Naturally Occurring Autoantibodies Against Alpha-synuclein That Inhibit The Aggregation And Cytotoxicity Of Alpha-synuclein

Abstract

The present invention refers to human antibodies which are directed against .alpha.-Synuclein (.alpha.-Syn) and their use in medicine and diagnosis.

Description

[0001] This application is divisional of Ser. No. 13/582,489, filed Nov. 12, 2012, which is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2011/053188, filed Mar. 3, 2011, which claims the benefit of European Patent Application No. 10155373.3 filed on Mar. 3, 2010, the disclosure of which is incorporated herein in its entirety by reference.

[0002] The present invention refers to human antibodies which are directed against .alpha.-Synuclein (.alpha.-Syn) and their use in medicine and diagnosis.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disorder globally as it affects about 1% of the population over 65 years old worldwide. It is clinically characterized by resting tremor, slowness of movement, muscular rigidity and impairment of postural reflexes. The progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions termed Lewy bodies (LBs) and Lewy neurites are the neuropathological hallmarks of PD.

[0004] .alpha.-Synuclein (.alpha.-Syn) has been identified as the major component of such inclusions and it is found in the brains of PD patients and patients with other degenerative disorders such as the LB variant of Alzheimer's disease, dementia with LBs and both glial and neuronal cytoplasmic inclusions of multiple system atrophy. .alpha.-Syn has become a primary target of interest both because point mutations in the .alpha.-Synuclein gene and dosage effects caused by gene triplication have been linked to familial PD and because over-expression of .alpha.-Syn in neuronal cell lines and transgenic mice has been shown to lead to the formation of similar inclusions.

[0005] .alpha.-Syn consists of 140 amino acids, primarily expressed at presynaptic terminals in the central nervous system. It is divided into three distinct regions. The N-terminal region contains six imperfect repeats of the consensus sequence KGKEGV which may facilitate protein-protein interactions. The central region is known as the non-amyloid component ("NAC region") and may be essential for the aggregation of the peptide. The acidic C-terminal region is most likely responsible for the chaperone function of .alpha.-Syn. Though the specific role of .alpha.-Syn is still unknown, ample evidence suggests that over-expression disturbs normal cell function, resulting in decreased neurite outgrowth and cell adhesion. The mechanism that leads to the accumulation of .alpha.-Syn and subsequent neurodegeneration is still subject to ongoing research. Abnormal accumulation of .alpha.-Syn oligomers in the synaptic terminals and axons is now believed to be a key event in the pathogenesis of PD. Current research is focused on finding new approaches aiming at the reduction of abnormal accumulation of .alpha.-Syn.

[0006] In recent years, one effective approach in reducing neuronal accumulation of .alpha.-Syn aggregates has been immunization. It was hypothesized to have a potential role in the treatment of PD. One group was able to show that active immunization against human .alpha.-Syn resulted in a significant reduction of .alpha.-Syn aggregates in neuronal cell bodies and synapses of immunoresponsive transgenic mice as compared to untreated animals (Masliah et al., 2005). More recently, a human single-chain antibody fragment against oligomeric .alpha.-Syn was isolated from a phage display antibody library. This antibody fragment was able to bind oligomeric forms of .alpha.-Syn and inhibited both aggregation and toxicity of .alpha.-Syn in vitro (Emadi et al., 2007).

[0007] We were able to identify and isolate naturally occurring autoantibodies that bind to .alpha.-Syn (.alpha.-Syn-Abs) from human sera and from commercial IgG preparations (IVIG). These autoantibodies may be involved in the metabolism and clearance of .alpha.-Syn oligomers. Thus, a treatment with .alpha.-Syn-autoantibodies may be a beneficial therapeutic approach for PD patients.

[0008] Thus, a first aspect of the invention is a human antibody which is directed against an epitope between amino acids 60-100, for example between amino acids 60-95, or between amino acids 73-82 and/or between amino acids 91-100, particularly between amino acids 74-79 and/or between amino acids 92-97, of human .alpha.-Synuclein (.alpha.-Syn) or a fragment of such an antibody.

[0009] The antibody is suitable for use in medicine, particularly human medicine, more particularly for the treatment of neurodegenerative disorder such as Parkinson's disease. Furthermore, the antibody is suitable for use as a diagnostic agent, particularly as an agent for the diagnosis of a neurodegenerative disorder, such as Parkinson's disease.

[0010] A further aspect of the invention is a nucleic acid molecule encoding the antibody optionally in operative linkage to an expression control sequence.

[0011] A further aspect of the present invention is a recombinant cell which comprises the nucleic acid molecule. The cell may be used for the preparation of the antibody.

[0012] Still a further aspect of the present invention is a pharmaceutical composition comprising the antibody, the nucleic acid molecule or the recombinant cell together with a pharmaceutically acceptable carrier.

[0013] Still a further aspect of the present invention is a method for the treatment of a neurodegenerative disorder, comprising administering an antibody as described above to a subject, particularly a human subject in need thereof. This subject is suffering from a neurodegenerative disorder, such as Parkinson's disease or in risk of developing a neurodegenerative disorder, such as Parkinson's disease.

[0014] The present invention refers to a human antibody directed against .alpha.-Syn or a fragment thereof. The term "human antibody" encompasses fully human or humanized antibodies. Human antibodies may be prepared from genetically engineered animals, e.g. animals comprising a xenogenic immune system or from antibody display libraries according to known techniques. Humanized antibodies may be prepared by humanization of monoclonal antibodies according to known techniques.

[0015] Preferably, the human antibody of the invention is a naturally occurring human auto-antibody. Such an antibody may be isolated from sera of human donors or from commercial immunoglobulin preparations such as IVIG by immunochromatography with immobilized .alpha.-Syn. A human autoantibody preparation may be heterogeneous or homogenous. A heterogeneous preparation of autoantibodies may comprise a plurality of different autoantibody species. Such a preparation is obtainable by isolation from the sera of human donors, e.g. by immunochromatography as described above. A homogeneous autoantibody preparation may be obtained by recombinant manufacture of a single autoantibody species as herein described in detail below.

[0016] The inventors found that IgG specific for .alpha.-Syn can be isolated from peripheral human blood. Thus, actively anti-.alpha.-Syn IgG secreting cells of the B-cell lineage must be circulating within the blood and lymphatic system. Moreover, B-cells presenting anti-.alpha.-Syn on their cell surface must also be part of the blood B-cell system. Each of those anti-.alpha.-Syn B-cells is producing only one single specific antibody, which is translated from two separate mRNAs: one being the rearranged transcript displaying the antibody heavy chain and the other displaying the light chain. These mRNA molecules contain all the information required for the generation of anti-.alpha.-Syn antibodies. After preparing RNA from B-cells, the mRNA within the sample can be used as substrate for cDNA preparation which then can be used as template for "universal" IgG specific PCR reactions.

[0017] A "universal" but specific PCR can be achieved by choosing up-stream primers within the leader region of the IgG which do not discriminate for the antibody nucleotide sequence amplified, but differ significantly from sequences found in other cDNA than IgG related ones. The downstream primer may be situated at the beginning of the constant domain of the heavy or the light chain. Within these regions, conserved nucleotide sequences can be found, which allow for an immunoglobulin subtype specific--but CDR independent--amplification of the Ig cDNA nucleotide sequence.

[0018] To get access to an optimal IgG mRNA substrate, RNA may be isolated from B-cells derived from blood donation buffy coats. After preparation of the peripheral blood mononuclear cells (PBMC), B-cells specific for .alpha.-Syn may be enriched. Subsequently, the mRNA may be reverse-transcribed into cDNA, e.g. by oligo-dT priming. The cDNA may be used as substrate for PCR. These PCRs may generate fragments of the variable domains of heavy and light chains from various B-cells, thus yielding a mixture of information on these molecules from different cells.

[0019] To be able to generate information on single HC/LC molecules, the PCR products may then be inserted into plasmids and transformed into bacterial cells. Colony-PCR products of the right size may be sequenced and the nucleotide information may be translated into the required amino acid information. Methods for insertion of PCR products into suitable plasmids, transformation of bacterial cells, isolation of plasmids therefrom, and performing colony PCR as well as sequencing reactions are well known in the art.

[0020] The antibodies of the invention may be of various immunoglobulin (Ig) types, for example of the IgA-, IgD-, IgE-, IgG- or IgM-type, preferably of the IgG- or IgM-type including, but not limited to the IgG1-, IgG2-, IgG3-, IgG4-, IgM1- and IgM2-type. In one preferred embodiment, the antibody is of the IgG1-type.

[0021] The term "antibody" particularly refers to molecules comprising at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. Each heavy and light chain may comprise a variable and a constant domain. The antigen-binding site may be formed from the variable domains of a heavy and a light chain. A variable region (also referred to as variable domain) comprises complementarity determining regions (CDRs), e.g. a CDR1, a CDR2 and a CDR3 region, and framework regions (FRs) flanking the CDRs. The term "complementarity determining region" is readily understood by the skilled person (see, for example, Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y., 1988; incorporated herein by reference in its entirety) and refers to the stretches of amino acids within the variable domain of an antibody that primarily make contact with the antigen and determine antibody specificity. This region is also known as the hypervariable region.

[0022] The invention also encompasses fragments of human antibodies, e.g. portions of the above-mentioned antibodies which comprise at least one antigen-binding site. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab').sub.2 fragments, Fv fragments, diabodies or single chain antibody molecules and other fragments as long as they exhibit the desired capability of binding to .alpha.-Syn.

[0023] The term "bind" or "binding" of an antibody means an at least temporary interaction or association with or to a target antigen, e.g. .alpha.-Syn, comprising fragments thereof containing an epitope.

[0024] Preferably, the antibody or the fragment of the invention binds to an epitope on .alpha.-Syn, which is located between amino acid residues 60 and 100 or between amino acid residues 60 and 95 of human .alpha.-Syn (SWISS Prot: P37840/SEQ ID NO:1). More preferably, the antibody binds to an epitope between amino acids 73-82 and/or 91-100 of human .alpha.-Syn. Most preferably, the epitope bound by the antibody or fragment according to the invention is located between amino acids 74-79 and/or 92-97, of human .alpha.-Syn.

[0025] The antibodies of the present invention may bind to monomeric .alpha.-Syn, to aggregated .alpha.-Syn or preferably to both of monomeric and aggregated, e.g. di-, tri- or tetrameric .alpha.-Syn. The antibody may also react with oligomeric, particularly tetrameric .beta.-Syn and/or .gamma.-Syn aggregates.

[0026] In certain embodiments of the present invention, the antibody may comprise specific heavy chain complementarity determining regions CDR1, CDR2, and/or CDR3 as described below. Accordingly, in one embodiment, the .alpha.-Syn antibody comprises a heavy chain (HC) complementarity determining region 1 (CDR1) having the consensus sequence GFTX.sup.1SX.sup.2X.sup.3X.sup.4X.sup.5X.sup.6 (SEQ ID NO.: 27). Within this consensus sequence, X.sup.1 may be F or V, X.sup.2 may be D or S, X.sup.3 may be A, N, or Y, X.sup.4 may be A, G, W, or Y, X.sup.5 may be I or M, and X.sup.6 may be H, N, or S. Preferably, the HC CDR1 has the sequence as shown in any one of SEQ ID NOs.: 28, 29, 30, 31, 32, 33, and 34.

[0027] In a further embodiment, the antibody comprises a heavy chain complementarity determining region 2 (CDR2) having the sequence as shown in any one of SEQ ID NOs.: 35, 36, 37, 38, 39, 40, 41, 42, and 43.

[0028] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64.

[0029] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 1 (CDR1) having the consensus sequence GGSISSGGYXWS (SEQ ID NO.: 65). Within this consensus sequence, X may be S or Y. Preferably, the HC CDR1 has the sequence as shown in any one of SEQ ID NOs.: 66 and 67.

[0030] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 2 (CDR2) having the consensus sequence YIYXSGSTYYNPSLKS (SEQ ID NO.: 68). Within this consensus sequence, X may be H or Y. Preferably, the HC CDR2 has the sequence as shown in any one of SEQ ID NOs.: 69 or 70.

[0031] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 71, 72, 73, 74, and 75.

[0032] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 1 (CDR1) having the sequence as shown in any one of SEQ ID NOs.: 76, 77, 78, 79, and 80.

[0033] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 2 (CDR2) having the consensus sequence X.sup.1IX.sup.2PX.sup.3X.sup.4GX.sup.5X.sup.6X.sup.7YAQKFQG (SEQ ID NO.: 81). Within this consensus sequence, X.sup.1 may be G, I, or W, X.sup.2 may be I, N, or T, X.sup.3 may be I, N, or S, X.sup.4 may be F, G, H, or S, X.sup.5 may be A, G, S or T, X.sup.6 may be A or T, and X.sup.7 may be N or S. Preferably, the HC CDR2 has the sequence as shown in any one of SEQ ID NOs.: 82, 83, 84, and 85.

[0034] In yet a further embodiment, the antibody comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 86, 87, 88, 89, 90, and 91.

[0035] The antibody according to the invention may also comprise specific light chain (LC) complementarity determining regions CDR1, CDR2, and/or CDR3.

[0036] Accordingly, in one embodiment, the antibody comprises a light chain complementarity determining region 1 (CDR1) having the sequence as shown in SEQ ID NO.: 92.

[0037] In a further embodiment, the antibody comprises a light chain complementarity determining region 2 (CDR2) having the sequence as shown in SEQ ID NO.: 93.

[0038] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 3 (CDR3) having the consensus sequence MQALQX.sup.1X.sup.2X.sup.3T (SEQ ID NO.: 94). Within this consensus sequence, X.sup.1 may not be present or may be T, X.sup.2 may be F or P, and X.sup.3 may be R, W, or Y. Preferably, the LC CDR3 has the sequence as shown in any one of SEQ ID NOs.: 95, 96, 97, and 98.

[0039] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 1 (CDR1) having the consensus sequence RASQSVSSX.sup.1X.sup.2LA (SEQ ID NO.: 99). Within this consensus sequence, X.sup.1 may not be present or may be S, and X.sup.2 may be N or Y. Preferably, the LC CDR1 has the sequence as shown in any one of SEQ ID NOs.: 100, 101, and 102.

[0040] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 2 (CDR2) having the consensus sequence VASX.sup.2RAT (SEQ ID NO.: 103). Within this consensus sequence, X.sup.1 may be D or G, and X.sup.2 may be N, S, or T. Preferably, the LC CDR2 has the sequence as shown in any one of SEQ ID NOs.: 104, 105, 106.

[0041] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 107, 108, 109, 110, 111, 112, 113, 114, 145, 146, and 147.

[0042] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 1 (CDR1) having the consensus sequence RX.sup.1SQX.sup.2IX.sup.3X.sup.4X.sup.5X.sup.6 (SEQ ID NO.: 115).

