HK40030984B - Anti-alpha-synuclein antibodies - Google Patents

Description

Anti-α synuclein antibody

Invention Field

This invention relates to anti-α-synuclein antibodies and methods of using them to treat synucleinosis. In particular, this invention relates to anti-human α-synuclein antibodies and their use in the treatment of Parkinson's disease.

Background of the Invention

Alpha synuclein is a small, soluble protein of 140 amino acids in a fundamentally different form. It is primarily found in presynaptic nerve endings, and while its exact function is unknown, researchers believe it plays a central role in multiple neurodegenerative processes.

Over the past 15 years, alpha synuclein has been shown to play a crucial role in the pathogenesis of all forms of Parkinson's disease. Genetic mutations or multiplexing of the alpha synuclein gene cause familial early-onset Parkinson's disease (PD). Interestingly, in families with multiplexing, the pathogenic effect is clearly dependent on the gene dosage. Doublexing causes a relatively early-onset form of PD (~47 years old) with a normal disease course, while triplexing is associated with a very early age of onset (~33 years old) and a very rapid disease course. In all forms of Parkinson's disease, alpha synuclein is a major component of the Rui body (a key pathological marker of the disease).

During the course of this disease, the pathological condition of the body of Ruij expands, and α-synuclein has been proposed to function as a prion-like protein, misfolding to form toxic oligomers and aggregates that can spread from affected neurons to unaffected neurons (Olanow C.W et al., Movement Disorders, Vol. 28, No. 1, 2013). Current therapies do not stop the spread of the disease and only help treat symptoms associated with the progressive loss of motor neuron-dependent activity. In 2014, Tran H.T. et al. (Cell Reports 7, 2054-2065, 2014) showed that intraperitoneal administration of a monoclonal antibody against misfolded α-synuclein to mice that had been pre-injected with α-synuclein pre-formed fibrils via the striatum alleviated the pathological condition of the body of Ruij, improved the loss of dopaminergic neurons in the substantia nigra, and improved motor impairment. Therefore, there remains a need for passive immunotherapy that can exert a therapeutic effect in PD and other alpha synucleinopathies.

Invention Summary

The present invention addresses the above-mentioned need by providing an anti-α-synuclein antibody according to the embodiments described below.

Implementation Scheme 1: An antibody or its antigen-binding fragment that binds to α-synuclein, wherein the antibody comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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.

Implementation Scheme 2: The antibody or its antigen-binding fragment according to Implementation Scheme 1 binds to α-synuclein at an epitope containing residues E123, Y125, E126, M127, P128, S129, E130 and E131 as relating to SEQ ID NO: 10, wherein the epitope optionally includes A124 and G132.

Implementation Scheme 3: An antibody or antigen-binding fragment thereof according to any one of Implementation Scheme 1 or 2, wherein the antibody or antigen-binding fragment thereof prevents α-synuclein aggregation induced by α-synuclein fibrils.

Implementation Scheme 4: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 3, wherein the antibody or antigen-binding fragment thereof is capable of binding as a monomer and in the α-synuclein in the fibrils.

Implementation Scheme 5: The antibody or its antigen-binding fragment according to any of the foregoing implementation schemes has a higher binding affinity for α-synuclein in fibrils than for α-synuclein as a monomer, characterized in that the dissociation constant ( _KD ) for monomeric α-synuclein is at least 10 times that for α-synuclein in fibrils.

Implementation Scheme 6: An antibody or its antigen-binding fragment according to any of the foregoing implementation schemes, having a _KD of 300 pM or less for α-synuclein in fibrils.

Implementation Scheme 7: The antibody or its antigen-binding fragment according to any of the foregoing implementation schemes does not bind to β-synuclein and/or γ-synuclein.

Implementation Scheme 8: An antibody or its antigen-binding fragment according to any of the foregoing implementation schemes, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

Implementation Scheme 9: An antibody according to any of the foregoing implementation schemes, wherein the antibody is a full-length antibody.

Implementation Scheme 10: The antibody according to Implementation Scheme 9, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P.

Implementation Scheme 11: An antigen-binding fragment according to any one of Implementation Schemes 1 to 8, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ , scFv, dAb or _VHH .

Implementation Scheme 12: An antibody or antigen-binding fragment thereof according to any of the foregoing implementation schemes, wherein the antibody or antigen-binding fragment thereof comprises:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

Implementation Scheme 13: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:23; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:25.

Implementation Scheme 14: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises:

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

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

c. Light chains containing SEQ ID NO:17 and heavy chains containing SEQ ID NO:29 or 37.

Implementation Scheme 15: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 11, wherein said antibody or antigen-binding fragment comprises:

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

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

c. Light chains containing SEQ ID NO:21 and heavy chains containing SEQ ID NO:25 or 33.

Implementation Scheme 16: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 11, wherein the antibody or antigen-binding fragment thereof comprises:

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

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

c. Light chains containing SEQ ID NO:21 and heavy chains containing SEQ ID NO:29 or 37.

Implementation Scheme 17: An antibody or its antigen-binding fragment, wherein:

a. Competing with the antibody or its antigen-binding fragment according to any of the foregoing embodiments to bind to α-synuclein; and/or

b. Cross-blocking of the antibody or its antigen-binding fragment according to any of the foregoing embodiments, or cross-blocking of the binding of the antibody or its antigen-binding fragment according to any of the foregoing embodiments to α-synuclein; and/or

c. Binding α-synuclein to the same epitope as the antibody or its antigen-binding fragment according to any of the foregoing embodiments; and/or

d. Containing 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

e. Contains a light chain variable region that has at least 80% identity or similarity to the sequence according to SEQ ID NO:15 or SEQ ID NO:19.

Implementation Scheme 18: An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 16.

Implementation Scheme 19: The isolated polynucleotide according to Implementation Scheme 18, wherein the polynucleotide encodes:

a. Light chain variable region, wherein the polynucleotide:

i. 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. Basically composed of SEQ ID NO:16 or SEQ ID NO:20;

b. Heavy chain variable region, wherein the polynucleotide:

i. With SEQ ID NO:24 or SEQ ID NO:28 or SEQ ID NO:32

Or at least 90% identical to SEQ ID NO:36; or

ii. Contains SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32

Or SEQ ID NO:36; or

iii. Basically composed of SEQ ID NO:24 or SEQ ID NO:28 or SEQ ID NO:32 or SEQ ID NO:36;

c. The light chain, wherein the polynucleotide is:

i. 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. Basically composed of SEQ ID NO:18 or SEQ ID NO:22;

d. Heavy chain, wherein the polynucleotide is:

i. With SEQ ID NO:26 or SEQ ID NO:30 or SEQ ID NO:34

Or at least 90% identical to SEQ ID NO:38; or

ii. Contains SEQ ID NO:26, SEQ ID NO:30, or SEQ ID NO:34

Or SEQ ID NO:38; or

iii. It is basically composed of SEQ ID NO:26 or SEQ ID NO:30 or SEQ ID NO:34 or SEQ ID NO:38.

Implementation Scheme 20: A cloning or expression vector comprising one or more polynucleotides according to any one of Implementation Scheme 18 or 19.

Implementation Scheme 21: Host cell, comprising:

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

b. One or more expression carriers according to implementation scheme 20.

Implementation Scheme 22: A method for generating an antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 17, comprising culturing a host cell according to Implementation Scheme 21 under conditions suitable for generating the antibody or an antigen-binding fragment thereof and isolating the antibody or an antigen-binding fragment thereof.

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

Implementation Scheme 24: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 17 or a pharmaceutical composition according to Implementation Scheme 23, for use in a therapy.

Implementation Scheme 25: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 17 or a pharmaceutical composition according to Implementation Scheme 23, for use in the treatment of one or more synucleinosis.

Implementation Scheme 26: An antibody or antigen-binding fragment thereof having the use according to Implementation Scheme 25, wherein the synucleinosis is selected from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd's dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd's variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1).

Implementation Scheme 27: An antibody or antigen-binding fragment thereof having the use according to Implementation Scheme 26, wherein the synucleinosis is Parkinson's disease.

Implementation Scheme 28: A method for treating synucleinosis in a patient, comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 17 or a pharmaceutical composition according to Implementation Scheme 23.

Implementation Scheme 29: According to the method of Implementation Scheme 29, the synucleinosis is selected from Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd's dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd's variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA) and type 1 brain iron accumulation neurodegeneration (NBIA-1), preferably Parkinson's disease.

Implementation Scheme 30: An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1 to 16 is used in the diagnosis of α-synucleinosis, preferably in the diagnosis of Parkinson's disease.

Brief description of the attached diagram

Figure 1. (A) SDS-PAGE of α-synuclein expression samples. Superdex 75 size exclusion chromatography for human α-synuclein (3) treated with TEV protease, with His tag (1) and after His tag removal (2). Protein molecular weight standard SeeBluePlus2 (Invitrogen) (M). (B) SDS-PAGE of human α-synuclein (4) purified from Expi 293 supernatant as wild-type untagged protein. Protein molecular weight standard SeeBluePlus2 (Invitrogen) (M).

Figure 2. (A) Analysis of fibrils by the JC-1 assay for monomers without fluorescence and fibrils with maximum fluorescence at 540 nm. (B) Typical examples of random coil spectra (wavelength 1646 ^cm⁻¹ ) of monomeric human α-synuclein and interstitial β-sheet formation (wavelength 1625–1630 ^cm⁻¹ ) in recombinant human α-synuclein fibrils.

Figure 3. ELISA binding assay. ELISA assay of rabbit 6470 IgG1 binding to recombinant human α-synuclein monomers and fibrils, as well as the human α-synuclein peptide PVDPDNEAYE.

Figure 4. (A) shows the Western blots of the binding of rabbit 6470 IgG1 to human α-synuclein and human β-synuclein. 1, human α-synuclein; 2, human α-synuclein (rPeptide); 3, human β-synuclein (rPeptide); standard, MagicMark XP. (B) shows the predicted NMR chemical shift changes of the 6470 epitope on human α-synuclein.

Figure 5. Inhibition of binding of 6470IgG to immobilized α-synuclein (for each of the peptides tested, the left and right columns represent monomers and fibrils, respectively).

Figure 6. Schematic illustration of 6470Fab complexed with peptides 123-132.

Figure 7. Schematic illustration of the contact between the 6470 Fab heavy chain and peptides 123-132. Peptide residues are directly labeled, and 6470 variable heavy chain residues are labeled with vH-residue numbers.

Figure 8. Schematic illustration of the contact between the 6470 Fab light chain and peptides 123-132. Peptide residues are directly labeled, and 6470 variable light chain residues are labeled with vL-residue numbers.

Figure 9. Humanization of the light chain. 6470 is a rabbit variable light chain sequence. 6470gL3 is a humanized graft of the 6470 variable light chain, using the IGKV1-16 human line as the recipient scaffold. CDRs are shown in bold/underlined. Donor residues are shown in bold/italic and shaded: Q48 and Q72. Mutations in CDRL1 N33R are shown in bold/underlined and shaded.

Figure 10. Humanization of the heavy chain. 6470 is the rabbit variable heavy chain sequence. 6470gH23 and gH36 are humanized grafts of the antibody 6470 variable heavy chain, using the IGHV3-23 human line as the recipient 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 mutations S56N and N102H in CDRH2 and CDRH3 are shown in bold/underlined and shaded, respectively.

Figure 11. Stress at the air-liquid interface. 6470 antibody and mutant in three pre-prepared buffer solutions at 3 and 24 hours after vortex oscillation.

Figure 12. Immunohistochemistry. Immunoreactivity in brain slices from PD patients (A-E) and non-PD patients (F-H). (A-C) In the temporal cortex of PD patients, antibody 6470 labeled neuropil and Rui body-like structures in the gray matter (white arrows). (D, E) Antibody 6470 labeled Rui body-like features in the substantia nigra of PD patients (white arrows). (F, G) In non-PD temporal cortex tissue, 6470 also labeled neuropil, but no Rui body-like structures were observed. (H) No Rui body-like structures were observed in the substantia nigra of non-PD individuals; black arrows point to non-specific markers. Scale bar = 50 μm.

Figure 13. Cell-based aggregation assay (HEK cells). The antibody of this invention inhibits α-synuclein aggregation induced by α-synuclein fibrils, exhibiting an _IC50 of less than 5 nM. Error bars represent the standard error of the measurement (SEM, N=3, n=9). In the legend, FL at the end of each antibody name signifies "full length".

Figure 14. Cell-based aggregation assay (primary neurons). The representative antibody according to the invention inhibits α-synuclein aggregation induced by α-synuclein fibrils in mouse primary neurons expressing endogenous levels of α-synuclein, with an _IC50 of less than 4 nM. Error bars represent the standard error of the measurement (SEM, N=4, n=18).

Figure 15. Immunohistochemical photographs of the pathological features of α-synuclein in different brain regions of male C57Bl/6J wild-type mice (A) and SNCA-OVX mice (B) injected with mouse or human PFF, respectively (arrows).

Figure 16. Quantification of α-synuclein pathology in different brain regions (A: cerebral cortex; B: striatum; C: amygdala; and D: substantia nigra) of wild-type C57Bl/6J mice injected with mouse PFF.

Figure 17. Pharmacokinetic characteristics of α-synuclein antibodies: A. 6470 antibody in wild-type mice; B. 6470 and comparative antibodies in cynomolgus monkeys.

Invention Details

The present disclosure will now be described with reference to particular non-limiting aspects and embodiments thereof, and with reference to certain accompanying drawings and examples.

Unless otherwise specified, technical terms are used in their ordinary meaning. If a particular meaning is to be conveyed to certain terms, then the definition of the term will be given in the context in which the term is used.

When the term "comprising (including)" is used in this specification and claims, it does not exclude other elements. For the purposes of this disclosure, the term "consisting of..." is considered a preferred embodiment of the term "comprising (including)...".

When referring to a singular noun, the use of an indefinite or definite article such as "a," "an," or "the" includes the plural form of that noun, unless otherwise specified.

As used herein, the term "treatment" refers to achieving a desired pharmacological and/or physiological effect. This effect may be preventative, in terms of completely or partially preventing the disease or its symptoms, and/or therapeutic, in terms of partially or completely curing the disease and/or adverse effects attributable to the disease. Therefore, treatment encompasses any treatment of a disease in mammals, particularly in humans, and includes: (a) preventing the disease from occurring in subjects who may be predisposed to it but have not yet been diagnosed with it; (b) suppressing the disease, even if its development ceases; and (c) alleviating the disease, i.e., causing its remission.

"Therapeutic effective dose" refers to an amount of anti-alpha synuclein antibody or its antigen-binding fragment that is sufficient to produce therapeutic effect on a disease when administered to a mammal or other subject. Therapeutic effective dose will vary depending on the anti-alpha synuclein antibody or its antigen-binding fragment, the disease and its severity, and the age, weight, etc., of the subject to be treated.

Throughout this specification, the term "isolated" means that the antibody, antigen-binding fragment, or polynucleotide may exist in a physical environment different from the environment in which it may occur in nature. The present invention provides an antibody or antigen-binding fragment thereof that binds to α-synuclein, wherein the antibody comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Independently, 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 present invention provides an antibody or antigen-binding fragment thereof that binds to α-synuclein, wherein the antibody comprises:

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6.

α-synuclein (or α-syn; a-synuclein; a-syn or any other known synonym) is the general name for this protein and includes, but is not limited to, alternative splicing variants, mutants, and α-synucleins from other species (mice, monkeys, etc.). Unless otherwise stated, when it is desired or explicitly referred to as human α-synuclein, such α-synuclein is contained in the sequence given in SEQ ID NO:10 or Uniprot P37840.

As used herein, the term "antibody" generally refers to a complete (full) antibody, i.e., an element comprising two heavy chains and two light chains. The antibody may further comprise additional binding domains, such as the molecule DVD-Ig disclosed in WO 2007/024715, or the so-called (FabFv) ₂ Fc described in WO2011/030107. Therefore, the antibody as used herein includes bivalent, trivalent, or quadrivalent full-length antibodies.

Antigen-binding fragments of antibodies include single-chain antibodies (i.e., full-length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., _VH or _VL or _VHH ), scFv, bivalent, trivalent or tetravalent antibodies, bi-scFv, double-chain antibodies, trisomic antibodies, triple-chain antibodies, quadruple-chain antibodies, 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 generating and preparing 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 form was first disclosed in WO2009/040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010/035012. Other antibody fragments used in this invention include the Fab and Fab' fragments described in international patent applications WO2005/003169, WO2005/003170, and WO2005/003171. Multivalent antibodies may contain multiple specificities, such as being bispecific, or may be monospecific (see, for example, WO 92/22583 and WO05/113605). One such example of the latter is Tri-Fab (or TFM) described in WO92/22583.

Alternative antigen-binding fragments include Fabs linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, while another scFv or dsscFv increases half-life by binding to, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015/197772, which, by reference, is incorporated herein by reference in its entirety and particularly relevant to the discussion of antibody fragments.

A typical Fab' molecule comprises a heavy chain and a light chain pair, wherein the heavy chain contains a variable region VH, a constant structural domain CH1, and a native or modified hinge region, and the light chain contains a variable region VL and a constant structural domain CL. The dimer of Fab' according to this disclosure produces F(ab') ₂ , wherein dimerization can, for example, be achieved through the hinge.

The antibody or its antigen-binding fragment according to the present invention binds to the epitope of α-synuclein.

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

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

It binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130, and E131, which are associated with SEQ ID NO:10, wherein the epitopes optionally include A124 and G132.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Independently, 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 α-synuclein comprises:

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6,

It binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130, and E131, which are associated with SEQ ID NO:10, wherein the epitopes optionally include A124 and G132.

Within this invention, the term "epitope" is used interchangeably for both conformational and linear epitopes, wherein a conformational epitope consists of a discontinuous portion of the primary amino acid sequence of an antigen, while a linear epitope is formed by a sequence of continuous amino acids.

The epitope can be identified using any suitable epitope mapping method known in the art in conjunction with any of the antibodies provided by the present invention. Examples of such methods include screening peptides of varying lengths derived from full-length α-synuclein for binding to the antibody or a fragment thereof, and identifying the smallest fragment containing the sequence of the epitope recognized by the antibody that can specifically bind to the antibody. α-synuclein peptides can be produced synthetically or by proteolytic digestion of α-synuclein protein. The peptide binding to the antibody can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by the antibody of the present invention. Typically, when epitope determination is performed by X-ray crystallography, the amino acid residues of the antigen within the CDR are considered as the amino acid residue portion of the epitope. Once identified, the epitope can be used to prepare fragments of the antibody of the present invention and, if desired, as an immunogen to obtain additional antibodies binding to the same epitope.

