (p62 is known to coaggregate in human Lewy bodies) and Amytracker (for protein aggregates in general) were performed on free-floating brain sections. VR6470 has been shown to reduce Amytracker-stained aggregated proteins and 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 FOV of pSer129 alpha-synuclein signal was used to quantify abnormal alpha-synuclein aggregation on the ipsilateral striatum, cortex, basolateral amygdala complexes, and substantia nigra. Regions of interest (ROIs) were manually delineated and automatic quantification of pSer129 alpha-synuclein signal in various 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 area of the label reflects the magnitude of pSer129 alpha-synuclein pathology, which covers different areas of the brain. All quantifications were blinded until the end of the statistical analysis.

Data analysis was performed for % label area (ie the ratio between the pSer129 signal area in µm ² and the area of interest in µm ² ). The area of the mark in % was reassessed 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 tonsil: 6-10 slices -0.58 to -2.06; substantia nigra: 6-8 slices -2.54 to -3.88), and AUC was calculated separately for each subject.

One-way ANOVA was used for statistical analysis. The ANOVA was followed by multiple pairwise comparisons between means without any regard for multiple comparisons (**p<0.01 and *p<0.05). The data were subjected to a logarithmic transformation to meet the criteria of normality and homoscedasticity. Plots represent the geometric means of the untransformed data.

As shown in FIGS. 15A and 15B, antibody 6470 (comprising the variable regions of SEQ ID NOs: 17 and 33 and the mouse IgG1 constant region) markedly reduced alpha-synuclein pathology (i.e., alpha-synuclein pSer129 signal) compared to three controls. groups in four different ipsilateral regions of the brain, including the striatum, cerebral cortex, amygdala, and substantia nigra, one month after administration of mouse PFFs to wild type C57Bl/6J male mice (Fig. 15A) and three months after administration of human PFFs to mice. male SNCA-OVX (Fig. 15B).

In FIG. 16 shows the quantitation of alpha-synuclein phosphorylated at Ser129 (AUC% marker area) in the ipsilateral cortex, striatum, amygdala, and substantia nigra in C57Bl/6J wild-type mice, respectively. The negative control antibody and C-terminal reference antibody did not reduce alpha-synuclein pathology compared to the vehicle group. In contrast, antibody 6470 significantly reduced the level of pathology (i.e. pSer129alpha-synuclein signal) in the cortex, striatum, amygdala, and substantia nigra (p<0.01) compared to three control groups of C57Bl/6J mice treated with mouse PFFs. Among wild-type C57Bl/6J mice tested, the 6470 group showed significant reductions in pSer129 alpha-synuclein levels in four different structures, three of which were distal from the injection site (cortex, substantia nigra, and amygdala).

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

These results confirm that the antibodies containing the structural features of the present invention are able to prevent in vivo the 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 . The reference antibody, which binds to the very C-terminus of alpha-synuclein with high affinity and which is effective in preventing 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 alpha synucleinopathies, for example, which are characterized by an increase in Ser129 phosphorylation, including Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse body disease Lewy body variant Alzheimer's 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 Mouse

Male C57/Bl6 mice (n=3 per drug) were injected intravenously as a single dose of 2 mg/kg of the 6470gL3gH36 IgG4P antibody (comprising SEQ ID NOS: 17 and 33; referred to as 6470 in FIG. 17 and henceforth).

Blood samples were taken (at 0.083, 1, 4, 8, 24, 72, 120, 168 and 336 hours post-injection) from the tail vein and allowed to coagulate at room temperature. Serum was isolated after centrifugation, which was then frozen and stored until analysis. 6470 was quantified by LC-MS/MS. Serum samples from the study were thawed and quantitated against a calibration curve generated with the 6470 antibody or reference antibody added at varying concentrations to control mouse sera. Before injecting 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 enzyme (Promega, UK) at 37°C. Digestion was stopped by adding formic acid to the samples to lower the pH and then desalted using a Waters HLB SPE plate. The resulting eluent was 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 (Schimadzu) connected to a Triple Quad 6500 mass spectrometer. , 2.6 μm), which was maintained at 50°C. A linear gradient of 5-70% acetonitrile in 0.1% formic acid was applied over 6 minutes and then increased to 95% acetonitrile in 0.1% formic acid over 0.8 minutes at a flow rate of 0.6 ml/min. The mass spectrometer was set to perform a multiple reaction monitoring analysis to detect multiple transitions of 6470 or 5811 peptides with a dwell time of 50 milliseconds per transition. Data analysis was performed using Analyst 1.6 software.