[0043] Within this consensus sequence, X.sup.1 may be A or M, X.sup.2 may be G or S, may be R or S, X.sup.4 may be N or S, X.sup.5 may be D, W, or Y, and X.sup.6 may be A or G. Preferably, the LC CDR1 has the sequence as shown in any one of SEQ ID NOs.: 116, 117, 118, 119, 120, 121, and 122.

[0044] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 2 (CDR2) having the sequence as shown in any one of SEQ ID NOs.: 123, 124, 125, 126, and 127.

[0045] In yet a further embodiment, the antibody comprises a light chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 and 144.

[0046] The antibody of the present invention may preferably comprise a specific combination of CDRs (i.e. of CDR1, CDR2, and CDR3) within one heavy chain.

[0047] Accordingly, in one preferred embodiment, the antibody comprises a heavy chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 28, 29, 30, 31, 32, 33, and 34, CDR2 is selected from the sequences shown in SEQ ID NOs.: 35, 36, 37, 38, 39, 40, 41, 42, and 43, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64.

[0048] In a further preferred embodiment, the HC CDR1 is selected from the sequences shown in SEQ ID NOs.: 66 and 67, HC CDR2 is selected from the sequences shown in SEQ ID NOs.: 69 and 70, and HC CDR3 is selected from the sequences shown in SEQ ID NOs.: 71, 72, 73, 74, and 75.

[0049] In a further preferred embodiment, the HC CDR1 is selected from the sequences shown in SEQ ID NOs.: 76, 77, 78, 79, and 80, HC CDR2 is selected from the sequences shown in SEQ ID NOs.: 82, 83, 84, and 85, and HC CDR3 is selected from the sequences shown in SEQ ID NOs.: 86, 87, 88, 89, 90, and 91.

[0050] Most preferably, the antibody of the invention comprises a heavy chain comprising three CDRs, wherein the combination of CDR1, CDR2, and CDR3 is selected from those shown in Table 1, Table 2 and Table 3. It is understood that each line of each of these Tables represents one specific combination of a CDR1, a CDR2, and a CDR3.

TABLE-US-00001 TABLE 1 Specific CDR combinations CDR1 CDR2 CDR3 GFTFSSYGMH VIWYDGSNKYYADSVKG DWGIVDTAMVPY (SEQ ID NO: 32) (SEQ ID NO: 39) YYYYGMDV (SEQ ID NO: 50) GFTFSSYGMH VIWYDGSNKYYADSVKG DRRGIAATAGYY (SEQ ID NO: 32) (SEQ ID NO: 39) YGMDV (SEQ ID NO: 49) GFTFSSYGMH VIWYDGSNKYYADSVKG DRGFGYCSSTSC (SEQ ID NO: 32) (SEQ ID NO: 39) HTEDAFDI (SEQ ID NO: 47) GFTFSSYGMH VISYDGSNKYYADSVKG ERYYYMDV (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 53) GFTFSSYGMH VISYDGSNKYYADSVKG QDIAAAAPYYFDY (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 60) GFTFSSYGMH VISYDGSNKYYADSVKG AMVRGVTKPFDY (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 44) GFTFSSYGMH VISYDGSNKYYADSVKG GGDYYDSSGYYLPWY (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 54) GFTFSSYGMH VISYDGSNKYYADSVKG DLVDYDSSGYYPDY (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 46) GFTFSSYGMH AISGSGGSTYYADSVKG AYYYYDSSGYGY (SEQ ID NO: 32) (SEQ ID NO: 35) (SEQ ID NO: 45) GFTFSSYAMH VISYDGSNKYYAD EAPSSGWYPYY (SEQ ID NO: 30) SVKG YYMDV (SEQ ID NO: 40) (SEQ ID NO: 51) GFTFSSYAMH VISYDGSNKYYADSVKG YCSSTSCSSEYFGH (SEQ ID NO: 30) (SEQ ID NO: 40) (SEQ ID NO: 63) GFTFSSYAMH VISYDGSNKYYADSVKG GVVPAAESWFDP (SEQ ID NO: 30) (SEQ ID NO: 40) (SEQ ID NO: 57) GFTFSSYAMH VISYDGSNKYYADSVKG QDIAAAAPYYFDY (SEQ ID NO: 30) (SEQ ID NO: 40) (SEQ ID NO: 60) GFTFSSYAMH VISYDGSNKYYADSVKG YYYDSSAVEGDAFDI (SEQ ID NO: 30) (SEQ ID NO: 40) (SEQ ID NO: 64) GFTFSSYAMS AISGSGGSTYYA DWGIVDTAMVPY (SEQ ID NO: 31) DSVKG YYYGMDV (SEQ ID NO: 35) (SEQ ID NO: 50) GFTFSSYAMS AISGSGGSTYY DRRGIAATAG (SEQ ID NO: 31) ADSVKG YYYGMDV (SEQ ID NO: 35) (SEQ ID NO: 49) GFTFSSYAMS AISGSGGSTY DRHPGYCSSTS (SEQ ID NO: 31) YADSVKG CFVRYFDY (SEQ ID NO: 35) (SEQ ID NO: 48) GFTFSSYAMS AISGSGGSTYYADSVKG GGDYYDSSGYYLPWY (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 54) GFTFSSYAMS AISGSGGSTYYADSVKG KTYYYYDSSGYGY (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 59) GFTFSSYAMS AISGSGGSTYYADSVKG QDIAAAAPYYFDY (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 60) GFTFSSYAMS AISGSGGSTYYADSVKG SGASLRAFDI (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 61) GFTFSSYAMS AISGSGGSTYYADSVKG SGYYYPLDY (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 62) GFTFSSYWMS NIKQDGSEKYYV EHRGGYYDILTG (SEQ ID NO: 33) DSVKG YTKHGGSNDY (SEQ ID NO: 37) (SEQ ID NO: 52) GFTFSSYWMS NIKQDGSEKYYVDSVKG DLVDYDSSGYYPDY (SEQ ID NO: 33) (SEQ ID NO: 37) (SEQ ID NO: 46) GFTFSSYWMS NIKQDGSEKYYV GTDTESVAAPY (SEQ ID NO: 33) DSVKG YYYMDV (SEQ ID NO: 37) (SEQ ID NO: 55) GFTFSSYWMS NIKQDGSEKYYADSVKG ERYYYMDV (SEQ ID NO: 33) (SEQ ID NO: 36) (SEQ ID NO 53) GFTFSDYYMS YISSSGGTIYYADSVKG GVAGRFDY (SEQ ID NO: 29) (SEQ ID NO: 42) (SEQ ID NO: 56) GFTFSDYYMS YISSSSSYTNYADSVKG YYYDSSAVEGDAFDI (SEQ ID NO: 29) (SEQ ID NO: 43) (SEQ ID NO: 64) GFTFSDAWIN RIKSKTDGGTTDY KDGSGSYYHY (SEQ ID NO: 28) AAPVKG YYYVMDV (SEQ ID NO: 38) (SEQ ID NO: 58) GFTVSSNYMS VIYSGGSTYYADSVKG SGASLRAFDI (SEQ ID NO: 34) (SEQ ID NO: 41) (SEQ ID NO: 61)

TABLE-US-00002 TABLE 2 Specific CDR combinations CDR1 CDR2 CDR3 GGSISSGGYSWS YIYHSGSTYYNPSLKS GTEYCTNGACYMG (SEQ ID NO: 66) (SEQ ID NO: 69) YYYYYMDV (SEQ ID NO: 74) GGSISSGGYSWS YIYHSGSTYYNPSLKS GTEYCTNGVCYMG (SEQ ID NO: 66) (SEQ ID NO: 69) YYYYYMDV (SEQ ID NO: 75) GGSISSGGYSWS YIYHSGSTYYNPSLKS AGYYYYYMDV (SEQ ID NO: 66) (SEQ ID NO: 69) (SEQ ID NO: 71) GGSISSGGYSWS YIYHSGSTYYNPSLKS AHPVRGSGSYYNR (SEQ ID NO: 66) (SEQ ID NO: 69) NYYYYYMDV (SEQ ID NO: 72) GGSISSGGYYWS YIYYSGSTYYNPSLKS GSREGYGDRIDY (SEQ ID NO: 67) (SEQ ID NO: 70) (SEQ ID NO: 73) GGSISSGGYYWS YIYYSGSTYYNPSLKS GTEYCTNGVCYMG (SEQ ID NO: 67) (SEQ ID NO: 70) YYYYYMDV (SEQ ID NO: 75)

TABLE-US-00003 TABLE 3 Specific CDR combinations CDR1 CDR2 CDR3 GYTFTGYYMH WINPNSGGTNYAQKFQG DSGSSGWYVPYWYFDL (SEQ ID NO: 79) (SEQ ID NO: 85) (SEQ ID NO: 88) GYTFTGYYMH WINPNSGGTNYAQKFQG PIGGGPSGWYE (SEQ ID NO: 79) (SEQ ID NO: 85) TSCFDP (SEQ ID NO: 89) GYTFTGYYMH WINPNSGGTNYAQKFQG AKDYDFWRGSTG (SEQ ID NO: 79) (SEQ ID NO: 85) MRYLDV (SEQ ID NO: 86) GYTFTGYYMH WINPNSGGTNYAQKFQG DKRCSSTSCQPYY (SEQ ID NO: 79) (SEQ ID NO: 85) YYYMDV (SEQ ID NO: 87) GYTFTGYYMH WINPNSGGTNYAQKFQG TSYGDSSSSSYY (SEQ ID NO: 79) (SEQ ID NO: 85) YYYGMDV (SEQ ID NO: 90) GYTFTSYYMH IINPSGGSTSYAQKFQG DSGSSGWYVPYWYFDL (SEQ ID NO: 80) (SEQ ID NO: 83) (SEQ ID NO: 88) GYIITNYYIH IITPSHGATNYAQKFQG AKDYDFWRGST (SEQ ID NO: 78) (SEQ ID NO: 84) GMRYLDV (SEQ ID NO: 86) GYIIANYYIH IITPSHGATNYAQKFQG AKDYDFWRGST (SEQ ID NO: 77) (SEQ ID NO: 84) GMRYLDV (SEQ ID NO: 86) GGTFSSYAIS GIIPIFGTANYAQKFQG VDYSNYVVDY (SEQ ID NO: 76) (SEQ ID NO: 82) (SEQ ID NO: 91)

[0051] According to the present invention, it is further preferred that the antibody comprises a specific combination of CDRs (i.e. of CDR1, CDR2, and CDR3) within one light chain.

[0052] Thus, in one preferred embodiment, the antibody comprises a light chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein LC CDR1 has the sequence shown in SEQ ID NO.: 92, LC CDR2 has the sequence shown in SEQ ID NO.: 93, and LC CDR3 is selected from the sequences shown in SEQ ID NOs.: 95, 96, 97, and 98.

[0053] In a further preferred embodiment, the LC CDR1 is selected from the sequences shown in SEQ ID NOs.: 100, 101, and 102, LC CDR2 is selected from the sequences shown in SEQ ID NOs.: 104, 105, and 106, and LC CDR3 is selected from the sequences shown in SEQ ID NOs.: 107, 108, 109, 110, 111, 112, 113, 114, 145, 146, and 147.

[0054] In a further preferred embodiment, the LC CDR1 is selected from the sequences shown in SEQ ID NOs.: 116, 117, 118, 119, 120, 121, and 122, LC CDR2 is selected from the sequences shown in SEQ ID NOs.: 123, 124, 125, 126, and 127, and LC CDR3 is selected from the sequences shown in SEQ ID NOs.: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, and 144.

[0055] Most preferably, the antibody of the invention comprises a light chain comprising three CDRs, wherein the combination of CDR1, CDR2, and CDR3 is selected from those shown in Table 4, Table 5 and Table 6. It is understood that each line of each of these Tables represents one specific combination of a CDR1, a CDR2, and a CDR3.

TABLE-US-00004 TABLE 4 Specific CDR combinations CDR1 CDR2 CDR3 RSSQSLLHSNGYNYLD LGSNRAS MQALQTPYT (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 97) RSSQSLLHSNGYNYLD LGSNRAS MQALQTPWT (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 96) RSSQSLLHSNGYNYLD LGSNRAS MQALQTPRT (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 95) RSSQSLLHSNGYNYLD LGSNRAS MQATQFRT (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 98)

TABLE-US-00005 TABLE 5 Specific CDR combinations CDR1 CDR2 CDR3 RASQSVSSSYLA GASSRAT QQYGSSWT (SEQ ID NO: 101) (SEQ ID NO: 105) (SEQ ID NO: 109) RASQSVSSYLA DASNRAT QQRSNWPPYT (SEQ ID NO: 102) (SEQ ID NO: 104) (SEQ ID NO: 108) RASQSVSSYLA DASNRAT QQRSNWPPT (SEQ ID NO: 102) (SEQ ID NO: 104) (SEQ ID NO: 107) RASQSVSSNLA GASTRAT QQYNNWYT (SEQ ID NO: 100) (SEQ ID NO 106) (SEQ ID NO: 114) RASQSVSSNLA GASTRAT QQYNNWWT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 113) RASQSVSSNLA GASTRAT QQYNNWPRT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 112) RASQSVSSNLA GASTRAT QQYNNWPLT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 110) RASQSVSSNLA GASTRAT QQYNNWPPMYT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 111) RASQSVSSNLA GASTRAT QQYGSSPRT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 147) RASQSVSSNLA GASTRAT QQRSNWPPYT (SEQ ID NO: 100) (SEQ ID NO: 106) (SEQ ID NO: 108)

TABLE-US-00006 TABLE 6 Specific CDR combinations CDR1 CDR2 CDR3 RASQGISNYLA AASSLQS QQYNSYPVT (SEQ ID NO: 117) (SEQ ID NO: 123) (SEQ ID NO: 137) RASQGISNYLA AASSLQS QQYNSYPYT (SEQ ID NO: 117) (SEQ ID NO: 123) (SEQ ID NO: 139) RASQGISNYLA AASSLQS QQYNSYPWT (SEQ ID NO: 117) (SEQ ID NO: 123) (SEQ ID NO: 138) RASQGISNYLA AASSLQS LQHNSYPFT (SEQ ID NO: 117) (SEQ ID NO: 123) (SEQ ID NO: 131) RASQGISNYLA AASSLQS LQHNSYPVT (SEQ ID NO: 117) (SEQ ID NO: 123) (SEQ ID NO: 132) RASQGISSYLA AASTLQS QQLNSYPLFT (SEQ ID NO: 119) (SEQ ID NO: 124) (SEQ ID NO: 135) RASQGISSWLA AASSLQS LQDYNYPYT (SEQ ID NO: 118) (SEQ ID NO: 123) (SEQ ID NO: 130) RASQGISSWLA AASSLQS QQANSFPIT (SEQ ID NO: 118) (SEQ. ID NO: 123) (SEQ ID NO: 133) RASQGISSWLA AASSLQS QQYNSYPVT (SEQ ID NO: 118) (SEQ ID NO: 123) (SEQ ID NO: 137) RMSQGISSWLA AASSLQS QQANSFPLT (SEQ ID NO: 121) (SEQ ID NO: 124) (SEQ ID NO: 134) RMSQGISSYLA AASSLQS QQANSFPLT (SEQ ID NO: 122) (SEQ ID NO: 124) (SEQ ID NO: 134) RASQSISSWLA KASSLES QQYNSYSRKYT (SEQ ID NO: 120) (SEQ ID NO: 127) (SEQ ID NO: 140) RASQGIRNDLG AASSLQS LGDYNYPYT (SEQ ID NO: 116) (SEQ ID NO: 123) (SEQ ID NO: 128) RASQGIRNDLG AASSLQS LQHNSYPFT (SEQ ID NO: 116) (SEQ ID NO: 123) (SEQ ID NO: 131) RASQGIRNDLG AASTLVS LQDNNYPRT (SEQ ID NO: 116) (SEQ ID NO: 125) (SEQ ID NO: 129) RASQGIRNDLG DASNLET QQYDNLPPFT (SEQ ID NO: 116) (SEQ ID NO: 126) (SEQ ID NO: 136)

[0056] As described above, the complementarity determining regions (CDRs) of an antibody may be flanked by framework regions (FRs). A heavy or light chain of an antibody containing three CDRs contains e.g. four FRs.