In one embodiment, epitopes of the antibody or its antigen-binding fragment are determined by X-ray crystallography, wherein an α-synuclein peptide comprising residues 123 to 132 relating to SEQ ID NO: 10 is used.

Preferably, the antibody or its antigen-binding fragment according to the invention prevents α-synuclein aggregation induced by α-synuclein fibrils.

In this particular context, the term “prevent” (and its grammatical variations) is used interchangeably with the term “inhibit” and refers to the effect of the antibody according to the invention relating to α-synuclein aggregation induced by α-synuclein fibrils. This effect can be preventative, in completely or partially preventing aggregation; or in completely or partially reducing, i.e., blocking further progression of aggregation that has already begun, or completely or partially reducing the occurrence of further aggregation; or in completely or partially reversing aggregation that has already occurred.

Without being bound by theory, it is assumed that the antibody or its antigen-binding fragment according to the present invention binds to:

i) α-synuclein in monomeric form, and prevents α-synuclein from forming oligomers and aggregates; and/or

ii) α-synuclein in the form of oligomers and fibrils, and prevents α-synuclein from spreading between neurons; and/or

iii) α-synuclein in the form of oligomers and/or fibrils, and prevents α-synuclein aggregation induced by α-synuclein fibrils, preferably endogenous α-synuclein aggregation.

In this article, the terms “fibril,” “fibril form,” or “in fibrils” used with respect to α-synuclein are intended to refer to the non-monomeric forms of α-synuclein, including α-synuclein oligomers, which can constitute a dispersed variety within and between brain structures.

Therefore, in one embodiment, the antibody or its antigen-binding fragment binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

It prevents α-synuclein aggregation induced by α-synuclein fibrils. Preferably, the antibody or its antigen-binding fragment binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as described in SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

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

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6,

Furthermore, it prevents α-synuclein aggregation induced by α-synuclein fibrils. Preferably, the antibody or its antigen-binding fragment binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as described in SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

In one embodiment, the antibody or antigen-binding fragment according to the invention is capable of binding to α-synuclein as a monomer and in fibrils. In one embodiment, the antibody or antigen-binding fragment has a stronger binding affinity for α-synuclein in fibrils than for α-synuclein as a monomer. This is characterized by a dissociation constant (K_{_D} ) for monomeric α-synuclein that is at least 10 times greater than the dissociation constant for α-synuclein in fibrils.

In one embodiment, the antibody or antigen-binding fragment according to the invention has a dissociation constant (K_{_D} ) of less than 15 nM for monomeric α-synuclein. In another embodiment, the antibody or antigen-binding fragment according to the invention has a dissociation constant (K_{_D} ) of less than 10 nM for α-synuclein in fibrils. In a preferred embodiment, the antibody or antigen-binding fragment according to the invention has a dissociation constant (K_{_D} ) of less than 300 pM for α-synuclein in fibrils.

As used herein, the term "K_{_D}" refers to the dissociation constant obtained from the ratio of K _{_d} to _Ka (i.e., K _{_d} / _Ka ) and expressed as a molar concentration (M). K_{_d} and _Ka refer to the dissociation rate and association rate of a specific antigen-antibody (or its antigen-binding fragment) interaction, respectively. The K_{_D} value of an antibody can be determined using methods well-established in the art. One method for determining the K_{_D} of an antibody is by using surface plasmon resonance, such as a system described, for example, in the embodiments herein, in which isolated natural or recombinant α-synuclein, its suitable fusion protein/peptide, or its fibrils are used. In one embodiment, affinity is measured using recombinant human α-synuclein, as described in the embodiments herein. For surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase moving along a flow cell. If a ligand binds to the immobilized target, the local refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to produce apparent rate constants for the associated and dissociated phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)).

In one embodiment, the antibody or antigen-binding fragment according to the invention has a higher binding affinity (i.e., a smaller _KD ) for α-synuclein in fibrils compared to α-synuclein as a monomer. The term "affinity" refers to the strength of the interaction between the antibody or antigen-binding fragment and α-synuclein.

In one embodiment, the antibody or antigen-binding fragment of the present invention has an _IC50 of less than 10 nM for blocking α-synuclein aggregation induced by α-synuclein in fibrils, preferably, the antibody or antigen-binding fragment of the present invention has an _IC50 of less than 5 nM for blocking α-synuclein aggregation induced by α-synuclein in fibrils. Examples of cell-based aggregation assays are disclosed in the embodiments.

As used herein, the term "IC ₅₀ " refers to the half-maximal inhibitory concentration, which is a measure of the efficacy of a substance, such as an antibody, in inhibiting a particular biological or biochemical function (in this invention, aggregation induced by α-synuclein (preferably α-synuclein in protofibrils)). _{IC 50} is a quantitative measure indicating how much of a particular substance is needed to inhibit a given biological process by half.

In one embodiment, in an in vitro assay, the antibody or antigen-binding fragment of the present invention has an _IC50 of less than 10 nM for blocking α-synuclein aggregation induced by α-synuclein in fibrils, preferably, the antibody or antigen-binding fragment of the present invention has an _IC50 of less than 5 nM for blocking α-synuclein aggregation induced by α-synuclein in fibrils.

The antibody or its antigen-binding fragment according to the present invention does not bind to β-synuclein and/or γ-synuclein, and is specific to α-synuclein.

As used herein, “specific” is intended to mean an antibody that recognizes only the antigen it is specific to, or an antibody that has a significantly higher binding affinity for the antigen it is specific to (e.g., α-synuclein) than for the antigens it is not specific to (γ- and β-synuclein), for example, a binding affinity of at least 5, 6, 7, 8, 9, or 10 times higher.

The antibodies according to the invention can be obtained using any suitable method known in the art. α-synuclein polypeptides/proteins, including fusion proteins, expressed (recombinantly or naturally) by cells can be used to generate antibodies that specifically recognize α-synuclein. The polypeptide can be a "mature" polypeptide or a biologically active fragment or derivative thereof.

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

Peptides used for immunizing a host can be prepared from genetically engineered host cells containing an expression system using processes well known in the art, or they can be recovered from natural biological sources. In this application, the term "peptide" includes peptide, polypeptide, and protein. These are used interchangeably unless otherwise stated. α-synuclein polypeptides or fragments thereof may, in some cases, be part of a larger protein (e.g., a fusion protein, such as one fused with an affinity tag).

Antibodies against the α-synuclein peptide can be obtained, wherein immunization of animals is essential, which is performed by administering the peptide to animals, preferably non-human animals, using well-known and conventional experimental protocols (see, for example, Handbook of Experimental Immunology, D.M. Weir (ed.), Volume 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, cattle, 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 hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), three-source hybridoma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R Liss, Inc., 1985).

The antibodies used in this invention can also be produced by using a monolymphocyte antibody method, which involves cloning and expressing immunoglobulin variable region cDNA produced from a single lymphocyte selected for the production of specific antibodies, by means of, 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 assays that measure binding to α-synuclein and/or assays that measure inhibition of α-synuclein fibrillation in the presence of the antibody or its fragments.

The antibody or antigen-binding fragment according to the invention comprises complementarity-determining regions (CDRs), three from the heavy chain and three from the light chain. Typically, the CDRs are in the framework and together form the variable region. By convention, the CDRs in the heavy chain variable region of the antibody or antigen-binding fragment are designated CDR-H1, CDR-H2, and CDR-H3, and the CDRs in the light chain variable region are designated CDR-L1, CDR-L2, and CDR-L3. They are sequentially numbered in the direction from the N-terminus to the C-terminus of each chain.

CDRs are conventionally numbered according to the system conceived by Kabat et al., described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter, "Kabat et al. (ibid.)"). This numbering system is used in this specification unless otherwise specified.

Kabat residue naming does not always directly correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence can contain fewer or more amino acids than would be found in a strict Kabat number, corresponding to shortening or insertion of structural components (whether framework regions or complementarity-determining regions (CDRs)) in a basic variable domain structure. For a given antibody, the correct Kabat number of a residue can be determined by comparing homologous residues in the antibody sequence to a “standard” Kabat-numbered sequence.

According to the Kabat numbering system, the CDR of the variable domain in the heavy chain is located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, A.M.J.Mol.Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise specified, “CDR-H1” as used herein is intended to refer to residues 26 to 35, as described by the combination defined by the Kabat numbering system and Chothia’s topological loop.

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

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

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

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

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

In one embodiment, the antibody or antigen-binding fragment according to the invention may be included in the framework region of the animal in which the antibody was generated. For example, if the antibody is generated in a rabbit, it will include the CDR defined above and the framework region of a rabbit antibody, such as an antibody containing a light chain variable region according to SEQ ID NO:11 (whose nucleotide sequence is shown in SEQ ID NO:12) and a heavy chain variable region according to SEQ ID NO:13 (whose nucleotide sequence is shown in SEQ ID NO:14).

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

Chimeric antibodies are typically produced using recombinant DNA methods. DNA can be modified by replacing the corresponding non-human (e.g., mouse) H and L constant regions with coding sequences for the human L and H chains (Morrison; PNAS 81, 6851 (1984)).

Human antibodies contain variable regions or full-length heavy or light chains that are either "products" or "derived from" a specific germline sequence if the variable regions or full-length chains of the antibody are obtained from a system using a human germline immunoglobulin gene. Such systems include immunizing transgenic mice carrying a human immunoglobulin gene with a target antigen, or screening a phage library of human immunoglobulin genes displayed with a target antigen. Human antibodies or fragments thereof that are either "products" 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 sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is sequence-closest (i.e., has the greatest % identity) to the sequence of the human antibody. Human antibodies that are either "products" or "derived from" a specific human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, due to, for example, naturally occurring somatic mutations or the deliberate introduction of site-directed mutations. However, typically, the selected human antibody is at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared with germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In some cases, the human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99%, identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, human antibodies derived from a specific human germline sequence will exhibit a difference of no more than 10 amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, the human antibody may exhibit a difference of no more than 5, or even no more than 4, 3, 2, or 1 amino acid from the amino acid sequence encoded by the germline immunoglobulin gene.

Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be prepared via hybridoma methods, using human myeloma or mouse-human heterologous myeloma cell lines (Kozbor, J Immunol, (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both utilizing human variable region libraries (Winter G, (1994) Annu Rev Immunol 12:433-455; Green LL, (1999) J Immunol Methods 231:11-23).

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof according to the present disclosure is humanized.

Therefore, the antibody or its antigen-binding fragment binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

The antibody or its antigen-binding fragment thereof is humanized. Preferably, the humanized antibody or its antigen-binding fragment prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably, binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Independently, 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 its antigen-binding fragment binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

It also prevents α-synuclein aggregation induced by α-synuclein profilosomes and binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130 and E131 as described in SEQ ID NO:10, wherein 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 its antigen-binding fragment binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6,

It also prevents α-synuclein aggregation induced by α-synuclein fibrils and binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130 and E131, which are related to SEQ ID NO:10.

As used herein, the term “humanized” antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment wherein the heavy chain and/or light chain comprises one or more CDRs from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) transplanted into the variable region framework of the heavy chain and/or light chain of a recipient antibody (e.g., 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 of the specific residues from any of the CDRs described above are transferred to the human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specific residues from one or more of the CDRs described above are transferred to the human antibody framework. In another embodiment, only the specific residues from each of the CDRs described above are transferred to the human antibody framework.

When transplanting a CDR, any suitable recipient variable region scaffold sequence can be used, taking into account the class/type of the donor antibody from which the CDR is derived, including mouse, primate, and human scaffold regions.

Suitably, the humanized antibody according to the invention has a variable domain comprising a human recipient framework region and one or more CDRs specifically provided herein. Thus, in one embodiment, a blocking humanized antibody for binding α-synuclein, preferably human α-synuclein, is provided, wherein the variable domain comprises a human recipient framework region and a non-human donor CDR.

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

In the humanized antibody or antigen-binding fragment thereof according to the present invention, the recipient heavy chain and light chain do not necessarily need to be derived from the same antibody, and may, if desired, comprise a complex chain having a framework region derived from different chains.

The suitable framework region of the light chain of the humanized antibody or its antigen-binding fragment according to the invention is derived from human lineage IGKV1-16 JK4, which has SEQ ID NO:39 and its nucleotide sequence is shown in SEQ ID NO:40.

The suitable framework region of the heavy chain of the humanized antibody or its antigen-binding fragment according to the invention is derived from human lineage IGHV3-23 JH4, which has the sequence shown in SEQ ID NO:41 and its nucleotide sequence shown in SEQ ID NO:42.

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

- The sequences 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, wherein the light chain framework region is derived from hominid 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, wherein the heavy chain framework region is derived from human germline IGHV3-23 JH4.

In the humanized antibody or antigen-binding fragment according to the invention, the framework region may not have the exact same sequence as that of the recipient antibody. For example, unusual residues may be replaced with residues more frequently occurring for that type or class of recipient chain. Alternatively, selected residues in the recipient framework region may be altered so that they correspond to residues found at the same positions in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to restore affinity to the donor antibody. Experimental protocols for selecting residues in the recipient framework region that may require alteration are described in WO91/09967.

Therefore, in one embodiment, residues 1, 2, 3, 4, 5, 6, 7 or 8 in the framework are replaced by alternative amino acid residues.

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

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

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

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

Preferably, in the humanized heavy chain variable region according to the 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. Furthermore, preferably, in the humanized heavy chain variable region of the humanized antibody or its antigen-binding fragment according to the invention, residue 24 is valine, residue 47 is tyrosine, residue 48 isoleucine, residue 49 is glycine, residue 78 is valine, and residue 97 is arginine.

In a preferred embodiment of the invention, the antibody or its antigen-binding fragment binds to α-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 its antigen-binding fragment comprises:

- Contains the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:23; or

- A light chain variable region containing SEQ ID NO:15 and a heavy chain variable region containing SEQ ID NO:27 or 35; or

- Contains a light chain variable region of SEQ ID NO:19 and a heavy chain variable region of 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 present invention provides an antibody or antigen-binding fragment thereof comprising a sequence that is 80% similar to or identical to the sequences disclosed herein, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a portion or all of a related sequence (e.g., a variable domain sequence, a CDR sequence, or a variable domain sequence excluding a CDR). In one embodiment, the related sequence is SEQ ID NO:15. In one embodiment, the related sequence is SEQ ID NO:23 or SEQ ID NO:31.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human α-synuclein, comprising a light chain, wherein a variable domain of the light chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:15 or SEQ ID NO:19, and/or the variable domain of the heavy chain comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given 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 α-synuclein, wherein the antibody or antigen-binding fragment thereof has a light chain variable domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to the sequence given in SEQ ID NO:15, but wherein the antibody or antigen-binding fragment thereof has 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.

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to human α-synuclein, wherein the antibody or antigen-binding fragment thereof has a heavy chain variable domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar to the sequence given in SEQ ID NO:31, but wherein the antibody or antigen-binding fragment thereof has 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.

As used herein, “identity” means that at any particular position in the aligned sequences, the amino acid residues are identical between the sequences. As used herein, “similarity” means that at any particular position in the aligned sequences, the amino acid residues are of a similar type between the sequences. For example, leucine can be substituted with isoleucine or valine. Other amino acids that can frequently be substituted with another amino acid include, but are not limited to:

- Phenylalanine, tyrosine, or tryptophan (amino acids with aromatic side chains);

- Lysine, arginine, and histidine (amino acids with basic side chains);

- Aspartic acid and glutamic acid (amino acids with acidic side chains);

- Asparagine and glutamine (amino acids with amide side chains); and

- Cysteine and methionine (amino acids with sulfur-containing side chains).

The degree of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991; BLAST data available from NCBI). ^TM software (Altschul, SF et al., 1990, J.Mol.Biol. 215: 403-410; Gish, W. & States, DJ 1993, Nature Genet. 3: 266-272; Madden, TL et al., 1996, Meth.Enzymol. 266: 131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25: 3389-3402; Zhang, J. & Madden, TL 1997, Genome Res. 7: 649-656).

In one embodiment, the antigen-binding fragment according to the invention may be, but is not limited to: Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single-domain antibody (e.g., _VH or _VL or _VHH ), scFv, dsscFv, bivalent, trivalent or tetravalent antibody, bi-scFv, double-chain antibody, triple-chain antibody, quadruple-chain antibody, 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 generating and preparing 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 used in this invention include the Fab and Fab' fragments described in WO2005/003169, WO2005/003170, and WO2005/003171. Multivalent antibodies may contain multiple specificities, such as being bispecific, or may be monospecific (see, for example, WO 92/22853, WO05/113605, WO2009/040562, and WO2010/035012).

Alternative antigen-binding fragments include Fabs linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, while another scFv or dsscFv increases half-life by binding to, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015/197772, which, by reference, is incorporated herein by reference in its entirety and particularly relevant to the discussion of antibody fragments.

In another embodiment, the antibody or antigen-binding fragment according to the invention is part of a fusion protein that binds to α-synuclein, said fusion protein comprising, for example, the fused antigen-binding fragment of the invention, as a Fab or Fab' fragment, and one or two single-domain antibodies (dAbs) directly or indirectly linked thereto, such as those described in WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492 and WO2011/086091, all of which are incorporated herein by reference. In one embodiment, said fusion protein comprises two domain antibodies, for example, as a pair of variable heavy chain (VH) and variable light chain (VL) linked optionally by disulfide bonds.

In one embodiment, the Fab or Fab' element of the fusion protein has the same or similar specificity as the single-domain antibody. In another embodiment, the Fab or Fab' has a different specificity than the single-domain antibody, i.e., the fusion protein is multivalent. In one embodiment, the multivalent fusion protein according to the invention has an albumin-binding site, for example, a VH/VL pair provides an albumin-binding site thereon.

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

In one embodiment, the antibody is a full-length antibody, preferably selected from IgG1 and IgG4 or IgG4P.

Therefore, the present invention provides a full-length humanized antibody that binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

The humanized antibody prevents α-synuclein aggregation induced by α-synuclein fibrils and preferably binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130, and E131 as described in SEQ ID NO: 10, wherein the epitopes optionally include A124 and G132, and wherein the antibody is an IgG4P isoform.

