WO2015004163A1

WO2015004163A1 - Anti-tau monoclonal antibodies

Info

Publication number: WO2015004163A1
Application number: PCT/EP2014/064658
Authority: WO
Inventors: Ann DE VOS; Eugeen Vanmechelen; Joris Winderickx; Jeff VAN DEN BRANDE
Original assignee: Adx Neurosciences; Katholieke Universiteit Leuven, K.U. Leuven R & D
Priority date: 2013-07-08
Filing date: 2014-07-08
Publication date: 2015-01-15
Also published as: GB201312226D0

Abstract

The invention provides for tau antibodies, antibody-like scaffolds or antibody fragments with improved binding capacity for tau antigen. In particular, the invention provides for improved compositions, methods and kits comprising such antibodies, antibody-like scaffolds or antibody fragments.

Description

ANTI-TAU MONOCLONAL ANTIBODIES

Field of the invention This invention relates generally to the field of immunodiagnostics. The invention provides for tau antibodies, antibody-like scaffolds or antibody fragments with improved binding capacity for tau antigen. In particular, the invention provides for improved compositions, methods and kits comprising such antibodies, antibody-like scaffolds or antibody fragments. Background of the invention

Tau is a m i crot ubu 1 e- as soc i ated protein expressed in the central nervous system with a primary function to stabilize microtubules. There are six major isoforms of tau expressed mainly in the adult human brain, which are derived from a single gene by alternative splicing. Under pathological conditions, the tau protein becomes hyperphosphorylated (hPtau ). resulting in a loss of tubulin binding and destabilization of microtubules, combined with the aggregation and deposition of tau in pathogenic neurofibrillary tangles. Disorders related to tau - collectively referred to as ne u rode ge n erat i ve tauopathies - are part of a group of protein misfolding disorders including Alzheimer's disease (AD ) among others.

Tau and phosphorylated tau (Ptau) molecules are early biomarkers in Alzheimer^' s Disease (AD ) and several monoclonal antibodies showing reactivity to human tau/Ptau molecules have been developed. Research based assays comprising such antibodies for AD characterizations have been described. Thus far. few of these assays seem to be capable of measuring the low tau concentrations present in cerebrospinal fluid and/or plasma (Andreasson et al. Biomarkers Med 2012;6: 1- 13).

The use of immunoassays for the detection of a wide range of biological compounds in medical context is well known. Sensitivity of the assays depends on specific interactions between antibody molecules (or fragments thereof) and analytes to be detected in a sample.

There are two aspects to be noted. First, the specificity of the antibodies for the analyte must be high in order to discriminate between the analyte and other materials present in a sample. Second, the affinity properties of the antibodies or fragments thereof must be such that tight binding to the analyte is achieved.

While affinity and specificity are being evaluated during the screening of potential interesting monoclonal antibodies, screening for bioconjugation properties of the antibodies is (usually) not done. Bioconjugation of antibodies is important for labeling efficiency of the antibodies, eg the amount of biotin which can be coupled to the antibody and when the antibody is immobilized by bioconjugation onto a surface. In particular the availability of functional groups, that is amine groups, sugar chains, thiol groups within the antibody sequence can be crucial for antibody-orientation on a surface (Aslam & Dent. Eds. Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences ( acmillan Reference Ltd. London. UK, 1998). Improvement effects on antibody orientation as high as 200 have been reported to improve assay performance, but are usually 2- 10 fold depending on the affinity and specificity of the antibodies (Trill ing et al. Biosens Bioelectron 2013; 40:2 19-226; Taj i ma et al. Anal Chem 2011 ;83: 1969- 1976).

Tau antibodies with the epitope around 2 1 8-224 have been described as bei ng capable of capturing efficiently circulating tau molecules from biological fluids such as cerebrospinal fluid (CSF) and plasma (Vandermeeren et al. J Neurochem 1993:61 : 1 828- 1 834; Vanmechelen et al. Neurosci Lett 2000;285:49-52).

However, there is a continuing need for better immunodiagnostic methods that enable more sensitive detection of AD and other tauopathies in more comple samples than CSF. such as e.g. serum.

Summary of the invention

The present invention meets the need mentioned above by providing tau antibodies, antibody-like scaffolds or antibody fragments with superior immobil izing capacity and tau binding affinity, compositions comprising such antibodies, antibody-like scaffolds or antibody fragments; nucleic acids encoding such tau antibodies, antibody-like scaffolds or functional antibody fragments; and cell lines and hybridomas secreting them. The invention further provides methods and kits for the detection of tau and for the in vitro diagnosis of tauopathies using these tau antibodies, antibody-like scaffolds or antibody fragments. The provided monoclonal antibodies allow for an improved detection of tau antigen.

The present invention relates at least in part to the following embodiments:

Embodiment (1): an isolated antibody, antibody-like scaffold or antibody fragment, characterized in that:

it specifically binds to tau;

it has an improved analytical sensitivity for tau compared to e.g. antibody Tau-5 when bioconjugated.

Embodiment (2): an isolated nucleic acid comprising a polynucleotide encoding the antibody, antibody-like scaffold or antibody fragment characterized in that said antibody, antibody-like scaffold or antibody fragment:

specifically binds to tau;

has an improved analytical sensitivity for tau compared to e.g. antibody Tau 5 when bioconjugated.

Embodiment (3): an isolated cell l ine producing the antibody, antibody-like scaffold or antibody fragment characterized in that said antibody, antibody-like scaffold or antibody fragment:

specifically binds to tau;

Embodiment (4): use of an antibody, antibody-l ike scaffold, or antibody fragment in the detection of tau or in the in vitro diagnosis of a taiiopathy characterized in that said antibody, antibody-l ike scaffold or antibody fragment:

specifically binds to tau;

has an improved analytical sensitivity for tau compared to e.g. antibody Tau 5 when bioconjugated. Embodiment (5): a method for detecting tau in a sample or for the in vitro diagnosis or monitoring of a tauopathy in a subject, comprising the steps of:

contacting an antibody, antibody-l ike scaffold or antibody fragment with a sample under conditions suitable for producing an antigen-antibody complex, and detecting the formation of said antigen-antibody complex;

wherein said antibody, antibody-like scaffold or antibody fragment:

specifically binds to tau;

Embodiment (6): a kit for the detection of tau or for the in vitro diagnosis or monitoring of a tauopathy in a subject, comprising an antibody, antibody-like scaffold or antibody fragment that:

- specifically binds to tau;

Embodiment (7): a kit to discriminate early stage Alzheimer's dementia, especially from other types of dementia in a subject, comprising an antibody, antibody-like scaffold or antibody fragment that:

specifically binds to tau;

Embodiment (8): an isolated antibody or antibody fragment characterized in that it is secreted by the cell line selected from the group consisting of hybridoma cell line ADx202 deposited under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection under No. LMBP 10376CB on 19 September 2013.

Embodiment (9): an isolated antibody, antibody-like scaffold or antibody fragment characterized in that the light chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 10, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 11 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 12; and wherein a heavy chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 15, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 16 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 17.

Embodiment (10): an isolated antibody, antibody- like scaffold or antibody fragment comprising at least one CDR triplet selected from the group consisting of

- a CDR triplet H1/H2/H3; and

- a CDR triplet L1/L2/L3 ;

wherein HI has an amino acid sequence chosen from SEQ ID NO. 15, H2 has an amino acid sequence chosen from SEQ ID NO. 16, H3 has an amino acid sequence chosen from SEQ ID NO. 17, LI has an amino acid sequence chosen from SEQ ID NO. 10, L2 has an amino acid sequence chosen from SEQ ID NO. 11, and L3 has an amino acid sequence chosen from SEQ ID NO. 12.

Embodiment (11): an isolated nucleic acid comprising a polynucleotide encoding an antibody, antibody-like scaffold or antibody fragment characterized in that the light chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 10, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 11 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 12; and wherein a heavy chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 15, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 16 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 17.

Embodiment (12): an isolated nucleic acid comprising a polynucleotide encoding the antibody, antibody-like scaffold or antibody fragment according to embodiment 2 comprising the nucleic acid sequence of SEQ ID NO. 19 and/or SEQ ID NO. 20. Figures

Figu e 1 : (A) Graph illustrating the count of beads coupled with ADx202; the Y-axis represents the count, the X-axis represents the diameter in μηι.

(B) Graph illustrating the count of beads coupled with Tau- 5; the Y-axis represents the count, the X-axis represents the diameter in μηι.

These data illustrate that beads are efficiently recovered upon coupling.

Figure 2: Graph representing MAGPIX median fluorescence intensity

(MFI) measurement of beads coupled with ADx202 and Tau -5 using a phycoerythrin- labeled anti-mouse IgG detection antibody (anti-mouse-PE). The X-axis represents the concentration of the ph ycoery th rin-1 abel ed anti-mouse IgG detection antibody: the Y-axis represents the MFI measurement. These data illustrate that the amount of antibody coupled per bead is higher for Tau5 than for ADx202.

Figure 3: Graph representing MAGPIX median fluorescence intensity (MFI) measurement of beads coupled with ADx202 and Tau-5 using antigen human tau-441 . recombinant e.coli; rPeptide (=rPeptide441) and biotinylated ADx215 detection antibody. The X-axis represents the concentration of rPeptide441 in pg/mL: the Y-axis represents the MFI measurement. These data illustrate that ADx202 captures rPeptide tau441 more efficiently then Tau5.

Figure 4: DNA and amino acid sequence of ADx202 light chain variable region. Figure 5: DNA and amino acid sequence of ADx202 heavy chain variable region.

Detailed description of the invention

The present invention relates to tau and tau antibodies directed towards tau.