[0057] In one embodiment, CDRs 1, 2, and 3 of the heavy chain of an inventive antibody are flanked by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 3, 4, 5, and 6. An antibody chain containing these FRs is sometimes referred to herein as HC.sub.v Type 1. According to the invention, the HC CDRs may also be flanked by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 7, 8, 9, and 10 (sometimes referred to as HC.sub.v Type 2), or, in another embodiment, by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 11, 12, 13, and 14 (sometimes referred to as HC.sub.v Type 3).

[0058] Similarly, the CDRs of the light chain may be flanked by FRs. In one embodiment, LC CDRs 1, 2 and 3 are flanked by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 15, 16, 17, and 18, or, in another embodiment, by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 19, 20, 21, and 22. In yet another embodiment, LC CDRs are flanked by four FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 23, 24, 25, and 26.

[0059] Variants of these framework regions are also within the scope of the present invention. In particular, FRs may contain amino acid substitutions at specific positions. For example, in HC.sub.v Type 1 FR1 (SEQ ID NO.: 3), the amino acid (aa) at position 1 may also be E or G instead of the Q shown in the consensus sequence. Further possible substitutions include those at positions 5 (L instead of V, abbreviated V>L), 10 (G>D), 11 (V>L), 13 (Q>K), 16 (R>G), 19 (R>G), and 23 (A>V) of SEQ ID NO.: 3.

[0060] In HC.sub.v Type 1 FR2 (SEQ ID NO.: 4), possible aa substitutions include those at positions 1 (W>R), 2 (V>I), and 14 (A>G/S; i.e. A can be replaced by G or S) of SEQ ID NO.: 4.

[0061] In HC.sub.v Type 1 FR3 (SEQ ID NO.: 5), possible aa substitutions include those at positions 8 (N>D), 9 (A>T), 12 (S>A), and 22 (T>S) of SEQ ID NO.: 5.

[0062] In HC.sub.v Type 1 FR4 (SEQ ID NO.: 6), possible aa substitutions include those at positions 1 (W>V), 3 (Q>K), 4 (G>E), 6 (T>L/M), 8 (T>N), 25 (S>C), 27(K>R), 31 (G>E), and 32 (G>S) of SEQ ID NO.: 6.

[0063] Some amino acid substitutions may be linked, e.g. substitutions at positions 11, 13, and 16 of SEQ ID NO: 3, substitutions at positions 8 and 22 of SEQ ID NO: 5, substitutions at positions 9 and 12 of SEQ ID NO: 5, substitutions at positions 25 and 27 of SEQ ID NO: 6, or substitutions at positions 31 and 32 of SEQ ID NO: 6.

[0064] In HC.sub.v Type 2 FR1 (SEQ ID NO.: 7), possible aa substitutions include those at positions 2 (L>V), 9 (S>P), and 23 (A>T) of SEQ ID NO.: 7. The substitutions at positions 2 and 9 may be linked.

[0065] In HC.sub.v Type 2 FR2 (SEQ ID NO.: 8), e.g. the amino acid at position 6 may be substituted (P>H).

[0066] In HC.sub.v Type 2 FR3 (SEQ ID NO.: 9), possible aa substitutions include those at positions 8 (R>T) and 16 (K>R) of SEQ ID NO.: 9.

[0067] In HC.sub.v Type 2 FR4 (SEQ ID NO.: 10), possible aa substitutions include those at positions 3 (K>Q), 6 (T>L), 25 (S>C) and 27 (K>R) of SEQ ID NO.: 10. The substitutions at positions 3, 6 and 25 may be linked.

[0068] In HC.sub.v Type 3 FR1 (SEQ ID NO.: 11), e.g. the amino acid at position 16 may be substituted (A>S).

[0069] In HC.sub.v Type 3 FR3 (SEQ ID NO.: 13), possible aa substitutions include those at positions 1 (W>R), 4 (M>I), 6 (R>A), 8 (T>E), 10 (I>T), 13 (A>V), 16 (E>H), 19 (R>S), 23 (D>E) and 32 (R>T) of SEQ ID NO.: 13. The substitutions at positions 6, 8, 10, 19, and 23, or substitutions at positions 6, 10, 13, 16, 19, and 23 may be linked.

[0070] In HC.sub.v Type 3 FR4 (SEQ ID NO.: 14), possible aa substitutions include those at positions 3 (R>P/K/Q), 6 (L>T), 25 (C>S), 27 (R>K), 29 (T>A), 31 (E>G), 32 (S>G), 48 (V>G), 49 (T>R), and 50 (V>G) of SEQ ID NO.: 14. The substitutions at positions 31 and 32, or substitutions at positions 48 and 49 may be linked.

[0071] In LC.sub.v Type 1 FR1 (SEQ ID NO.: 15), e.g. the amino acid at position 7 may be substituted (S>T).

[0072] In LC.sub.v Type 1 FR3 (SEQ ID NO.: 17), e.g. the amino acid at position 20 may be substituted (S>G).

[0073] In LC.sub.v Type 1 FR4 (SEQ ID NO.: 18), possible aa substitutions include those at positions 6 (K>R), 7 (L>V), 9 (I>S) and 14 (A>T) of SEQ ID NO.: 18.

[0074] In LC.sub.v Type 2 FR1 (SEQ ID NO.: 19), possible aa substitutions include those at positions 1 (E>K), 4 (L>M), 9 (G>A), 13 (L>V) and 22 (S>P) of SEQ ID NO.: 19. The substitutions at positions 4 and 13 may be linked.

[0075] In LC.sub.v Type 2 FR3 (SEQ ID NO.: 21), possible aa substitutions include those at positions 4 (D>A), 14 (D>E), 18 (T>I), 21 (R>S), 23 (E>Q), 24 (P>S), and 25 (E>K) of SEQ ID NO.: 21. The substitutions at positions 23 and 24 may be linked.

[0076] In LC.sub.v Type 2 FR4 (SEQ ID NO.: 22), possible aa substitutions include those at positions 3 (Q>G), 6 (K>R), and 7 (V>L) of SEQ ID NO.:22. The substitutions at positions 6 and 7 may be linked.

[0077] In LC.sub.v Type 3 FR1 (SEQ ID NO.: 23), possible aa substitutions include those at positions 1 (D>A/V), 3 (Q>W), 5 (T>A), 10 (S>T/L), 11 (L>V), and 22 (T>S) of SEQ ID NO.: 23.

[0078] In LC.sub.v Type 3 FR2 (SEQ ID NO.: 24), possible aa substitutions include those at positions 2 (F>Y), 5 (K>R), 7 (G>R), and 12 (S>L/R) of SEQ ID NO.: 24.

[0079] In LC.sub.v Type 3 FR3 (SEQ ID NO.: 25), possible aa substitutions include those at positions 5 (K>R), 14 (D>E), 17 (L>F), 20 (S>T), 21 (S>T), 25 (E>D), 27 (F>I/S), and 29 (T>N) of SEQ ID NO.: 25. The substitutions at positions 20 and 21 may be linked.

[0080] In LC.sub.v Type 3 FR4 (SEQ ID NO.: 26), possible aa substitutions include those at positions 3 (Q>P), 7 (L>V), and 8 (E>D) of SEQ ID NO.: 26.

[0081] Preferably, the framework regions of either the heavy or the light chain show a maximum exchange of two amino acids, one amino acid, or are identical to the consensus sequence.

[0082] It is particularly preferred that the antibody of the invention comprises a heavy chain comprising a specific combination of framework regions and complementarity determining regions.

[0083] Thus, in one embodiment of the invention, said antibody heavy chain comprises FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 3, 4, 5, and 6 (i.e. 3-6) or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 28, 29, 30, 31, 32, 32, 33, and 34 (28-34), CDR2 is selected from the sequences shown in SEQ ID NOs.: 35, 36, 37, 38, 39, 40, 41, 42, and 43 (35-43), and CDR3 is selected from the sequences shown in SEQ ID NOs.: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64 (44-64).

[0084] In another embodiment, said antibody heavy chain comprises FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 7, 8, 9, and 10 (7-10) or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 66 and 67, CDR2 is selected from the sequences shown in SEQ ID NOs.: 69 and 70, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 71, 72, 73, 74, and 75 (71-75).

[0085] In yet another embodiment, said antibody heavy chain comprises FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 11, 12, 13, and 14 (11-14) or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 76, 77, 78, 79, and 80 (76-80), CDR2 is selected from the sequences shown in SEQ ID NOs.: 82, 83, 84, and 85 (82-85), and CDR3 is selected from the sequences shown in SEQ ID NOs.: 86, 87, 88, 89, 90, and 91 (86-91).

[0086] In a most preferred embodiment, the antibody heavy chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 3, 4, 5, and 6 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 1, where each line of the Table represents one specific CDR1, 2, 3 combination.

[0087] In a further most preferred embodiment, the antibody heavy chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 7, 8, 9, and 10 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 2.

[0088] In yet a further most preferred embodiment, the antibody heavy chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 11, 12, 13, and 14 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 3.

[0089] Likewise, it is particularly preferred that the antibody of the invention comprises a light chain comprising a specific combination of FRs and CDRs. Accordingly, in one embodiment of the invention, said antibody light chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 15, 16, 17, and 18 (15-18) or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 has the sequence shown in SEQ ID NO.: 92, CDR2 has the sequence shown in SEQ ID NO.: 93, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 95, 96, 97, and 98 (95-98).

[0090] In another embodiment, said antibody light chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 19, 20, 21, and 22, or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 100, 101, and 102, CDR2 is selected from the sequences shown in SEQ ID NOs.: 104, 105, and 106, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 107, 108, 109, 110, 111, 112, 113, 114, 145, 146, and 147.

[0091] In another embodiment, said antibody light chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 23, 24, 25, and 26 or variants thereof, and further comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 116, 117, 118, 119, 120, 121, and 122, CDR2 is selected from the sequences shown in SEQ ID NOs.: 123, 124, 125, 126, and 127, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, and 144.

[0092] In a most preferred embodiment, the antibody light chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 15, 16, 17, and 18 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 4, where each line of the Table represents one specific CDR1, 2, 3 combination.

[0093] In a further most preferred embodiment, the antibody light chain comprises FRs consisting of a consensus sequence comprising SEQ ID NOs.: 19, 20, 21, and 22 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 5.

[0094] In yet a further most preferred embodiment, the antibody light chain comprises FRs consisting of a consensus sequence as shown in SEQ ID NOs.: 23, 24, 25, and 26 or variants thereof as defined supra, and further comprises a specific combination of CDR1, CDR2, and CDR3 as shown in Table 6.

[0095] In a further preferred embodiment, the antibody of the present invention is characterized by a light chain sequence (SEQ ID NO:2) or a variant thereof:

TABLE-US-00007 1 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY 51 GASSRATGIP DRFSGSGSGT DFTLTISSLQ SEDFATYYCR LTEEKGWMYL 101 GYTFGQGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA 151 KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC 201 EVTHQGLSSP VTKSFNRGEC

[0096] The light chain sequence above comprises a constant domain (aa111-220) and a variable domain (aa1-110). The variable domain comprises Framework (FR) and CDR sequences. The CDR sequences are located from aa23-35 (LCDR1), aa51-57 (LCDR2) and aa90-102 (LCDR3).

[0097] The term "variant" as used hereinabove, particularly includes amino acid sequences which differ from the indicated sequence by partial or complete deletion of the constant domain and/or by partial or complete exchange of FR sequences. Further, the term "variant" also includes amino acid sequences which differ from the indicated CDR sequences by substitution, deletion or addition of one or two amino acids, preferably by substitution, deletion or addition of one amino acid.

[0098] The antibody of the present invention may be coupled to a heterologous group, e.g. an effector group. Such an antibody conjugate is especially suitable for therapeutic applications. The term "effector group" may refer to a cytotoxic group, such as a radioisotope or radionuclide, a toxin, a therapeutic group or another effector group known in the art. Alternatively, the antibody of the invention may be coupled to a labelling group. Such an antibody conjugate is particularly suitable for diagnostic applications. As used herein, the term "labelling group" refers to a detectable marker, e.g. a radiolabelled amino acid or biotinyl moiety, a fluorescent marker, an enzyme or any other type of marker which is known in the art.

[0099] The antibody of the present invention is suitable for use in medicine, particularly for use in human medicine. The antibody may be used in the treatment of a neurodegenerative disorder, which deposits .alpha.-Syn, for example Parkinson's disease or Dementia with Lewy bodies (DLB). More preferably, the disorder is Parkinson's disease. The treatment may comprise a passive immune therapy thereby reducing and/or inhibiting detrimental effects of .alpha.-Syn aggregate formation in the nervous system, particularly in the central nervous system of the subject to be treated. These detrimental effects may include cytotoxicity, particularly neurotoxicity.

[0100] Furthermore, the antibody of the invention may be used as a diagnostic agent, for example for the diagnosis of neurodegenerative disorders, such as PD or DLB. More preferably, the antibody of the invention may be used as a diagnostic agent for PD.

[0101] The invention also refers to a nucleic acid molecule encoding the antibody as described above. The term "nucleic acid molecule" encompasses DNA, e.g. single- or double-stranded DNA, or RNA. The DNA may be of genomic, cDNA or synthetic origin, or a combination thereof. The nucleic acid molecule of the invention may be in operative linkage to an expression control sequence, i.e. to a sequence which is necessary to effect the expression of coding nucleic acid sequences. Such expression control sequences may include promoters, enhancers, ribosomal binding sites and/or transcription termination sequences. Specific examples of suitable expression control sequences are known in the art.