In SEQ ID NO:44, Xaa is asparagine (Asn; N) or arginine (Arg; R). Independently, 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 a preferred embodiment, the full-length humanized antibody binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46,

Furthermore, it prevents α-synuclein aggregation induced by α-synuclein profilosomes and preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as described in SEQ ID NO:10, wherein said epitopes optionally comprise A124 and G132, and wherein said antibody is an IgG4P isoform, wherein in SEQ ID NO:44, Xaa is aspartic acid (Asn; N), in SEQ ID NO:45, Xaa is serine (Ser; S), and in SEQ ID NO:46, Xaa is aspartic acid (Asn; N).

In a most preferred embodiment, the full-length humanized antibody binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6,

It also prevents α-synuclein aggregation induced by α-synuclein fibrils and preferably binds to α-synuclein at epitopes containing residues E123, Y125, E126, M127, P128, S129, E130 and E131 as relating to SEQ ID NO:10, wherein the epitopes optionally include A124 and G132.

Those skilled in the art will also understand that antibodies can undergo a wide variety of post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody and the culture conditions. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and aspartic acid deamidation. A common modification is the loss of a carboxyl-terminal basic residue (e.g., lysine or arginine), attributed to the action of carboxypeptidase (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Therefore, the C-terminal lysine of the antibody heavy chain may be absent.

In one implementation, during post-translational modification, the C-terminal amino acid is cleaved from the antibody.

In one implementation, during post-translational modification, the N-terminal amino acid is cleaved from the antibody.

In one embodiment, the antibody or its antigen-binding fragment comprises a light chain variable region according to 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 according to 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 according to SEQ ID NO:17 and a heavy chain according to SEQ ID NO:25 or SEQ ID NO:33. In yet another embodiment, the antigen-binding fragment is Fab', comprising a light chain variable region according to 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 its antigen-binding fragment comprises a light chain variable region according to 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 according to 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 according to SEQ ID NO:17 and a heavy chain according to SEQ ID NO:29 or SEQ ID NO:37. In yet another embodiment, the antigen-binding fragment is Fab', comprising a light chain variable region according to 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 its antigen-binding fragment comprises a light chain variable region according to 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 according to 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 according to SEQ ID NO:21 and a heavy chain according to SEQ ID NO:29 or SEQ ID NO:37. In yet another embodiment, the antigen-binding fragment is Fab', comprising a light chain variable region according to 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 its antigen-binding fragment comprises a light chain variable region according to 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 according to 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 according to SEQ ID NO:21 and a heavy chain according to SEQ ID NO:25 or SEQ ID NO:33. In yet another embodiment, the antigen-binding fragment is Fab', comprising a light chain variable region according to 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 α-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 α-synuclein aggregation induced by α-synuclein fibrils, and more preferably, the antibody binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO:10, wherein the epitopes optionally comprise A124 and G132.

In another preferred embodiment, the antibody binds to α-synuclein and is a full-length IgG4 antibody comprising: a light chain comprising SEQ ID NO:17 and a heavy chain comprising SEQ ID NO:33. More preferably, the antibody prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably, the antibody binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 related to SEQ ID NO:10, wherein the epitopes optionally comprise A124 and G132.

Furthermore, the present invention also provides an antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to the present invention for binding to α-synuclein.

Therefore, the present invention provides an antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of the present invention for binding α-synuclein by cross-blocking the antibody or antigen-binding fragment of the present invention or being cross-blocked by the antibody or antigen-binding fragment of the present invention; and particularly, such an antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:27 or SEQ ID NO:35, and a light chain variable region comprising SEQ ID NO:15 or SEQ ID NO:19.

In another embodiment, the antibody or its antigen-binding fragment competes with the antibody or its antigen-binding fragment according to the invention for binding α-synuclein at the same epitope, and in particular, competes with the antibody or its antigen-binding fragment having 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 for binding α-synuclein at an epitope comprising at least residues M127, P128, S129, E130 and E131 (preferably residues E123, Y125, E126, M127, P128, S129, E130 and E131) as relating to SEQ ID NO:10.

In one embodiment, such an antibody or antigen-binding fragment thereof competes with an antibody or fragment thereof according to the 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.

Competitive antibodies can be identified using any suitable method in the art, such as by using a competitive ELISA or BIAcore assay, wherein the binding of a cross-blocking antibody to human α-synuclein prevents the binding of the antibody of the present invention, and vice versa. Such competitive assays can use isolated natural or recombinant α-synuclein or suitable fusion proteins/peptides. In one example, competition is measured using recombinant human α-synuclein (SEQ ID NO: 10). In one example, according to the embodiments herein, competition is measured using recombinant human α-synuclein tagged at the N-terminus or C-terminus (e.g., a 6xHis-tagged fusion with a TEV recognition site). In another example, competition is measured using recombinant human α-synuclein fibrils.

In one embodiment, the competitive antibody is wholly human or humanized. In one embodiment, the competitive antibody has an affinity for human α-synuclein of 100 pM or less, preferably 50 pM or less.

Biomolecules, such as antibodies or fragments, contain acidic and/or basic functional groups, thereby imparting a positive or negative net charge to the molecule. The total amount of "observed" charge will depend on the absolute amino acid sequence of the entity, 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 solvent surface can reach a surface without carrying a net charge. In one example, the anti-α-synuclein antibody or its antigen-binding fragment according to the invention can be modified to have a suitable isoelectric point. This can result in antibodies and/or fragments with more robust properties, particularly suitable solubility and/or stability properties and/or improved purification characteristics.

Therefore, in one aspect, the present invention provides a humanized antibody or antigen-binding fragment thereof that binds to an α-synuclein and is modified to have an isoelectric point different from that of the originally identified antibody. For example, the antibody can be modified by replacing amino acid residues, for example, replacing acidic amino acid residues with one or more basic amino acid residues. Alternatively, basic amino acid residues can be introduced or acidic amino acid residues can be removed. Alternatively, if the molecule has an unacceptably high pI value, then acidic residues can be introduced to lower the pI, if necessary. Importantly, when manipulating the pI, care must be taken to maintain the desired activity of the antibody or fragment. Thus, in one embodiment, the modified antibody or antigen-binding fragment thereof has the same or substantially the same activity as the "unmodified" antibody or fragment.

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

It will be appreciated that the affinity of the antibodies provided by the present invention can be modified by using any suitable method known in the art. Therefore, the present invention also relates to variants of the antibody molecules of the present invention having improved affinity for α-synuclein, particularly human α-synuclein. Such variants can be obtained through a number of affinity maturation protocols, including CDR mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand truncation (Marks et al., Bio/Technology, 10, 779-783, 1992), using a mutant strain of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA mixing (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (ibid.) discussed these affinity maturation methods.

Within this invention, affinity maturation is performed via IOTA (WO2014198951).

If desired, antibodies or antigen-binding fragments thereof according to the invention can be conjugated to one or more effector molecules. It will be appreciated that the effector molecules may comprise a single effector molecule or two or more such molecules linked together to form a single portion that can be attached to an antibody or antigen-binding fragment of the invention. In cases where it is desired to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody fragment is linked directly or by a conjugating agent to the effector molecule. Techniques 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., editors, 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, the ligation can be achieved using recombinant DNA procedures, such as those described in WO 86/01533 and EP0392745.

As used herein, the term "effect molecule" includes, for example, antitumor agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, especially radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

Examples of effector molecules can include cytotoxic agents, which include any agent that is harmful to cells (e.g., kills cells). Examples include compressoritine, dolalastatin, epokine, astrosaponins, maytansine compounds, spongiformin, lisodium, levofloxacin, bacitracin, hammetrine, paclitaxel, pinocembrin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxetine dione, mitoxantrone, styracin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues.

Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminoimide), alkylating agents (e.g., nitrogen mustard, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II)(DDP)(cisplatin)), anthracyclines (e.g., daunorubicin (formerly doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly actinomycin), bleomycin, photomycin, aztracin (AMC), calcitrinin or pyromycin), and antimitotic agents (e.g., vinblastine and vinblastine).

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

Other effector molecules include proteins, peptides, and enzymes. Target enzymes include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, and transferases. Target proteins, polypeptides, and peptides include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator, thrombotic agents or anti-angiogenic agents such as thrombosin or endostatin, or biological response regulators such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors and immunoglobulins.

Other effector molecules may include, for example, detectable substances useful in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (for use in positron emission tomography) and non-radioactive paramagnetic metal ions. For information on metal ions that can be conjugated to antibodies used as diagnostic agents, see U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptoavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; and suitable radionuclides include ^125I , ^131I , ^111In , and ^99Tc .

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

When the effector molecule is a polymer, it can typically be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyolefin, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.

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

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

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

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

As used herein, “derivative” is intended to include reactive derivatives, such as thiohydroxy-selective reactive groups like maleimide. These reactive groups can be attached to the polymer directly or via linker segments. It will be appreciated that residues of such groups may, in some cases, serve as linker groups between the antibody fragment and the polymer, becoming part of the product.

The size of the polymer can be varied as desired, but will typically be in the range of 500 Da to 50,000 Da, for example 5,000 to 40,000 Da, or even 20,000 to 40,000 Da in terms of average molecular weight. In particular, the polymer size can be selected based on the intended use of the product, such as its ability to target certain tissues, like tumors, or its ability to prolong the circulating half-life (see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545 for a review). Thus, for example, in cases where the product is intended to leave the circulation and penetrate tissue (e.g., for use in the treatment of tumors), it may be advantageous to use a low molecular weight polymer (e.g., with a molecular weight of approximately 5,000 Da). For applications where the product remains in the circulation, it may be advantageous to use a higher molecular weight polymer (e.g., with a molecular weight in the range of 20,000 Da to 40,000 Da).

Suitable polymers include polyalkylene polymers, such as polyethylene glycol, or especially methoxy polyethylene glycol or its derivatives, which have a molecular weight in the range of about 15,000 Da to about 40,000 Da.

In one example, an antibody or antigen-binding fragment according to the invention is attached to a polyethylene glycol (PEG) moiety. In a particular embodiment, the antigen-binding fragment and PEG molecule according to the invention can be attached via any available amino acid side chain or terminal amino acid functional group (e.g., any free amino, imino, thiohydroxy, hydroxy, or carboxyl group) located in the antibody fragment. Such amino acids may be naturally present in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see, for example, US 5,219,996; US 5,667,425; WO98/25971; WO2008/038024). In one example, the antibody molecule of the invention is a modified Fab fragment, wherein the modification involves adding one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

Suitablely, PEG molecules are covalently linked via a thiohydroxyl group located on at least one cysteine residue in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to a sulfur atom on a cysteine residue in the fragment. This covalent link will typically be a disulfide bond, or particularly a sulfur-carbon bond. When using a thiohydroxyl group as the attachment site for a suitably activated effector molecule, thiohydroxyl-selective derivatives such as maleimide and cysteine derivatives may be used. In the preparation of the polymer-modified antibody fragment described above, an activated polymer may be used as the starting material. The activated polymer may be any polymer containing a thiohydroxyl reactive group (e.g., α-halocarboxylic acid or ester, such as iodoacetamide, diimide, such as maleimide, vinyl sulfone, or disulfide). Such starting materials may be commercially available (e.g., from Nektar (formerly Shearwater Polymers Inc.), Huntsville, AL, USA), or may be prepared from commercially available starting materials using conventional chemical procedures. Special PEG molecules include 20K methoxy-PEG-amine (available from Nektar (formerly Shearwater); Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar (formerly Shearwater)).

In one embodiment, the antibody is a modified Fab fragment, Fab' fragment, or diFab that is PEGylated, i.e., has PEG (polyethylene glycol) covalently attached thereto, for example according to the methods disclosed in EP 0948544 or EP1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed.), Plenum Press, New York; "Poly(ethyleneglycol) Chemistry and Biological A... "Applications", J. Milton Harris and S. Zalipsky (eds.), American Chemical Society, Washington DC; and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54: 531-545. In one example, PEG is attached to a cysteine residue in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently linked to a single thiohydroxy group in the modified hinge region. Lysine residues can be covalently linked to this maleimide group, and to each of the amine groups on the lysine residue, a methoxy polyethylene glycol polymer with a molecular weight of approximately 20,000 Da can be attached. Therefore, the total molecular weight of PEG attached to the Fab fragment can be approximately 40,000 Da.

The particular PEG molecule includes 2-[3-(N-maleimide)propionamido]ethylamide of lysine modified with N,N’-bis(methoxy polyethylene glycol, MW 20,000), also known as PEG2MAL40K (available from Nektar (formerly Shearwater)).

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

That is, each PEG is approximately 20,000 Da.

Therefore, in one embodiment, the PEG is 2,3-di(methylpolyoxyethylene-oxy)-1-{[3-(6-maleimino-1-oxohexyl)amino]propoxy}hexane (2-arm branched PEG, _-CH2 )3NHCO( _CH2 ) ₅ -MAL, Mw 40,000, referred to as SUNBRIGHT GL2-400MA3.

The following are further alternative PEG-effect molecules:

This information is available from Dr. Reddy, NOF, and Jenkem.

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

In one embodiment, a PEGylated (e.g., PEG as described herein) antibody is provided, wherein attachment is performed by a cysteine amino acid residue at or about amino acid 226 in the chain, such as amino acid 226 of the heavy chain (as numbered sequentially), such as amino acid 223 of SEQ ID NO:33.

In one embodiment, this disclosure provides a Fab’PEG molecule comprising one or more PEG polymers (e.g., 1 or 2 polymers, such as a 40 kDa polymer).

Fab'-PEG molecules according to this disclosure may be particularly advantageous because they have a half-life independent of the Fc fragment. In one embodiment, a Fab' is provided that is conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule. In one embodiment, an scFv is provided that is conjugated to a polymer such as a PEG molecule, a starch molecule, or an albumin molecule. In one embodiment, a Fab or Fab' according to this disclosure 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 proteins are described in US 8,017,739 (which is incorporated herein by reference).

The present invention also provides isolated polynucleotides encoding antibodies or antigen-binding fragments thereof according to the invention. The isolated polynucleotides according to the invention may comprise synthetic DNA (which is produced, for example, by chemical treatment), cDNA, genomic DNA, or any combination thereof.

Standard molecular biology techniques can be used to prepare DNA sequences encoding the antibodies or antigen-binding fragments of the present invention. The desired DNA sequence can be synthesized completely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used, depending on the application.

In one embodiment, the isolated polynucleotide encoding according to the present invention is:

a. Light chain variable region, wherein the polynucleotide:

i. 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. Basically composed of SEQ ID NO:16 or SEQ ID NO:20;

b. Heavy chain variable region, wherein the polynucleotide:

i. With SEQ ID NO:24 or SEQ ID NO:28 or SEQ ID NO:32

Or at least 90% identical to SEQ ID NO:36; or

ii. Contains SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32

Or SEQ ID NO:36; or

iii. Basically composed of SEQ ID NO:24 or SEQ ID NO:28 or SEQ ID NO:32 or SEQ ID NO:36;

c. The light chain, wherein the polynucleotide is:

i. 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. Basically composed of SEQ ID NO:18 or SEQ ID NO:22;

d. Heavy chain, wherein the polynucleotide is:

i. With SEQ ID NO:26 or SEQ ID NO:30 or SEQ ID NO:34

Or at least 90% identical to SEQ ID NO:38; or

ii. Contains SEQ ID NO:26, SEQ ID NO:30, or SEQ ID NO:34

Or SEQ ID NO:38; or

iii. Basically composed of SEQ ID NO:26 or SEQ ID NO:30 or SEQ ID NO:34 or SEQ ID NO:38;

e. Light chain variable region, wherein the polynucleotide:

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

ii. Contains SEQ ID NO:12; or

iii. It is basically composed of SEQ ID NO:12;

f. Heavy chain variable region, wherein the polynucleotide:

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

ii. Contains SEQ ID NO:14; or

iii. It is basically composed of SEQ ID NO:14.

In one embodiment, the present invention provides isolated polynucleotides encoding a heavy chain of the antibody Fab' fragment or IgG1 or IgG4 antibody of the present invention, said heavy chain comprising the sequence given in SEQ ID NO: 24, 28, 32 or 36. Isolated polynucleotides are also provided encoding a light chain of the antibody Fab' fragment or IgG1 or IgG4 antibody of the present invention, said light chain comprising the sequence given in SEQ ID NO: 16 or 20.

In another embodiment, the present invention provides isolated polynucleotides encoding the heavy and light chains of the IgG4(P) antibody of the present invention, wherein the polynucleotide encoding the heavy chain is contained in the sequence given in SEQ ID NO: 26, 30, 34 or 38 and the polynucleotide encoding the light chain is contained in the sequence given in SEQ ID NO: 18 or 22.

The present invention also provides cloning or expression vectors comprising one or more polynucleotides described herein. In one example, the cloning or expression vector according to the invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

The general methods, transfection methods, and culture methods by which the vector can be constructed are well known to those skilled in the art. In this regard, see “Current Protocols in Molecular Biology”, 1999, F.M. Ausubel (ed.), Wiley Interscience, New York; and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Host cells are also provided, which contain one or more isolated polynucleotides according to the invention, or one or more clones or expression vectors containing one or more isolated polynucleotide sequences encoding antibodies of the invention. Any suitable host cell/vector system can be used for the expression of polynucleotide sequences encoding antibodies of the invention or their antigen-binding fragments. Bacterial (e.g., *Escherichia coli*) and other microbial systems can be used, or eukaryotic (e.g., mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells.

Suitable types of Chinese hamster ovaries (CHO cells) used in this invention may include CHO and CHO-K1 cells, including dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which can be used in conjunction with DHFR selective labeling, or CHOK1-SV cells, which can be used in conjunction with glutamine synthetase selective labeling. Other cell types useful in antibody expression include lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, and COS cells. Host cells can be stably transformed or transfected using the isolated polynucleotide sequences or expression vectors according to this invention.

In one embodiment, the host cell according to the invention is a CHO-DG44 cell stably transfected with an expression vector containing the isolated polynucleotide sequence of the invention (preferably, containing the isolated polynucleotide sequence according to SEQ ID NO:18 and 26 or SEQ ID NO:18 and 34 or SEQ ID NO:18 and 30 or SEQ ID NO:18 and 38).

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

The antibody or its antigen-binding fragment may contain only heavy-chain or light-chain polypeptides; in this case, only the heavy-chain or light-chain polypeptide coding sequence is needed to transfect the host cell. To generate an antibody or its antigen-binding fragment containing both heavy and light chains, the cell line can be transfected using 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, comprising sequences encoding both light and heavy-chain polypeptides.