(hyper iphosphorylated tau and their aggregates. The present invention provides isolated tau antibodies, antibody-like scaffold and antibody fragments, characterized in that upon bio- conjugation the tau antibodies, antibody-like scaffold and antibody fragments show an improved capturing capacity for tau compared to existing tau antibodies, antibody-l ike scaffold and antibody fragments. The invention can be implemented in a number of ways, including as a method, an assay, a kit and a composition of matter. In general, the order of the steps of disclosed methods may be altered within the scope of the invention. Embodi ments will be discussed with reference to the accompanying figures, which depict one or more exemplary embodiments. Embodiments may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, shown in the figures and/or described below. Rather, these exemplary embodiments are provided to allow a complete disclosure that conveys the principles of the invention, as set forth in the claim, to those skilled in the art. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the invention is not unnecessarily obscured. Unless indicated or defined otherwise, all terms used have their usual meaning in the art. which will be clear to the skilled person. Reference is further made to the standard handbooks, such as ( Sambrook.J. and Russell. D.W. (2001). Molecular cloning: A Laboratory Manual.. F.Ausubel. ed. (Cold Spring Harbor. NY: Cold Spring Harbor Laboratory Press; Delves. P.. Martin. S.. Burton. D.. and Roitt.I. (2006). Roitt's Essential Immunology. Wiley-Blackwell; Krebs.J.E.. Goldstein. E.S.. and

Kilpatrick.S.T. (2009). Lewin's Genes X. Jones & Bartlett Publishers ), as well as to the general background art cited herein.

As used in the specification and the attached claims, the use of "a," "an^" and "the^" include references to plural subject matter referred to unless the context clearly dictates otherwise. Thus, for example, reference to "a protein^" includes a single catalyst as well as a combination or mixture of two or more proteins, reference to "an antigen^" encompasses a combination or mixture of different antigens as well as a single antigen, and the like.

A term which is subsumed under another term may be embraced by the broader term or by the more narrow specific term as appropriate within the context of the use of that term. All terms used to describe the present invention are used within context.

Presently known antibody Tau-5 is an antibody regularly, but not exclusively, used as capture moiety that binds to tau in sandwich immunoassays and allows quantification of total amounts of protein tau in the range of 20-2000 pg/ml. which is suitable for e.g. CSF quantification. Antibody Tau-5 is a commercially available anti-tau IgGl antibody specific to 45-68 kDa proteins identified as tau proteins and does not cross-react with tubul in or other microtubule-associated proteins (Carmel et al .. J. Biol . Chem. 1996; 271 :32789). Antibody Tau- 5 reacts with phosphorylated as well as non-phosphorylated forms of tau and stains human neurofibrillary tangles, neutrophil threads and neuritic plaques associated with Alzheimer^'s disease.

Surprisingly, in the present invention an antibody. ADx202, was isolated and characterized which has improved reactivity characteristics when compared to the reactivity characteristics of antibody Tau-5. The present invention thus relates to any antibody which has reactivity characteristics in common with the antibody ADx202 described in the example section. As shown in the example section, the antibodies, antibody-like scaffolds, or antibody fragments of the present invention specifically bind to tau and are further characterized by an improved analytical sensitivity for tau compared to antibody Tau5 when bioconjugated to a magnetic surface. The antibodies of the invention (ADx202 or any other similar antibodies ) may be selected from a range of antibodies obtained by direct immunization with full-length human tau (longest isoform. 2N4R; 441 amino acids ) purified as a N-terminal His6 fusion product from the Apho85Aadhl yeast deletion strain as illustrated in the example section. In one embodiment, the present invention relates to isolated antibodies, antibody-like scaffolds or antibody fragments characterized in that they:

specifically bind to tau;

have an improved analytical sensitivity for tau compared to antibody Tau-5 when bioconjugated.

More particularly, the antibodies are tau-antibodies. preferably monoclonal antibodies, more preferably mouse monoclonal antibodies. In preferred embodiments, antibodies of the invention have been selected for their specifically binding to tau in general and have not been selected for their specifically binding to non-phosphorylated tau. ( h y er ) phos phory 1 ated tau. monomers or aggregates in particular. In a preferred embodiment, the antibody of the invention is secreted by the hybridoma cell line deposited under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection under No. 10376CB. This hybridoma cell line and the secreted monoclonal antibody will hereinafter be referred to as ADX202 and ADx202 respectively. In preferred embodiments, the antibodies have the binding characteristics of antibody ADx202. Most preferably, the antibody is antibody ADx202. Thus, the present invention relates to antibody ADx202 or other si milar antibodies.

By '"tau antibody^" is meant any antibody directed towards tau antigen.

The terms "antibody^" and "antibodies^" are well known in the art and refer to proteins also known as immunoglobulins that bind to antigens. It is to be understood that these terms encompass conventional vertebrate antibodies like IgA. IgD, IgE, IgG. IgM. IgT. IgX and IgY. composed of at least two heavy and two light chains, as well as antibodies only composed of two heavy chains (VHH antibodies. IgNAR, heavy-chain antibodies, single-domain antibodies or nanobodies ). and single-chain antibodies. In the case of conventional antibodies, the antigen- binding sites are contributed to by the variable domains of both the heavy and light chains (VH and VL). The term "variable domain" refers to the part or domain of an antibody which is partially or fully responsible for antigen binding. Generally, variable domains will be amino acid sequences that essentially consist of 4 framework regions ( FRl to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), or any suitable fragment of such an amino acid sequence which usually contains at least some of the amino acid residues that form at least one of the CDR's. Such variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. Each CDR may contribute to a greater or lesser extent to antigen binding by the antibody. .Single domain antibodies or heavy-chain antibodies can be found in camel ids and sharks, and each of the antigen-binding sites of these antibodies is formed by a single heavy chain variable domain (VHH) only. Therefore, only three CDRs contribute to a greater or lesser extent to each antigen-binding site. Single chain antibodies (scFv) are derived from conventional antibodies by translational fusion of the VH and VL domains, separated by a flexible linker, into a single antigen-binding domain. Framework sequences of an antibody may be altered without altering the antigenic specificity of the antibody, or in order to change the binding affinity of the antibody. Furthermore, conventional antibodies may switch classes or isotypes without substantially affecting antigen-binding characteristics.

By the term "antibody fragment or functional antibody fragment " are used

interchangeably to denote a fragment of an antibody that largely retains antigen-binding capacity of the antibody from which it is derived. Therefore, a tau-specific antibody fragment of the invention is capable of preferentially binding to tau antigen. Antigen-binding capacity is determined by the variable domain or domains, more particularly by 1 , 2, 3, 4, 5 or 6 CDRs located in the VH and/or VL domains in the case of conventional and single-chain antibodies, and 1 , 2 or 3 CDRs in the case of single-domain antibodies. Preferred antibody fragments of the invention therefore comprise antigen-binding sites comprising 1 , 2, 3, 4, 5 or 6 CDRs. Two or more CDRs may be physically separated from each other by connecting regions to provide a framework structure for the CDRs. More preferred antibody fragments of the invention comprise antigen-binding sites comprising 1 or 2 variable domains. Examples of antibody fragments are well -known to the skilled person and include the monovalent antigen-binding fragments (Fab ), bivalent F(ab')₂ fragments. Fv fragments (e.g. single chain antibodies scFv ). miniaturized antibodies, single-domain antibody fragments l ike nanobodies (Nelson, 2010). Antibody fragments of the invention may be obtained by enzymatic or chemical proteolysis, or by recombinant DNA technology techniques well known to the skilled person.

"Antibody-derived scaffold or antibody-like scaffolds^" are modified antibodies that largely retains antigen-binding capacity of the antibody or antibody fragments from which it is derived. Examples of antibody-derived scaffolds or antibody-like scaffolds are for domain antibody (dAb ) that selectively or preferentially bind the same epitope as a natural antibody for instance dAb with fully human frameworks, for instance dAb fused to a human Fc domain or for instance nanobodies engineered in a molecule that has an IgG-like circulating half- l ife in humans or antibody fragments with retained antigen-binding capacity or domain antibody with active scaffolds for controlled and cell delivery. In a preferred embodiment of the invention, the antibody of the invention is monoclonal.

The term "monoclonal antibody^" is well recognized in the art and refers to an antibody or a homogenous population of antibodies that is derived from a single clone. Individual antibodies from a monoclonal antibody population are essentially identical, in that minor naturally occurring mutations may be present. Antibodies from a monoclonal antibody population show a homogenous binding specificity and affinity for a particular epitope. In one embodiment, the isolated antibody or antibody fragment of the present invention comprises at least one CDR having an amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO. 12 and SEQ ID NO. 14 to SEQ ID NO. 17, or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO. 12 and SEQ ID NO. 14 to SEQ ID NO. 17.

In a particular embodiment, the isolated antibody or antibody fragment of the present invention is characterized in that the light chain variable region comprises in a CDRl region an amino acid sequence as set out in SEQ ID NO: 10, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 11 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 12; and wherein a heavy chain variable region comprises in a CDRl region an amino acid sequence as set out in SEQ ID NO: 15, in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 16 and in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 17, or an wherein the light chain variable region comprises in a CDRl region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 10, in a CDR2 region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO.11, in a CDR3 region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO.12, or wherein the heavy chain variable region comprises in a CDRl region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO.15, in a CDR2 region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO 16, in a CDR3 region amino acid which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO 17. In a particular embodiment, the antibody or antibody fragment comprises at least one CDR triplet selected from the group consisting of

- a CDR triplet H1/H2/H3; and

- a CDR triplet L1/L2/L3;

wherein HI has an amino acid sequence chosen from SEQ ID NO. 15 or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 15, H2 has an amino acid sequence chosen from SEQ ID NO. 16 or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 16, H3 has an amino acid sequence chosen from SEQ ID NO. 17 or an amino acid sequence which has at least 80, 81, 82,

83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 17, LI has an amino acid sequence chosen from SEQ ID NO. 10 or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 10, L2 has an amino acid sequence chosen from SEQ ID NO. 11 or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 11, and L3 has an amino acid sequence chosen from SEQ ID NO. 12 or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to SEQ ID NO. 12. In a particular embodiment, the antibody or antibody fragment comprises at least one variable domain having an amino acid sequence selected from the group consisting of SEQ ID NO. 9, 10, 11, 12, 14, 15, 16 and 17 or an amino acid sequence which has at least 80, 81, 82, 83,

84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 9, 10, 11, 12, 14, 15, 16 and 17.