[0102] The nucleic acid molecule of the invention may be located on a vector which may additionally contain a replication origin and/or a selection marker gene. Examples of vectors are e.g. plasmids, cosmids, phages, viruses etc.

[0103] Further, the invention refers to a recombinant cell, which comprises the nucleic acid molecule as described above. The nucleic acid molecule may be introduced into the recombinant cell by transformation, transfection or transduction according to any method known in the art. The recombinant cell may e.g. be a prokaryotic or eukaryotic cell. Preferably, the cell is a mammalian cell, e.g. a hamster, rabbit, or human cell. Preferably, the cell is a human cell.

[0104] The antibody of the invention may be prepared by a method, wherein the cell as described above is cultured under conditions which allow expression of the antibody encoding nucleic acid molecule. The antibody may be collected from the cultured cell or the culture supernatant. Preferably, the antibody is prepared from a mammalian, particularly from a human cell.

[0105] Still a further aspect of the present invention relates to a pharmaceutical composition comprising the antibody, the nucleic acid molecule or the recombinant cell as described above together with a pharmaceutically acceptable carrier. The term "carrier" includes agents, e.g. diluents, stabilizers, adjuvants or other types of excipients that are non-toxic to the cell or mammal to be exposed thereto at the dosages and concentrations employed. Often, the pharmaceutically acceptable carrier is an aqueous pH buffered solution, which is useful for drug delivery, particularly for the delivery of antibody molecules. The pharmaceutical composition may be formulated by mixing the active agent with carriers and optionally other agents that are usually incorporated into the formulation. For example, the composition may be formulated in the form of lyophilized formulations, aqueous solutions, dispersions or solid preparations.

[0106] The present invention also encompasses the administration of the pharmaceutical composition to a subject in need thereof, particularly a human patient suffering from a neurodegenerative disorder, such as Parkinson's disease or DLB. Depending on the type and the severity of the condition to be treated about 1 .mu.g/kg to 15 mg/kg of the active ingredient may be administered to a patient in need thereof, e.g. by one or more separate administrations or by continuous infusion. A typical daily dosage might range from about 1 .mu.g/kg to about 100 mg/kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition to be treated, the treatment is sustained until a desired suppression of the disease or the symptoms occurs. The composition may be administered by any suitable route, for example, by parenteral, subcutaneous, intranasal, intravascular, intravenous, intraarterial, or intrathecal injection or infusion.

[0107] The active agent according to the present invention may be administered together with other active agents, particularly active agents useful for the treatment of neurodegenerative disorders, such as PD or DLB.

[0108] Furthermore, the present invention relates to a diagnostic method comprising determining the amount and/or localization of .alpha.-Syn in the patient tissue or a patient sample. In this embodiment, the antibody of the present invention preferably carries a labelling group as described above.

[0109] Finally, the present invention relates to kits for diagnosis or treatment of neurodegenerative disorders comprising at least one antibody and/or nucleic acid molecule and/or cell as described above. In addition, the kit further comprises at least one other active agent or further components.

[0110] The present invention shall be explained in more detail by the following figures and examples.

FIG. LEGENDS

[0111] FIG. 1: Naturally occurring .alpha.-Syn-Abs were isolated from the serum of a single donor (30.6 mg/ml starting material) and from a commercially available IVIG preparation (Octagam; 10 mg/ml starting material) using affinity chromatography. The fractions that resulted from a representative experiment are depicted.

[0112] FIG. 2: The fractions that resulted from the chromatography column were analyzed in an .alpha.-Syn ELISA. The main fractions (MF) with high IgG content were compared to the peripheral fractions (PF) that contained less IgG. As a negative control, the flow through (FT) from the affinity purification was analyzed. Samples were added to .alpha.-Syn-coated wells of an ELISA plate. Bound antibodies were detected with a HRP-conjugated goat anti-human IgG antibody followed by Tetramethylbenzimide (TMB)/peroxidase colour reaction that was detected at 450 nm (Pierce Biotechnologies). A. Fractions containing .alpha.-Syn-Abs compared to fractions without .alpha.-Syn-Abs and to flow through of the affinity chromatography. B. .alpha.-Syn-Abs isolated from IVIG versus .alpha.-Syn-Abs isolated from the serum of a single donor.

[0113] FIG. 3: SDS-Page and Western blot analysis of naturally occurring .alpha.-Syn-Abs.

[0114] Different amounts of recombinant .alpha.-Syn (5 .mu.g, 2.5 .mu.g, 1 .mu.g, 0.5 .mu.g, 0.25 .mu.g, and 0.1 .mu.g), .beta.-Syn (5 .mu.g, 2.5 .mu.g, 1 .mu.g, and 0.5 .mu.g) and .gamma.-Syn (5 .mu.g, 2.5 .mu.g, 1 .mu.g, and 0.5 .mu.g) were separated on a 4-12% gradient mini gel (Invitrogen) and detected by: A. Naturally occurring .alpha.-Syn-Abs isolated from IVIG, and B. Naturally occurring .alpha.-Syn-Abs isolated from the serum of a single donor.

[0115] FIG. 4: Immunoprecipitation of .alpha.-Syn by .alpha.-Syn-Abs affinity-purified using IVIG. Naturally occurring .alpha.-Syn-Abs isolated from IVIG, positive control with a commercially available monoclonal .alpha.-Synuclein antibody (clone Syn 211, Biosource) and a negative control (flow through from the affinity purification) were immunoprecipitated with .alpha.-Syn and subjected to Western blot analysis. Data shown are from a representative experiment.

[0116] FIG. 5: Surface plasmon resonance analysis of affinity-purified polyclonal .alpha.-Syn-Abs (analyte) to .alpha.-Syn was done on BIACORE 2000 (Biacore AB) at 25.degree. C. A. Interaction analysis of immobilized .alpha.-, .beta.- and .gamma.-Syn with affinity-purified .alpha.-Syn-Abs (pAB). Plot of sensograms of antibody binding to .alpha.- (red; top graph), to 6- (blue; middle graph) and .gamma.-Syn (green; bottom graph). B. Interaction analysis of immobilized .alpha.-, .beta.- and .gamma.-Syn with a monoclonal antibody against human .alpha.-Syn (mAB; Syn 211 Biosource). Plot of sensograms of antibody binding to .alpha.- (red; top graph), to .beta.- (blue; middle graph) and .gamma.-Syn (green; bottom graph).

[0117] FIG. 6: Immunohistochemical detection of .alpha.-Syn in a brain sample of a patient with Parkinson's disease (PD). A. Immunostain of a brain sample of a human PD case (left panel) using the naturally occurring .alpha.-Syn-Ab. B. Immunostain (positive control) of a brain sample of a human PD case (right panel) incubated with a commercially available .alpha.-Syn monoclonal antibody (mAb) (MBL clone 211). C. Immunostain of a brain sample of a transgenic mouse model using the affinity-purified .alpha.-Syn-Ab. D. Immunostain (positive control) of a brain sample of a transgenic mouse model incubated with a commercially available .alpha.-Syn-mAb (MBL clone 211). LB stands for Lewy body, LN for Lewy neurites and LB Ii for Lewy body like inclusions.

[0118] FIG. 7: A. .alpha.-Syn sequence. Epitope Mapping of .alpha.-Syn-nAbs using Dot Blot analysis (B.) and a Peptide Array (C.). Results are summarized in D. with the relevant sequences highlighted in bold characters and relevant amino acids as determined by an alanine scan in boxes.

[0119] FIG. 8: .alpha.-Syn specific IgG1 heavy chain variable domain amino acid sequences. All amino acid sequences displayed as consensus sequence for the anti-.alpha.-Synuclein HC.sub.v molecules including the associated CDR regions were found in the shown arrangement in vivo. The consensus sequence reflects the ones found most often from the alpha synuclein specific B-cells analysed. Positions with amino acid variants indicating additional specific antibody subtypes are highlighted. Possible amino acid exchanges are shown below the Anti-.alpha.-Synuclein HC.sub.v sequence. Here, anti-.alpha.-Synuclein HC.sub.v Type 1 sequences are shown. 48 sequences derived from 80 clones were analyzed. These 48 clones attributed to HC.sub.v Type 1 correspond to 60% of all analyzed IgG sequences. 4% of all Type 1 sequences are identical to the consensus sequence, 27% show a maximum exchange of 2 amino acids. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein HC.sub.v Type 1 antibodies in the combinations detected in blood derived B-cells are shown in Table 1.

[0120] FIG. 9: .alpha.-Syn specific IgG1 heavy chain variable domain amino acid. sequences. Further details are specified in the legend to FIG. 8. Here, anti-.alpha.-Synuclein HC.sub.v Type 2 sequences are shown. 13 sequences derived from 80 clones were analysed. These 13 clones attributed to HC.sub.v Type 2 correspond to 15% of all analyzed IgG sequences. 67% of all Type 2 sequences are identical to the consensus sequence; 17% show a maximum exchange of 1 amino acid. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein HC.sub.v Type 2 antibodies in the combinations detected in blood derived B-cells are shown in Table 2.

[0121] FIG. 10: .alpha.-Syn specific IgG1 heavy chain variable domain amino acid sequences. Further details are specified in the legend to FIG. 8. Here, anti-.alpha.-Synuclein HC.sub.v Type 3 sequences are shown. 19 sequences derived from 80 clones were analysed. These 19 clones attributed to HC.sub.v Type 3 correspond to 24% of all analyzed IgG sequences. 37% of all Type 3 sequences are identical to the consensus sequence, 10% show a maximum exchange of 2 amino acids. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein HC.sub.v Type 3 antibodies in the combinations detected in blood derived B-cells are shown in Table 3.

[0122] FIG. 11: Naturally occurring .alpha.-Syn specific kappa light chain variable domain amino acid sequences. All amino acid sequences displayed as consensus sequence for the anti-.alpha.-Synuclein LC.sub.v molecules including the associated CDR regions were found in the shown arrangement in vivo. The consensus sequence reflects the ones found most often from the alpha synuclein specific B-cells analysed. Positions with amino acid variants indicating additional specific antibody subtypes are highlighted. Possible amino acid exchanges are shown below the anti-.alpha.-Synuclein LC.sub.v sequence. Here, anti-.alpha.-Synuclein LC.sub.v Type 1 sequences are shown. 64 sequences derived from 125 clones were analyzed. These 64 clones attributed to LC, Type 1 correspond to 51% of all analyzed IgG sequences. 48% of all Type 1 sequences are identical to the consensus sequence, 50% show a maximum exchange of 1 amino acid. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein LC.sub.v Type 1 antibodies in the combinations detected in blood derived B-cells are shown in Table 4.

[0123] FIG. 12: Naturally occurring .alpha.-Syn specific kappa light chain variable domain amino acid sequences. Further details are specified in the legend to FIG. 11. Here, anti-.alpha.-Synuclein LC.sub.v Type 2 sequences are shown. 24 sequences derived from 125 clones were analyzed. These 24 clones attributed to LC.sub.v Type 2 correspond to 19% of all analyzed IgG sequences. 38% of all Type 2 sequences are identical to the consensus sequence. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein LC.sub.v Type 2 antibodies in the combinations detected in blood derived B-cells are shown in Table 5.

[0124] FIG. 13: Naturally occurring .alpha.-Syn specific kappa light chain variable domain amino acid sequences. Further details are specified in the legend to FIG. 11. Here, anti-.alpha.-Synuclein LC.sub.v Type 3 sequences are shown. 33 sequences derived from 125 clones were analysed. These 33 clones attributed to LC.sub.v Type 3 correspond to 26% of all analyzed IgG sequences. 36% of all Type 3 sequences are identical to the consensus sequence, 9% show a maximum exchange of 2 amino acids. Linked amino acid exchanges are shown within one box. CDR1/CDR2/CDR3 of anti-.alpha.-Synuclein LC.sub.v Type 3 antibodies in the combinations detected in blood derived B-cells are shown in Table 6.

[0125] FIG. 14: Effect of .alpha.-Syn-Abs on aggregation of .alpha.-Syn. The kinetics of .alpha.-Syn fibril formation was monitored by Thioflavin T (ThT) fluorescence. .alpha.-Syn (rPeptide; 4 mg/ml) was shaken at 37.degree. C. and 600 rpm. A. At the time points indicated, aliquots were taken and a ThT assay was performed. B. .alpha.-Syn was incubated with or without the .alpha.-Syn-Abs affinity-purified from IVIG or from the serum of a single donor (1 .mu.M; Serum Abs). The samples were incubated four days at 37.degree. C. and 600 rpm and aliquots were added to the ThT solution. ThT fluorescence intensity was measured at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. Samples were run in triplicates and plotted as means+/-SD. A comparison of the cells treated with .alpha.-Syn alone to cells treated with either .alpha.-Syn Abs from IVIG or .alpha.-Syn Abs from serum was performed using a t-test (* p<0.05).

[0126] FIG. 15: Effect of the affinity-purified .alpha.-Syn-Abs on .alpha.-Syn-induced cytotoxicity. SH-SY5Y cells were treated with 10 .mu.M aliquots of .alpha.-Syn aggregated with and without 1 .mu.M affinity-purified IVIG .alpha.-Syn-Abs or a synthetic antibody against a growth factor receptor protein as a non-specific control. Samples were run in triplicates and plotted as means+/-SD. A comparison between every group in the assay was performed using the ANOVA Tukey test (** p<0.001), n.s. is not specific.

EXAMPLES

1. Materials and methods

1.1 Isolation and Purification of Naturally Occurring .alpha.-Synuclein Antibodies

[0127] Naturally occurring .alpha.-Synuclein antibodies (.alpha.-Syn-Abs) were isolated using affinity chromatography. A column was packed with NH.sub.2-activated resin (PIERCE Biotechnology, Rockford, Ill.), labelled with recombinant .alpha.-Syn (rPeptide, Bogart, Ga.; 1 mg/2 ml drained resin) and equilibrated and washed with phosphate buffered saline (pH 7.4). After passing either purified human intravenous immunoglobulin G (IVIG) or IgG fraction from the plasma of a healthy donor through the column, sixteen fractions were eluted with glycine buffer at pH 2.8 and collected. The main fractions that contain the greatest amount of .alpha.-Syn-Abs as well as the peripherical fractions that contain low amounts of .alpha.-Syn-Abs were pooled and their binding capacity was tested using an .alpha.-Syn-ELISA.