Therefore, methods are provided for culturing host cells and expressing antibodies or fragments thereof, isolating the latter, and optionally purifying them to provide isolated antibodies or fragments. In one embodiment, the method further includes the step of conjugating an effector molecule with the isolated antibody or fragment, for example, with a PEG polymer (particularly as described herein).

Therefore, in one embodiment, a purified anti-α-synuclein antibody or a fragment thereof is provided, such as a humanized antibody or a fragment thereof, particularly an antibody or a fragment thereof according to the invention, which is in a substantially purified form and is particularly free of or substantially free of endotoxins and/or host cell proteins or DNA.

Generally, "virtually no endotoxin" means an antibody product with an endotoxin content of 1 EU/mg or less, such as 0.5 or 0.1 EU/mg products.

Typically, "substantially no host cell protein or DNA" means a host cell protein and/or DNA content of 400 μg/mg antibody product or less, such as 100 μg/mg or less, especially 20 μg/mg, depending on the case.

Since the antibodies of the present invention are useful in the treatment, diagnosis and/or prevention of pathological conditions such as alpha synucleinosis, the present invention also provides pharmaceutical or diagnostic compositions comprising the antibody or antigen-binding fragment thereof according to the present invention, and one or more pharmaceutically acceptable carriers, excipients or diluents in combination therewith.

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

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46.

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

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6.

In one embodiment, the antibody or antigen-binding fragment according to the invention is the sole active ingredient. In another embodiment, the antibody or antigen-binding fragment according to the invention is combined with one or more other active ingredients. Alternatively, the pharmaceutical composition comprises the antibody or antigen-binding fragment according to the invention as the sole active ingredient, and it can be administered to a patient individually (e.g., simultaneously, sequentially, or separately) in combination with other reagents, drugs, or hormones.

In another embodiment, the pharmaceutical composition comprises an antibody or an antigen-binding fragment thereof, which includes a light chain variable region of SEQ ID NO:15 or 19 and a heavy chain variable region of SEQ ID NO:23, 27, 31 or 35, such as SEQ ID NO:15 and SEQ ID NO:23 or SEQ ID NO:15 and SEQ ID NO:31.

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

Suitablely, the pharmaceutical composition according to the invention can be administered to a patient to determine the required therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent required to treat, improve, or prevent the targeted disease or condition, or to demonstrate a detectable therapeutic or preventative effect. For any antibody, the therapeutically effective amount can be initially estimated in a cell culture assay or in an animal model (typically rodents, rabbits, dogs, pigs, or primates). Animal models can also be used to determine suitable concentration ranges and routes of administration. Such information can then be used to determine the dosage and route of administration useful for human use.

The precise therapeutically effective dose for human subjects will depend on the severity of the disease state, the subject's overall health status, age, weight and sex, diet, timing and frequency of administration, drug combination, sensitivity to response, and tolerance/response to the therapy. This dose can be determined through routine laboratory testing and is within the judgment of the clinician. Typically, the therapeutically effective dose will be from 0.01 mg/kg to 500 mg/kg, for example from 0.1 mg/kg to 200 mg/kg, for example 100 mg/kg. Pharmaceutical compositions can be conveniently presented in unit dose form, which contains a predetermined amount of the active agent/dosage of the present invention.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally comprise liquids such as water, saline, glycerin, and ethanol. Additionally, excipients, such as wetting agents, emulsifiers, or pH buffers, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, and suspensions for patient ingestion.

Suitable forms for administration include those suitable for parenteral administration (e.g., by injection or infusion, such as by bolus or continuous infusion), intravenous, inhalable, or subcutaneous forms. When the product is intended for injection or infusion, it can be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier, and it can contain formulation agents such as suspending agents, preservatives, stabilizers, and/or dispersants. Alternatively, the antibody or its antigen-binding fragment according to the invention can be in a dry form, which is intended for reconstitution with a suitable sterile liquid prior to use. A solid form suitable for dissolving or suspending in a liquid carrier prior to injection can also be prepared.

Once formulated, the compositions of the present invention can be administered directly to a subject. Therefore, the use of antibodies or antigen-binding fragments thereof according to the present invention in the preparation of medicaments is provided herein.

The subjects to be treated may be animals. Preferably, the pharmaceutical composition according to the invention is suitable for administration to human subjects.

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

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46.

Preferably, the antibody or its antigen-binding fragment is humanized and prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

In a preferred embodiment, the antibody or antigen-binding fragment thereof for use in therapy, or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is an antibody or antigen-binding fragment thereof that binds to α-synuclein and comprises:

a. Light chain variable region, which includes:

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

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:5; and

vi. Contains CDR-H3 with SEQ ID NO:6.

Preferably, the antibody or its antigen-binding fragment is humanized and prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

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

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

a. Light chain variable region, which includes:

i. A CDR-L1 containing SEQ ID NO:44;

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

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

b. Heavy chain variable region, which includes:

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

v. Containing CDR-H2 with SEQ ID NO:45; and

vi. Contains CDR-H3 with SEQ ID NO:46.

Preferably, the antibody or its antigen-binding fragment is humanized and prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

In a preferred embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition comprising the antibody or its antigen-binding fragment, is used for the treatment of one or more α-synucleinosis disorders, wherein the antibody or its antigen-binding fragment binds to α-synuclein and comprises:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

Preferably, the antibody or its antigen-binding fragment is humanized and prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

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

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

Preferably, the antibody or its antigen-binding fragment is humanized and prevents α-synuclein aggregation induced by α-synuclein fibrils, and more preferably binds to α-synuclein at epitopes comprising residues E123, Y125, E126, M127, P128, S129, E130, and E131 as relating to SEQ ID NO: 10, wherein the epitopes optionally comprise A124 and G132.

Alternatively, the antibody or its antigen-binding fragment, or a pharmaceutical composition comprising the antibody or its antigen-binding fragment, is used in a therapeutic manner or for use in the treatment of one or more α-synucleinopathies, and is such an antibody or its antigen-binding fragment comprising:

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

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

c. Light chains containing SEQ ID NO:17 or SEQ ID NO:21 and heavy chains 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 invention, the method for treating one or more α-synucleinosis in a patient comprises administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to α-synuclein and comprises:

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

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

c. Light chains containing SEQ ID NO:17 or SEQ ID NO:21 and heavy chains containing SEQ ID NO:25 or SEQ ID NO:29 or SEQ ID NO:33 or SEQ ID NO:37.

The α-synucleinopathy of the present invention includes, but is not limited to: Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Leiblioid dementia (DLB), diffuse Leiblioid disease (DLBD), Leiblioid variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1). Preferably, the α-synucleinopathy is Parkinson's disease (PD).

In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition comprising the antibody or its antigen-binding fragment, is used in the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1) (preferably, Parkinson's disease (PD)), and is such an antibody or its antigen-binding fragment comprising:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

In another embodiment, the antibody or its antigen-binding fragment, or a pharmaceutical composition comprising the antibody or its antigen-binding fragment, is used in the treatment of Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1) (preferably, Parkinson's disease (PD)), and is such an antibody or its antigen-binding fragment comprising:

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

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

c. Light chains containing SEQ ID NO:17 or SEQ ID NO:21 and heavy chains 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 treating Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd dementia (DLB), diffuse Lloyd disease (DLBD), Lloyd variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and brain iron accumulation neurodegeneration type 1 (NBIA-1) (preferably, Parkinson's disease (PD)) in a patient, comprising administering to the patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

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

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

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

c. Light chains containing SEQ ID NO:17 or SEQ ID NO:21 and heavy chains containing SEQ ID NO:25 or SEQ ID NO:29 or SEQ ID NO:33 or SEQ ID NO:37.

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

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

The use of the anti-α-synuclein antibody or its antigen-binding fragment as a diagnostically active reagent or in diagnostic assays is also part of this invention, for example, for the diagnosis of α-synuclein diseases such as Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd's dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd's variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1).

Preferably, the diagnosis can be performed on biological samples. "Biological samples" includes a wide variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. 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 biopsy samples or tissue cultures or cells and their progeny derived therefrom. The definition also includes samples that have been manipulated in any way after acquisition, such as by treatment with reagents, solubilization, or enrichment for certain components (e.g., polynucleotides).

Preferably, the diagnostic test can be performed on a biological sample that does not come into contact with a human or animal body. Such diagnostic tests are also known as in vitro tests. In vitro diagnostic tests can rely on in vitro methods for detecting α-synuclein in biological samples already obtained from an individual, comprising the steps of: i) contacting the biological sample with an anti-α-synuclein antibody or its antigen-binding fragment as described herein; and ii) detecting the binding of the anti-α-synuclein antibody or its antigen-binding fragment to α-synuclein. One or more α-synucleinopathy disorders can be identified by comparing the detected α-synuclein level or the presence of a specific post-translational modification of α-synuclein with a suitable control. Therefore, such a detection method can be used to determine whether a subject has α-synucleinopathy or is at risk of developing α-synucleinopathy, including determining the stage (severity) of α-synucleinopathy.

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

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

Preferably, the present invention provides an antibody or antigen-binding fragment thereof for use in the diagnosis of α-synucleinosis, preferably in the diagnosis of Parkinson's disease, wherein the antibody or antigen-binding fragment thereof binds to α-synuclein and comprises:

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

b. Containing the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:31; or

c. The light chain containing SEQ ID NO:17 and the heavy chain containing SEQ ID NO:33.

The sequences included in this invention are shown in Table 1:

Table 1

The invention will now be further described by way of examples and with reference to the embodiments illustrated in the accompanying drawings.

Example

Example 1: Expression of human α-synuclein monomers and fibrils

A gene encoding human α-synuclein was synthesized and subcloned into the vector pMH 10His TEV (containing the CMV promoter) using standard molecular biology techniques to create a modified vector that produces α-synuclein with an N-terminal 10His-TEV tag. Following the manufacturer's protocol, the resulting vector was transfected into Expi293F cells using an Expi293™ Expression System (Invitrogen). The α-synuclein protein accumulated in the culture medium (from which it was recovered) was purified using an immobilized metal ion affinity chromatography HisTrap excel column (GE Healthcare). The column was washed with 25 mM TrisHCl, 300 mM NaCl, pH 8.0, and the protein was eluted using a stepwise gradient of 500 mM imidazole in the same buffer. The 10His tag was removed using the TEV protease. The sample was then concentrated and desalted, and the cleaved protein was applied back onto a HisTrap excel column, with the cleaved α-synuclein collected in the effluent. The α-synuclein was further purified by gel filtration on a HiLoad 26/600 Superdex 75 column (GE Healthcare), and endotoxins were removed via a Proteus NoEndo cassette (Generon). The purified α-synuclein was confirmed to be monomeric using SECMALS (Figure 1A).

Wild-type (untagged) human α-synuclein was also expressed in Expi293F cells. The protein was recovered from the culture medium via anion exchange using a HiTrap Q column (GE Healthcare). The column was washed with 20 mM TrisHCl pH 8.0 and eluted using a sodium chloride gradient up to 400 mM. The fraction was concentrated and desalted using a HiPrep 26/10 column (GE Healthcare) and eluted with 20 mM TrisHCl pH 8.0. The protein was further purified using a MonoQ10/100GL column, eluted with a sodium chloride gradient up to 400 mM in 20 mM TrisHCl pH 8.0, followed by gel filtration on a HiLoad 26/600 Superdex 75 column (GE Healthcare) and elution in PBS pH 7.4 (Figure 1B).

Wild-type (unlabeled) α-synuclein monomers were used to prepare α-synuclein fibrils, which were obtained by continuously agitating purified recombinant α-synuclein monomers (9–10 mg/mL, in PBS pH 7.4) in a Vortemp 56 shaking incubator (Labnet) at 1200 rpm and 37 °C for 10 days. Fibrillation was assessed by JC-1 assay (Lee et al., Biochem. J. 2009, 418, 311-323) and C-Fourier transform infrared spectroscopy of the solution. Unincorporated monomers in the fibril solution were assessed by ultracentrifugation and by passing through a 100 kDa cutoff membrane followed by gel electrophoresis. In further studies, only fibrils with a JC-1 response >15, low amounts of soluble monomers (<5%), and FTIR spectra with major absorbance between 1625 and 1630 ^cm⁻¹ were used (Figure 2). The prepared fibrils were stored at -80 °C.

Example 2: Immunization and Antibody Isolation

Many immunization strategies using various species and immunogens were implemented. Antibody 6470 was derived from female New Zealand white rabbits (>2kg) that had been subcutaneously immunized with rabbit Fc fusion protein (containing human α-synuclein residues 68-140 fused with rabbit Fc) (SEQ ID NO:43).

Following the manufacturer's experimental protocol, the rabbit Fc fusion protein of α-synuclein (68-140) for immunization was expressed in Expi293F cells using the Expi293 ^™ Expression System (Invitrogen). The protein was purified from the supernatant using affinity chromatography on a MabSelectSure column (GE Healthcare). The column was equilibrated with 50 mM glycine/sodium glycine (pH 8.8) buffer and eluted with a gradient of 0.1 M citrate (pH 2.0) in the same buffer. The protein fraction was neutralized with 2 M Tris HCl (pH 8.5), concentrated, and further purified by gel filtration on a HiLoad 26/600 Superdex 200 column (GE Healthcare) (equilibrated and eluted in PBS pH 7.4). Rabbits received an initial immunization containing 500 μg of the fusion protein emulsified in an equal volume of complete Freund's adjuvant (CFA). Rabbits were given two booster injections at 21-day intervals using incomplete Freund's adjuvant (IFA), with blood collected from the ear 14 days after immunization. Immunization was completed 14 days after the final booster, during which single-cell suspensions of spleen, bone marrow, and peripheral blood mononuclear cells were prepared and frozen at -80°C in 10% dimethyl sulfoxide (DMSO) in fetal bovine serum (FCS).

B cell culture

B cell cultures were prepared using a method similar to that described by Tickle et al., 2015. J Biomol Screen: 20(4), 492-497. Briefly, B cells derived from lymph nodes or spleen cells of immunized animals were cultured for seven days at 37°C in a 5% _CO2 atmosphere in barcoded 96-well tissue culture plates containing 200 μl/well of RPMI 1640 medium supplemented with 10% FCS (Sigma Aldrich), 2% HEPES (Sigma Aldrich), 1% L-glutamine (Gibco BRL), 1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol (Gibco BRL), 1% activated human PBMC supernatant (BSS), and X-ray irradiated mutant EL4 murine thymoma cells (5 × ^10⁴ /well) (Gibco BRL). Cultures were established using B cells from all immunized animals, with a total of approximately 1.7 × ^10⁹ B cells sampled.

6470, the antibody according to the invention, is generated from activated lymph node-derived B cells cultured at a density of approximately 5000 cells/well. In addition to spleen cells, lymph nodes are used for antibody discovery, thus providing us with alternative B cell sources from which novel antibodies can be sampled and identified. Antibodies with relevant sequences were identified from B cells derived from lymph nodes rather than the spleen. Approximately 9.6 × ^10⁷ cells were sampled from rabbits immunized with the C-terminal protein of human α-synuclein.

Initial screening

The presence of human α-synuclein-specific antibodies in B cell culture supernatant was determined using Superavidin ^™ beads (Bangs Laboratories) coated with biotinylated recombinant human α-synuclein full-length monomers as the target antigen, employing a uniform fluorescence-based binding assay. The recombinant human α-synuclein described herein was biotinylated using a 3-fold molar excess of biotin. A low molar excess of biotin was used to avoid complete modification of all seven lysine residues within the α-synuclein molecule. The α-synuclein monomer was incubated with biotin at 40°C, and free biotin was removed the following day using a Zeba ^™ spin desalting column. Screening involved transferring 10 μl of supernatant from barcoded 96-well tissue culture plates to barcoded 384-well blackwall assay plates containing biotinylated recombinant human α-synuclein monomers (10 μl/well) immobilized on Superavidin beads using an Agilent Bravo liquid handling system. Binding was revealed using a goat anti-rabbit IgG Fcγ-specific Alexafluor647 conjugate (Jackson). Plates were read on a TTP Labtech Mirrorball to identify wells containing α-synuclein-specific IgG.

Secondary screening

Following initial screening, positive supernatants were combined onto 96-well barcoded master plates using a Beckman Coulter Biomek NXP hit-picking robot, and B cells in the cell culture plates were frozen at -80°C. The master plates were then screened using either biotinylated recombinant human α-synuclein monomers or biotinylated recombinant human α-synuclein protofibrils in a streptavidin capture ELISA assay. This screening was performed to identify wells showing binding to both monomeric and protofibril-bound recombinant human α-synuclein and to exclude any false-positive wells showing off-target binding to Superavidin ^™ beads. Given the insoluble nature of protofibrils, conventional ELISA coating protocols using proteins in solution were not recommended. A minimal biotinylation protocol was chosen to preserve protofibril structure and promote effective coating of protofibrils on streptavidin-pre-coated ELISA plates.

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

Biotinylated monomers or biotinylated fibrils in PBS were captured on 384- _well Maxisorp plates coated with streptavidin in carbonate-coated buffer ( _dH₂O + 0.16% _Na₂CO₃ + 0.3% _NaHCO₃ ). The plates were blocked with 1% w/v PEG/PBS and then incubated with 10 μl/well of B cell culture supernatant (diluted 1:1 with blocking buffer). HRP-conjugated goat anti-rabbit IgG Fc secondary antibody (Stratech Scientific Ltd/Jackson ImmunoResearch) was added to the plates, followed by visualization of binding with TMB substrate (3,3',5,5'-tetramethylbenzidine, from EMD Millipore; 10 μl/well). Density was measured at 630 nM using a BioTek Synergy2 microplate reader. The initial binding assay identified 640 hits, and after ELISA screening, 491 of these hits showed both binding monomers and fibrils of recombinant human α-synuclein.

B cell supernatants exhibiting the strongest ELISA binding signal for recombinant fibrils were selected for further analysis by surface plasmon resonance to identify those with optimal dissociation rates on recombinant human α-synuclein monomers, recombinant human α-synuclein fibrils, and recombinant mouse α-synuclein fibrils. Supernatants from 80 different B cell strains were tested, with nine wells showing dissociation rates (kd) <1 × ^10⁻⁵ on recombinant human fibrils. Of these, seven showed dissociation rates (kd) <1 × ^10⁻⁵ on recombinant mouse fibrils, and two showed dissociation rates (kd) <1 × ^10⁻⁵ on recombinant human monomers. All nine supernatants were selected for variable region recovery.