These data demonstrate that the 6470 antibody exhibited 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; WO2013/063516).

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 LC-MS/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 Table 12, in Cynomolgus monkeys, the 6470 antibody 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 pharmacokinetic characteristics 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)

--->

SEQUENCE LIST

<110> YUSB Biopharma SRL

<120> Antibodies

<130> PF0130-WO

<150>GB1720975.0

<151> 2017-12-15

<160> 49

<170>PatentIn version 3.5

<210> 1

<211> 13

<212> Protein

<213> Oryctolagus cuniculus

<400> 1

Gln Ala Ser Gln Ser Val Tyr Lys Asn Asn Tyr Leu Ala

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<211> 7

<212> Protein

<213> Oryctolagus cuniculus

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Gly Ala Ser Thr Leu Ala Ser

15

<210> 3

<211> 12

<212> Protein

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Ala Gly Tyr Lys Gly Gly Arg Asn Asp Gly Phe Ala

1.5 10

<210> 4

<211> 10

<212> Protein

<213> Oryctolagus cuniculus

<400> 4

Gly Ile Asp Leu Ser Ser His Asp Met Tyr

1.5 10

<210> 5

<211> 16

<212> Protein

<213> Oryctolagus cuniculus

<400> 5

Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

1.5 10 15

<210> 6

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<213> Oryctolagus cuniculus

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Ile His Tyr Gly Asn Ser Gly Gly Leu

15

<210> 7

<211> 13

<212> Protein

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<220>

<223> CDR-L1 N33R

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Gln Ala Ser Gln Ser Val Tyr Lys Asn Arg Tyr Leu Ala

1.5 10

<210> 8

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H2 S56N

<400> 8

Ala Ile Tyr Ala Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

1.5 10 15

<210> 9

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H3 N102H

<400> 9

Ile His Tyr Gly His Ser Gly Gly Leu

15

<210> 10

<211> 140

<212> Protein

<213> Homo sapiens

<400> 10

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

1.5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

<210> 11

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<212> Protein

<213> Oryctolagus cuniculus

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Ala Ile Val Met Thr Gln Thr Pro Ser Ser Lys Ser Val Ala Val Gly

1.5 10 15

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

20 25 30

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

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

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

65 70 75 80

Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 12

<211> 336

<212> DNA

<213> Oryctolagus cuniculus

<400> 12

gccatcgtga tgacccagac tccatcttcc aagtctgtcg ctgtgggaga cacagtcacc 60

atcaattgcc aggccagtca gagtgtttat aagaacaact acttagcctg gtttcaacag 120

aaaccagggc agcctcccaa acaactgatc tatggtgcgt ccactctggc atctggggtc 180

ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagcgatgtg 240

gtgtgtgacg atgctgccac ttactactgt gcaggatata aaggtggtcg taatgatggt 300

tttgctttcg gcggagggac cgaggtggtg gtcaaa 336

<210> 13

<211> 114

<212> Protein

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

1.5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser His Asp

20 25 30

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

35 40 45

Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ile His

85 90 95

Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 14

<211> 342

<212> DNA

<213> Oryctolagus cuniculus

<400> 14

cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60

tgcacagtct ctggaatcga cctcagtagc cacgacatgt attgggtccg ccaggctcca 120

gggaaggggc tggaatacat tggagccatt tatgctagtg gtagcacata ctacgcgagc 180

tgggcgaaag gccgattcac catctccaag acctcgacca cggtggatct gaaaatgacc 240

agtctgacaa ccgaggacac ggccacctat ttctgtgcca gaattcatta tggtaatagt 300

ggtgggttgt ggggccaagg caccctggtc accgtctcga gt 342

<210> 15

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<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gL3 VL

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

1.5 10 15

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

20 25 30

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

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 16

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gL3 VL nucl.