1.2 .alpha.-Syn-ELISA

[0128] A 96-well ELISA plate was coated with recombinant .alpha.-Syn (rPeptide)dissolved in coating buffer (1.7 mM H.sub.2PO.sub.4.times.H.sub.2O; 98 mM Na.sub.2HPO.sub.4.times.H.sub.2O; 0.05% sodium azide, pH 7.4). After blocking the plate with SuperBlock blocking buffer (PIERCE Biotechnology), .alpha.-Syn-Abs samples were loaded overnight at 4.degree. C. An appropriate secondary antibody, goat anti-human IgG H+L peroxidase conjugate (Calbiochem; Merck KGaA, Darmstadt, Germany), was incubated for one hour. Tetramethylbenzimide (TMB, Calbiochem) was added, and the reaction was stopped with 2N H.sub.2SO.sub.4. Finally, measurement was carried out in an ELISA plate reader (Multiskan Ex, Thermo, Waltham, Mass.) at 450 nm.

1.3 Protein Gel Analysis

[0129] Samples were mixed with 4.times.LDS sample buffer (Invitrogen, Karlsruhe, Germany) with DTT, boiled for 5 min and subjected to polyacrylamide gel electrophoresis. Samples were separated on NUPAGE Bis-Tris 4-12%, 1 mm gels (Invitrogen) in MES running buffer at 160 V according to the manufacturer's instructions. Once separated, the proteins were either visualized using silver staining or subjected to a Western blot analysis.

1.4 Western Blot Analysis

[0130] After being separated, the proteins were transferred to a PVDF membrane at 160 mA for 45 min. Membranes were blocked using RotiBlock (Roth, Karlsruhe, Germany) for 2 h at room temperature and were then probed overnight with either affinity-purified .alpha.-Syn-Abs 1:20,000 in RotiBlock or monoclonal .alpha.-Syn-Abs (clone Syn211, Invitrogen) 1:20,000 in RotiBlock, as indicated. The membranes were then washed three times in 1.times.phosphate buffered saline with 0.05% Tween 20 (PBST) and incubated with the appropriate secondary antibody, goat anti-human or goat anti-mouse (Pierce Biotechnology), at a concentration of 1:100,000 in PBST for 1 h at room temperature. Proteins were visualized using SuperSignal West Dura (PIERCE Biotechnology).

1.5 Immunoprecipitation of .alpha.-Synuclein by Affinity-Purified .alpha.-Syn-Abs

[0131] The reaction mixture of .alpha.-Synuclein was incubated with affinity-purified .alpha.-Syn-Abs, monoclonal .alpha.-Syn-Abs (as positive control, clone Syn 211, Invitrogen), phosphate buffered saline (PBS), and flow through from the affinity chromatography (as negative control) at 4.degree. C. overnight. Protein G was added and incubated at 4.degree. C. overnight to precipitate the IgG/.alpha.-Syn complex.

[0132] The precipitates were centrifuged and washed five times with PBS before loading a 12% SDS gel. In the Western blot, .alpha.-Syn monoclonal antibody clone 211 (Invitrogen) and HRP-conjugated goat anti-human IgG were used and this was then followed by detection with West Dura Super Signal (PIERCE Biotechnology).

1.6 Surface Plasmon Resonance

[0133] Surface Plasmon Resonance (SPR) analysis of the affinity-purified polyclonal .alpha.-Syn-Abs binding to .alpha.-Synuclein was done on BIACORE 2000 (GE Healthcare, Freiburg, Germany). Ligand immobilization of .alpha.-, .beta.- and .gamma.-Synuclein (Sigma Aldrich, Munich, Germany) resuspended in 1.times.PBS; 10-20 .mu.g/ml) was performed by amino coupling to 10,000 units (RU) on different flow cells (Fc2-4) of the CM5 sensor chip. .beta.- and .gamma.-Synuclein were used as negative controls. The SPR-signal of the reference flow cell (Fc1) was automatically subtracted from the sensograms of any other flow cell. Interaction analysis was performed by injection of analyte samples (IVIG, 75-150 .mu.g/ml) or .alpha.-Syn monoclonal antibody clone Syn 211 (Invitrogen, 10 .mu.g/ml as positive control; 20 .mu.l/min) diluted in running buffer (1.times.PBS/0.005% P20). The sensor chip was cleaned from immune-complexes by the injection of 5-20 .mu.l of regeneration solution (25 mM NaOH). Sensogram evaluation was performed using the BIA evaluation 3.2 RCI.

1.7 Epitope Mapping

[0134] In order to map detailed epitopes of the affinity-purified .alpha.-Syn-Abs, we used four peptides that spanned the whole sequence of .alpha.-Syn. The .alpha.-Syn-Abs were tested for binding capability to peptides Syn 1-60, Syn 61-140, Syn 1-95, Syn 96-140 (Sigma Aldrich) using Western blot and surface plasmon resonance as described above, with .alpha.-Syn-Abs as the ligand and the Synuclein truncations as analytes.

[0135] In an additional set of experiments, we represented .alpha.-synuclein as an array of overlapping peptides using the SPOT technology of multiple peptide synthesis. Specifically, a series of decamer peptides was assembled in an array format on a nitrocellulose membrane by walking through the entire .alpha.-synuclein sequence with a sliding window of six amino acids (FIG. 7). A total of 23 peptide spots were generated to cover the entire .alpha.-synuclein sequence. These spots were numbered sequentially from 1-23. In addition to the overlapping peptides we added a version with an amino acid randomly changed, numbered 25-47 as well as a scrambled version, numbered 49-71 of the same peptides. We performed an alanin scan of the NAC region of .alpha.-synuclein. The corresponding peptides were added to the membrane.

[0136] Subsequently, the .alpha.-synuclein peptide array was probed with .alpha.-Syn-nAbs and spots on the array produced binding signals.

1.8 Immunohistochemistry

[0137] In order to determine the cellular and subcellular localization of the binding partners of the .alpha.-Syn-Abs in the brain of transgenic mice (Thy1)-h[A30P] (Kahle et al., 2004), immunohistochemical experiments were conducted using brain samples of patients with Parkinson's disease and a neuroblastoma cell line (SH-SY5Y). The histopathological staining properties of .alpha.-Syn-Abs were compared to those of commercially available paraffin permeable antibodies against .alpha.-Synuclein (clone 211, MBL, Woburn, Mass., USA). Briefly, antigen retrieval was carried out by incubation in 70% formic acid for 20 min. After quenching of the endogenous peroxidase by 3% H.sub.2O.sub.2 in Methanol for 20 min the primary antibodies were incubated in a 1:100 dilution at 37.degree. C. in a humid chamber for 1 hr. As a detection system, the Vectastain Elite ABC kit or the Vectastain M.O.M. kit (Biozol, Eching, Germany) was used according to the manufacturer's instructions.

1.9 .alpha.-Synuclein Fibril Formation

[0138] The generation of fibrillated .alpha.-Syn was performed as described previously (Herrera et al., 2008). Briefly, recombinant .alpha.-Syn (rPeptide) was diluted in 10 mM Tris buffer of pH 7.4 and shaken at 37.degree. C. and 600 rpm. At different time points (0, 2, 4 and 8 days), aliquots were taken and measured using the Thioflavin T fibrillation assay. In order to determine the ideal .alpha.-Syn concentration for the assay, several concentrations (4, 2, 1 mg/ml) were tested after four days of incubation.

1.10 Thioflavin T Fibrillation Assay

[0139] In order to determine the amount of fibrillated .alpha.-Syn, a fluorometric experiment was performed as described previously (Herrera et al., 2008). Recombinant .alpha.-Syn (rPeptide) was incubated as described above. In this case, 10 .mu.l aliquots were added to 80 .mu.50 mM glycine puffer of pH 8.5 and 10 .mu.l 100 .mu.M Thioflavin T solution (Sigma Aldrich) and fluorescence was measured spectrofluorometrically using a Tecan reader Infinite M200 (Crailsheim, Germany) at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. Samples were run in triplicates and plotted as means+/-SD. Each experiment was performed at least three times.

1.11 Cell Culture and Toxicity Assay

[0140] Human neuroblastoma cells (SH-SY5Y) were maintained in RPM11640 (Lonza, Cologne, Germany) supplemented with 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin antibiotic mix and grown in a 5% CO.sub.2 atmosphere at 37.degree. C. Cells were harvested and plated in 96-well plates coated with poly-L-Lysin at 20,000 cells per well per 100 .mu.l of medium. Cells were then treated with .alpha.-Syn alone or .alpha.-Syn pre-incubated with .alpha.-Syn-Abs for four days at 37.degree. C. and 600 rpm. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich) reagent was resuspended in 5 mg/ml with deionised water and diluted in cell culture media to 0.5 mg/ml. Treatment was removed from the cells and 200 .mu.MTT solution was added and incubated for 2-4 hours at 37.degree. C. After removal of the MTT solution, cells were treated with 200 .mu.l DMSO to reduce the tetrazolium salt into the insoluble, purple coloured formazane. Readings were taken at 570 nm after another 30 min incubation at room temperature and again in the dark using a Tecan reader. Samples were run in triplicates and plotted as means+/-SD. Each experiment was performed at least three times.

1.12 De Novo Amino Acid Sequencing of One Purified Human Antibody Against Human .alpha.-Synuclein

1.12.1 Reduction and Alkylation of Disulfide Bonds

[0141] Protein samples were resolubilized in 50 mM triethylammonium bicarbonate (TEAB) buffer prior to reduction by addition of tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 5 mM and incubation at 37.degree. C. for 20 min. Subsequently iodoacetamide to a 10 mM final concentration was added and the sample was incubated at room temperature for another 20 mins in the dark.

1.12.2 SDS-PAGE

[0142] Separation of antibody light chain molecules and heavy chains molecules was performed by SDS-PAGE according to Laemmli et al. For subsequent proteolytic cleavage of the light chain IgG molecules small gel spots were cut and washed according to Proteome Factory's (Berlin, Germany) in-gel digestion protocol.

1.12.3 Enzymatic Cleavage

[0143] Alkylated peptides were used for enzymatic cleavage with trypsin, chymotrypsin, glutamic-C protease, clostripain, LysC or proteinase K. Therefore a small aliquot was diluted with 10 volumes of the suitable buffer for the enzymatic cleavage. Incubation time was varied in order to produce overlapping peptides by each protease.

[0144] Buffer solutions: Trypsin, Thermolysin, LysC: 50 mM ammonium bicarbonate, 10% acetonitrile (v/v) Chymotrypsin: 100 mM Tris-HCl, 10 mM CaCl.sub.2, 5% ACN (v/v), pH 8.0

[0145] Proteinase K: 100 mM Tris-HCl, 10 mM CaCl.sub.2, 5% ACN (v/v), pH 8.0 Glutamic-C protease: 50 mM Tris-HCl, 0.5 mM Glu-Glu, pH 8.0 Clostripain: 50 mM Tris-HCl, 10 mM CaCl.sub.2, 10% ACN (v/v), 20 mM DTT, pH 8.5

1.12.4 Mass Spectrometry (MS)

[0146] For carrying out nanoLC-ESI-MS/MS high resolution MS, the HPLC system was coupled to an Advion NanoMate 100 chip-electrospray system (Advion, Ithaca, N.Y.), and detection was performed on a Finnigan LTQ-FT mass spectrometer (ThermoFisher, Bremen, Germany) equipped with a 6T magnet. Peptides from enzymatic cleavage were acidified with formic acid and applied to nanoLC-ESIMS/MS. After trapping and desalting the peptides on enrichment column (Zorbax SB C18, 0.3.times.5 mm, Agilent) using 1% acetonitrile/0.5% formic acid solution for five minutes peptides were separated on Zorbax 300 SB C18, 75 .mu.m.times.150 mm column (Agilent, Waldbronn) using an acetonitrile/0.1% formic acid gradient from 5% to 40% acetonitrile within 40 to 115 minutes. MS overview spectra were automatically taken in FT-mode (+/-3 ppm) according to manufacturer's instrument settings for nanoLC-ESI-MSMS analyses, peptide fragmentation and detection was accomplished in the instrument's LTQ ion trap with an accuracy of +/-0.3 Da.

1.12.5 Database Search

[0147] The peptide masses and fragmentation data was searched against a human antibody sequenced derived from the NCBInr (National Center for Biotechnology Information, Bethesda, USA) database utilizing the MASCOT search engine (Matrix Science, London). Positive identification of peptides were annotated for the generation of sequence candidates.

[0148] Unassigned data was extracted and used for subsequent de novo peptide sequencing and searching of amino acid permutated human antibody peptide amino acid sequences.

1.12.6 Search Parameters

[0149] MS parent ion accuracy +/-3 ppm

[0150] MSMS fragment ion accuracy +/-0.3 Da

[0151] Fixed modification: Carbamidomethylation (Cys)

[0152] Denovo sequencing

[0153] Binning of duplicate spectra and peaks:

[0154] Binning MS 0.00015%

[0155] Binning MSMS 0.15 Da

[0156] Sequencing parameters:

[0157] Tolerance MS 0.0003%

[0158] Tolerance MSMS 0.3 Da

[0159] Candidate sequences were subjected to the Basic Local Alignment Search Tool (BLAST)

[0160] BLAST search parameters:

[0161] BLAST matrix: PAM30

[0162] Expect value: 10

[0163] Database: Human antibody sequences derived from NCBInr

[0164] HPLC separation of peptides

[0165] HPLC separation of LysC digested peptides was performed by using an Agilent 1100 HPLC system with a Zorbax 300SB-C8 column (150.times.2.1 mm) and a micro fraction collector for automatic peak fractionation. Solvent A was 0.1% TFA in water and solvent B 0.1% TFA in acetonitrile. The gradient started at 0% B for 5 minutes followed by increasing concentrations of B to 10 at 10 min, 40% at 55 min, 60% at 65 min and 100% at 70 minutes.

1.12.7 N-Terminal Edman Sequencing

[0166] N-terminal Edman sequencing of HPLC separated peptides was performed by an ABI Procise Model 49.times.protein sequencer using peptide fractions spotted on to Biobrene treated glass fiber discs.

2. Results

2.1 Isolation of Naturally Occurring .alpha.-Syn-Abs from IVIG and Serum of a Single Donor

[0167] We purified human .alpha.-Syn-Abs from IVIG and from the serum of a single donor by using an affinity column coated with recombinant .alpha.-Syn (FIG. 1). The resulting fractions with the highest IgG content were pooled and their binding properties to .alpha.-Syn were tested using an ELISA. We found that the resulting main fractions had a strong anti-.alpha.-Syn signal as compared to both the flow through IgG and peripheral fractions without .alpha.-Syn-Abs (FIG. 2a). There was a 2.5-fold increase in the fraction containing affinity-purified .alpha.-Syn-Abs as compared to the peripheral fractions without the antibody and a 4-fold increase as compared to the flow through of the affinity column. In FIG. 2b we show that .alpha.-Syn-Abs isolated from IVIG as well as from the serum of a single donor are dose-dependently binding .alpha.-Syn in an ELISA. The detection limit lies between 125 and 62.5 ng/ml for both affinity purified .alpha.-Syn-Abs.