Variable area recycling

To allow for the recovery of antibody variable region genes from the selection of the target supernatant, a deconvolution step was necessary to enable the identification of antigen-specific B cells in a given well containing a heterogeneous population of B cells. This was achieved using a fluorescence focusing method (Clargo et al., 2014. MAbs:6(1), 143-159). Briefly, immunoglobulin-secreting B cells from positive wells were mixed with streptavidin beads (New England Biolabs) coated with biotinylated recombinant human α-synuclein fibrils (which were produced using a 1:50 mixture, as described above) and a 1:1200 final dilution of goat anti-rabbit Fcγ fragment-specific FITC conjugate (Jackson). After static incubation at 37°C for 1 hour, antigen-specific B cells could be identified by the presence of a fluorescent halo around the B cell. These individual B cell clones, identified using an Olympus microscope, were then selected using an Eppendorf micromanipulator and placed in PCR tubes.

The antibody variable region gene was recovered from single cells by reverse transcription (RT)-PCR using heavy and light chain variable region-specific primers. Two rounds of nested 2° PCR were performed, with restriction sites incorporated at the 3' and 5' ends, allowing the variable region to be cloned into rabbit IgG (VH) or rabbit κ (VL) mammalian expression vectors. Anti-α-synuclein antibody genes from five 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), and the recombinant antibody was expressed in 30 ml volumes in 125 ml Erlenmeyer flasks. After 5–7 days of expression, the supernatant was harvested and purified using affinity chromatography.

ELISA screening of transient supernatant

The purified antibodies were then subjected to further screening via ELISA. Biotinylated recombinant human α-synuclein monomers and fibrils were captured on ₃₈₄ -well Maxisorp plates (ThermoScientific/Nunc) coated with streptavidin in carbonate-coated buffer ( _dH₂O + 0.16% _Na₂CO₃ + 0.3% _NaHCO₃ ). Separate plates were also coated with biotinylated peptides corresponding to residues 117 to 126 of human α-synuclein according to SEQ ID NO: 10 (peptide PVDPDNEAYE) to examine whether transient supernatant bound to this region or a different region of the molecule. Plates were blocked with 1% w/v PEG/PBS and then incubated with several dilutions of purified transient supernatant. HRP-conjugated goat anti-rabbit IgG Fc secondary antibody (Stratech Scientific Ltd/JacksonImmunoResearch) was added to the plates, followed by visualization of binding using TMB substrate (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. Data for 6470 are shown in Figure 3. As can be seen, 6470 shows binding to both monomeric and protofibrillary recombinant human α-synuclein, but not to peptide 117–126.

The antibodies (IgG) were then tested in a cell-based aggregation assay, as described later in Example 7. The binding kinetics of all antibodies that showed activity in the cell assay were subsequently determined by surface plasmon resonance. The antibodies were tested in IgG and Fab form to determine antibody affinity (bivalent binding) and affinity (monovalent binding), respectively.

Example 3: Antibody Characterization

Biacore Dynamics

Interaction kinetics were determined using surface plasmon resonance (SPR) on a Biacore T200 instrument. Three different ligands (recombinant full-length human α-synuclein monomer, purified recombinant human α-synuclein fibrils, and purified recombinant mouse α-synuclein fibrils, prepared as described herein) were immobilized on three separate flow cells on a CM5 chip surface using amine coupling chemistry. The three ligands were prepared in 10 mM NaAc, pH 3.5 and immobilized onto the separate flow cell surfaces at a flow rate of 10 μl/min to achieve the following immobilization levels: approximately 30 response units (RU) for the α-synuclein monomer; approximately 40 RU for the human α-synuclein fibrils; and approximately 300 RU for the mouse α-synuclein fibrils. HBS-EP+ (GE healthcare Bio-Sciences AB) buffer was used as the running buffer for both ligand immobilization and kinetic assays. Then, the binding of monoclonal 6470 rabbit IgG1 (containing SEQ ID NO: 47 and 48) and monoclonal 6470 rabbit Fab (containing SEQ ID NO: 47 and 49) to the three ligands was measured. Monoclonal IgG or Fab antibodies were injected into three flow cells at a flow rate of 100 μl/min at seven different concentrations ranging from 800 nM to 0.195 nM, with a contact time of 3 minutes and a dissociation time of 30 minutes. Surface regeneration was achieved by a single injection of 50 mM HCl at 10 μl/min for 90 seconds and another injection of 50 mM HCl at 10 μl/min for 60 seconds. Data were analyzed using a bivalent analyte model with Biacore T200 evaluation software (version 3.0), assuming no bulk contribution (RI = 0) and total Rmax for the IgG form, and a 1:1 model with flexible bulk contribution (local RI) and total Rmax.

The kinetic values for the binding of both IgG and Fab to the immobilized target are shown in Table 2. The IgG form showed a significantly selective affinity for human α-synuclein fibrils compared to its affinity for human α-synuclein monomers, because the dissociation constant KD for human fibrils was more than 10-fold lower.

Table 2

Binding with β-synuclein

The binding of antibodies against human α-synuclein to human β-synuclein was tested using Western blotting with rPeptide β-synuclein. One microgram of synuclein was electrophoresed on a 4–12% Bis/Tris gel and blotted onto a PVDF membrane. The membrane was blocked in PBS containing 3% BSA and 0.1% Tween 20. 6470 rabbit IgG1 antibody was added to the blocked blot and incubated at room temperature for 1 hour. The blot was washed with PBS and 0.1% Tween 20 and incubated for 1 hour with a secondary antibody-HRP conjugate (anti-rabbit H+L HRP conjugate, Bethyl, A120-101P). The blot was thoroughly washed with PBS, PBS, and water containing 0.1% Tween 20. Chemiluminescence was measured after adding ECL Western blotting substrate (Pierce). As shown in lane 3 of Figure 4(A), 6470 rabbit IgG1 does not bind to human β-synuclein.

Epitope plotting

NMR

Human α-synuclein was cloned into the pET28a expression vector, enabling the protein to be expressed without any tag. The construct was transformed into *E. coli* BL21(DE3) cells (Stratagene) and the cells were grown in a medium with a composition defined by ^C13- labeled DL-glucose and ^N15 -labeled ammonium sulfate, with or without deuterium oxide ( _D₂O ). Expression was induced with 300 mM IPTG at OD₆₀₀ = 1, and the culture was incubated at 30 °C for 4 h. Cells were precipitated into granular pellets and lysed by three freeze-thaw cycles in 100 ml of lysis buffer (20 mM Tris/HCl pH 8.0, 25 units of benzonase (Merck Millipore), a complete EDTA-free protease inhibitor mixture (2 tablets, Roche), and 10 mg of lysozyme (Sigma)). The lysate was clarified by centrifugation at 18,000 rpm and passed through a 0.22 μm filter (Stericup, Millipore). The sterile lysate was loaded onto a MonoQ 10/100GL (GE Healthcare) column equilibrated with 20 mM Tris/HCl pH 8.0, 5CV, and eluted with a gradient of up to 500 mM NaCl in the same buffer. After a 5-fold dilution in 20 mM Tris/HCl pH 8.0, further purification of the purest fraction was repeated on a MonoQ 10/100GL column. The purest fraction was collected, concentrated using a 10 kDa MWCO centrifuge (Centriprep, Millipore), purified by size exclusion on a HiLoad 26/600 Superdex 75 column (GE Healthcare), and eluted with 25 mM sodium phosphate buffer, 100 mM NaCl (pH 6.4). Fractions from a Superdex 75 column were collected and sodium azide (0.02% final concentration) and AEBSF (10 μM final concentration) were added. The final protein concentration was approximately 5 mg/ml.

Rabbit 6470Fab (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 as a His-tagged entity in CHO SXE and purified from the supernatant by His-tagged affinity chromatography, wherein the protein was bound from the supernatant to HisTrap Excel (GE Healthcare) and eluted with 250 mM imidazole in PBS. The eluted pool was loaded onto a HiTrap GammaBind Plus Sepharose (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). Eluted Fab-His protein was buffer-exchanged onto a HiPrep 26/10 desalting column to NMR buffer (25 mM sodium phosphate, pH 6.4, 100 mM NaCl). The Fab-His protein fraction was concentrated and supplemented with the protease inhibitor AEBSF (10 μM final concentration) and sodium azide (0.02% final concentration) before sterilization via filtration through a Millex GV 0.22 μm filter. For crystallography, the concentrated 6470 Fab-His was purified by preparative size exclusion chromatography on a HiLoad 26/600 Superdex 75 (GE Healthcare) column equilibrated and eluted with 25 mM sodium phosphate, pH 6.4, and 100 mM NaCl. The purity of the final pool was tested at >99% purity on UPLC-SEC. The final pool was then sterilized via a Millex GV 0.22 mm filter.

Main chain allocation of α-synuclein

Typically, NMR samples were 350 μL in a 5 mm Shigemi tube containing either 360 μM of human α-synuclein labeled with ^13C / ^15N or 430 μM of human α-synuclein labeled with ^2H / ^13C / ^15N . Buffer conditions were 100 mM NaCl, 25 mM sodium phosphate (pH 6.4), 10 μM AEBSF, and 0.02% _NaN₃ . All experiments were recorded at 20 °C on a 600 MHz Bruker AVIII or 800 MHz Bruker AVII spectrometer (equipped with cryogenically cooled probes). Sequential linking of backbone NMR signals of protein residues was performed using a 3D(H)N(CA)NNH experiment (Weisemann et al., 1993, 3D Triple-resonance NMR techniques for these quential assignment of NH and 15N resonances in 15N-and 13C-labeled proteins. J. Biomol. NMR 3), H _N (i)-N(i)-N(i±1), recorded in ¹⁵ N, ¹⁵ N, and ¹ H dimensions with spectral widths of 28, 28, and 10 ppm and acquisition times of 117 (F1), 117 (F2), and 140 (F3) ms, respectively, with 8 scans/increments and a 1.5 s relaxation delay. Non-uniform sampling was employed, with a sampling density of 10% (4000 out of 40000 hypercomplex points), resulting in a total acquisition time of 2.75 days. Improved 3D triple-resonanceNMR techniques applied to a 31kDa protein. J.Magn.Reson.96, 432-440; Salzmann et al., 1998. TROSY in triple-resonance experiments: new perspectives for sequentialNMR assignment of large proteins.Proc.Natl.Acad.Sci.USA.95,13585-90) and TROSY-HNCACB (Wittekind and Mueller, 1993HNCACB, a High-Sensitivity 3D NMR Experiment toCorrelate Amide-Proton and Nitrogen Resonances with the Alpha-and Beta-CarbonResonances 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) were used to confirm sequential linkage and identify residue types. TROSY-HNCA experiments were recorded in ¹³ C, ¹⁵ N, and ¹ H dimensions with spectral widths of 23, 28, and 10 ppm and acquisition times of 12.1 (F1), 21.7 (F2), and 100 (F3) ms (8 scans/increments, 1.5 s relaxation delay, 1 day total acquisition time). TROSY-HNCACB experiments were recorded in ¹³ C, ¹⁵ N, and ¹ H dimensions with spectral widths of 56, 28, and 10 ppm and acquisition times of 8.2 (F1), 21.7 (F2), and 100 (F3) ms (8 scans/increments, 1.5 s relaxation delay, 1.7 days total acquisition time). The carbonyl assignment of the main chain was obtained from TROSY-HNCA spectra recorded at spectral widths of 10, 29, and 10 ppm in ^{the 13} C, ¹⁵ N, and ¹ H dimensions, and acquisition times of 80 (F1), 21.7 (F2), and 150 (F3) ms (8 scans/increments and a 1.5 s relaxation delay). (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). Non-uniform sampling was employed, with a sampling density of 15% (1208 out of 8050 hypercomplex points), resulting in a total acquisition time of 19 hours. NMR spectra were processed using NMRPipe (Delaglio et al., 1995 NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-93), where linear prediction was used to extend the effective acquisition time for nitrogen by up to 1 fold. Non-uniformly sampled data were reconstructed using the Harvard iterative soft thresholding method (Hyberts et al., 2012), where the data was reconstructed to the next Fourier number, increasing the indirect acquisition time by up to 60%. Data analysis was performed using Sparky (Goddard and Kneller, DGSPARKY 3. In., University of California, San Francisco), which resulted in the allocation of amide proton and nitrogen resonances for 133 residues (corresponding to 99% of the residues, excluding proline residues and N-terminal methionine).

Mapping of the binding sites of 6470Fab was performed using a 150 μM sample of human α-synuclein labeled with ² H/ ¹³ C/ ¹⁵ N (containing a 10% molar excess of unlabeled 6470Fab). The sample was prepared in the same buffer as described above for the backbone partitioning of α-synuclein. The chemical shifts of 1H, 15N, and 13C were determined by comparing the TROSY-HNCO spectra recorded on the α-synuclein/Fab complex (Grzesiek and Bax, 1992 Improved 3D triple-resonance NMR techniques applied to a 31kDa protein. J. Magn. Reson. 96, 432-440; Salzmann et al., 1998. ^TROSY intriple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc. Natl. Acad. Sci. USA. ⁹⁵ , ^13585-90 ) with equivalent control spectra recorded for free α-synuclein. The control TROSY-HNCO experiments for free α-synuclein were recorded in ¹³ C, ¹⁵ N, and ¹ H dimensions with spectral widths of 10, 28, and 10 ppm and acquisition times of 80 (F1), 22 (F2), and 150 (F3) ms (16 scans/increments, 1.5 s relaxation delay). Non-uniform sampling was used, with a sampling density of 25% (2013 out of 8050 hypercomplex points), resulting in a total acquisition time of 2.7 days. The TROSY-HNCO experiments for the α-synuclein/Fab complex were recorded in ¹³ C, ¹⁵ N, and ¹ H dimensions with spectral widths of 10, 28, and 10 ppm and acquisition times of 80 (F1), 21.7 (F2), and 80 (F3) ms (32 scans/increments, 1.5 s relaxation delay). Non-uniform sampling was employed, with a sampling density of 25% (1119 out of 4477 hypercomplex points), resulting in a total acquisition time of 2.8 days. NMR spectra were processed using NMRPipe (Delaglio et al., 1995, NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-93), where NUS data reconstruction was performed using mddnmr (Analysis of non-uniformly sampled spectra with Multi-Dimensional Decomposition. Prog. Nucl. Magn. Reson. Spectrosc., 59, pp. 271-292). The effective acquisition time for the nitrogen dimension increased by up to 1 time during data reconstruction.

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

_ΔδHN , _ΔδN , and _ΔδC represent the differences in chemical shifts at ^1H , ^15N , and ^13C , respectively. αN and αC correspond to scaling factors of 0.2 and 0.35, respectively, and are used to explain the differences in the range of chemical shifts for the amide proton, nitrogen, and carbonyl groups.

To identify Fab binding sites (epitopes) on α-synuclein, histograms of combinatorial minimum shifts against protein sequences are used to reveal regions of α-synuclein containing significantly impaired signals. Residues whose combinatorial chemical shifts for a single amino acid exceed the average of the combinatorial chemical shifts for all amino acids plus a threshold of standard deviation from that average are selected as potential contact residues at the Fab binding site for further evaluation.

Significantly affected residues were identified if their minimum displacement was at least greater than the average of all calculated displacements plus one standard deviation. Four different thresholds were applied to identify residues bound by Fab. Residues involved at the binding site were scored with increasing stringency as follows: those with a minimum displacement greater than the average of all calculated displacements plus one standard deviation (>0.018925); those with a minimum displacement greater than the average of all calculated displacements plus two standard deviations (>0.032049); those with a minimum displacement greater than the average of all calculated displacements plus three standard deviations (>0.045174); and those with a minimum displacement greater than the average of all calculated displacements plus four standard deviations (>0.058299). Proline residues could not be identified in this analysis because they do not contain amide protons.

Therefore, with increased rigor, the epitopes for 6470Fab are defined as follows: the average of all calculated displacements plus one standard deviation: D121, N122, E123, A124, Y125, E126, M127, S129, E130, Y133, Q134, D135, and Y136; the average of all calculated displacements plus two standard deviations: E123, A124, Y125, E126, M127, S129, E130, D135, and Y136; the average of all calculated displacements plus three standard deviations: Y125, M127, S129, and D135; and the average of all calculated displacements plus four standard deviations: M127, S129, and D135.

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

Peptide mapping

Further characterization of the epitope bound by 6470 was performed using short (typically 9-mers or 10-mers) peptides representing and covering the C-terminal region of human α-synuclein. These peptides were used in a competitive surface plasmon resonance assay to test whether any of them could inhibit the binding of the antibody to monomeric α-synuclein or pre-formed α-synuclein fibrils immobilized on a Biacore chip. The peptide exhibiting the maximum level of inhibition was then selected for co-crystallization studies with the antibody to confirm the accurate epitope.

Peptides were supplied by Peptide Protein Research Ltd., Bishop’s Waltham, U.K., and synthesized via Fmoc solid-phase peptide chemistry according to the method of Atherton and Sheppard (see Atherton, E., Sheppard, R.C. (1989). Solid Phasepeptide synthesis: a practical approach. Oxford, England: IRL Press). N- and C-terminal peptides were capped with acetyl and amide groups, respectively, except in the case of peptides representing the N-terminus and C-terminus of α-synuclein (where the amino and carboxyl groups remained free, respectively). Stock solutions of peptides were prepared in 10 mM DMSO. A complete list of peptides is shown in Table 3.

Table 3

Recombinant human α-synuclein monomers and pre-formed α-synuclein fibrils were immobilized on a CM5 chip using a Biacore 3000 instrument (GE Healthcare). After activation of carboxymethyl dextran by injecting 100 μl of a fresh 1:1 (v/v) mixture of 50 mM N-hydroxysuccinimide and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide at a flow rate of 10 μl/min HBS-EP (GE Healthcare) (as run buffer), coupling was achieved by injecting 100 μl of monomer and fibrils at 5 μM in 10 mM acetate at pH 5.0 into separate flow cells. A reference flow cell was activated in the same manner, and all flow cell surfaces were then deactivated with a 50 μl pulse of 1 M ethanolamine hydrochloride (pH 8.5).