In an alternative embodiment, the antibody or antibody fragment comprises at least one CDR having an amino acid sequence selected from the group consisting of ID NO. 9 to SEQ ID NO. 12 and SEQ ID NO. 14 to SEQ ID NO. 17, or an amino acid sequence which has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to an amino acid sequence selected from the group consisting of ID NO. 9 to SEQ ID NO. 12 and SEQ ID NO. 14 to SEQ ID NO. 17, characterized in that it preferentially binds to tau and has an improved analytical sensitivity for tau compared to antibody Tau-5 when bioconjugated. Tau is a microtubule-associated protein (MAP) synthesized in neurons. Six major isoforms of tau having different physiological roles are derived from a single gene by alternative spl icing (Goedert. Spillantini et al. 1989 ). The isoforms can contain 0, 1 . or 2 N-terminal insertions (denoted as ON, IN and 2N isoforms. respectively) encoded by exons 2 and 3, and further 0 or 1 extra C-terminal microtubule-binding domain encoded by ex on 10 (denoted as 3R and 4R, respectively). As such, the isoforms are denoted as 0N/3R, 0N/4R, 1N/3R, 1N/4R, 2N/3R, and 2N/4R. For instance in an embodiment of present invention the isoform is microtubule- associated protein tau isoform 1 [Homo sapiens] with the NCBI Reference Sequence:

NPJ358519.3 as deposited with accession number NPJ358519 w on 26-JUN-2011 (SEQ ID NO: 1 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 2 [Homo sapiens] with NCBI Reference

Sequence: NPJJ05901.2 as deposited under accession number NP_005901 NPJ776088 date 26 June 20 1 1 (SEQ ID NO: 2 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 3 [Homo sapiens] with the NCBI Reference Sequence: NP__058518.1 date 26 June 201 1 as deposited with the accession number NP_058518, version NP_058518.1 GL8400711 (SEQ ID NO : 3 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 4 [Homo sapiens] with NCBI Reference Sequence: NP_058525.1 date 26 June 201 1 as deposited with the accession number NPJJ58525, version NP_058525.1

G 1: 84007 1 5 (SEQ ID NO: 4 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 5 [Homo sapiens] with NCBI Reference Sequence: NP_001116539.1 date 26 June 201 1 as deposited with the accession number NPJ)01 1 16539 version NPJJO l 1 16539. 1 G 1: 178557736 (SEQ ID NO: 5 in this application ). For instance in another embodiment of present invention the isoform is

microtubule-associated protein tau isoform 6 [Homo sapiens] with NCBI Reference Sequence: NPJJO l 1 1 6538.2 date 26 June 201 1 as deposited with the accession number NPJJO l 1 16538 version NPJJO l 1 1 6538.2 GL294862258 (SEQ ID NO: 6 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 7 [Homo sapiens] with NCBI Reference Sequence: NPJXJ l 1901 80. 1 date 26 June 20 1 1 as deposited with accession number NPJXJ l 1901 80 version NPJJO l 1901 80. 1 GL322303720 (SEQ ID NO: 7 in this application ). For instance in another embodiment of present invention the isoform is microtubule-associated protein tau isoform 8 [Homo sapiens] with NCBI Reference

.Sequence: NP_001 1901 8 1 . 1 date 26 June 201 1 as deposited with accession number

NPJJ01 1 901 8 1 version NP_001 1 901 8 1 . 1 GL322303747 (SEQ ID NO: 8 in this application ) As used herein, "tau", "tau antigen^", "tau protein^", "tau isoform^". "tau molecule^", "tau variant^", "tau mutant^", "tau homologue^" and "tau isoform^" are used interchangeable to denote a polypeptide or protein that is encoded by at least one exon of a tau gene, irrespective of whether post-translational modifications are present or not. Such gene can encode a protein of the tau protein family mentioned above and derivatives thereof. .Such proteins are characterized as one family among a larger number of protein families which co- purify with microtubules during repeated cycles of assembly and disassembly (Shelanski et al. (1973) Proc. Natl. Acad. Sci. USA. 70. 765-768), and known as microtubule-associated-proteins (MAPs). The tau family in addition is characterized by the presence of a characteristic N -terminal segment which is shared by all members of the family, sequences of ~50 amino acids inserted in the N -terminal segment. which are developmental ly regulated in the brain, a characteristic tandem repeat region consisting of 3 or 4 tandem repeats of 3 1 -32 amino acids, and a C-terminal tail. A tau protein can in an embodiment comprise the amino acid sequence of "T40" with the sequence described in Goedert. M.. Spillantini. M. G.. Jakes, R., Rutherford, D.. and Crowther. R . A. Neuron. 3[4], 519-526, 1 989. Under pathological conditions, the tau protein becomes hyperphosphorylated (Ptau ). resulting in a loss of tubulin binding and destabilization of microtubules, combined with the aggregation and deposition of tau in pathogenic neurofibrillary tangles. The longest isoform. tau-2N/4R, is 441 amino acids long and has 85 putative phosphorylation sites, the majority of which are located in and adjacent to the miciOtubule-bi tiding domains. As used herein, "phosphoryiated tau^" and "phospho-tau^" are used interchangeably to denote tau protein of which at least one amino acid is phosphory ated. By "hyperphosphorylated tau^" (hPtau ) is meant tau protein of which at least two amino acids are phosphoryiated.

"Tau aggregate^", "aggregated tau^". "tau oligomer^", "oligomeric tau^". "ol igomeric form of tau^". and "tau conformer^" are used interchangeably to denote protein structures comprising more than one tau molecule, as opposed to "monomeric tau^" and "tau monomers^". As such, these terms include but are not limited to dinners, trimers. tetramers. pentamers. hexamers. heptamers. octamers. enneamers. decamers. dodecamers. icosamers. triacontamers. tetracontamers. or higher-order oligomers and mul timers of tau. non-limiting examples of which are granular aggregates, paired hel ical filaments (PHF or PHFtau ). straight filaments and neurofibrillary tangles (NFT) ( Mandelkow et al .. 2003; Drevves. 2004)(Drewes 2004). The monomers in tau aggregates can be in any form of tau. as described above. Individual monomers in tau aggregates may be homogenous, in that all monomers of an aggregate are alike, or heterogenous, in that individual aggregates comprise different forms of tau. The monomers in tau aggregates may be covalently linked to each other, or non-covalently by weak intermolecular forces, including but not limited to hydrophobic or hydrophylic interactions, hydrogen bonding, salt bridges, or van der Waals forces. A population of aggregates can be homogenous, in that all individual aggregates in that population are al ike, or heterogenous, in that individual aggregates in the population may differ from others.

By "phosphoryiated tau aggregates^'* is meant aggregates of phosphoryiated tau or hyper phosphorylated tau.

Tau aggregates may be soluble or insoluble. In a particular embodiment, the

phosphory ated tau aggregates are soluble. By "soluble^" is meant that the tau aggregates will dissolve in fluid. The term "fluid" includes bodily fluids l ike CSF. blood, plasma, serum, urine, etc.. physiological solutions, know to those skilled in the art and including but not limited to physiological salt solutions, and may comprise additional agents like buffering agents.

detergents, surfactants, sugars, chelating agents, enzyme inhibitors, reducing agents, oxidizing agents, etc. By "insoluble^" is meant that the tau aggregates will precipitate out of the fluid.

The phrase "preferabl y b i nd( s )^" or "specifically bind(s )^" or "bind(s) specifically^" when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule. The phrases "preferably bind(s ) to^" or "specifically binds to^" refers to a binding reaction that is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. .Specific binding to a target protein under such conditions may require a binding moiety that is selected for its specificity for a particular target antigen. A variety of assay formats may be used to select l igands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background. For instance antibodies and antibody fragments of the invention preferentially bind to tau. whereby by "preferentially binding^", "preferentially recognizing^" or "preferentially reacting with^" is meant that the antibodies or antibody fragments show greater binding capacity for tau as compared to any other antigen. The binding capacity of an antibody or antibody fragment to an antigen is reflective of its affinity (strength of single binding ) and/or avidity (combined strength of multiple bond interactions ) for that antigen. Typically, specific binding between two entities, such as a ligand and a receptor, means a binding affinity of at least about 10⁶ M^{" 1}. 10 M ' . 10⁸ M^{" 1}. 10⁹ M^{" 1}. or 10¹⁰ M^{" 1}. 10¹¹ M^{" 1}. 10¹² M^{" 1}. 10° M ' . 10¹⁴ M^{" 1}. or 10¹⁵ M^"

1

'Tauopathy^" is a class of degenerative diseases resulting from the pathological aggregation of tau protein in cells in case of neurodegenation of the human brain, and in case of type 2 diabetes tauopathy in the β-cells. Frequent concomitant manifestation of type 2 diabetes mellitus (T2DM) and Alzheimer^' s disease ( AD ) has been recently demonstrated by epidemiological studies. There are functional similarities between β-cells and neurons, such as secretion on demand of highly specific molecules in a tightly controlled fashion. An additional similarity represents the age-related alteration of hyperphosphorylated tau in AD patients. .Similarly.