2.2 Binding Specificity of the Affinity-Purified .alpha.-Syn-Abs from IVIG and Serum

[0168] The purified .alpha.-Syn-Abs from IVIG and that from single donor serum were able to detect the monomeric form of recombinant .alpha.-Syn peptide at 19 kDa on Western blots in a dose dependent manner. It also detected aggregated .alpha.-Syn species (FIGS. 3a and 3b). The signals at 38, 57 and 76 kDa correspond to the approximate sizes of dimeric, trimeric and tetrameric forms of .alpha.-Syn, respectively. A tetrameric form of .beta.- and .gamma.-Syn corresponding to the 76 kDa band was clearly recognized by .alpha.-Syn-Abs from IVIG and from serum.

[0169] In order to further confirm the ability of the naturally occurring .alpha.-Syn-Abs to bind .alpha.-Syn, we performed immunoprecipitation of recombinant .alpha.-Syn peptide (FIG. 4). We were able to confirm that the affinity-purified .alpha.-Syn-Abs do bind .alpha.-Syn peptide. The negative control, consisting of the same immunoprecipitation reaction with the column flow-through alone, did not bind to the recombinant .alpha.-Syn peptide. The reactions with protein G alone or with non-specific antibodies (Interleukin-1 antibody) were also negative (data not shown).

[0170] Furthermore, we obtained SPR data on the interaction of affinity-purified .alpha.-Syn-Abs from IVIG with .alpha.-, .beta.- and .gamma.-Synuclein immobilized via amino coupling (FIG. 5). Data demonstrated that the affinity-purified .alpha.-Syn-Abs from IVIG bind to .alpha.-Syn, to some extent as well as to .beta.-Syn, but not to .gamma.-Syn. After 20 seconds, the antibody dissociates to 50% from .alpha.-Syn and 100% from the .beta.-isoform. In comparison, a monoclonal antibody (clone Syn 211, Biosource) against human .alpha.-Syn bound particularly strong to .alpha.-Syn but bound very little or not at all to .beta.-Syn and .gamma.-Syn.

2.3 Affinity-Purified Antibodies Recognize .alpha.-Synuclein in Lewy Bodies--Immunohistochemical Experiments

[0171] In order to display the anatomical and histopathological localization of .alpha.-Syn in Lewy bodies using affinity-purified .alpha.-Syn-Abs, brain samples from PD patients and from an .alpha.-Syn transgenic (Thy1)-h[A30P] mouse model were analyzed using immunohistochemistry. In human PD patient samples, the affinity-purified .alpha.-Syn-Abs recognized the same structures as the monoclonal anti-human .alpha.-Syn-Ab (FIG. 6); these included a halo around a weaker core in Lewy bodies and drilled roots in Lewy neurites as well as in somatodendritic deposits. In the .alpha.-Syn transgenic mouse model there are no Lewy bodies present but rather only so called "Lewy body like inclusions" have been reported (Kahle et al., 2001). The immunohistochemical staining demonstrated that both the affinity purified .alpha.-Syn-Abs and the monoclonal .alpha.-Syn-Ab recognised these inclusions. Taken together, our results could conclusively demonstrate the specific binding of the affinity-purified antibodies to either recombinant human .alpha.-Syn or native human .alpha.-Syn.

2.4 Epitope Mapping

[0172] In order to further characterize the naturally occurring antibodies, we identified which epitopes of .alpha.-Syn are bound by .alpha.-Syn-Abs. Therefore, four truncated synthetic peptides spanning different regions of .alpha.-Syn were subjected to Western blot analysis (FIG. 7a). The .alpha.-Syn-Abs bound to the peptides corresponding to residues 1-60, 1-95 and 61-140 but not to the peptide corresponding to residues 96-140 (FIG. 7b).

[0173] Dot Blot analysis (FIG. 7) shows that the .alpha.-Syn-nAbs bind to the full-length .alpha.-synuclein but not to the albumin control. The strongest signal is observed with the peptide corresponding to residues 61-140. There is a very slight binding to the peptide corresponding to residues 1-60 and 1-95, and no binding to the peptide corresponding to residues 96-140 (FIG. 7).

[0174] Inspection of the decamer peptide array revealed positive signals for peptides 1, 7, 13 and 16. corresponding to the sequences listed in Table 7.

TABLE-US-00008 TABLE 7 List of .alpha.-synuclein peptides that showed significant binding to .alpha.-Syn-nAbs in the peptide array membrane. The missing aa sequences 2, 25, 26, 34, 36, and 61 were not positive and are therefore marked with --. Sequence One aa change scrambled 1 MDVFMKGLSK 25 -- 49 MMVGKSDLKF 2 -- 26 -- 50 LESVGAKVKG 7 VLYVGSKTKE 31 VLYVGSKT>MKE 55 VYSVLKKETG 13 GVTAVAQKTN 34 -- 61 -- 16 ATGFVKKDQL 36 -- 64 LKFTKDVQGA

[0175] The non-amyloid component (NAC) is located between .about.61 and 95 and seemed to be relevant in the binding of .alpha.-Syn-Abs to .alpha.-Syn. This data was confirmed by SPR data that also showed that the .alpha.-Syn fragments containing the NAC region had a higher binding specificity to .alpha.-Syn as compared to fragments that lacked this region (data not shown).

[0176] To validate results obtained from the Dot Blot analysis, we selectively synthesized an overlapping peptide array through the NAC region of .alpha.-synuclein (residues 61-100) of hexamer peptides with a sliding window of 3 amino acids. A total of 11 peptide spots were generated to cover the complete NAC region of .alpha.-synuclein. These spots were numbered sequentially from 73-83. In addition to the overlapping peptides, we added a scrambled version, numbered 85-95 as well as a version with an amino acid randomly changed, numbered 87-107. The .alpha.-synuclein peptide array was probed with .alpha.-Syn-nAbs and spots on the array produced binding signals. The peptide sequences corresponding to positive signals are shown in Table 8.

TABLE-US-00009 TABLE 8 List of .alpha.-synuclein NAC region peptides that showed significant binding to .alpha.-Syn-nAbs in the peptide array membrane. The corresponding sequence with one aa change and the scrambled version were negative and therefore marked with --. Sequence One aa change scrambled 77 VTAVAQ 89 -- 101 -- 83 TGFVKK 95 F>M TGMVKK 107 KTKGFV

2.5 .alpha.-Syn-Abs Inhibit the .alpha.-Syn Fibril Formation

[0177] We investigated whether purified .alpha.-Syn-Abs had an effect on .alpha.-Syn fibril formation by using Thioflavin T (ThT), a fluorescent reagent that specifically binds to fibrillar structures. Incubation of highly concentrated .alpha.-Syn (4 mg/ml) alone resulted in a time-dependent increase in fluorescence as the .alpha.-Syn began to aggregate (FIG. 14a). Incubation of .alpha.-Syn with 2 .mu.M affinity-purified .alpha.-Syn-Abs incubated for four days at 37.degree. C. and 600 rpm caused a significant decrease in ThT fluorescence, suggesting that the affinity-purified autoantibody was able to reduce fibril formation of .alpha.-Syn (FIG. 14b). The antibody isolated from serum did not show the same capacity for inhibiting fibril formation as compared to the antibody isolated from IVIG. When .alpha.-Syn was incubated with the column flow-through, a decrease in fibril formation was observed. The resulting data was analysed by a t-test using the GraphPad Software (GraphPad Software Inc., San Diego, Calif.).

2.6 Affinity-Purified .alpha.-Syn Antibodies Block Cytotoxicity of Aggregated .alpha.-Syn

[0178] We examined the cytotoxicity of pre-aggregated .alpha.-Syn samples on the human neuroblastoma cell line SH-SY5Y using an MTT assay. We tested whether the affinity-purified .alpha.-Syn-Abs had an effect on .alpha.-Syn-mediated cytotoxicity. Cells were treated with .alpha.-Syn and pre-incubated for four days with or without .alpha.-Syn-Abs. As depicted in FIG. 15, there was a significant increase in cell viability when cells were treated with .alpha.-Syn in the presence of .alpha.-Syn-Abs. Incubation with an unspecific synthetic antibody did not produce the same effect. The resulting data were analyzed with one way analysis of variance (ANOVA) using the SigmaStat Software (Systat Software GmbH, Erkrath, Germany).

2.7. Amino Acid Sequence Determination of IgG1 Variable Domains of Heavy and Light Chains of Naturally Occurring Antibodies Directed Against .alpha.-Synuclein

[0179] In order to obtain information on the amino acid sequence of the three complementarity determining regions (CDR) of each involved antibody chain as well as the relevant combination of the CDR1, CDR2, and CDR3 an RT-PCR/cDNA-sequencing approach using B-cell derived mRNA as template was performed.

[0180] Briefly, RNA was first isolated from B-cells enriched for anti-.alpha.-Syn, which were derived from blood donation buffy coats. Subsequently, cDNA was generated from the mRNA by oligo-dT priming. The cDNA was used as substrate for two types of PCR. The first PCR was specific for the variable domain of all human IgG1 heavy chains including a fragment of the adjoining constant domain. The second PCR was specific for the variable domain of human Kappa light chain plus a part of the neighboring sequences derived from the constant domain of the kappa light chain. These PCRs generate fragments of the variable domains of heavy and light chains from various B-cells thus demonstrating a mixture of information on these molecules from different cells. To be able to generate information on single HC/LC molecules, the PCR products were cloned into plasmids. Finally, colony-PCR products of the right size were sequenced and the nucleotide information translated into the required amino acid information. This information could be analyzed for its relationship to the expected IgG1 sequences as well as for statistical distribution of the number of sequences found within the samples.

[0181] The sequences determined for exemplary .alpha.-Syn specific light chains are shown in SEQ ID NOs.: 2, 148, 149, 150, and 151.

2.8. Statistical Evaluation

TABLE-US-00010 [0182] Statistical evaluation for IgG1 HC.sub.v amino acid sequences Number of sequences analysed: Sequences in total: 100 (100%) IgM related sequences: 11 (11%) IgG1 related but truncated: 9 (9%) IgG1 related sequences: 80 (80%) IgG1 HC.sub.v types defined due to sequence homologies: Type1: 48 (60%) Type2: 13 (16%) Type3: 19 (24%) Homology between Type1/Type2/Type3 consensus sequences (without CDR regions): Type1/Type 2: 76% Type 2/Type 3: 64% Type 1/Type 3: 71% Number of CDR sequences detected within IgG1 HC.sub.v: CDR1: 13 CDR2: 14 CDR3: 31 Type1: 6 Type1: 8 Type1: 20 Type2: 2 Type2: 2 Type2: 5 Type3: 5 Type3: 4 Type3: 6 No CDR region found within one type of .alpha.-Syn specific IgG1 HC.sub.v was found in another type as well. Number of CDR1/CDR2/CDR3 combinations detected: Total: 45 Type1: 30 within 48 clones => ratio 0.63 new combinations per clone Type2: 6 within 13 clones => ratio 0.46 Type3: 9 within 19 clones => ratio 0.47

[0183] The statistics presented here do not reflect the similarity by the detected amino acid sequences within the CDR groups. The combinations mentioned within the calculations above often differ by one single amino acid only.

TABLE-US-00011 Statistical evaluation for Kappa LC.sub.v amino acid sequences Number of sequences analysed: Sequences in total: 137 (100%) Non .kappa.LCv related sequences: 12 (9%) .kappa.LCv related but truncated: 0 (0%) .kappa.LCv related sequences: 125 (91%) .kappa.LCv types defined due to sequence homologies: Type1: 64 (51%) Type2: 24 (19%) Type3: 33 (27%) Not defined: 4 (3%) (not considered for further evaluation) Homology between Type1/Type2/Type3 consensus sequences (without CDR regions): Type1/Type2: 76% Type2/Type3: 74% Type1/Type3: 68% Number of CDR sequences detected within .kappa.LCv: CDR1: 11 CDR2: 9 CDR3: 25 Type1: 1 Type1: 1 Type1: 4 Type2: 3 Type2: 3 Type2: 8 Type3: 7 Type3: 5 Type3: 13 No CDR region found within one type of .alpha.-Syn specific .kappa.LCv was found in another type as well. Number of CDR1/CDR2/CDR3 combinations detected: Total: 30 Type1: 4 within 64 clones => ratio 0.06 new combinations per clone Type2: 10 within 24 clones => ratio 0.42 new combinations per clone Type3: 16 within 33 clones => ratio 0.48 new combinations per clone

[0184] The statistics presented here do not reflect the similarity by the detected amino acid sequences within the CDR groups. The combinations mentioned within the calculations above often differ by one single amino acid only.

3. Discussion

[0185] Neuropathologic and genetic studies as well as the development of transgenic animal models have provided evidence for the involvement of .alpha.-Synuclein (.alpha.-Syn) in the pathogenesis of Parkinson's disease (PD). It has become increasingly evident that the misfolded and aggregated species of .alpha.-Syn are known to be neurotoxic and subsequently lead to neurodegeneration. Thus, research has focused on finding new approaches to reducing abnormal accumulation of .alpha.-Syn.

[0186] In recent years, immunization has been shown to be effective in reducing neuronal accumulation of .alpha.-Syn aggregates. Masliah and colleagues also demonstrated that active immunization against human .alpha.-Syn reduced .alpha.-Syn aggregates in the brains of transgenic mice (Masliah et al., 2005). Papachroni and colleagues demonstrated for the first time the existence of autoantibodies (AAbs) against .alpha.-Syn that are positively correlated with the familial but not sporadic form of PD. In the study, they examined the presence of AAbs against .alpha.-Syn in the peripheral blood serum of PD patients and controls. They could detect such AAbs in 65% of all tested patients and they demonstrated that 90% of patients with familial PD tested positive for AAbs against .alpha.-Syn. Therefore, they hypothesized that these AAbs could be involved in the pathogenesis of .alpha.-Syn. (Papachroni et al., 2007).

[0187] Emadi and colleagues have isolated a human single-chain antibody fragment (scFv) against oligomeric .alpha.-Syn from a phage display antibody library (Emadi et al., 2007). They described binding only to oligomeric forms of .alpha.-Syn and inhibited both aggregation and toxicity of .alpha.-Syn in vitro. This approach is considered a significant advance toward a molecular therapy targeted against PD and other neurodegenerative conditions in which .alpha.-Syn aggregations represent a primary hallmark of disease.

[0188] In this study, we were able to isolate naturally occurring .alpha.-Syn antibodies from IVIG as well as from the serum of a healthy donor. Subsequent characterization by different assays including ELISA, Western blot analysis and surface plasmon resonance (SPR) analysis demonstrated that these .alpha.-Syn antibodies recognize several species of .alpha.-Syn. ELISA showed that antibodies purified from IVIG and from the serum of a single donor could both bind .alpha.-Syn. Western blot analysis demonstrated that the autoantibody was able to bind different species of .alpha.-Syn but not monomeric .beta.- and .gamma.-Synuclein. These results were confirmed using SPR analysis: the binding of the autoantibody was shown to be specific for monomeric .alpha.-Syn but not for monomeric .beta.- or .gamma.-Syn. Interestingly, oligomeric forms of all three synucleins were also recognized.