A peptide solution was prepared at 100 μM in run buffer, and a peptide blank control was prepared as a 1:100 dilution of DMSO in run buffer. A solution of 6470 rabbit Fab (containing SEQ ID NO: 47 and 49) was prepared at 50.5 nM in run buffer, followed by pre-incubation of 198 μL with 2 μL of blank control or diluted peptide to produce a final mixture of 50 nM Fab and 1 μM peptide or a control. Sensing patterns were recorded for each sample by injecting 30 μL of the mixture at 10 μL/min and recording a reporting point for 5 seconds before the end of the injection. At the end of each cycle, the chip was regenerated by 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 a percentage change in response units measured at the report point relative to the average of adjacent control cycles.

The inhibitory levels for each α-synuclein peptide are shown in Figure 5. Significant inhibition of 6470Fab against α-synuclein monomers or protofibrils was observed only for three peptides: AS121-130, AS123-132, and AS125-134, with the highest inhibition level observed for AS123-132, where the antibody bound to the monomer and protofibrils at 37% and 54%, respectively. Slightly lower inhibition levels were obtained for peptide AS125-134, at 34% and 52%, respectively, indicating that the major components of the epitope comprise residues 125 to 132. Peptide AS121-130 showed lower inhibition levels of 20% and 27%, respectively, suggesting that the common residues 125 to 130 contribute the most to the epitope for all three peptides.

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

X-ray crystallography

To prepare the complex, 1 ml of purified rabbit Fab (approximately 10 mg/ml) was mixed with α-synuclein peptide 123-132 (EAYEMPSEEG) at a Fab:peptide molar ratio of 1:2 and incubated at room temperature for 1 hour. Suitable crystal growth conditions were determined using a commercially available crystallizer (Qiagen) via the sitting drop vapor diffusion method. To produce diffraction-quality crystals, the hanging drop vapor diffusion method was used.

For the 6470Fab-peptide 123-132 complex, 1 μl of the protein solution was mixed with 1 μl of the reservoir solution (containing 1.6 M ammonium sulfate and 0.1 M Hepes buffer, pH 7.5). Crystals were harvested and then rapidly frozen in liquid nitrogen after a brief cryoprotective solution containing 1.6 M ammonium sulfate, 0.1 M Hepes buffer, pH 7.5, and 20% glycerol. Crystals were also harvested and then rapidly frozen in liquid nitrogen after a brief cryoprotective solution containing 0.2 M ammonium sulfate and 35% (v/v) polyethylene glycol 8000.

Diffraction data were collected from single crystals of Fab 123-132 at Diamond Synchrotron, Didcot, Oxfordshire, UK, on beamline i04-1, and processed using Mosflm, Aimless, and Truncate. The structure of the complex was resolved by molecular substitution using Phaser, with the coordinates of the inner Fab as the search model.

A cycle of refinement and model building was performed using CNS (Brunger et al., (2007) Nature Protocols 2, 2728-2733) and COOT (Emsley et al., (2004) Acta crystallographica. Section D, Biological crystallography 60, 2126-2132) until all refinement statistics converged for both models. Model geometry was validated using Molprobity43. Molecular visualizations were generated using Pymol44. Epitope information, as described below, was obtained by considering atoms within a distance of the Fab/peptide contact surface. Data collection and refinement statistics are shown in Tables 4A and 4B.

Table 4A

Table 4B

The values in parentheses refer to the high-resolution shell. Rsym = Σ|(I – <I>)|/Σ(I), where I is the observed cumulative intensity, <I> is the average cumulative intensity obtained from multiple measurements, and the sum includes all observed reflections. _Rwork = Σ||Fobs| – k|Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. _Rwork is calculated as _Rfree using 5% of the reflection data that is randomly selected and omitted from the refinement calculation.

The major contacts between heavy and light chain residues and peptides are shown in Table 5.

Table 5

In summary, the epitopes comprise residues E123, Y125, E126, M127, P128, S129, E130, and E131. Figure 6 shows 6470Fab in complex with peptides 123-132, and Figures 7 and 8 show the contacts between peptides 123-132 and the heavy and light chains of 6470Fab, respectively.

Example 4: Antibody humanization and affinity maturation

Rabbit antibody 6470 was humanized by transplanting a CDR from the rabbit V-region onto the human antibody V-region framework. To restore antibody activity, many framework residues from the rabbit V-region were also preserved in the humanized sequence. These residues were selected using the experimental protocol outlined by Adair et al. (1991) (WO91/09967). Alignments of the rabbit antibody (donor) V-region sequence with the human (recipient) V-region sequence are shown in Figures 9 and 10, along with the designed humanized sequence. The CDR transplanted from the donor to the recipient sequence was as defined by Kabat (Kabat et al., 1987), except for CDR-H1, where the combined Chothia/Kabat definition was used (see Adair et al., WO91/09967).

Genes encoding numerous variants of the heavy and light chain V-region sequences were designed and constructed by DNA2.0 Inc. using automated synthesis methods. Further variants of the heavy and light chain V-regions were created by modifying the VH and VK genes via oligonucleotide-guided mutagenesis, including, in some cases, mutations within the CDR. For transient expression in mammalian cells, the humanized light chain V-region gene was cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human κ chain constant region (Km3 allotype). The humanized heavy chain V-region gene was cloned into the UCB human γ-4 heavy chain expression vector pMhγ4PFL, which contains DNA encoding the human γ-4 heavy chain constant region with the hinge stabilization mutation S241P (Angal et al., Mol. Immunol. 1993, 30(1):105-8). Similarly, a chimeric 6470 containing rabbit V-regions (SEQ ID NO: 11 and 13) and human constant regions was prepared and used as a comparator antibody. The resulting heavy and light chain vectors were co-transfected into Expi293 ^™ suspension cells to give expression of a humanized recombinant antibody with human IgG4P.

Human V-region IGKV1-16 plus JK4 J-region (IMGT, http://www.imgt.org/) was selected as the recipient for the light chain CDR of antibody 6470. The light chain framework residues in the graft gL3 were all derived from human germline genes, except for residues 48 and 72 (related to SEQ ID NO:15), in which donor residues glutamine (Q48) and glutamine (Q72) were retained, respectively. The retention of residues Q48 and Q72 is essential for the full potency of this humanized antibody in binding to human α-synuclein fibrils (Figure 9 and Table 6).

Table 6

The human V-region IGHV3-23 plus the JH4 J-region (IMGT, http://www.imgt.org/) was selected as the recipient for the heavy chain CDR of antibody 6470. Similar to many rabbit antibodies, the VH gene of antibody 6470 is shorter than that of the selected human recipient. When aligned with the human recipient sequence, the VH region architecture 1 of antibody 6470 lacks N-terminal residues, which are retained in the humanized antibody (Fig. 10). The 6470 rabbit VH region architecture 3 also lacks two residues (75 and 76) in the loop between D and E of the β-sheet chain: in the humanized graft, this gap is filled with the corresponding residues from the selected human recipient sequence (lysine 75, K75; asparagine 76, N76) (Fig. 10). The heavy chain framework residues in grafts gH23 and gH36 are all derived from human germline genes, except for one or more residues from the group comprising residues 24, 47, 48, 49, 73, 78, and 97 (with respect to SEQ ID NO: 23 and 31), in which donor residues valine (V24), tyrosine (Y47), isoleucine (I48), glycine (G49), serine (S73), valine (V78), and arginine (R97) are retained, respectively. The retention of residues V24, Y47, I48, G49, and R97 is essential for the full potency of this humanized antibody in binding to human α-synuclein fibrils.

In addition, the humanized VH gene was cloned into the UCB human Fab-HIS expression vector pMhFab10HIS, which contains DNA encoding a human γ-1CH1 hinge domain with a C-terminal tag (ten histidine residues). The histidine tag facilitated the purification of the expressed Fab by affinity chromatography. The resulting heavy and light chain vectors were co-transfected into Expi293 ^™ suspension cells to produce the expression of the humanized recombinant antibody in Fab-HIS form.

Affinity maturation was performed using the IOTA method described in WO2014198951. Interfacial processes between 6470 rabbit Fab and the α-synuclein peptide EAYEMPSEEG (123-132) in the complex, determined by X-ray crystallography, were analyzed to identify mutations that could potentially improve the affinity of 6470 rabbit Fab for α-synuclein proteins. IOTA is a statistically significant tool for determining the probability of a given contact atom type at a protein interface or binding site.

To evaluate the effect of these mutations on the efficacy of the antibody in binding to human α-synuclein monomers or fibrils, the mutations were first investigated in Rabbit Fab 6470 (Table 7A). Interaction kinetics were determined on a Biacore T200 instrument using surface plasmon resonance (SPR) technology, as described in Example 3. Residue 33 in CDRL1 (related to SEQ ID NO: 11) was mutated from asparagine (N) to arginine (R) or lysine (K): the mutation of residue 33 to arginine resulted in increased affinity for α-synuclein (Table 7A). Mutating residue 55 in CDRH2 from serine (S) to asparagine (N), and residue 99 in CDRH3 from asparagine (N) to lysine (K), glutamine (Q), histidine (H), or tryptophan (W) (with respect to SEQ ID NO:13), results in increased affinity for α-synuclein (Table 7A). The mutation of asparagine in CDRH3 (N99H) also removes a potential deamidation site.

Table 7A

Finally, the newly identified mutations were also tested in the previously generated full-length humanized antibody (Table 6), and their selectivity for human fibrils was tested (Table 7B). Interaction kinetics were determined on a Biacore T200 instrument using surface plasmon resonance (SPR) technology, as described in Example 3. As shown in Table 7B, mutations at position 33 in the light chain (corresponding to SEQ ID NO: 19) and positions 56 and 102 in the heavy chain (corresponding to SEQ ID NO: 27 and 35) resulted in increased affinity for human fibrils, a favorable feature for antibodies that need to cross the blood-brain barrier to bind their targets. When antibodies are administered systemically, a significant amount may be lost because antibodies have a limited system to cross complex physiological barriers.

Table 7B

The expression variants of humanized antibody chains and combinations thereof were evaluated, and their potency relative to parental antibodies, their biophysical properties, and their suitability for downstream processing were assessed.

Example 5: Characterization of humanized antibodies

Biophysical characterization was performed on six humanized 6470IgG4P antibodies (sequences in Table 8 and Table 1).

Table 8

descriptor gL3gH23 gL3gH36 gL3-N33RgH23-S56N-N102H gL3-N33RgH36-S56N-N102H gL3gH23-S56N-N102H gL3gH36-S56N-N102H

All antibodies were screened based on thermal stability (Tm), experimental pI, hydrophobicity, solubility (PEG precipitation assay), and aggregation stability at the air/liquid interface to determine whether the mutation had any impact, particularly regarding affinity, stability, and exploitability.

The screening process also includes assessing chemical stability (deamidation, aspartic acid isomerization tendency), because the antibodies possess:

1. In heavy chain CDR3, the Asn(102)S moiety (deamidation) is only for gL3gH23 and gL3gH36;

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

3. The Asn(32)Asn(33) motif (deamidation) in the light chain CDR1, for all except the N33 mutant;

4. The Asp(99)G motif (Asp isomerization) in the light chain CDR3 is used for all antibodies.

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

Thermal stability ( _Tm ) measurement

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

The reaction mixture contained 5 μl of 30x orange dye (Invitrogen ^™ ) diluted with PBS from a 5000X stock solution and 45 μl of 0.12 mg/mL sample (in PBS pH 7.4). Approximately 10 μl of the mixture was distributed in quadruplicate to 384-well PCR plates and run on a 7900HT Rapid Real-Time PCR System (Applied Biosystems ^™ ). The PCR system heating device was set from 20°C to 99°C at an increase rate of 1.1°C/min. Fluorescence changes in the wells were monitored using a charge-coupled device. The intensity increases were plotted, and Tm was calculated using the inflection point of the slope, as described below.

Two unfolding transitions were observed for all antibodies. The first can be attributed to the Tm of the CH2 domain. According to the literature (Garber E, Demarest SJ. Biochem Biophys Res Commun. 2007 Apr 13, 355(3):751-7), the second can be attributed to the average Tm of the Fab unfolding domain and the CH3 domain. Table 9 summarizes the results.

Table 9

For the IgG4 molecule, the thermal stability is within the normal expected 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).

Experimental pI

Experimental pI of the 6470 antibody was obtained using the whole-capillary imaging cIEF iCE3 ^™ system (ProteinSimple).

Samples were prepared by mixing the following components: 30 μL sample (from a 1 mg/mL stock solution in HPLC-grade water), 35 μL 1% methylcellulose solution (Protein Simple), 4 μL pH 3-10 ampholyte (Pharmalyte), 0.5 μL 4.65 and 0.5 μL 9.77 synthetic pI standards (Protein Simple), and 12.5 μL 8M urea solution (Sigma-). The final volume was brought to 100 μL using HPLC-grade water. Before analysis, the mixture was briefly vortexed to ensure complete mixing and centrifuged at 10,000 rpm for 3 min to remove air bubbles. The sample was focused at 1.5 kV for 1 min, followed by 3 kV for 5 min, and A280 images of the capillary were acquired using ProteinSimple software. The resulting electrophoresis results were first analyzed using iCE3 software, and pI values (linear relationships between pI standards) were assigned. Then, the calibrated electrophoresis pattern was integrated using software (Waters).

The experimental pI for all 6470 antibodies was in the range of 8.4–9.2. There were slight differences in the ratio of charged species between molecules, but this is not unexpected for IgG4P molecules. All pIs were high, which will therefore aid the antibody preparation process.

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 times correspond to greater hydrophobicity.

The 2 mg/mL sample was diluted 1:2 with 1.6 M ammonium sulfate and PBS (pH 7.4). 5 μg (5 μL) of the sample was injected onto a Dionex ProPac ^™ HIC-10 column (100 mm x 4.6 mm) connected in tandem with an Agilent 1200 binary HPLC system equipped with a fluorescence detector. The separation was monitored by intrinsic fluorescence (excitation and emission wavelengths of 280 nm and 340 nm, respectively).

Samples were analyzed using buffer A (0.8 M ammonium sulfate, 100 mM phosphate, pH 7.4) and buffer B (100 mM phosphate, pH 7.4) with gradient elution as follows: (i) hold at 0% B for 2 min, (ii) linear gradient from 0 to 100% B (0.8 mL/min) over 30 min, and (iii) wash the column with 100% B for 2 min and reequilibrate at 0% B for 10 min before the next sample injection. The column temperature was maintained at 20 °C.

Standards exhibiting low and high hydrophobicity, along with controls, were also analyzed in the same running sequence to allow for the normalization of retention times (Table 11). The retention times (RT) of the samples were normalized for the low and high hydrophobic standards using the following equation:

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

Table 10

All 6470 antibodies and mutants exhibited similar normalized retention times and similar low hydrophobicity. Commercially available therapeutic antibodies tend to exhibit low hydrophobicity (Jain et al., “Biophysical properties of the clinical-stage antibody landscape” Proc Natl Acad Sci U S A. 2017 Jan 31, 114(5):944-949). Low hydrophobicity helps with stability (i.e., reduces aggregation) during preparation.

Solubility determination using the polyethylene glycol (PEG) precipitation assay

Colloidal stability was analyzed using a polyethylene glycol (PEG) precipitation assay. PEG is used to reduce protein solubility in a quantitatively deterministic manner by increasing the PEG concentration (w/v) and measuring the amount of protein remaining in solution. This assay is used to simulate the effect of high concentrations of solubility, rather than using conventional concentration methods. PEG-induced precipitation of antibody 6470 was investigated 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. Buffer exchange of samples was performed by dialysis if necessary, and the concentration was adjusted to 2 mg/mL. To minimize non-equilibrium precipitation, sample preparation consisted of a 1:1 volume ratio of 2× protein and 2× PEG solution. After mixing, the sample was incubated at 37 °C for 30 min to redissolve the non-equilibrium aggregates. After overnight incubation at 20°C, the sample was centrifuged for 60 minutes (4000g). Aliquots of the supernatant were transferred to a half-volume 96-well optical plate, and absorbance at 280 nm was measured using a BMG Labtech Omega LVIS A280 plate reader. The concentration data were plotted against PEG%, and the calculated midpoint (LogEC50) (generated by nonlinear curve fitting with a variable slope) was used as a measure of the sample's relative colloidal solubility. In this assay, a higher LogEC50 is equivalent to greater colloidal stability.

Results (not shown) indicate that for all 6470 antibodies, colloidal stability decreased with increasing buffer pH. Additionally, the following trend was observed from most soluble to less soluble: gL3gH23 and gL3gH36 > gL3gH23-S56N-N102H and gL3gH36-S56N-N102H > gL3-N33RgH23-S56N-N102H and gL3-N33RgH36-S56N-N102H.

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

The effect of stress at the air-liquid interface (aggregate measurement method)

When exposed to an air-liquid interface, proteins tend to unfold, where a hydrophobic surface is exposed to the hydrophobic environment (air) and a hydrophilic surface is exposed to the hydrophilic environment (water). Agitation of the protein solution creates a large air-liquid interface, which can drive aggregation. This assay is used to simulate the stress molecules would experience during preparation (e.g., ultrafiltration) and provides stringent conditions to attempt to distinguish different antibody molecules.

Stress was applied to 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 using vortexing with an Eppendorf Thermomixer Comfort ^™ . The aforementioned buffers were selected as the common pre-prepared buffer. Prior to vortexing, the concentration was adjusted to 1 mg/mL using an appropriate extinction factor (1.35 Abs 280 nm, 1 mg/mL, 1 cm path length), and absorbance at 280 nm, 340 nm, and 595 nm was obtained using a Varian50-Bio spectrophotometer to establish zero-time readings. Each sample was subaliquoted into 1.5 mL conical-type capped tubes (4 x 250 μL) and subjected to rigorous conditions to test robustness by vortexing at 1400 rpm at 25 °C for 24 hours. Time-dependent aggregation (turbidity) was monitored by measuring samples at 595 nm using a Varian Cary ^™ 50-Bio spectrophotometer at 3 and 24 hours after vortex oscillation. For each sample, the average absorbance value was plotted against time.

The results are illustrated in Figure 11. There was no difference in the 6470 antibody at 24 hours in any of the three buffers; however, a small difference in aggregation tendency was discernible 3 hours after vortexing, which appeared to be buffer-dependent.