<400> 16

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaacaact acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcgggagtg 180

ccgagccgct tctcgggatc tgggtccgga actcagttca ccctgactat ctcgtccctg 240

caacccgagg atttcgccac ctactactgc gccggctata agggaggacg gaacgacggc 300

ttcgcttttg gtggaggcac caaggtcgaa atcaag 336

<210> 17

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gL3 Light chain

<400> 17

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

1.5 10 15

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

20 25 30

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

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

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

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gL3 Light chain nuc.

<400> 18

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaacaact acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcgggagtg 180

ccgagccgct tctcgggatc tgggtccgga actcagttca ccctgactat ctcgtccctg 240

caacccgagg atttcgccac ctactactgc gccggctata agggaggacg gaacgacggc 300

ttcgcttttg gtggaggcac caaggtcgaa atcaagcgta cggtggccgc tccctccgtg 360

ttcatcttcc caccctccga cgagcagctg aagtccggca ccgcctccgt cgtgtgcctg 420

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact ccggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 19

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gL3 VL N33R

<400> 19

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

1.5 10 15

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

20 25 30

Arg Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Gln

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 20

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gL3 VL N33R nu.

<400> 20

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaaccgtt acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcgggagtg 180

ccgagccgct tctcgggatc tgggtccgga actcagttca ccctgactat ctcgtccctg 240

caacccgagg atttcgccac ctactactgc gccggctata agggaggacg gaacgacggc 300

ttcgcttttg gtggaggcac caaggtcgaa atcaag 336

<210> 21

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gL3 Light chain N33R

<400> 21

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

1.5 10 15

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

20 25 30

Arg Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Gln

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

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

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 22

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gL3 Light chain N33R nc.

<400> 22

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaaccgtt acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcgggagtg 180

ccgagccgct tctcgggatc tgggtccgga actcagttca ccctgactat ctcgtccctg 240

caacccgagg atttcgccac ctactactgc gccggctata agggaggacg gaacgacggc 300

ttcgcttttg gtggaggcac caaggtcgaa atcaagcgta cggtggccgc tccctccgtg 360

ttcatcttcc caccctccga cgagcagctg aagtccggca ccgcctccgt cgtgtgcctg 420

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact ccggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc agggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 23

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH23 VH

<400> 23

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> 6479gH23 VH n.c.

<400> 24

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtagcac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgacaact ctaaaaacac cgtgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 25

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH23 Heavy chain

<400> 25

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440

<210> 26

<211> 1332

<212> DNA

<213> Artificial sequence

<220>

<223> 6470gH23 Heavy chain n.c.

<400> 26

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtagcac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgacaact ctaaaaacac cgtgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag cgcttctaca 360

aagggcccct ccgtgttccc tctggcccct tgctcccggt ccacctccga gtctaccgcc 420

gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaatg ccaagaccaa gccggagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

1080

tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200

ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agcctgggca ag 1332

<210> 27

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH23 VH S56N N102H

<400> 27

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly His Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH23 VH S56N N102H nu.

<400> 28

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtaatac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgacaact ctaaaaacac cgtgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 29

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH23 Heavy chain S56N N102H

<400> 29

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly His Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440

<210> 30

<211> 1332

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH23 Heavy chain S56N N102H nc.

<400> 30

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtaatac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgacaact ctaaaaacac cgtgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag cgcttctaca 360

aagggcccct ccgtgttccc tctggcccct tgctcccggt ccacctccga gtctaccgcc 420

gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaatg ccaagaccaa gccggagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

1080

tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200

ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agcctgggca ag 1332

<210> 31

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH36 VH

<400> 31

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 32

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH36 VH n.c.

<400> 32

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtagcac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgactcca gcaaaaacac cctgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 33

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH36 Heavy chain

<400> 33

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440

<210> 34

<211> 1332

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH36 Heavy chain n.c.

<400> 34

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtagcac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgactcca gcaaaaacac cctgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag cgcttctaca 360

aagggcccct ccgtgttccc tctggcccct tgctcccggt ccacctccga gtctaccgcc 420

gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaatg ccaagaccaa gccggagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

1080

tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200

ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agcctgggca ag 1332

<210> 35

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH36 VH S56N N102H

<400> 35

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly His Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH36 VH S56N N102H nu.