alterations have been identified in β-cells of T2DM patients. The islet amyloid polypeptide has been associated with β-cell apoptosis. As a consequence of increasing age. the accumulation of highly modified proteins together with decreased regenerative potential might lead to increasing rates of apoptosis. Moreover, reduction of β-cell replication capabilities results in reduction of β- cell mass in mammals, simultaneously with impaired glucose tolerance. The new challenge is to learn much more about age-related protein modifications. This can lead to new treatment strategies for reducing the incidence of T2DM and AD ( Magdalena Maj et al World J Diabetes 2011 April 15; 2(4): 49-53). The best known tauopathy is Alzheimer's disease (AD ), where tau protein is deposited within neurons in the form of neurofibrillary tangles (NFTs). They were first described by the eponymous Alois Alzheimer in one of his patients suffering from the disorder. Tangles are formed by hyperphosphory ation of a microtubule-associated protein known as tau. causing it to aggregate in an insoluble form. The precise mechanism of tangle formation is not completely understood, and it is still controversial whether tangles are a primary causative factor in the disease or play a more peripheral role. AD is also classified as an amyloidosis because of the presence of senile plaques. The degree of aggregation of hyperphosphorylated tau protein (PHF, or "paired helical filaments" ) involvement in AD is defined by Braak stages. Braak stages I and II are used when NFT involvement is confined mainly to the transentorhinal region of the brain, stages III and IV when there's also involvement of limbic regions such as the

hippocampus, and V and VI when there's extensive neocortical involvement. This should not be confused with the degree of senile plaque involvement, which progresses differently. Other conditions in which neurofibrillary tangles are commonly observed include: Dementia pugilistica (chronic traumatic encephalopathy). Frontotemporal dementia and parkinsonism linked to chromosome 1 7 however without detectable β-amyloid plaques. Lytico-Bodig disease

( Park i n s o n - de m e n t i a complex of Guam ). Tangle-predominant dementia, with NFTs similar to AD. but without plaques, tends to appear in the very old. Gangl ioglioma and gangliocytoma. Meningioangiomatosis. Subacute sclerosing panencephalitis. As well as lead encephalopathy. tuberous sclerosis. Hallervorden-Spatz disease, and lipofuscinosis. In Pick's disease and corticobasal degeneration tau proteins are deposited in the form of inclusion bodies within swollen or "ballooned" neurons. Argyrophilic grai n disease (AGD ). another type of dementia, is marked by the presence of abundant argyrophilic grains and coiled bodies on microscopic examination of brain tissue. Some consider it to be a type of Alzheimer disease. It may co-exist with other tauopathies such as progressive supranuclear palsy and corticobasal degeneration. Some other tauopathies include: Frontotemporal dementia. Frontotemporal lobar degeneration. The non-Alzheimer's tauopathies are sometimes grouped together as "Pick's complex".

The antibodies of the present invention have improved reactivity characteristics compared to antibody Tau -5. which improved reactivity characteristics are attributable to the specific recognition of tau and the analytical sensitivity to tau of the antibody when bioconjugated. The antibodies of the present invention are characterized by the fact that they are suitable for use in immunoassays requiring a high analytical sensitivity for tau. such as accurate measurement of tau levels in the 0. 1 - 10 pg/ml range in body fluids such as serum and/or plasma. Accordingly. the invention relates to the use of an antibody, antibody-like scaffold, or antibody fragment in the detection of tau or in the in vitro diagnosis of a tauopathy characterized in that said antibody, antibody-like scaffold or antibody fragment: specifically binds to tau;

has an improved analytical sensitivity for tau compared to antibody Tau -5 when bioconjugated.

The antibody, antibody-like scaffold or antibody fragment of the present invention can be used in a method for diagnosis or detection of a neurological disorder, such as Alzheimer^' s disease, by detecting tau antigen present in a sample.

"Diagnosis^" is defined herein to include monitoring the state and progression of the disease, checking for recurrence of disease following treatment and monitoring the success of a particular treatment. The test may also have prognostic value. The prognostic value of the tests may be used as a marker of potential susceptibility to tauopathy. Thus patients at risk may be identified before the disease has a chance to manifest itself in terms of symptoms identifiable in the patient.

Diagnosis or detection of a tau-associated disease or condition or of a predi sposition to a tau-associated disease or condition in an individual may be achieved by detecting the immunospecific binding of a monoclonal antibody, antibody-like scaffold or antibody fragment thereof to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau antigen into contact with an antibody, antibody-like scaffold or antibody fragment which binds an epitope of the tau protein, allowing the antibody, antibody- like scaffold or antibody fragment to bind to the tau antigen to form an immunological complex, detecting the formation of the immunological complex and correlating the presence or absence of the immunological complex with the presence or absence of the tau antigen in the sample or specific body part or body area, optionally comparing the amount of said immunological complex to a normal control value, wherein an increase in the amount of said complex compared to a normal control value indicates that said individual is suffering from or is at risk of developing a tau-associated disease or condition. In a preferred embodiment, diagnosis or detection of a tau-associated disease or condition or of a

predisposition to a tau-associated disease or condition in an individual is achieved by detecting the immunospecific binding of a monoclonal antibody of the present invention to tau. Preferably, the antibody for use in the method of detection is ADx202, antibody-l ike scaffold or a functional antibody fragment thereof.

Immunological methods for detecting immunospecific binding include but are not limited to fluid or gel precipitation reactions, immuno diffusion (single or double ), agglutination assays, immuno-electrophoresis. radioimmunoassays (RIA), enzyme-linked immunosorbent assays ( ELLS A ). Western blots, dot blots, slot blots, l iposome immunoassays, line immunoassays (LI A ), complement-fixation assays. fluorescent immunoassays. Luminex™ xMAP™, i m m u n o fl u o re s ce n t flow cytometry, protein A immunoassays, or immuno PGR. An overview of different immunoassays is given in (Wild D. (201301), The Immunoassay Handbook 42nd edition. Nature Pr., London. UK) and (Ghindilis A.L., Pavlov A.R., Atanassov P.B. (eds.) (2002) Immunoassay Methods and Protocols. Humana Press. Totowa. NJ, US). Immunological detection methods further comprise i mm unoh i stochem i stiy . i mm unofl uorom icroscopy and immuno-electron microscopy.

More recently, microfluidic platforms that enable the miniaturization, integration and automation of biochemical assays have been developed. These miniaturized systems can carry out entire protocols traditionally performed in a laboratory. Sample pretreatment. sample/reagent transport, mixing, reaction, separation, detection, and product collection can all be performed automatically on a single Lab-on-a-Chip system. Microfluidic immunoassays have been implemented in both heterogeneous and homogeneous configurations. In heterogeneous configurations, antibodies are either immobil ized on the surface of the microfluidic device or on micron-dimension beads ("microbeads^" ) embedded in the device. In non-magnetic microbead- based immunoassays, a physical retention microstructure is necessary to facilitate the removal of unbound analyte or antibodies. The use of magnetic microbeads in microfluidic platforms for immunoassays is an emerging trend, as it eliminates the need for physical retention microstructures. In typical magnetic bead-based immunoassays, antibody-coated magnetic beads are immobilized on the device surface or directly on an integrated electrochemical sensor for the duration of the assay.

Another technology which makes use of magnetic beads is the xMAP technology by

Luminex corporation. Luminex discri mates the magnetic beads by color-coding the microspheres with two dyes. The ratio between these dyes determines the color-code or so-called bead region. Inside a Luminex-system. e.g. MAGPIX^®, a l ight source excites the internal dyes that identities each type of bead while another light source excites the reporter dye captured during the assay. Using this technology. xMAP^® allows multiplex ing of up to 50 analytes on the MAGPIX^®- system. 100 on the Luminex200^{1 M}-system and 500 on the FLEXMAP 3D^®-system. The technology platform allows building custom multiplexed immuno-assays by coupling oligonucleotides, antibodies or other proteins to the beads of a preferred bead region. Compared to traditional ELISA. the advantages of performing a multiplex assay are the generation of more data with less sample, reduced costs and labor, and in some cases, increased sensitivity of the immunoassay.

Thus, in one embodiment, the antibody, antibody-like scaffold or antibody fragment of the invention is used as a capture antibody and may be bound (e.g.. covalently or non-covalently. via hydrophobic or hydrophilic interactions, hydrogen bonding, or van der Waals forces ) to a solid phase, such as a bead, a plate, a membrane or a chip. Methods of coupling biomolecules. such as antibodies or antigens, to a solid phase are well known in the art. They can employ, for example, bifunctional linking agents, or the solid phase can be treated with a reactive group, such as an epoxide or an imidizole. that will bind the molecule on contact. It is to be understood that more than one antibody or antibody fragment of the invention can be used concomitantly to capture tau. The immobilized antibody or antibody fragment of the invention is then brought into contact with the sample to be tested for tau. Samples to be tested may include bodily samples such as CSF. blood, plasma, serum, urine, etc.. but also in vitro generated samples. After removal of unbound sample, the antigen-antibody complex can be detected by detection of the bound tau. This detection can be performed by using an antibody able to bind to non-aggregated tau.

aggregated tau. non-phoshorylated tau or ( h y per ) phos phory 1 ated tau. Alternatively, the whole antibody-antigen complex is detected.

In an alternative embodi ment, the capturing is done with an antibody able to bind to tau. non-aggregated tau. aggregated tau. non-phosphorylated tau. or phosphorylated tau. and the detection is performed by using an antibody, antibody-l ike scaffold or antibody fragment of the invention. Detection of the antigen-antibody complex can be performed by various methods known to the skilled person and involve detectable labels. The particular label or detectable group used in the assay is generally not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody or antibody fragment to the antigen. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the field of immunoassays and. in general, almost any label useful in such methods can be applied to the method of the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical,

immunochemical, electrical, optical, radiological or chemical means. Useful labels in the present invention include but are not limited to magnetic beads (e.g. DynabeadsTM ). fluorescent dyes (e.g. fluorescein isothiocyanate. texas red. rhodamine). radiolables (e.g. 3R, 1251, 35 S. 14C. or 32p), enzymes (e.g. horseradish peroxidase, alkaline phosphatase, luciferase. and others commonly used in an ELISA ). and colorimetric labels such as colloidal gold, colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc. ) beads. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, the ease of conjugation with the compound, stability requirements, the available instrumentation and disposal provisions. Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g. biotin ) is covalently bound to the antibody. The l igand then binds to an anti-ligand (e.g. streptavidin ) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-l igands can be used. Where a l igand has a natural anti- ligand. for example, biotin. thyrox ine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, a hapten ic or antigenic compound can be used in combination with an antibody. The antibodies can also be con jugated directly to signal- generating compounds, for example, by conjugation with an enzyme or fluorophore. Enzymes of interest will primarily be hydrolases, particularly phosphatases, esterases and glycosidases. or oxidoreductases. particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl. umbel l iferone. etc. Chemiluminescent compounds include luciferin. and 2.3-dihydrophtalazinediones. for example, luminol . A review of other labeling or signal producing systems is available in US patent No. 4.391 .904. Means for detecting labels are well known in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of a photographic film, by the use of electronic detectors such as charge coupled devices (CCDs ) or photomultipliers and the l ike. Similarly, enzyme labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Final ly simple colorimetric labels may be detected simply by observing the color associated with the label.