Deamidation/Asp isomerization stress study

Stress studies were conducted using 6470 antibodies gL3gH23 and gL3gH36 to determine the deamidation/Asp-isomerization propensity for four identified potential sequence adversaries: 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/ratio for deamidation and Asp-isomerization could not be predicted because it depends on the primary sequence and 3D structure, as well as solution properties (R C Stephenson and S Clarke (1989); K. Diepold et al., (2012); Jasmin F. Sydow et al., (2014); N.E. Robinson et al., (2004).

The baseline deamidation level (for unstressed samples) was also obtained—low levels indicate low susceptibility, but levels can vary due to different preparation batches/conditions.

Two 6470 antibodies were buffer exchanged to the following buffers: (i) a buffer known to favor the deamidation of Asn(N) residues (Tris, pH 8/37°C); and (ii) a buffer known to favor Asp isomerization (acetate, pH 5/37°C). The final concentration of the sample in each buffer was adjusted to ~6.5 mg/mL, and then divided into two aliquots, one stored at 4°C and the other at 37°C for up to 4 weeks. The aliquots were removed immediately (T0) and after 2 and 4 weeks of storage at -20°C.

Samples were thawed after 2 weeks and analyzed using trypsin digestion/mass spectrometry (MS) for chemical modification analysis as follows. Stressed protein samples were reduced with TCEP and alkylated with chloroacetic acid in Tris-HCl buffer at pH 8.0 containing 0.1% w/v Rapigest detergent. Trypsin (1:25 w/w) was added and the sample was digested overnight at room temperature. Protein hydrolysis was terminated by adding formic acid to 1% v/v and the sample was diluted to 0.5 mg/mL, followed by centrifugation to remove precipitated Rapigest ^™ . The resulting peptides were separated and analyzed on a Waters BEH C18 column interconnected with a Thermo Fusion ^™ mass spectrometer, using the +ve-ion, data-dependent orbitrap-orbitrap method with collision-induced dissociation (CID) fragmentation. LC-MS data were analyzed using Thermo Xcalibur ^™ and Pepfinder ^™ software.

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

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

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

The effects of chemical modifications (deamidation at Asn(102) on the heavy chain CDR3 and the formation of a succinimide intermediate at Asp(99) on the light chain CDR3) on affinity/antibody affinity were evaluated for recombinant full-length human α-synuclein monomers and purified recombinant human α-synuclein protofibrils. In this study, the fully deamidated product (Asn(102)Asp) and stress-applied material (pH 5/2 weeks/37°C) were used.

Example 6: Immunohistochemistry

Immunohistochemistry was performed by Asterand Bioscience (Royston, United Kingdom). First, frozen sections (10 μm) underwent an antigen retrieval procedure using Dako PT Link and EnVision FLEX TargetRetrieval Solutions (pH 6) at 97°C for 20 minutes with automatic heating and cooling. All subsequent incubation steps were performed at room temperature. Frozen sections were air-dried for 30 minutes, fixed for 10 minutes in 4% paraformaldehyde prepared in 1X PBS, washed with Dako EnVision ^™ FLEX wash buffer (Dako), and then loaded into Dako Autostainer Plus. Endogenous peroxidase activity was blocked by incubating the sections with Dako peroxidase blocking solution (Dako) for 5 minutes. The sections were then washed twice with 1X PBS before incubating with Dako CSA II protein blocking solution (Dako) for 10 minutes. The protein blocking solution was removed by air jetting, and the slides were incubated for 30 minutes with 6470 rabbit IgG1 (containing SEQ ID NO: 47 and 48) (0.05 μg/ml) diluted in Dako antibody diluent (Dako). After incubation, the slides were washed twice with 1X PBS, then incubated for 20 minutes with anti-rabbit Dako Flex polymer-HRP substrate (Dako), washed twice, and then incubated for 10 minutes with diaminobenzidine substrate (Dako). The chromogenic reaction was terminated by rinsing the slides with distilled water. After chromogenic reaction, the slides were removed from Dako Autostainer Plus and manually counterstained with hematoxylin, dehydrated in an ascending series of ethanol solutions, cleared in three xylene changes, and covered with coverslips under DPX mounting medium (Sigma-Aldrich). Digital images of the stained slides were obtained using an Aperio ScanScope AT Turbo system (Leica Biosystems). Antibody 6470 was tested on brain slices (1 slice/donor) from five different pS129-α-synuclein-positive donors and three different pS129-α-synuclein-negative donors. Antibody 6470 labeled neuropil and Rui body-like features in the temporal cortex and substantia nigra of PD patients (Fig. 12A-E). In non-PD brain tissue, antibody 6470 labeled neuropil in the temporal cortex, but Rui body-like structures were not 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 tissue from both PD and non-PD patients, while it binds only to pathological α-synuclein present in Rui bodies in PD patients.

Example 7: Cell-based aggregation assay

HEKFreestyle 293F cells (suspension cells) were prepared in Freestyle 293 expression medium (Invitrogen ^™ ) at a concentration of 0.7 × ^10⁶ cells/ml and cultured to a concentration of 300 × ^10⁶ cells/ml. Transfection was performed according to the manufacturer's instructions; briefly, 600 μg of pcDNA3.1(+) incorporating the α-synuclein gene was mixed in 20 ml of OptiMEM, while 293Fectin was diluted in OptiMEM medium (Invitrogen ^™ ) and incubated at room temperature for 5 min. The diluted DNA was added and incubated at room temperature for 20 min before being added dropwise to the cells (20 ml/flask). The cells were incubated at 37 °C, 125 rpm, and 8% _CO₂ for 24 h. Cells were used immediately or frozen in FBS + 10% DMSO at a concentration of 5 × ^10⁶ cells/ml.

If the cells have been previously frozen, thaw the flask and resuspend the cells in Freestyle 293 medium. Centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend the granular pellet at 2 × ^10⁶ cells/ml in Freestyle 293 medium (Life Technologies ^™ ) containing Pen/Strep (Invitrogen ^™ ). Add 20 μl of the cell suspension to each 384-well Grainer ^™ plate (to a total of approximately 40,000 cells/well). Add 150 nM of human α-synuclein fibrils (prepared as described in Example 1 herein) to each well, followed by the antibody to be detected in PBS (6470 gL3gH23; 6470 gL3gH36; 6470 gL3N33gH23 S56N N102; 6470 gL3N33gH36 S56N N102; 6470 gL3gH23 S56N N102; 6470 gL3gH36 S56N N102; all in full-length IgG4P, sequences in Table 1) (at various concentrations). Incubate the plates in a cell culture incubator at 37°C, 5% _CO2 , and 95% humidity for 2 days.

At the end of the second day, aspirate the culture medium from all wells and wash the plates, leaving 20 μl/well. Add approximately 50 μl of PBS to each well and centrifuge the plates at 500g for 5 minutes. Aspirate the supernatant from all wells using a plate washer, leaving 20 μl of culture medium in each well. Add Versene (Lonza ^™ ) (50 μl/well) and centrifuge the plates at 500g for 5 minutes, aspirating the supernatant to leave only 20 μl of culture medium/well. Replenish each well with 20 μl of 8% formaldehyde (16% solution in water, Life Technologies ^™ ) + 2% Triton X-100 (VWR ^™ ) in PBS. Incubate the plates at room temperature for 15 minutes, and then add 50 μl of FACS buffer consisting of HBSS (calcium- and magnesium-free VWR ^™ ) + 2% FBS + 2 mM EDTA (Life Technologies ^™ ). Centrifuge the plates at 2000g for 1 minute and aspirate the supernatant, leaving only 20 μl of culture medium in each well. Add 20 μl of FACS buffer containing a 1:300 dilution of anti-pSer129α-synuclein antibody (AbCam ^™ ) to each well. Incubate the plates at room temperature for 1 hour, then add 50 μl of FACS buffer to each well before centrifuging again at 2000g for 1 minute. Remove the supernatant before adding a 1:500 dilution of anti-rabbit secondary antibody (Life Technologies ^™ ) and DAPI (Life Technologies ^™ ) conjugated with Alexafluor647 to each well. Incubate the plates at room temperature in the dark for 1 hour, then add 50 μl of FACS buffer and centrifuge at 2000g for 1 minute. After washing, add more FACS buffer, and the plates are ready to be placed in a flow cytometer (BD FACS Canto II) for reading.

FACS data were analyzed using FlowJo software. First, gating was performed on live single cells using forward and side scattering. Second, DAPI+ events were gated, and their number was used as a measure of the number of live nucleated single cells. Finally, phosphoserine 129-α-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 relative to wells treated with only fibrils and without antibodies, and expressed as percentages.

The results are summarized in Figure 13, which shows the ability of the tested antibody to inhibit α-synuclein-induced aggregation on cells expressing α-synuclein. These data confirm that the antibody of the present invention can block α-synuclein-induced aggregation with an _IC50 of less than 5 nM.

The error bar represents the standard error of the measurement (SEM, N=3, n=9).

Example 8: Primary Neuron Aggregation Assay

Hippocampus tissue from E17 mouse embryos was dissected in dissection buffer (calcium and magnesium-free HBSS, 0.6% D-(+)-glucose, 20 mM Hepes). The dissection buffer was then removed and replaced with dissociation solution (calcium and magnesium-free HBSS, 0.6% D-(+)-glucose, 20 mM HEPES, 40 U/m Papain, 1 mg/ml DNase, 1 mM L-cysteine, 0.5 mM EDTA). After incubation at 37°C for 30 min, the dissociation buffer was removed and the hippocampus was washed three times with plating medium (Neurobasal ^™ medium, 2% B27 supplement, 1 mM GlutaMAX, 2.5% FBS, 50 U/ml penicillin-streptomycin). Tissue clumps were homogenized using a 1 ml pipette to obtain a single-cell suspension. Cells were diluted to the appropriate concentration in plating medium. Approximately 15,000 cells were seeded into each well of a 384-well plate coated with PDL. The cells were then maintained in a cell culture incubator at 37°C, 5% _CO2 , and 95% humidity.

The following day, 80% of the medium was replaced with FBS-free plating medium [Neurobasal ^™ medium, 2% B27 supplement, 1 mM GlutaMAX, 50 units/ml penicillin-streptomycin]. Seven days after plating, the medium was removed, leaving 20 μl in each well. 100 nM human α-synuclein fibrils (prepared as described in Example 1 herein) were added to each well, followed by the antibody 6470 (6470 gL 3 g H36 h IgG4P; VR6470 in Figure 14, which contains SEQ ID NO:17 and SEQ ID NO:33) in PBS to be tested (at various concentrations). The plates were incubated in a cell culture incubator at 37°C, 5% _CO2 , and 95% humidity for another 7 days. Fourteen days after plating, the medium was aspirated from all wells, leaving 20 μl/well. Each well was washed with 80 μl of Dulbecco phosphate-buffered saline (DPBS). Remove DPBS and incubate cells for 15 minutes in 40 μl fixation buffer (DPBS with 4% paraformaldehyde)/well. Then, remove the fixation buffer and wash the cells again with 80 μl of DPBS. Remove DPBS and replace with 40 μl permeation buffer (DPBS with 0.1% Triton X-100)/well. After 10 minutes, remove the permeation buffer and incubate cells for 1 hour in 40 μl blocking buffer (PBS with 1% BSA and 0.1% Triton X-100)/well. Then, remove the blocking buffer and replace with 40 μl/well of primary antibody solution (blocking buffer with 0.3% rabbit antiphosphoserine 129α-synuclein antibody (AbCam ^™ ab51253)). Incubate the antibody solution on the cells for 1 hour, followed by three washes (90 μl of PBS each). After the final wash, the PBS was removed and replaced with 40 μl of secondary antibody solution (0.1% anti-rabbit antibody conjugated with AlexaFluor647 in PBS containing 0.2% anti-β-III-tubulin antibody conjugated with AlexaFluor488). The secondary antibody solution was incubated on the cells for 1 hour, then removed and replaced with 40 μl of PBS containing 0.3% CellMask Blue ^™ . After incubation for 5 minutes, the wells were washed three times with 80 μl of PBS, then filled with 50 μl of PBS per well, and the plates were sealed with a clear plastic film.

Flat-panel imaging was performed using an Arrayscan flat-panel imager (ThermoFisher Scientific ^™ ). Images were analyzed using HCS Scan ^™ software from the same manufacturer. Neuronal density was monitored using β-III-tubulin signal. Fields of view that were sparse or showed damaged neuronal cell layers (reflected by a significant reduction in the area of β-III-tubulin signal) were excluded. Finally, the area/field of pSer129α synuclein signal was used to quantify pathological α-synuclein aggregation.

Phosphorylation at S129 of α-synuclein is considered to play an important role in the control of normal α-synuclein function and the regulation of its accumulation, LB formation and neurotoxicity. Under normal conditions, only a small fraction of α-synuclein is constitutively phosphorylated at S129 in the brain (Fujiwara H et al., (2002) Nat Cell Biol, 4, 160-164), while remarkable accumulation of pS129 has been observed in the brains of patients with α-synucleinopathy (Kahle PJ et al., (2000) Ann N Y Acad Sci, 920, 33-41; Okochi M et al., (2000) J Biol Chem, 275, 390-397; Anderson JP et al., (2006) J Biol Chem, 281, 29739-29752).

Data were normalized relative to wells treated with only fibrils and without antibodies, and are expressed as percentages. As shown in Figure 14, 6470gL3gH36 (labeled VR6470) inhibited α-synuclein aggregation induced by α-synuclein fibrils in primary mouse neurons expressing endogenous levels of α-synuclein. Standard error of the mean in the error bar table (SEM, N=4, n=18). These data confirm that 6470gL3gH36 can block α-synuclein fibril-induced aggregation in primary mouse neurons with an _IC50 of less than 4 nM.

Example 9: Evaluation of the efficacy of VR6470 in vivo

The antibody 6470gL3gH36 IgG4P (named VR6470 in this example and containing SEQ ID NO:17 and SEQ ID NO:33) was tested in wild-type male mice C57Bl/6J (Janvier, France) and in a transgenic model of α-synuclein knockout mice expressing human α-synuclein (hereinafter referred to as SNCA-OVX; Charles River, France).

C57Bl/6J and SNCA-OVX mice were injected with 6470 g L3g H36 IgG4P and preformed fibrils (PFFs) of the mouse (prepared as described herein in Example 1). A negative control antibody (101.4) and a loading material were also injected, along with a comparative anti-α-synuclein antibody (comparative C-term Ab) that binds to the last nine C-terminal residues of α-synuclein. This comparative antibody (which has a different CDR than the antibody according to the invention) exhibited binding characteristics comparable to the antibody of the invention. The comparative antibody has a higher affinity for α-synuclein and similar biophysical properties than the antibody of the invention. It was also equally effective in inhibiting α-synuclein aggregation in the HEK cell-based assay according to Example 8 (Table 11).

Table 11

Before direct administration into the brain of animals, the antibody and PFF were pre-incubated together on a shaker at room temperature for 30 minutes. An antibody/PFF mixture was prepared in PBS at a ratio of 1 μg PFF/10 μg antibody. PBS at pH 7.4 was used as the loading solution. A single injection was performed 24 hours prior to intracerebral administration.

The antibody was then administered intraperitoneally to mice at a dose of 30 mg/kg. A second intraperitoneal injection was given 7 days after the first, followed by the same administration regimen (30 mg/kg at a dose of 10 ml/kg, once per week intraperitoneal injection). Wild-type male C57Bl/6J mice underwent a total of 4 injections over 4 weeks, and SNCA-OVX mice underwent a total of 12 injections over 11 weeks. In both experiments, mice were randomly assigned to the treatment group without the researchers' knowledge of the treatment.

Animal experiments were conducted in accordance with European Directive 2010/63/EU and Belgian regulations. The experimental protocol (ASYN-IC-PARKINSON-MO) was approved by the Animal Ethics Committee of UCB Biopharma SPRL (LA1220040 and LA2220363). At the time of surgery, mice weighed between 25 and 30 g and were 17 weeks old. Mice were housed in cages (4 mice/cage, Macrolon 2). They were kept in a 12:12 light/dark cycle, with a light cycle at 06:00. The temperature was maintained at 20–21°C and the humidity at approximately 40%. All animals had free access to standard pelleted food and water before being assigned to experimental groups. Additional nutritional fortification and welfare (Enviro-dri, PharmaServ) were provided before and after surgery. Animal health was monitored daily by animal care personnel. Every effort was made to minimize suffering. Eradication was performed under anesthesia.

The surgery was performed under general anesthesia using a mixture of 50 mg/kg ketamine (Nimatek, Eurovet Animal Health B.V.) and 0.5 mg/kg medemidin (Domitor, Orion Corporation) administered intraperitoneally. Additionally, 2.5 mg/kg atemetic (Antisedan, Orion Corporation) was administered to support awakening. Recombinant purified PFF was thawed and sonicated at room temperature (Qsonica 500–20 kHz; 65% power, 30 pulses, 1 second on, 1 second off, 1 minute). The PFF was then premixed with the antibody for 30 minutes and agitated at room temperature for 30 minutes prior to brain injection. The solution (2 μl) was infused at a rate of 0.2 μl/min, and the needle was left in place for an additional 2.5 minutes before being slowly withdrawn. Inject unilaterally in the right striatum at the following coordinates: AP = +0.20 mm, ML = -2.00 mm, DV = -3.20 mm.

Following anesthesia, mice were infused via cardiac infusion for 9 minutes at a flow rate of 6 ml/min in ice-cold 0.9% PBS containing 10 U/ml heparin through the left ventricle. The right atrium was cut open as the outflow pathway. Subsequently, the animals were infused for 15 minutes at a flow rate of 6 ml/min in ice-cold 4% paraformaldehyde in PBS. The brain was post-fixed overnight at 4°C in PBS containing 4% paraformaldehyde (Day 0). The following morning (Day +1), the 4% paraformaldehyde was discarded, and the brain was washed in cold PBS and incubated overnight. The following day (Day +2), the brain was washed in PBS for at least 1 hour and transferred to PBS containing 15% sucrose and stored at 4°C until transport.

Brain sectioning was performed at Neuroscience Associates (TN, USA). First, the brain was treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freezing artifacts and then embedded in a gelatin matrix using a technique. After solidification, the blocks were rapidly frozen by immersion in isopentane cooled to -70°C with crushed dry ice and placed on the freezing stage of an AO860 microtome. The blocks were sectioned at 40 μm in the coronal plane. All sections were sequentially collected into 24 containers/blocks filled with antigen preservation solution (49% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone). Unstained sections were immediately stored at -20°C.