<400> 36

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtaatac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgactcca gcaaaaacac cctgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 37

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 gH36 Heavy chain S56N N102H

<400> 37

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

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Ser His

20 25 30

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

35 40 45

Gly Ala Ile Tyr Ala Ser Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ile His Tyr Gly His Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440

<210> 38

<211> 1332

<212> DNA

<213> Artificial sequence

<220>

<223> 6470 gH36 Heavy chain S56N N102H nc.

<400> 38

60

tcttgtgcag taagcggcat cgacctgtcc agccacgaca tgtattgggt acgtcaggca 120

ccgggtaaag gtctggaata catcggcgcc atttatgcta gtggtaatac atactacgcg 180

agctgggcga aaggccgttt caccatctcc cgtgactcca gcaaaaacac cctgtacctg 240

cagatgaact ctctgcgtgc ggaagacact gcggtttact attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag cgcttctaca 360

aagggcccct ccgtgttccc tctggcccct tgctcccggt ccacctccga gtctaccgcc 420

gctctgggct gcctggtcaa ggactacttc cccgagcccg tgacagtgtc ctggaactct 480

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtcgtgac cgtgccctcc tccagcctgg gcaccaagac ctacacctgt 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggaatc taagtacggc 660

cctccctgcc ccccctgccc tgcccctgaa tttctgggcg gaccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccaggaaga tcccgaggtc cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaatg ccaagaccaa gccggagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

1080

tccctgacct gtctggtcaa gggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggctcc 1200

ttcttcctgt actctcggct gaccgtggac aagtcccggt ggcaggaagg caacgtcttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agcctgggca ag 1332

<210> 39

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Human IGKV1-16 JK4 acceptor scaffold

<400> 39

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

1.5 10 15

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

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Human IGKV1-16 JK4 acceptor scaffold nucl.

<400> 40

gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgtc gggcgagtca gggcattagc aattatttag cctggtttca gcagaaacca 120

gggaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgccaacag tataatagtt accctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 41

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Human IGHV3-23 JH4 scaffold scavengers

<400> 41

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

1.5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 42

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Human IGHV3-23 JH4 acceptor framework nucl.

<400> 42

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaatacttt 300

gactactggg gccaaggaac cctggtcacc gtctcctca 339

<210> 43

<211> 306

<212> Protein

<213> Artificial sequence

<220>

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

<400> 43

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

1.5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Gly Lys

305

<210> 44

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-L1 X33

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa is Asn (N) or Arg (R)

<400> 44

Gln Ala Ser Gln Ser Val Tyr Lys Asn Xaa Tyr Leu Ala

1.5 10

<210> 45

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H2 X56

<220>

<221> MISC_FEATURE

<222>(7)..(7)

<223> Xaa is Ser (S) or Asn (N)

<400> 45

Ala Ile Tyr Ala Ser Gly Xaa Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

1.5 10 15

<210> 46

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR-H3 X102

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa is Asn (N) or His (H)

<400> 46

Ile His Tyr Gly Xaa Ser Gly Gly Leu

15

<210> 47

<211> 216

<212> Protein

<213> Oryctolagus cuniculus

<400> 47

Ala Ile Val Met Thr Gln Thr Pro Ser Ser Lys Ser Val Ala Val Gly

1.5 10 15

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

20 25 30

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

35 40 45

Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe

50 55 60

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

65 70 75 80

Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Gly Gly

85 90 95

Arg Asn Asp Gly Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

Arg Thr Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp

115 120 125

Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr

130 135 140

Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr

145 150 155 160

Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr

165 170 175

Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser

180 185 190

His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val

195 200 205

Gln Ser Phe Asn Arg Gly Asp Cys

210 215

<210> 48

<211> 437

<212> Protein

<213> Oryctolagus cuniculus

<400> 48

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

1.5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser His Asp

20 25 30

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

35 40 45

Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ile His

85 90 95

Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys

115 120 125

Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys

130 135 140

Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu

145 150 155 160

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

165 170 175

Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val

180 185 190

Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr

195 200 205

Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu

210 215 220

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

225 230 235 240

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser

405 410 415

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser

420 425 430

Arg Ser Pro Gly Lys

435

<210> 49

<211> 227

<212> Protein

<213> Artificial sequence

<220>

<223> 6470 Rabbit Fab-His heavy chain

<400> 49

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

1.5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser His Asp

20 25 30

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

35 40 45

Ala Ile Tyr Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

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

65 70 75 80

Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ile His

85 90 95

Tyr Gly Asn Ser Gly Gly Leu Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys

115 120 125

Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys

130 135 140

Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu

145 150 155 160

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

165 170 175

Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val

180 185 190

Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr

195 200 205

Val Ala Pro Ser Thr Cys Ser Lys Pro His His His His His His His His

210 215 220

His His His

225 22

<---

Claims (57)

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:1; 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:5, and iii. CDR-H3 containing SEQ ID NO:6. 2. An antibody or antigen-binding fragment thereof according to claim 1 that binds to an alpha-synuclein 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. 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 to alpha-synuclein both as a monomer and as 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 the form of fibrils compared to alpha-synuclein in the form of a monomer, characterized by a dissociation constant (K _D ) of 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 claim 9, 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 comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:31; or b. 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 comprising SEQ ID NO:15 and a heavy chain variable region comprising SEQ ID NO:23; or b. a light chain containing SEQ ID NO:17; and a heavy chain containing SEQ ID NO:25. 14. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-13. 15. The isolated polynucleotide according to claim 14, where the polynucleotide encodes: a. the variable region of the light chain, where the polynucleotide: i. at least 90% identical to SEQ ID NO:16; or ii. contains SEQ ID NO:16; or iii. essentially consists of SEQ ID NO:16; b. heavy chain variable region, where the polynucleotide: i. at least 90% identical to SEQ ID NO:24 or SEQ ID NO:32; or ii. contains SEQ ID NO:24 or SEQ ID NO:32; or iii. essentially consists of SEQ ID NO:24 or SEQ ID NO:32; c. light chain, where the polynucleotide: i. at least 90% identical to SEQ ID NO:18; or ii. contains SEQ ID NO:18; or iii. essentially consists of SEQ ID NO:18; d. heavy chain, where the polynucleotide: i. at least 90% identical to SEQ ID NO:26 or SEQ ID NO:34; or ii. contains SEQ ID NO:26 or SEQ ID NO:34; or iii. essentially consists of SEQ ID NO:26 or SEQ ID NO:34. 16. A cloning vector containing one or more polynucleotides according to any one of claims 14 or 15. 17. An expression vector containing one or more polynucleotides according to any one of claims 14 or 15. 18. A host cell for obtaining an antibody or antigen-binding fragment according to any one of claims 1-13, containing: a. one or more polynucleotides according to any one of claims 14 or 15 or b. one or more expression vectors according to claim 17. 19. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising culturing the host cell according to claim 18 under suitable conditions to obtain an antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof. 20. A pharmaceutical composition for the treatment of Parkinson's disease, comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and one or more pharmaceutically acceptable carriers, excipients or diluents. 21. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for use in the treatment of one or more synucleinopathies. 22. An antibody or antigen-binding fragment thereof for use according to claim 21, wherein the synucleinopathy is selected from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lewy body dementia (DLB), diffuse Lewy body disease (DLBD), variant Alzheimer's disease with Lewy bodies (LBVAD), combined Alzheimer's and Parkinson's disease, multiple system atrophy (MSA) and neurodegeneration with iron accumulation in the brain type 1 (NBIA-1). 23. An antibody or antigen-binding fragment thereof for use according to claim 22, wherein the synucleinopathy is Parkinson's disease. 24. A method of treating Parkinson's disease (PD) in a patient, comprising administering to said patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 20. 25. The method of claim 24 wherein Parkinson's disease is an idiopathic and hereditary form of Parkinson's disease. 26. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for use in diagnosing alpha synucleinopathy. 27. An antibody or antigen-binding fragment for use according to claim 26 for use in the diagnosis of Parkinson's disease.

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