Thus, in one specific embodiment, the antibodies of the present invention are suitable for capturing tau antigen from a sample under conditions to form a tau antigen-antibody complex detectable by a detector. The detector can be one or more antibodies, monoclonal or polyclonal antibodies, recognizing one or more tau-epitopes on the tau antigen-antibody complex.

As indicated, in a specific embodiment, a method and/or assay for detecting tau in a sample or for the in vitro diagnosis or monitoring of a tauopathy in a subject is provided, of which the method and/or assay comprises the steps:

contacting an antibody, antibody-like scaffold or antibody fragment with a sample under conditions suitable for producing an antigen-antibody complex, and detecting the formation of said antigen-antibody complex;

wherein said antibody, antibody-like scaffold or antibody fragment:

specifically binds to tau. and

Thus, in particular embodiments, the antibody, antibody- like scaffold or antibody fragment is used as a capture moiety that binds to tau. including variants thereof or peptides. The antibody, antibody l ike scaffold or antibody fragment may hereto be bioconjugated onto a surface, e.g. a microcarrier or nanoparticle. before contact with the sample. The capture moiety may be bioconjugated by any conventional means including one or more l inkages or by adsorptions. In one embodiment, one or more different types of capture moieties that bind to different epitopes of tau may be immobilized or bioconjugated onto a surface. Alternatively, bioconjugated surfaces may incorporate moieties that bind to other antigens in addition to the tau capture moieties as described. This is particularly suitable in case tau antigen needs to be detected in addition to other antigens linked with degenerative diseases. Non-l imiting examples include antibodies, antibody-like scaffolds or antibody fragments specifically binding to beta- ameloid antigen. Further, it may be beneficial to combine detection of tau antigen with the detection of certain nucleic acid signatures. In preferred embodiments as shown in the example section, the antibody, antibody-like scaffold or antibody fragment is bio-conjugated to a magnetic surface, e.g. a magnetic microcarrier or magnetic nanoparticle.

Alternatively, in particular embodiment, the antibody, antibody-like scaffold or antibody fragment of the present invention is used as a detector moiety that binds to Tau. including variants thereof or peptides.

The example section shows that the screening and evaluation of antibody affinity and specificity combined with antibody bioconjugation properties of potential interesting antibodies results in improved assay performance. Assay performance measures sensitivity and specificity. "Sensitivity^" is the percentage (%) of known-positive samples that test positive in an assay. "Specificity^" is the percentage (%) of known-negative samples that test negative in an assay. The "analytical sensitivity or detection limit" is the lowest concentration of analyte (i .e. tau antigen ) in a sample that can be distinguished from background, but not necessarily quantified.

"Analytical specificity or selectivity^" is the degree to which an assay does not cross-react with related antigens.

Bioconjugation is the process of coupling two biomolecules together in a covalent linkage. Common types of bioconjugation chemistry are amine coupling of lysine amino acid residues (typically through amine-reactive succinimidyl esters ), sulfhydryl coupling of cysteine residues (via a su 1 fh ydry 1 - react i ve maleimide ). and photochemically initiated free radical reactions, which have broader reactivity. The product of a bioconjugation reaction is a bioconjugate. In preferred embodiments, the antibodies of the invention are bioconjugated to a magnetic surface.

Antibodies are produced by cell lines. The present invention thus also provides isolated cell lines producing the antibody or antibody fragments of the present invention. Under "cell line^" is to be understood a homogenous population of eukaryotic cells which is genetically stable and can be cultured. Preferably, the cell l ine is of animal origin. More preferably, the cell l ine is immortal ized. Alternatively, the cell line is of plant or fungal origin. In one embodiment, the cell line of the invention is obtained by genetic transformation with a nucleic acid comprising a polynucleotide encoding the antibody or antibody fragment of the invention under suitable transcriptional and translational control elements, which are known to those skilled in the art, to allow efficient production of the antibody or antibody fragment. Preferably, the nucleic acid comprises the nucleic acid sequence as set out in SEQ ID NO. 19 and/or SEQ ID NO. 20. In a preferred embodiment, the cell line is a hybridoma cell line. ADX202, producing antibody ADx202, deposited under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection under No. 10376CB (name of depositor: Dr Eugeen Vanmechelen; address of the depositor: ADx NeuroSciences, Technologiepark Zwijnaarde 4, 9052 Gent-Zwijnaarde ). The term "hybridoma^" is well recognized in the art and refers to a cell l ine resulting from the fusion of a single an t i body- produc i n g cell clone and an immortal cell or tumor cell . As used throughout the text, the term ADx and ADX are used interchangeably.

Further provided are isolated nucleic acids comprising a polynucleotide encoding the tau antigen fragment binding to of one of the antibodies of the present invention. In a preferred embodiment, said polynucleotide comprises a nucleotide sequence encoding the tau antigen fragment binding to the antibody produced by cell line ADX 202 deposited under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms BCC M™/L M B P Collection under No. 10376CB.

The nucleic acid molecules according to the invention may, advantageously, be included in a suitable expression vector. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of a cell and subsequent selection of the transformed cells is well known to those skilled in the art. Any suitable technique may be employed. Examples are provided in Sambrook and Russell (2001), Molecular cloning: A Laboratory Manual, Cold Spring Harbour Laboratory. An expression vector, according to the invention, includes a vector comprising a nucleic acid according to the invention operably linked to one or more regulatory sequences, such as promoter regions, that are capable of effecting expression of antibodies, antibody-like scaffolds or antibody fragments encoded by the nucleic acid. A vector can include a large number of nucleic acids which can have a desired sequence inserted therein by, for example, using an appropriate restriction enzyme and ligating the sequence in the vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for expression. The vectors may be capable of replicating within a host environment and may also comprise one or more restriction sites for nucleases which permits them to be restricted in a selective manner at a particular location for insertion of a new nucleic acid molecule or sequence therein.

The vectors may be, for example, plasmid, virus or phagemid vectors. They may be provided with an origin of replication, a promoter for the expression of the peptide from the nucleic acid and/or a regulator of the promoter for example. The vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance gene.

Regulatory elements required for expression include promoter sequences to bind RNA polymerase and to direct an appropriate level of transcription initiation and also translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. However, the precise regulatory elements required for expression of a gene of interest may vary between different cell types but generally include 5' non-transcribing and non-translating regions which are required for initiation of translation and transcription. Such vectors may be obtained commercially or be assembled from known vectors using methods well known in the art.

Transcription of DNA by higher eukaryotes may be optimised by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase the level of transcription. In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including, in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue). The term "nucleic acid" also includes the complementary sequence to any single stranded sequence given regarding base variations.

A further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention. The cell or organism may be transformed or transfected using any suitable technique. Many examples are well known in the art, such as electroporation and use of liposomes.

Any suitable host cell or organism may be used, for example a prokaryotic or eukaryotic host cell. Examples include but are not limited to bacteria, yeasts, higher plant cells in culture, insect cells in culture and mammalian cells in culture.

The antibody of the present invention can be used in a method for monitoring residual disease, such as Alzheimer^'s disease, following treatment with a vaccine composition.

Monitoring minimal residual disease in an individual following treatment with a vaccine composition may be achieved by detecting the immunospecific binding of a monoclonal antibody or a functional fragment thereof to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau antigen into contact with an antibody which binds an epitope of the tau protein, allowing the antibody to bind to the tau antigen to form an immunological complex, detecting the formation of the immunological complex and correlating the presence or absence of the immunological complex with the presence or absence of the tau antigen in the sample or specific body part or body area, optionally comparing the amount of said immunological complex to a normal control value, wherein an increase in the amount of said aggregate compared to a normal control value indicates that said individual is still suffering from a minimal residual disease. In a preferred embodiment. Monitoring minimal residual disease in an individual following treatment with a vaccine composition is achieved by detecting the immunospecific binding of a monoclonal antibody of the present invention to aggregated tau. Preferably, the antibody for use in the method of detection is ADx202 or a functional fragment thereof. The antibody of the present invention can also be used in a method for predicting responsiveness of a patient to a treatment with a vaccine composition. Predicting responsiveness of a patient to a treatment with a vaccine composition may be achieved by detecting the immunospecific binding of a monoclonal antibody or a functional fragment thereof to an epitope of the tau protein in a sample or in situ, which includes bringing the sample or a specific body part or body area suspected to contain the tau antigen into contact with an antibody which binds an epitope of the tau protein, allowing the antibody to bind to the tau antigen to form an immunological complex, detecting the formation of the immunological comple and correlating the presence or absence of the immunological complex with the presence or absence of the tau antigen in the sample or specific body part or body area, optionally comparing the amount of said immunological complex before and after onset of the treatment, wherein a decrease in the amount of said complex indicates that said individual has a high potential of being responsive to the treatment. In the alternative, the method for predicting responsiveness of a patient to a treatment with a vaccine composition may detect that there is no decrease in the amount of the immunological complex before and after onset of the treatment and thus indicate that the individual has low potential of being responsive to the treatment. In a preferred embodiment, predicting responsiveness to a treatment with a vaccine composition in an idividual is be achieved by detecting the immunospecific binding of a monoclonal antibody of the present invention to aggregated tau. Preferably, the antibody for use in the method of detection is ADx202 or a functional fragment thereof.