Free-floating sections were stained by immunohistochemistry with pSer129α-synuclein antibody (mouse anti-α-synuclein (pSer129) biotin – (Wako-010-26481)) diluted 1:30,000. All incubation solutions, starting with blocking serum, used Tris-buffered saline (TBS) with Triton X-100 as the loading medium; all washes were performed with TBS. Endogenous peroxidase activity was blocked by treatment with 0.9% hydrogen peroxide, and nonspecific binding was blocked with 1.26% normal whole serum. After washing, sections were stained overnight with primary antibody at room temperature. The loading solution contained 0.3% Triton X-100 for permeation. After washing, sections were incubated with the anti-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) at room temperature for one hour. After rinsing, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) and 0.0015% hydrogen peroxide to produce visible reaction products, placed on gelatin-coated (with an adhesive layer) slides, air-dried, lightly stained with thionine, dehydrated in alcohol, made clear in xylene, and covered with a coverslip using Permount.

Fluorescent immunohistochemistry and Amytracker (typically used for protein aggregates) staining were performed on floating brain slices for p62/SQSTM1 (p62 is known to co-aggregate in Ruines in humans). VR6470 showed a reduction in the number of aggregated proteins stained with Amytracker and co-localization with pS129. This indicates that the VR6470 antibody reduces not only phosphosynuclein but also synuclein aggregates (data not shown).

Quantification of pSer129α synuclein signal in each field of view was used to quantify pathological α-synuclein aggregations in the ipsilateral striatum, cortex, basolateral amygdala, and substantia nigra. Target regions (ROIs) were manually delineated, and automated quantification of pSer129α synuclein signals in different brain regions was performed using VisioPharm 6 software (VisioPharm). For quantification of pSer129α synuclein signals, a linear Bayesian algorithm was used, providing values for the signal area (labeled area in ^μm² ). The labeled area reflects the amount of pSer129α synuclein pathology covering different brain regions. All quantifications were performed anonymously until the completion of statistical analysis.

Data analysis was performed on the percentage area of the marker (i.e., the ratio between the area of pSer129 signal in ^μm² and the area of the target region in ^μm² ). The percentage area of the marker was repeatedly assessed for multiple brain slices localized in a head-to-tail manner (striatum: 13–14 slices, from +1.1 to -0.94 at the anterior fontanelle; cortex: 13–14 slices, from +1.1 to -0.94; basolateral amygdala: 6–10 slices, from -0.58 to -2.06; substantia nigra: 6–8 slices, from -2.54 to -3.88), and the AUC was calculated separately for each subject tested.

For statistical analysis, consider one-way ANOVA. Multiple pairwise comparisons within the means are then performed in the ANOVA, without any adjustment for multiplicity (with ** for p < 0.01; and * for p < 0.05). The data are log-transformed to meet the criteria for normality and homoscedasticity. The plot shows the geometric mean of the untransformed data.

As shown in Figures 15A and 15B, one month after administration of mouse PFF to male C57Bl/6J wild-type mice (Figure 15A) and three months after administration of human PFF to male SNCA-OVX mice (Figure 15B), the 6470 antibody (corresponding to 6470gL3gH36 IgG4P) significantly reduced α-synuclein pathology (i.e., pSer129α-synuclein signaling) in four different ipsilateral brain regions (including the striatum, cerebral cortex, amygdala, and substantia nigra) compared to the three control groups.

Figure 16 shows the quantification (AUC of % label area) of phosphorylated α-synuclein at Ser129 in the ipsilateral cortex, striatum, amygdala, and substantia nigra of C57Bl/6J wild-type mice. Negative control and comparative C-terminal antibodies did not reduce α-synuclein pathology compared to the feed-treated groups. Conversely, the 6470 antibody significantly reduced the levels of pathology (i.e., pSer129α-synuclein signaling) in the cortex, striatum, amygdala, and substantia nigra compared to the three control groups of C57Bl/6J mice injected with mouse PFF (p < 0.01). When tested in C57Bl/6J wild-type mice, the 6470-treated groups showed significantly reduced levels of pSer129α-synuclein in four different structures (of which, three regions distal to the injection site (cortex, substantia nigra, and amygdala)).

In SNCA OVX mice injected with human PFF, the 6470 antibody significantly reduced the levels of pathological conditions in the cortex and striatum compared to mice that received the loading, the negative control antibody (101.4), and the comparator C-terminal antibody. In SNCA-OVX mice, 6470 showed significantly reduced levels of pSer129 in at least two distinct brain structures (cortex and striatum), at least one of which (cerebral cortex) was located remotely from the injection site.

These results confirm that antibodies incorporating the structural features of the present invention, such as 6470gL3gH36 IgG4P, can prevent the appearance of phosphorylated α-synuclein at Ser129 in vivo.

Furthermore, the results demonstrated that not all antibodies binding to α-synuclein in the C-terminal region were effective in vivo. The comparative antibody (which binds precisely to the C-terminus of α-synuclein with high affinity and is effective in preventing α-synuclein aggregation in cell-based assays) failed to prevent Ser129 phosphorylation in vivo.

Therefore, the antibodies of the present invention can be used to treat α-synucleinosis, such as when characterized by increased Ser129 phosphorylation, including Parkinson's disease (PD) (including idiopathic and hereditary forms of Parkinson's disease), Lloyd dementia (DLB), diffuse Lloyd disease (DLBD), Lloyd variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and type 1 brain iron accumulation neurodegeneration (NBIA-1).

Example 10: Pharmacokinetics of Antibody 6470 in Mice

Male C57/Bl6 mice (n = 3/drug) were injected intravenously with antibody 6470gL3gH36 IgG4P (containing SEQ ID NO: 17 and 33; in Figure 17 and hereinafter referred to as 6470) at a single dose of 2 mg/kg.

Blood samples were collected from the tail vein (0.083, 1, 4, 8, 24, 72, 120, 168, and 336 hours after injection) and allowed to coagulate at room temperature. Serum was separated after centrifugation and then frozen until analysis. Quantification of 6470 was performed by LC-MS/MS. Serum samples from this study were thawed and quantified against calibration lines prepared using 6470 incorporated into control mouse serum at different concentrations or against a comparative antibody. Before injecting samples into the LC-MS/MS system, serum was denatured, reduced, and alkylated using acetonitrile (VWR, UK), TCEP-Tris(2-carboxyethyl)phosphonic acid hydrochloride (Sigma, UK), and iodoacetamide (Sigma, UK), respectively. The alkylated samples were then reconstituted in 100 mM ammonium bicarbonate buffer (Sigma, UK) and digested overnight at 37°C using trypsin (Promega, UK). Digestion was terminated by adding formic acid to the sample to lower the pH, followed by desalting 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 a 95/5 water/acetonitrile solution containing 0.1% formic acid and injected into an LC-MS/MS system. LC-MS/MS analysis was performed using a Schimadzu Prominence HPLC system coupled to an AB Sciex QTrap 6500 triple quadruple mass spectrometer. The digested sample was injected via an autosampler onto a reverse-phase HPLC column (Phenomenex Aeris C18 peptide column, 100 x 2.1 mm, 2.6 μm) maintained at 50 °C. A linear gradient of 5–70% acetonitrile in 0.1% formic acid was applied for 6 min, followed by a ramp-up to 95% acetonitrile in 0.1% formic acid over 0.8 min at a flow rate of 0.6 mL/min. The mass spectrometer was configured to run multiple reaction monitoring analysis to detect multiple transitions of peptides 6470 or 5811 at a sampling time of 50 ms per transition. Data analysis was performed using Analyst software version 1.6.

These data demonstrate that antibody 6470 exhibits very good pharmacokinetic properties in mice (Table 12 and Figure 17A), based on the measured low clearance values. These appear to be superior to the typical range cited for human IgG drugs administered to mice (3–16 ml/day/kg; Deng et al., 2011 mabs 3:1 61–66).

The pharmacokinetic properties of antibody 6470 were also investigated in cynomolgus monkeys and compared with existing antibodies. Male cynomolgus monkeys (n=3 or n=6/drug) were administered a single dose of antibody 6470 gL3gH36 IgG4P (6470) at a dose of 2 or 3 mg/kg and another comparator, an anti-α-synuclein antibody (anti-α-synuclein IgG1 antibody that binds to α-synuclein within amino acid range 118-126; WO2013/063516), via intravenous injection.

Blood samples were acquired at multiple time points (0.083, 1, 3, 6, 24, 48, 96, 168, 240, 336, 504, 576, and 672 hours after injection and allowed to coagulate at room temperature. Serum was separated after centrifugation and then frozen until analysis. Samples were thawed and analyzed using LC/ESI MS/MS. For the 6470, the method described earlier in this embodiment was used, with quantification performed by establishing a standard curve in cynomolgus monkey serum. For the comparator antibody, equine myoglobin was used as an internal standard, and quantification was performed by comparing the signal with the internal standard signal. For preparation, the sample was mixed with the internal standard. The sample was then denatured, alkylated, and thus subjected to overnight enzymatic digestion (trypsin). After digestion, the sample was diluted, and characteristic peptides for all analytes were analyzed by LC-MS/MS. Samples were prepared only once and injected twice (once for each method).

Concentration-time characteristics were analyzed using Pharsight Phoenix 6 through non-compartmental analysis to derive clearance and half-life pharmacokinetic parameters for each individual animal. Mean and standard deviation parameters were recorded for each molecule.

As shown in Figure 17B and Table 12, antibody 6470 also exhibited superior pharmacokinetic properties in cynomolgus monkeys, demonstrating low clearance. Its pharmacokinetic behavior appeared to be superior to the typical range cited for human IgG drugs administered to cynomolgus monkeys (5–12 ml/day/kg; Deng et al., 2011 mabs 3:161–66), as in mice.

The rapid clearance observed in cynomolgus monkeys for the comparative antibody is consistent with published human data (JAMANeurology 2018, 75, 10:1206-14). Antibody 6470 outperformed the comparative antibody in both exposure and clearance compared to the comparative antibody, which exhibited poor, atypical pharmacokinetic characteristics and parameters.

Table 12

sequence list

<110> UCB Biopharma Sprl

<120> Anti-α-synuclein antibody

<130> PF0130-WO

<150> GB1720975.0

<151> 2017-12-15

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 13

<212> PRT

<213> Oryctolagus cuniculus

<400> 1

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

1 5 10

<210> 2

<211> 7

<212> PRT

<213> Hollow Rabbit

<400> 2

Gly Ala Ser Thr Leu Ala Ser

1 5

<210> 3

<211> 12

<212> PRT

<213> Hollow Rabbit

<400> 3

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

1 5 10

<210> 4

<211> 10

<212> PRT

<213> Hollow Rabbit

<400> 4

Gly Ile Asp Leu Ser Ser His Asp Met Tyr

1 5 10

<210> 5

<211> 16

<212> PRT

<213> Hollow Rabbit

<400> 5

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

1 5 10 15

<210> 6

<211> 9

<212> PRT

<213> Hollow Rabbit

<400> 6

Ile His Tyr Gly Asn Ser Gly Gly Leu

1 5

<210> 7

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR-L1 N33R

<400> 7

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

1 5 10

<210> 8

<211> 16

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> CDR-H3 N102H

<400> 9

Ile His Tyr Gly His Ser Gly Gly Leu

1 5

<210> 10

<211> 140

<212> PRT

<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 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

<211> 112

<212> PRT

<213> Hollow Rabbit

<400> 11

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> Hollow Rabbit

<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 acaagttca ctctcaccat cagcgatgtg 240

gtgtgtgacg atgctgccac ttactactgt gcaggatata aaggtggtcg taatgatggt 300

tttgctttcg gcggagggac cgaggtggtg gtcaaa 336

<210> 13

<211> 114

<212> PRT

<213> Hollow Rabbit

<400> 13

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> Hollow Rabbit

<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

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> 6470 gL3 VL

<400> 15

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 Nucleic Acid

<400> 16

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaacaact acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcggggagtg 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> PRT

<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 nucleic acid

<400> 18

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaacaact acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcggggagtg 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 cccaggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc aggggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 19

<211> 112

<212> PRT

<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 Nucleic Acid

<400> 20

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaaccgtt acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcggggagtg 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> PRT

<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 nucleic acid

<400> 22

gacattcaga tgacccagtc cccttcatca ctgtccgcga gcgtgggcga cagagtgacc 60

attacgtgcc aagccagcca gtccgtgtac aagaaccgtt acctggcctg gttccagcaa 120

aagcccggga aggcgccaaa acagcttatc tacggtgcat ccactctcgc ctcggggagtg 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 cccaggaatc cgtcaccgag caggactcca aggacagcac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600

gtgacccacc aggggcctgtc cagccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 23

<211> 117

<212> PRT

<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 Nucleic Acid

<400> 24

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 25

<211> 444

<212> PRT

<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 nucleic acid

<400> 26

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat 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 gcccagagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 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> PRT

<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 Nucleic Acid

<400> 28

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 29

<211> 444

<212> PRT

<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 Nucleic acid

<400> 30

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat 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 gcccagagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 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> PRT

<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 Nucleic Acid

<400> 32

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat attgcgcgcg tattcattat 300

ggtaatagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 33

<211> 444

<212> PRT

<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 nucleic acid

<400> 34

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat 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 gcccagagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 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> PRT

<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 Nucleic Acid

<400> 36

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat attgcgcgcg tattcattat 300

ggtcacagtg gtgggttgtg gggtcagggt actctggtta ccgtctcgag c 351

<210> 37

<211> 444

<212> PRT

<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 Nucleic acid

<400> 38

gaggttcagc tgctggagtc tggaggcggg cttgtccagc ctggagggag cctgcgtctc 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 gcggtttat 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 gcccagagag gaacagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc cagcatcgaa aagaccatct ccaaggccaa gggccagccc 1020

cgcgagcccc aggtgtacac cctgccccct agccaggaag agatgaccaa gaaccaggtg 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> PRT

<213> Artificial Sequence

<220>

<223> Human IGKV1-16 JK4 Recipient Framework

<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 recipient structure nucleic acid

<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> PRT

<213> Artificial Sequence

<220>

<223> Human IGHV3-23 JH4 Recipient Framework

<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 recipient structure nucleic acid

<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> PRT

<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 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> PRT

<213> Artificial Sequence

<220>

<223> CDR-L1 X33

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa is either 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> PRT

<213> Artificial Sequence

<220>

<223> CDR-H2 X56

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> Xaa is either 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> PRT

<213> Artificial Sequence

<220>

<223> CDR-H3 X102

<220>

<221> MISC_FEATURE

<222> (5)..(5)

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

<400> 46

Ile His Tyr Gly Xaa Ser Gly Gly Leu

1 5

<210> 47

<211> 216

<212> PRT

<213> Hollow Rabbit

<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 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 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> PRT

<213> Hollow Rabbit

<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> PRT

<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

210 215 220

His His His

225

Claims (23)

1. An antibody or an antigen-binding fragment thereof that binds to α-synuclein, wherein the antibody comprises: a. Light chain variable region, which includes: i. A CDR-L1 consisting of SEQ ID NO: 1; ii. CDR-L2 consisting of SEQ ID NO: 2; and iii. CDR-L3 consisting of SEQ ID NO: 3; and b. Heavy chain variable region, which includes: i. CDR-H1 consisting of SEQ ID NO: 4; ii. CDR-H2 consisting of SEQ ID NO: 5; and iii. CDR-H3 consisting of SEQ ID NO: 6. 2. The antibody or antigen-binding fragment of claim 1, which binds to α-synuclein at an epitope comprising residues E123, Y125, E126, M127, P128, S129, E130 and E131 as relating to SEQ ID NO: 10. 3. The antibody of claim 2 or its antigen-binding fragment, wherein the epitope further comprises A124 and G132. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof prevents α-synuclein aggregation induced by α-synuclein fibrils. 5. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is capable of binding as a monomer and in the fibrils of α-synuclein. 6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, having a higher binding affinity for α-synuclein in fibrils than for monomeric α-synuclein, characterized in that the dissociation constant ( _KD ) for monomeric α-synuclein is at least 10 times that for α-synuclein in fibrils. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, having a _KD of 300 pM or less for α-synuclein in fibrils. 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein it does not bind to β-synuclein. 9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a full-length antibody. 11. The antibody of claim 10 or an antigen-binding fragment thereof, wherein the full-length antibody is selected from IgG1, IgG4 or IgG4P. 12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ₂ or scFv. 13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises: a. The light chain variable region consisting of SEQ ID NO: 15 and the heavy chain variable region consisting of SEQ ID NO: 31; or b. The light chain consisting of SEQ ID NO: 17 and the heavy chain consisting of SEQ ID NO: 33. 14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises: a. The light chain variable region consisting of SEQ ID NO: 15 and the heavy chain variable region consisting of SEQ ID NO: 23; or b. The light chain consisting of SEQ ID NO: 17 and the heavy chain consisting of SEQ ID NO: 25. 15. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14. 16. The isolated polynucleotide of claim 15, wherein the polynucleotide encodes: a. Light chain variable region, wherein the polynucleotide: i. Composed of SEQ ID NO: 16; and b. Heavy chain variable region, wherein the polynucleotide: i. Composed of SEQ ID NO: 32; or c. The light chain, wherein the polynucleotide is: i. Composed of SEQ ID NO: 18; and d. Heavy chain, wherein the polynucleotide is: i. Composed of SEQ ID NO: 34. 17. A cloning or expression vector comprising one or more polynucleotides according to any one of claims 15 or 16. 18. Host cell, which contains: a. One or more polynucleotides according to any one of claims 15 or 16, or b. One or more expression vectors according to claim 17. 19. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14, comprising culturing a host cell according to claim 18 under conditions suitable for producing the antibody or an antigen-binding fragment thereof and isolating the antibody or the antigen-binding fragment thereof. 20. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14 and one or more pharmaceutically acceptable carriers, excipients or diluents. 21. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 20 in the preparation of a medicament for use in the treatment of one or more synucleinosis disorders, wherein the synucleinosis disorder is selected from Parkinson's disease (PD), Lloyd's dementia (DLB), diffuse Lloyd's disease (DLBD), Lloyd's variant of Alzheimer's disease (LBVAD), combined Alzheimer's disease and Parkinson's disease, multiple system atrophy (MSA), and brain iron accumulation neurodegeneration type 1 (NBIA-1). 22. The use according to claim 21, wherein the synucleinosis is Parkinson's disease. 23. The use according to claim 22, wherein the Parkinson's disease is idiopathic Parkinson's disease or a hereditary form of Parkinson's disease.