[0189] Our attempt to characterize the linear binding epitope of our .alpha.-Syn-Abs indicated that the non amyloidal component (NAC) region might be of importance. The NAC Region was originally identified as a component of Alzheimer's amyloid plaques. Deletions of this regions have been shown to be crucial for the aggregation of .alpha.-Syn in vitro (Ueda et al., 1993, Conway et al., 1998, Takeda et al., 1998) and through their toxicity to dopaminergic neurons in a Drosophila model (Periquet et al., 2007).

[0190] We were able to determine the cellular and subcellular localization of the binding partners of the .alpha.-Syn-Abs in the brains of PD patients as well as in the brains of transgenic mice by immunohistochemistry. The functionality of the affinity-purified .alpha.-Syn antibodies was tested using a thioflavin T assay and we were able to show that it can inhibit fibrillation of .alpha.-Syn. Also, a cell viability assay demonstrated that the affinity-purified .alpha.-Syn-Abs were able to increase cell viability and reduce .alpha.-Syn-induced cytotoxicity. These results are consistent with the effects of the antibodies against oligomeric forms of .alpha.-Syn as published in Emadi et al., 2007.

[0191] Taken together, these data suggest that the isolated .alpha.-Syn antibodies could have a therapeutic application in controlling the aggregation of .alpha.-Synuclein and consequently in the progression of PD.

REFERENCES

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Masliah E, Rockenstein E, Adame A, Alford M, Crews L, Hashimoto M, Seubert P, Lee M, Goldstein J, Chilcote T, Games D, Schenk D (Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease. Neuron 46:857-868.2005). [0205] 14. Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L (Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science (New York, N.Y. 287:1265-1269.2000). [0206] 15. Murphy R C, Messer A (A single-chain Fv intrabody provides functional protection against the effects of mutant protein in an organotypic slice culture model of Huntington's disease. Molecular Brain Research 121:141-145.2004). [0207] 16. Papachroni K K, Ninkina N, Papapanagiotou A, Hadjigeorgiou G M, Xiromerisiou G, Papadimitriou A, Kalofoutis A, Buchman V L (Autoantibodies to alpha-synuclein in inherited Parkinson's disease. Journal of neurochemistry 101:749-756.2007). 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Takenouchi T, Hashimoto M, Hsu L J, Mackowski B, Rockenstein E, Mallory M, Masliah E (Reduced neuritic outgrowth and cell adhesion in neuronal cells transfected with human alpha-synuclein. Molecular and cellular neurosciences 17:141-150.2001). [0215] 24. Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero D A, Kondo J, Ihara Y, Saitoh T (Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proceedings of the National Academy of Sciences of the United States of America 90:11282-11286.1993). [0216] 25. Wakabayashi K, Hayashi S, Kakita A, Yamada M, Toyoshima Y, Yoshimoto M, Takahashi H (Accumulation of alpha-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy. Acta neuropathologica 96:445-452.1998). [0217] 26. Zarranz J J, Alegre J, Gomez-Esteban J C, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J, Rodriguez O, Atares B, Llorens V, Gomez Tortosa E, del Ser T, Munoz D G, de Yebenes J G (The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Annals of neurology 55:164-173.2004). [0218] 27. Zhou W, Hurlbert M S, Schaack J, Prasad K N, Freed C R (Overexpression of human alpha-synuclein causes dopamine neuron death in rat primary culture and immortalized mesencephalon-derived cells. Brain research 866:33-43.2000).

Sequence CWU 1

1

1641140PRTHomo sapiensMISC_FEATURE(1)..(140)alpha-Synuclein 1Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90 95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile 100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala 130 135 140 2220PRTHomo sapiensMISC_FEATURE(1)..(220)light chain sequence 2Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Arg Leu Thr Glu Glu Lys Gly 85 90 95 Trp Met Tyr Leu Gly Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 325PRTHomo sapiens 3Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 414PRTHomo sapiens 4Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 532PRTHomo sapiens 5Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 650PRTHomo sapiens 6Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 1 5 10 15 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 20 25 30 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 35 40 45 Thr Val 50 725PRTHomo sapiens 7Gln Leu Gln Leu Gln Glu Ser Gly Ser Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser 20 25 814PRTHomo sapiens 8Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 932PRTHomo sapiens 9Arg Val Thr Ile Ser Val Asp Arg Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 1050PRTHomo sapiens 10Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 1 5 10 15 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 20 25 30 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 35 40 45 Thr Val 50 1125PRTHomo sapiens 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 1214PRTHomo sapiens 12Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 1332PRTHomo sapiens 13Trp Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 1450PRTHomo sapiens 14Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 1 5 10 15 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 20 25 30 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 35 40 45 Thr Val 50 1523PRTHomo sapiens 15Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys 20 1615PRTHomo sapiens 16Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 1732PRTHomo sapiens 17Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 1816PRTHomo sapiens 18Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 1 5 10 15 1923PRTHomo sapiens 19Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 2015PRTHomo sapiens 20Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 2132PRTHomo sapiens 21Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 2216PRTHomo sapiens 22Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 1 5 10 15 2323PRTHomo sapiens 23Asp 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 20 2415PRTHomo sapiens 24Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile Tyr 1 5 10 15 2532PRTHomo sapiens 25Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 2616PRTHomo sapiens 26Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 1 5 10 15 2710PRTArtificialanti-alpha-Synuclein HCv Type 1, CDR1, consensus sequence 27Gly Phe Thr Xaa Ser Xaa Xaa Xaa Xaa Xaa 1 5 10 2810PRTHomo sapiens 28Gly Phe Thr Phe Ser Asp Ala Trp Ile Asn 1 5 10 2910PRTHomo sapiens 29Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser 1 5 10 3010PRTHomo sapiens 30Gly Phe Thr Phe Ser Ser Tyr Ala Met His 1 5 10 3110PRTHomo sapiens 31Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 3210PRTHomo sapiens 32Gly Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 10 3310PRTHomo sapiens 33Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser 1 5 10 3410PRTHomo sapiens 34Gly Phe Thr Val Ser Ser Asn Tyr Met Ser 1 5 10 3516PRTHomo sapiens 35Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 3617PRTHomo sapiens 36Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 3717PRTHomo sapiens 37Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly 3819PRTHomo sapiens 38Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly 3917PRTHomo sapiens 39Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 4017PRTHomo sapiens 40Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 4116PRTHomo sapiens 41Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 4217PRTHomo sapiens 42Tyr Ile Ser Ser Ser Gly Gly Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 4317PRTHomo sapiens 43Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 4412PRTHomo sapiens 44Ala Met Val Arg Gly Val Thr Lys Pro Phe Asp Tyr 1 5 10 4512PRTHomo sapiens 45Ala Tyr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Gly Tyr 1 5 10 4614PRTHomo sapiens 46Asp Leu Val Asp Tyr Asp Ser Ser Gly Tyr Tyr Pro Asp Tyr 1 5 10 4720PRTHomo sapiens 47Asp Arg Gly Phe Gly Tyr Cys Ser Ser Thr Ser Cys His Thr Glu Asp 1 5 10 15 Ala Phe Asp Ile 20 4819PRTHomo sapiens 48Asp Arg His Pro Gly Tyr Cys Ser Ser Thr Ser Cys Phe Val Arg Tyr 1 5 10 15 Phe Asp Tyr 4917PRTHomo sapiens 49Asp Arg Arg Gly Ile Ala Ala Thr Ala Gly Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 5020PRTHomo sapiens 50Asp Trp Gly Ile Val Asp Thr Ala Met Val Pro Tyr Tyr Tyr Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 5116PRTHomo sapiens 51Glu Ala Pro Ser Ser Gly Trp Tyr Pro Tyr Tyr Tyr Tyr Met Asp Val 1 5 10 15 5222PRTHomo sapiens 52Glu His Arg Gly Gly Tyr Tyr Asp Ile Leu Thr Gly Tyr Thr Lys His 1 5 10 15 Gly Gly Ser Asn Asp Tyr 20 538PRTHomo sapiens 53Glu Arg Tyr Tyr Tyr Met Asp Val 1 5 5415PRTHomo sapiens 54Gly Gly Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Pro Trp Tyr 1 5 10 15 5517PRTHomo sapiens 55Gly Thr Asp Thr Glu Ser Val Ala Ala Pro Tyr Tyr Tyr Tyr Met Asp 1 5 10 15 Val 568PRTHomo sapiens 56Gly Val Ala Gly Arg Phe Asp Tyr 1 5 5712PRTHomo sapiens 57Gly Val Val Pro Ala Ala Glu Ser Trp Phe Asp Pro 1 5 10 5817PRTHomo sapiens 58Lys Asp Gly Ser Gly Ser Tyr Tyr His Tyr Tyr Tyr Tyr Val Met Asp 1 5 10 15 Val 5913PRTHomo sapiens 59Lys Thr Tyr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Gly Tyr 1 5 10 6013PRTHomo sapiens 60Gln Asp Ile Ala Ala Ala Ala Pro Tyr Tyr Phe Asp Tyr 1 5 10 6110PRTHomo sapiens 61Ser Gly Ala Ser Leu Arg Ala Phe Asp Ile 1 5 10 629PRTHomo sapiens 62Ser Gly Tyr Tyr Tyr Pro Leu Asp Tyr 1 5 6314PRTHomo sapiens 63Tyr Cys Ser Ser Thr Ser Cys Ser Ser Glu Tyr Phe Gly His 1 5 10 6415PRTHomo sapiens 64Tyr Tyr Tyr Asp Ser Ser Ala Val Glu Gly Asp Ala Phe Asp Ile 1 5 10 15 6512PRTArtificialanti-alpha-Synuclein HCv Type 2, CDR1, consensus sequence 65Gly Gly Ser Ile Ser Ser Gly Gly Tyr Xaa Trp Ser 1 5 10 6612PRTHomo sapiens 66Gly Gly Ser Ile Ser Ser Gly Gly Tyr Ser Trp Ser 1 5 10 6712PRTHomo sapiens 67Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser 1 5 10 6816PRTArtificialanti-alpha-Synuclein HCv Type 2, CDR2, consensus sequence 68Tyr Ile Tyr Xaa Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 6916PRTHomo sapiens 69Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 7016PRTHomo sapiens 70Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 7110PRTHomo sapiens 71Ala Gly Tyr Tyr Tyr Tyr Tyr Met Asp Val 1 5 10 7222PRTHomo sapiens 72Ala His Pro Val Arg Gly Ser Gly Ser Tyr Tyr Asn Arg Asn Tyr Tyr 1 5 10 15 Tyr Tyr Tyr Met Asp Val 20 7312PRTHomo sapiens 73Gly Ser Arg Glu Gly Tyr Gly Asp Arg Ile Asp Tyr 1 5 10 7421PRTHomo sapiens 74Gly Thr Glu Tyr Cys Thr Asn Gly Ala Cys Tyr Met Gly Tyr Tyr Tyr 1 5 10 15 Tyr Tyr Met Asp Val 20 7521PRTHomo sapiens 75Gly Thr Glu Tyr Cys Thr Asn Gly Val Cys Tyr Met Gly Tyr Tyr Tyr 1 5 10 15 Tyr Tyr Met Asp Val 20 7610PRTHomo sapiens 76Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser 1 5 10 7710PRTHomo sapiens 77Gly Tyr Ile Ile Ala Asn Tyr Tyr Ile His 1 5 10 7810PRTHomo sapiens 78Gly Tyr Ile Ile Thr Asn Tyr Tyr Ile His 1 5 10 7910PRTHomo sapiens 79Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His 1 5 10 8010PRTHomo sapiens 80Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5 10 8117PRTArtificialanti-alpha-Synuclein HCv Type 3, CDR2, consensus sequence 81Xaa Ile Xaa Pro Xaa Xaa Gly Xaa Xaa Xaa Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 8217PRTHomo sapiens 82Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 8317PRTHomo sapiens 83Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 8417PRTHomo sapiens 84Ile Ile Thr Pro Ser His Gly Ala Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 8517PRTHomo sapiens 85Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 8618PRTHomo sapiens 86Ala Lys Asp Tyr Asp Phe Trp Arg Gly Ser Thr Gly Met Arg Tyr Leu 1 5 10 15 Asp Val 8719PRTHomo sapiens 87Asp Lys Arg Cys Ser Ser Thr Ser Cys Gln Pro Tyr Tyr Tyr Tyr Tyr 1 5 10 15 Met Asp Val 8816PRTHomo sapiens 88Asp Ser Gly Ser Ser Gly Trp Tyr Val Pro Tyr Trp Tyr Phe Asp Leu 1 5 10 15 8917PRTHomo sapiens 89Pro Ile Gly Gly Gly Pro Ser Gly Trp Tyr Glu Thr Ser Cys Phe Asp 1 5 10 15 Pro 9019PRTHomo sapiens 90Thr Ser Tyr Gly Asp Ser Ser Ser Ser Ser Tyr Tyr Tyr Tyr Tyr Gly 1 5 10 15 Met Asp Val 9110PRTHomo sapiens 91Val Asp Tyr Ser Asn Tyr Val Val Asp Tyr 1 5 10 9216PRTHomo sapiens 92Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 937PRTHomo sapiens 93Leu Gly Ser Asn Arg Ala Ser 1 5 949PRTArtificialanti-alpha-Synuclein LCv Type 1, CDR3, consensus sequence 94Met Gln Ala Leu Gln Xaa Xaa Xaa Thr 1 5 959PRTHomo sapiens 95Met Gln Ala Leu Gln Thr Pro Arg Thr 1 5 969PRTHomo sapiens 96Met Gln Ala Leu Gln Thr Pro Trp Thr 1 5 979PRTHomo sapiens 97Met Gln Ala Leu Gln Thr Pro Tyr Thr 1 5 988PRTHomo sapiens 98Met Gln Ala Thr Gln Phe Arg Thr 1 5