The invention also provides peptides representing an epitope of the tau protein, which epitope is recognized by an antibody according the present invention. The peptides of the present invention find their application in various methods and tests, such as but not limited to methods for diagnosis or the detection of a tau-associated disease or condition or of a predisposition to a tau-associated disease or condition in an individual, methods for monitoring residual disease, such as Alzheimer^' s disease, following treatment with a vaccine composition, or methods for predicting responsiveness of a patient to a treatment with a vaccine composition. The peptides may be used as suitable controls to ensure that the methods and tests are working properly. The peptides may for instance be used as positive controls, as internal standards, as calibrators, or for quantification purpose. The invention also provides kits which may be used in order to carry out the methods of the invention. The kits may incorporate any of the preferred features mentioned in connection to the various methods and uses of the invention described herein. Thus, the invention provides a kit for detecting a tau-associated disease or condition or of a predisposition to a tau-associated disease or condition in a body sample of an individual, and comprises at least one or more antibodies of the present invention, preferably the antibody ADx202 or antibodies having the binding characteristics of antibody ADx202. In particular, the test kit comprises a container holding a packaged combination of reagents in predetermined amounts, such as one or more antibodies according to the present invention, with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and co- factors required by the enzyme. Further additives may be included such as stabil izers, buffers and the like. The kit comprising one or more antibodies of the present invention may be used to discriminate for instance early stage Alzheimer^'s dementia form other types of dementia in an individual. The kit comprising one or more antibodies of the present invention may be used to identify tau. The kit may incorporate suitable controls to ensure that the method and test is working properly. The kit may for that purpose incorporate one or more peptides of the present invention. Preferably, the kit incorporates a peptide. Characteristics of the one or more antibodies and of the one or more peptides are summarized elsewhere in the detailed description and in the experimental part below.

Examples

1 . Generation of antibody ADx202

For the generation of novel mAbs, full-length human tau (longest isoform. 2N4R; 441 amino acids ) was purified as a N-terminal His6 fusion product from the Apho85Aadhl yeast deletion strain using conventional i l MAC chromatography. Tau expressed in this yeast strain has been characterized extensively with respect to (hyper- (phosphorylation, conformational change and aggregation (Vandebroek et al. Biochemistry 2005;44: 11466- 11475; Vandebroek et al. J Biol Chem 2006:28 1 :253888-25397; Vanhelmont et al. FEMS Yeast Res 20 10: 10:992- 1005). 8 BALB/c mice were immunized with 5 μg tau and titers were determined. The spleen of the mouse with the highest titer was removed and fused to SP2/0 myeloma cells in a 1/1 ratio. Positive hybridoma^' s were selected based on different human tau isolates ( yeast derived human tau. e.coli derived human tau. etc. ) and on yeast extracts. Upon expansion and further cloning, tau positive hybridoma^' s were further characterized on western-blots. ADx202 reacted as Tau-5 on different tau forms and was found to be a pan-like antibody such as Tau-5 not dependent on phosphorylation or hyperphosphorylation for optimal recognition.

2. Procedure coating/coupling

ELISA

ΙΟΟμΙ ADx201 (harvested by In Vivo) or ADx202 (harvested by PharmAbs ) at different concentrations were added in PBS buffer (Lonza. nr BE17-512F) and incubated overnight at 4°C in a microtiter plate (F8 Maxisorp. cat nr 735-0048, VWR ). The plate was washed 1 x with wash buffer (PBS buffer with Tween-20 added (0.05%)).

MagPix

The stock microspheres (region 1 5. Luminex Corps, cat nr MC I 001 5-01 ; lot nr 30661 ; concentration (determined with a Scepter system by Merck Mill i pore ) 1 .027. 1 0 /niL) were resiispended by vortex (10"), sonicated (3'), and 2.5xlOE6 of the microspheres were transferred to an Eppendoif Protein Safe-Lock micro-tube (cat nr EPPE0030120.086. VWR ). The reaction tube with microspheres was placed into a magnetic separator for 2 minutes in order to wash the beads with 100μ L distilled water (vortex 10", sonication 20"). Subsequently, the water was removed and the beads were re-suspended in 80μ ί Monobasic Sodium Phosphate buffer (lOOmM, pH6.2). 2.5μΕ of a 50mg/mL, freshly prepared. Sulfo-NHS (cat nr 24520. Thermo Scientific ) solution was added to the microspheres. After brief vortex ing. 20μ Ε of a 50mg/mL, freshly prepare. EDC (cat nr 77 149. Thermo Scientific ) solution, was also added to the beads. Following 10^" vortex ing and 20^" sonication. the mixture was incubated for 20^' at RT. in the dark, by rotation at 15rpm. Afterwards, the reaction tube with microspheres was placed into the magnetic separator for 2 minutes, the supernatant was removed and the beads were washed with 1 25μ Ε of MES buffer (50mM, pH5.0). After repeating this washing, the beads were re- suspended in 50μ ί of MES buffer. The capture antibody (62,5μg) (see listed below ) was added to the re-suspended beads and the mix was brought to a total volume of 250μΕ MES buffer. The coupling reaction was mixed by vortex ing (10") and sonication (20") and further incubated overnight (17h30) at RT in the dark, on the rotator (15rpm). The beads were washed once with 250μ Ε PBS-TB (PBS pH7.4, 0.1% BSA, 0.02% Tween-20 ). followed by two washes with SOO L of PBS-TB and re-suspended in 500μ ί PBS pH7.4 with 0.1% BSA ). The final concentration of the coupled beads was determined using a Scepter-system (Merck Millipore ).

Capture antibodies:

1 . ADx202: PharmAbs (production in serum -free medium ); concentration l,08mg/mL

2. Tau -5: Covance (via Eurogentec; cat nr SIG-39413-0500; lot nr 10FC00934); concentration 0,5mg/mL Results count of coupled beads are illustrated in Figures 1 A to 1 B and had the following associated characteristics:

For ADx202 (Figure I A ):

Low marker (μπι) 3; high marker (μm) 13.01 : corrected concentration 4,397E+06/mL (dilution factor 10: system^'s read -out 4,397E+05/mL); mean cell diameter (μπι) 6.63: mean cell volume (pL) 0,15.

For Tau-5 (Figure 1 B ):

Low marker (μπι) 3: high marker (μm) 1 3.01 : corrected concentration 5,343E+06/mL (dilution factor 10; system^'s read-out 5,343E+05/mL); mean cell diameter (μπι) 6.49; mean cell volume (pL) 0.14. 3. Confirmation of antibody coating/coupling

MagPix

After vortex ing (10"), sonicating (3') and vortex ing (5"), a working solution of beads (coupled with ADx201 . ADx202 or Tau-5) was prepared in assay buffer (PBS (Lonza. nr BE 17- 512F) + 0.1% BSA + 0.3% Pluronic F- 1 27 + 0.077% CHAPS ), with a final bead concentration of 40000 per mL. In parallel, a 2-fold serial dilution of phycoerythrin-labeled anti-mouse IgG detection antibody (cat nr 1 15- 1 16-072; lot nr 101 858. Jackson ImmunoResearch ) was made in assay buffer (from 4 to 0.0625 g/mL). 50μ Ι.. of the working solution of the beads was al iquoted into the appropriate wells (after washing the plate with 200μ L of assay buffer during 10' on the shaker) and 50μΕ of the diluted detection antibody was added. Following incubation for I h at room temperature (RT) (in the dark, on a plate shaker), the beads were washed 3x using the link program "MAGPIXW" on the ELx50-system ( BioTek ) and resuspended in 100μ L drive fluid (cat nr. MPXDF-4PK. Luminex Corps. ). After 5' shaking at RT (in the dark ). 75μ 1_ of each well was analyzed on the MagPix system ( Luminex corps. ).

Link program "MAGPIXW^":

• Soak program:

1 . Soak duration: 60sec

2. Shake before soak?: No

• Wash program:

o Method:

1 . umber of cycles: 3

2. Wash format: Plate

3. Soak/shake: Yes

4. Soak duration: 60 sec

5. Shake before soak: No

6. Prime after soak: No

o Dispense:

1 . Dispense volume: 20C^L/well

2. Dispense flow rate: 5

3. Dispense height: 130 ( 16.5 1 mm )

4. Horizontal disp pos: 00 (0mm )

5. Bottom Wash first? : No

6. Prime before start?: No

o Aspiration:

1 . Aspirate height: 35 (4.445mm )

2. Horizontal Asp Pos: 24 (1,097mm)

3. Travel rate: 06 ( 1 0.7 mm/sec )

4. Aspiration delay: 0

5. Crosswire Aspir: No

6. Final Aspir: YES

7. Final Aspir delay: 0 (0msec )

Results are shown in Figure 2. 4. Functional assay

MagPix

The plate is washed with 200_,u L of assay buffer (PBS pH7.4 (Lonza. nr BE17-512F); 0,1% BSA. 0.3% Pluronic F- 127. 0,077% CHAPS) during 10' on the shaker. Meanwhile, after vorte ing (10"), sonicating (3') and vortexing (5"), a working solution of beads (coupled with ADx202 or Tau-5) was prepared in assay buffer, with a final bead concentration of 160000 per niL. 50μL· of this working solution of beads was aliquoted into the appropriate wells and following 1 ^' on a handheld magnet, the well content was discarded (the beads retained in the wells due to the magnet). Ί5μL· of a dilution series of rPeptide441

(human tau-441. recombinant e.coli; rPeptide. cat nr T- 1001 -2 ) was added in the wells, combined with 25μL· of the diluted detection antibody at a concentration of

(biotinylated ADx215, ratio 32/1 (biotin/Ab). batch 1010201 2( E)). Following incubation for 3h at room temperature (RT) (in the dark, on a plate shaker), the beads were washed 3x using the link program "MAGPIXW" on the ELx50-system (BioTek) (wash buffer: PBS + 0.05%

Tween-20 ) and resuspended in/ incubated with 100μ L SA-PE (Streptavidin-phycoeiythrin. Moss substrates, cat nr SAPE-OO I B. lot nr 1 1332 1 14 ) (lh, dark. RT. shaker). The beads were washed again (link program "MAGPIXW^" on the ELx50-system (BioTek) ) and resuspended in 100μ L drive fluid (cat nr. MPXDF-4PK. Luminex Corps. ). After 5' shaking at RT (in the dark). 75μ L of each well was analyzed on the MagPix system (Luminex corps. ).

Results are represented in Figure 3. 4. Cloning of Antibody Light Chain and Heavy Chain Variable regions

Antibody heavy chain and light variable region genes from the hybridoma cells were cloned and the DNA sequences and location of the complementary determining regions (CDRs) determined (Chothia C, Lesk AM (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol 196: 901-917; Kettleborough CA, Saldanha J, Ansell KH, Bendig MM (1993) Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction. Eur J Immunol 23: 206-211; Orlandi R, Gussow DH, Jones PT, Winter G (1989) Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A 86: 3833-3837). Total RNA was prepared from 3.9*10e6 hybridoma cells ( 1 vial) using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 50 mL water and checked on a 1.2% agarose gel. VH and VK cDNAs were prepared using reverse transciptase with IgG and kappa constant region primers. The first strand cDNAs were amplified by PCR using a large set of signal sequence primers. The amplified DNAs were gel-purified and cloned into the vector pGem T Easy (Promega). The VH and VK clones obtained were screened for inserts of the expected size. The DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Kabat EW, T.; Perry, H.; Gottesman, K.; Foeller, C. (1991) Sequences of proteins of Immunological Interest. US department of Health and Human Services). Sequences are shown in Figures 4 and 5 and protein sequences are represented by SEQ ID NO. 9 to SEQ ID NO. 18, and nucleic acid sequences are represented by SEQ ID NO. 19 and SEQ ID NO. 20

Sequence ID 1

organ ism=" Homo sapiens" /db_xref="taxon:9606" /chromosome=" 17" /map=" 17q2 1 . 1 "

Protein 1 ..758

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg

61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag

121 hvtqepesgk vvqegtlrep gppglshqlm sgmpgapllp egpreatrqp sgtgpedteg

1 8 1 grhapellkh qllgdlhqeg pplkgaggke rpgskeevde drdvdesspq dsppskaspa

241 qdgrppqtaa reatsipgfp aegaiplpvd flskvsteip asepdgpsvg rakgqdaple

301 ftfhveitpn vqkeqahsee hlgraafpga pgegpeargp slgedtkead lpepsekqpa

361 aaprgkpvsr vpqlkarmvs kskdgtgsdd kkaktstrss aktlknrpcl spkhptpgss

42 1 dpliqpsspa vcpeppsspk yvssvtsrtg ssgakemklk gadgktkiat prgaappgqk

48 1 gqanatripa ktppapktpp ssgeppksgd rsgysspgsp gtpgsrsrtp slptpptrep

541 kkvavvrtpp kspssaksi qtapvpmpdl knvkskigst enlkhqpggg kvqiinkkld

60 1 Isnvqskcgs kdnikhvpgg gsvqivykpv dlskvtskcg signihhkpg ggqvevksek

66 1 ldfkdrvqsk igsldnithv pgggnkkiet hklttVenak aktdhgaeiv ykspvvsgdt

72 1 sprhlsnvss tgsidmvdsp qlatladevs aslakqgl

Sequence ID 2

organ ism=" Homo sapiens" /db_x ret^*=" tax on : 9606 " /chromosome=" 17" /map=" 17q2 1 . 1 " /

Protein 1 ..441

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg

61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag

1 2 1 hvtqarmvsk skdgtgsddk kakgadgktk iatprgaapp gqkgqanatr ipaktppapk

1 8 1 tppssgeppk sgdrsgyssp gspgtpgsrs rtpslptppt repkkvavvr tppkspssak

241 si qtapvpm pdlknvkski gstenlkhqp gggkvqii nk kldlsnvqsk cgskdnikhv

301 pgggsvqivy kpvdlskvts kcgslgnihh kpgggqvevk sekldfkdrv qskigsldni

361 thvpgggnkk iethklttVe nakaktdhga eivykspvvs gdtsprhlsn vsstgsidmv

42 1 dspqlatlad evsaslakqg 1

Sequence ID 3

/organism="Homo sapiens" / db_xref="taxon:9606" / chromosome=" 1 7" /map=" 1 7q2 1 . 1 " / Protein 1 ..383 1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkaeeagi gdtpsledea

61 aghvtqarmv skskdgtgsd dkkakgadgk tkiatprgaa ppgqkgqana tripaktppa

121 pktppssgep pksgdrsgys spgspgtpgs rsrtpslptp ptrepkkvav vrtppkspss

1 8 1 aksi qtapv pmpdlknvks kigstenlkh qpgggkvqii nkkldlsnvq skcgskdnik

241 hvpgggsvqi vykpvdlskv tskcgslgni hhkpgggqve vksekldfkd rvqskigsld

301 nithvpgggn kkiethkltf renakaktdh gaeivykspv vsgdtsprhl snvsstgsid

361 mvdspqlatl adevsaslak qgl

Sequence ID 4

organism="Homo sapiens" /db_xref="taxon:9606" /chromosome=" 17" /map=" 17q2 I . I " /

Protein 1..352

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkaeeagi gdtpsledea

6 1 aghvtqarmv skskdgtgsd dkkakgadgk tkiatprgaa ppgqkgqana tripaktppa

12 1 pktppssgep pksgdrsgys spgspgtpgs rsrtpslptp ptrepkkvav vrtppkspss

1 8 1 aksiiqtapv pmpdlknvks kigstenlkh qpgggkvqiv ykpvdlskvt skcgslgnih

241 hkpgggqvev ksekldfkdr vqskigsldn ithvpgggnk kiethklttr enakaktdhg

301 aeivykspvv sgdtsprhls nvsstgsidm vdspqlatla devsaslakq gl

Sequence ID 5

organ ism=" Homo sapiens" /db_xref="taxon:9606" /chromosome=" 17" /map=" 1 7q2 1 . 1 " Protein 1 ..41 2

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg

61 setsdakstp taeaeeagig dtpsledeaa ghvtqarmvs kskdgtgsdd kkakgadgkt

12 1 kiatprgaap pgqkgqanat ripaktppap ktppssgepp ksgdrsgyss pgspgtpgsr

1 8 1 srtpslptpp trepkkvavv rtppkspssa ksiiqtapvp mpdlknvksk igstenlkhq

241 pgggkvqi in kkldlsnvqs kcgskdiiikh vpgggsvqiv ykpvdlskvt skcgslgnih

301 hkpgggqvev ksekldfkdr vqskigsldn ithvpgggnk kiethklttr enakaktdhg

361 aeivykspvv sgdtsprhls nvsstgsidm vdspqlatla devsaslakq gl

Sequence ID 6

organ ism=" Homo sapiens" /db_xref="taxon:9606" /chromosome=" 17" /map=" 17q2 1 . 1 " Protein 1 ..776

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg 61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag 121 hvtqepesgk vvqegflrep gppglshqlm. sgmpgapllp egpreatrqp sgtgpedteg 1 8 1 grhapellkh qllgdlhqeg pplkgaggke rpgskeevde drdvdesspq dsppskaspa 241 qdgrppqtaa reatsipgfp aegaiplpvd flskvsteip asepdgpsvg rakgqdaple 301 ftfhveitpn vqkeqahsee hlgraafpga pgegpeargp slgedtkead lpepsekqpa 361 aaprgkpvsr vpqlkarmvs kskdgtgsdd kkaktstrss aktlknrpcl spkhptpgss 42 1 dpliqpsspa vcpeppsspk yvssvtsrtg ssgakemklk gadgktkiat prgaappgqk 48 1 gqanatripa ktppapktpp ssatkqvqrr pppagprser geppksgdrs gysspgspgt 541 pgsrsrtpsl ptpptrepkk vavvrtppks pssaksrlqt apvpmpdlkn vkskigsten 601 Ikhqpgggkv qiinkkldls nvqskcgskd nikhvpgggs vqivykpvdl skvtskcgsl 661 gnihhkpggg qvevksekld fkdrvqskig sldnithvpg ggnkkiethk IttVenakak 72 1 tdhgaeivyk spvvsgdtsp rhlsnvsstg sidmvdspql atladevsas lakqgl

Sequence ID 7

/organ i sm=" Homo sapiens" /db_x ret^*=" tax on : 9606 " /chromosome=" 17"

/map=" 17q21.1 "

Protein I ...38 1

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg 61 setsdakstp taeaeeagig dtpsledeaa ghvtqarmvs kskdgtgsdd kkakgadgkt 1 2 1 kiatprgaap pgqkgqanat ripaktppap ktppssgepp ksgdrsgyss pgspgtpgsr 1 8 1 srtpslptpp trepkkvavv rtppkspssa ksi qtapvp mpdlknvksk igstenlkhq 241 pgggkvqivy kpvdlskvts kcgslgnihh kpgggqvevk sekldfkdrv qskigsldni 30 1 thvpgggnkk iethkltfre nakaktdhga eivykspvvs gdtsprhlsn vsstgsidmv 36 1 dspqlatlad evsaslakqg 1

Sequence ID 8

organism=" Homo sapiens" /db_xret^*="taxon:9606" /chromosome=" 1 7" /map=" Protein 1 410