9912PRTArtificialanti-alpha-Synuclein LCv Type 2, CDR1, consensus sequence 99Arg Ala Ser Gln Ser Val Ser Ser Xaa Xaa Leu Ala 1 5 10 10011PRTHomo sapiens 100Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 10112PRTHomo sapiens 101Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 10211PRTHomo sapiens 102Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 1037PRTArtificialanti-alpha-Synuclein LCv Type 2, CDR2, consensus sequence 103Xaa Ala Ser Xaa Arg Ala Thr 1 5 1047PRTHomo sapiens 104Asp Ala Ser Asn Arg Ala Thr 1 5 1057PRTHomo sapiens 105Gly Ala Ser Ser Arg Ala Thr 1 5 1067PRTHomo sapiens 106Gly Ala Ser Thr Arg Ala Thr 1 5 1079PRTHomo sapiens 107Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5 10810PRTHomo sapiens 108Gln Gln Arg Ser Asn Trp Pro Pro Tyr Thr 1 5 10 1098PRTHomo sapiens 109Gln Gln Tyr Gly Ser Ser Trp Thr 1 5 1109PRTHomo sapiens 110Gln Gln Tyr Asn Asn Trp Pro Leu Thr 1 5 11111PRTHomo sapiens 111Gln Gln Tyr Asn Asn Trp Pro Pro Met Tyr Thr 1 5 10 1129PRTHomo sapiens 112Gln Gln Tyr Asn Asn Trp Pro Arg Thr 1 5 1138PRTHomo sapiens 113Gln Gln Tyr Asn Asn Trp Trp Thr 1 5 1148PRTHomo sapiens 114Gln Gln Tyr Asn Asn Trp Tyr Thr 1 5 11511PRTArtificialanti-alpha-Synuclein LCv Type 3, CDR1, consensus sequence 115Arg Xaa Ser Gln Xaa Ile Xaa Xaa Xaa Leu Xaa 1 5 10 11611PRTHomo sapiens 116Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10 11711PRTHomo sapiens 117Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala 1 5 10 11811PRTHomo sapiens 118Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 11911PRTHomo sapiens 119Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala 1 5 10 12011PRTHomo sapiens 120Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 12111PRTHomo sapiens 121Arg Met Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 12211PRTHomo sapiens 122Arg Met Ser Gln Gly Ile Ser Ser Tyr Leu Ala 1 5 10 1237PRTHomo sapiens 123Ala Ala Ser Ser Leu Gln Ser 1 5 1247PRTHomo sapiens 124Ala Ala Ser Thr Leu Gln Ser 1 5 1257PRTHomo sapiens 125Ala Ala Ser Thr Leu Val Ser 1 5 1267PRTHomo sapiens 126Asp Ala Ser Asn Leu Glu Thr 1 5 1277PRTHomo sapiens 127Lys Ala Ser Ser Leu Glu Ser 1 5 1289PRTHomo sapiens 128Leu Gly Asp Tyr Asn Tyr Pro Tyr Thr 1 5 1299PRTHomo sapiens 129Leu Gln Asp Asn Asn Tyr Pro Arg Thr 1 5 1309PRTHomo sapiens 130Leu Gln Asp Tyr Asn Tyr Pro Tyr Thr 1 5 1319PRTHomo sapiens 131Leu Gln His Asn Ser Tyr Pro Phe Thr 1 5 1329PRTHomo sapiens 132Leu Gln His Asn Ser Tyr Pro Val Thr 1 5 1339PRTHomo sapiens 133Gln Gln Ala Asn Ser Phe Pro Ile Thr 1 5 1349PRTHomo sapiens 134Gln Gln Ala Asn Ser Phe Pro Leu Thr 1 5 13510PRTHomo sapiens 135Gln Gln Leu Asn Ser Tyr Pro Leu Phe Thr 1 5 10 13610PRTHomo sapiens 136Gln Gln Tyr Asp Asn Leu Pro Pro Phe Thr 1 5 10 1379PRTHomo sapiens 137Gln Gln Tyr Asn Ser Tyr Pro Val Thr 1 5 1389PRTHomo sapiens 138Gln Gln Tyr Asn Ser Tyr Pro Trp Thr 1 5 1399PRTHomo sapiens 139Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 14011PRTHomo sapiens 140Gln Gln Tyr Asn Ser Tyr Ser Arg Lys Tyr Thr 1 5 10 1419PRTHomo sapiens 141Leu Gln Asp Tyr Asn Tyr Pro Leu Thr 1 5 1429PRTHomo sapiens 142Gln Gln Tyr Asn Ser Tyr Leu Tyr Thr 1 5 1439PRTHomo sapiens 143Gln Gln Leu Asn Ser Tyr Pro Arg Thr 1 5 1449PRTHomo sapiens 144Leu Arg Asp Tyr Asn Tyr Pro Leu Thr 1 5 14514PRTHomo sapiens 145Arg Leu Thr Glu Glu Lys Gly Trp Met Tyr Leu Gly Tyr Thr 1 5 10 14610PRTHomo sapiens 146Gln Gln Arg Ser Asn Trp Pro Pro Ile Thr 1 5 10 1479PRTHomo sapiens 147Gln Gln Tyr Gly Ser Ser Pro Arg Thr 1 5 148118PRTHomo sapiens 148Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro 115 149118PRTHomo sapiens 149Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro 115 150124PRTHomo sapiens 150Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Val Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Asn Cys Gly Cys Thr Ile Cys Leu His Leu Pro 115 120 151124PRTHomo sapiens 151Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Tyr Gln 100 105 110 Thr Asn Cys Gly Cys Thr Ile Cys Leu His Leu Pro 115 120 15210PRTArtificialdecamer peptide spot No. 1 152Met Asp Val Phe Met Lys Gly Leu Ser Lys 1 5 10 15310PRTArtificialdecamer peptide spot No. 7 153Val Leu Tyr Val Gly Ser Lys Thr Lys Glu 1 5 10 15410PRTArtificialdecamer peptide spot No. 13 154Gly Val Thr Ala Val Ala Gln Lys Thr Asn 1 5 10 15510PRTArtificialdecamer peptide spot No. 16 155Ala Thr Gly Phe Val Lys Lys Asp Gln Leu 1 5 10 15610PRTArtificialdecamer peptide spot No. 31 156Val Leu Tyr Val Gly Ser Lys Met Lys Glu 1 5 10 15710PRTArtificialdecamer peptide spot No. 49 157Met Met Val Gly Lys Ser Asp Leu Lys Phe 1 5 10 15810PRTArtificialdecamer peptide spot No. 50 158Leu Glu Ser Val Gly Ala Lys Val Lys Gly 1 5 10 15910PRTArtificialdecamer peptide spot No. 55 159Val Tyr Ser Val Leu Lys Lys Glu Thr Gly 1 5 10 16010PRTArtificialdecamer peptide spot No. 64 160Leu Lys Phe Thr Lys Asp Val Gln Gly Ala 1 5 10 1616PRTArtificialhexamer peptide spot No. 77 161Val Thr Ala Val Ala Gln 1 5 1626PRTArtificialhexamer peptide spot No. 83 162Thr Gly Phe Val Lys Lys 1 5 1636PRTArtificialhexamer peptide spot No. 95 163Thr Gly Met Val Lys Lys 1 5 1646PRTArtificialhexamer peptide spot No. 107 164Lys Thr Lys Gly Phe Val 1 5

Claims

1. A human antibody which is directed against an epitope between amino acids 60-100, preferably between amino acids 73-82 and/or 91-100, more preferably between amino acids 74-79 and/or 92-97, of human o Synuclein (.alpha.-Syn), or a fragment or derivative of said antibody.

2. The antibody of claim 1 which is a naturally occurring human autoantibody or a fragment or derivative thereof.

3. The antibody of claim 1 which binds monomeric .alpha.-Syn, aggregated .alpha.-Syn, or both monomeric and aggregated .alpha.-Syn.

4. The antibody of claim 1, which comprises a heavy chain complementarity determining region 1 (CDR1) having the consensus sequence GFTX.sup.1SX.sup.2X.sup.3X.sup.4X.sup.5X.sup.6 (SEQ ID NO.: 27), preferably having the sequence as shown in any one of SEQ ID NOs.: 28-34.

5. The antibody of claim 1, which comprises a heavy chain complementarity determining region 2 (CDR2) having the sequence as shown in any one of SEQ ID NOs.: 35-43.

6. The antibody of claim 1, which comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 44-64.

7. The antibody of claim 1, which comprises a heavy chain complementarity determining region 1 (CDR1) having the consensus sequence GGSISSGGYXWS (SEQ ID NO.: 65), preferably having the sequence as shown in any one of SEQ ID NOs.: 66 and 67.

8. The antibody of claim 1, which comprises a heavy chain complementarity determining region 2 (CDR2) having the consensus sequence YIYXSGSTYYNPSLKS (SEQ ID NO.: 68), preferably having the sequence as shown in any one of SEQ ID NOs.: 69 and 70.

9. The antibody of claim 1, which comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 71-75.

10. The antibody of claim 1, which comprises a heavy chain complementarity determining region 1 (CDR1) having the sequence as shown in any one of SEQ ID NOs.: 76-80.

11. The antibody of claim 1, which comprises a heavy chain complementarity determining region 2 (CDR2) having the consensus sequence X.sup.1 IX.sup.2PX.sup.3X.sup.4GX.sup.5X.sup.6X.sup.7YAQKFQG (SEQ ID NO.: 81), preferably having the sequence as shown in any one of SEQ ID NOs.: 82-85.

12. The antibody of claim 1, which comprises a heavy chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 86-91.

13. The antibody of claim 1, which comprises a light chain complementarity determining region 1 (CDR1) having the sequence as shown in SEQ ID NO.: 92.

14. The antibody of claim 1, which comprises a light chain complementarity determining region 2 (CDR2) having the sequence as shown in SEQ ID NO.: 93.

15. The antibody of claim 1, which comprises a light chain complementarity determining region 3 (CDR3) having the consensus sequence MQALQX.sup.1X.sup.2X.sup.3T (SEQ ID NO.: 94), preferably having the sequence as shown in any one of SEQ ID NOs.: 95-98.

16. The antibody of claim 1, which comprises a light chain complementarity determining region 1 (CDR1) having the consensus sequence RASQSVSSX X.sup.2LA (SEQ ID NO.: 99), preferably having the sequence as shown in any one of SEQ ID NOs.: 100-102.

17. The antibody of claim 1, which comprises a light chain complementarity determining region 2 (CDR2) having the consensus sequence X.sup.1ASX.sup.2RAT (SEQ ID NO.: 103), preferably having the sequence as shown in any one of SEQ ID NOs.: 104-106.

18. The antibody of claim 1, which comprises a light chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 107-114, 145, 146, and 147.

19. The antibody of claim 1, which comprises a light chain complementarity determining region 1 (CDR1) having the consensus sequence RX.sup.1SQX.sup.2IX.sup.3X.sup.4X.sup.5LX.sup.6 (SEQ ID NO.: 115), preferably having the sequence as shown in any one of SEQ ID NOs.: 116-122.

20. The antibody of claim 1, which comprises a light chain complementarity determining region 2 (CDR2) having the sequence as shown in any one of SEQ ID NOs.: 123-127.

21. The antibody of claim 1, which comprises a light chain complementarity determining region 3 (CDR3) having the sequence as shown in any one of SEQ ID NOs.: 128-144.

22. The antibody of claim 1, which comprises a heavy chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 28-34, CDR2 is selected from the sequences shown in SEQ ID NOs.: 35-43, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 44-64.

23. The antibody of claim 1, which comprises a heavy chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 66-67, CDR2 is selected from the sequences shown in SEQ ID NOs.: 69-70, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 71-75.

24. The antibody of claim 1, which comprises a heavy chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 76-80, CDR2 is selected from the sequences shown in SEQ ID NOs.: 82-85, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 86-91.

25. The antibody of claim 1, which comprises a light chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 has the sequence shown in SEQ ID NO.: 92, CDR2 has the sequence shown in SEQ ID NO.: 93, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 95-98.

26. The antibody of claim 1, which comprises a light chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 100-102, CDR2 is selected from the sequences shown in SEQ ID NOs.: 104-106, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 107-114, 145, 146, and 147.

27. The antibody of claim 1, which comprises a light chain comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 116-122, CDR2 is selected from the sequences shown in SEQ ID NOs.: 123-127, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 128-144.

28. The antibody of claim 1, wherein the complementarity determining regions (CDRs) of the heavy chain are flanked by framework regions consisting of a consensus sequence as shown in: (i) SEQ ID NOs.: 3-6, or (ii) SEQ ID NOs.: 7-10, or (iii) SEQ ID NOs.: 11-14; or variants thereof.

29. The antibody of claim 1, wherein the complementarity determining regions (CDRs) of the light chain are flanked by framework regions consisting of a consensus sequence as shown in: (i) SEQ ID NOs.: 15-18, or (ii) SEQ ID NOs.: 19-22, or (iii) SEQ ID NOs.: 23-26; or variants thereof.

30. The antibody of claim 1, which comprises a heavy chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 3-6 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 28-34, CDR2 is selected from the sequences shown in SEQ ID NOs.: 35-43, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 44-64.

31. The antibody of claim 1, which comprises a heavy chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 7-10 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 66-67, CDR2 is selected from the sequences shown in SEQ ID NOs.: 69-70, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 71-75.

32. The antibody of claim 1, which comprises a heavy chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 11-14 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 76-80, CDR2 is selected from the sequences shown in SEQ ID NOs.: 82-85, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 86-91.

33. The antibody of claim 1, which comprises a light chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 15-18 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 has the sequence shown in SEQ ID NO.: 92, CDR2 has the sequence shown in SEQ ID NO.: 93, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 95-98.

34. The antibody of claim 1, which comprises a light chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 19-22 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 100-102, CDR2 is selected from the sequences shown in SEQ ID NOs.: 104-106, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 107-114, 145, 146 and 147.

35. The antibody of claim 1, which comprises a light chain comprising framework regions consisting of a consensus sequence as shown in SEQ ID NOs.: 23-26 or variants thereof, further comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 is selected from the sequences shown in SEQ ID NOs.: 116-122, CDR2 is selected from the sequences shown in SEQ ID NOs.: 123-127, and CDR3 is selected from the sequences shown in SEQ ID NOs.: 128-144.

36. The antibody of claim 1 for use in medicine, particularly in human medicine.

37. The antibody of claim 1 for use in the treatment of a neurodegenerative disorder, particularly Parkinson's disease or Dementia with Lewy bodies.

38. The antibody of claim 1 for use in passive immune therapy.

39. The antibody of claim 1 for use as a diagnostic agent.

40. A nucleic acid molecule encoding the antibody of claim 1 optionally in operative linkage to an expression control sequence.

41. A recombinant cell which comprises the nucleic acid molecule of claim 40.

42. A method of preparing an antibody of claim 1 comprising the steps: culturing a recombinant cell which comprises a nucleic acid molecule encoding the antibody of claim 1 optionally in operative linkage to and expression control sequence under conditions which allow expression of the antibody encoding nucleic acid molecule, and collecting the antibody from the cell or the culture supernatant.

43. A pharmaceutical composition comprising the antibody of claim 1, together with a pharmaceutically acceptable carrier.

44. The composition of claim 43 for use in passive immune therapy.

45. A pharmaceutical composition comprising the nucleic acid molecule of claim 40 with a pharmaceutically acceptable carrier.

46. A pharmaceutical composition comprising the recombinant cell of claim 41 together with a pharmaceutically acceptable carrier.

47. A method of treating a neurodegenerative disease in a patient in need of such treatment, comprising administering an effective amount of an antibody of claim 1 to said patient.