1 maeprqefev medhagtygl gdrkdqggyt mhqdqegdtd aglkesplqt ptedgseepg 61 setsdakstp taedvtaplv degapgkqaa aqphteipeg ttaeeagigd tpsledeaag 1 2 1 hvtqarmvsk skdgtgsddk kakgadgktk iatprgaapp gqkgqanatr ipaktppapk 1 8 1 tppssgeppk sgdrsgyssp gspgtpgsrs rtpslptppt repkkvavvr tppkspssak 241 srlqtapvpm pdlknvkski gstenlkhqp gggkvqivyk pvdlskvtsk cgslgnihhk

301 pgggqvevks ekldfkdrvq sk igsldnit hvpgggnkki ethkltfren akaktdhgae

361 ivykspvvsg dtsprhlsnv sstgsidmvd spqlatlade vsaslakqgl

Amino acid sequence of ADx202 light chain variable region SEQ ID NO 9:

DIVMSOSPSSLAVSVGEKVIMSCKSSOSLLYSTNOKNFLAWYOOKPGOSPKLLIYWAST RESGVPDRFTGSGSGTDFTLTISSLKAEDLAVYYCOOYYSYPYTFGGGTKLEIKRADAA PTV

Amino acid sequence of CDR1 of ADx202 light chain variable region SEQ ID NO 10: KSSQSLLYSTNQKNFLA

Amino acid sequence of CDR2 of ADx202 light chain variable region SEQ ID NO 11 : WASTRES

Amino acid sequence of CDR3 of ADx202 light chain variable region SEQ ID NO 12: QQYYSYPYT

Constant region of ADx202 light chain SEQ ID NO 13:

RADAAPTV

Amino acid sequence of ADx202 heavy chain variable region SEQ ID NO 14:

OIOLVOSGPELKKPGETVKISCKASGYTFTDYSMNWVKOAPGOGLKWVGWINTETGEP AYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDFFDFWGOGTSLTVSSAKT TP

Amino acid sequence of CDR1 of ADx202 heavy chain variable region SEQ ID NO 15: GYTFTDYSMN

Amino acid sequence of CDR2 of ADx202 heavy chain variable region SEQ ID NO 16: WINTETGEPAYADDFKG Amino acid sequence of CDR3 of ADx202 heavy chain variable region SEQ ID NO 17: DFFDF Constant region of ADx202 heavy chain SEQ ID NO 18 :

AKTTP

REFERENCES: ANDREASSON, U., E. VANMECHELEN, ET AL. (2012). "ANALYTICAL ASPECTS OF

MOLECULAR ALZHEIMER'S DISEASE BIOMARKERS . " BIOMARK MED 6(4): 377-389.

ASLAM, M. AND A. DENT (1998). BIOCONJUGATION: PROTEIN COUPLING TECHNIQUES FOR THE BIOMEDICAL SCIENCES, MACMILLAN REFERENCE.

CARMEL, G., E. M. MAGER, ET AL. (1996). "THE STRUCTURAL BASIS OF MONOCLONAL ANTIBODY ALZ50'S SELECTIVITY FOR ALZHEIMER'S DISEASE PATHOLOGY. " J BIOL CHEM

271(51): 32789-32795.

DELVES, P. J., S. J. MARTIN, ET AL. (2011). ROUT'S ESSENTIAL IMMUNOLOGY, WILEY.

DREWES, G. (2004). "MARKING TAU FOR TANGLES AND TOXICITY." TRENDS BIOCHEM SCI 29(10): 548-555.

GHINDILIS, A. L., A. R. PAVLOV, ET AL. (2003). IMMUNOASSAY METHODS AND PROTOCOLS.

HUMANA PRESS.

GOEDERT, M., M. G. SPILLANTINI, ET AL. (1989). "MULTIPLE ISOFORMS OF HUMAN

MICROTUBULE-ASSOCIATED PROTEIN TAU: SEQUENCES AND LOCALIZATION IN

NEUROFIBRILLARY TANGLES OF ALZHEIMER'S DISEASE." NEURON 3(4): 519-526.

MAJ, M., A. ILHAN, ET AL. (2011). "AGE RELATED CHANGES IN PANCREATIC BETA CELLS: A

PUTATIVE EXTRA-CEREBRAL SITE OF ALZHEIMER'S PATHOLOGY." WORLD J DIABETES 2(4): 49-53.

SAMBROOK, J. AND D. W. RUSSELL (2001). MOLECULAR CLONING: A LABORATORY MANUAL, COLD SPRING.

TAJIMA, N., M. TAKAI, ET AL. (2011). "SIGNIFICANCE OF ANTIBODY ORIENTATION

UNRAVELED: WELL-ORIENTED ANTIBODIES RECORDED HIGH BINDING AFFINITY." ANAL CHEM 83(6): 1969-1976.

TRILLING, A. K.. M. M. HARMSEN, ET AL. (2013). "THE EFFECT OF UNIFORM CAPTURE MOLECULE ORIENTATION ON BIOSENSOR SENSITIVITY: DEPENDENCE ON ANALYTE PROPERTIES." BIOSENS BIOELECTRON 40(1): 219-226. VANDEBROEK, T., T. VANHELMONT, ET AL. (2005). "IDENTIFICATION AND ISOLATION OF A HYPERPHOSPHORYLATED, CONFORMATIONALLY CHANGED INTERMEDIATE OF HUMAN PROTEIN TAU EXPRESSED IN YEAST." BIOCHEMISTRY 44(34): 11466-11475.

VANDERMEEREN, M., M. MERCKEN, ET AL. (1993). "DETECTION OF TAU PROTEINS IN NORMAL AND ALZHEIMER'S DISEASE CEREBROSPINAL FLUID WITH A SENSITIVE SANDWICH ENZYME-LINKED IMMUNOSORBENT ASSAY." J NEUROCHEM 61(5): 1828-1834. VANHELMONT, T., T. VANDEBROEK, ET AL. (2010). "SERINE-409 PHOSPHORYLATION AND OXIDATIVE DAMAGE DEFINE AGGREGATION OF HUMAN PROTEIN TAU IN YEAST." FEMS YEAST RES 10(8): 992-1005.

VANMECHELEN, E., H. VANDERSTICHELE, ET AL. (2000). "QUANTIFICATION OF TAU PHOSPHOR YLATED AT THREONINE 181 IN HUMAN CEREBROSPINAL FLUID: A SANDWICH ELISA WITH A SYNTHETIC PHOSPHOPEPTIDE FOR STANDARDIZATION." NEUROSCI LETT 285(1): 49-52.

WILD. D. G. (2013). THE IMMUNOASSAY HANDBOOK: THEORY AND APPLICATIONS OF LIGAND BINDING, ELISA AND RELATED TECHNIQUES, ELSEVIER SCIENCE.

List of abbreviatons

2N/4R longest isoform of protein tau. with 2 -terminal inserts (2N) and 4 microtubule binding regions (4R)

Mg/mL m icrogram/m i 11 i 1 iter; 10^~6 gram/ 10^"3 liter

μηι micrometer: 10^~6 meter

Ab antibody

AD Alzheimer^'s disease

AGD Argyrophil ic grain disease

BSA bovine serum albumin

CCD' s charge coupled devices

CDR l -3 complementarity determi ning regions 1 to 3 of an antibody

CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-propane- sulfonate

CSF cerebrospinal fluid

dAb domain antibody

DNA Deoxyribonucleic acid

EDC 1 -ethyl- 3 - (3 -dimethylaminopropyl)carb odiimide

ELISA enzyme-linked immunosorbent assays

Fab antigen-binding fragment of an antibody

FR 1 -4 framework regions 1 to 4 of an antibody

hPTau hyperphosphorylated tau

HRP horse radish peroxidase

IgG/A/... immunoglobulin isotype G/A/...

kDa kilo Dalton: 10³ Dalton

LI A line immunoassays

mAb(s) monoclonal antibody(ies )

MAP microtubule-associated protein

MES 2-(N-morpholino)ethanesulfonic acid

MFI median fluorescent intensity

NFT neurofibrillary tangles

OD optical density

pg/mL picogram/millilite 10^{" "} gram/ 10^" liter PBS phosphate buffered saline

PGR polymerase chain reaction

PHF paired hel ical filaments

Fl^'au phosphorylated tau

RIA radio-immunoassays

RT room temperature

scFv single chain antibodies

S/N ratio signal to noise

SA-PE streptavidin-phycoerythrin conjugate

Sulfo-NHS N-hydroxysulfosuccinimide

T2DM type 2 diabetes mellitus

TMB 3,3' ,5,5'-Tetramethylbenzidine

V_H heavy chain of an antibody

V_HH antibody with two heavy chains only

V_L light chain of an antibody

Claims

1 . A(n isolated) tau antibody, antibody-like scaffold or antibody fragment thereof, that: specifically binds to tau;

has an improved analytical sensitivity for tau compared to antibody Tau-5 when bioconjugated.

2. An (isolated) antibody, antibody-like scaffold or antibody fragment wherein the light chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 10, and/or in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 11 and/or in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 12; and/or wherein a heavy chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 15, and/or in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 16 and/or in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 17.

3. An (isolated) antibody, antibody- like scaffold or antibody fragment comprising at least one CDR triplet selected from the group consisting of

- a CDR triplet H1/H2/H3; and

- a CDR triplet L1/L2/L3;

wherein HI has an amino acid sequence as set out in SEQ ID NO. 15, H2 has an amino acid sequence as set out in SEQ ID NO. 16, H3 has an amino acid sequence as set out it SEQ ID NO. 17, LI has an amino acid sequence as set out in SEQ ID NO. 10, L2 has an amino acid sequence as set out in SEQ ID NO. 11, and L3 has an amino acid sequence as set out in SEQ ID NO. 12.

4. The antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 3. which is a monoclonal antibody secreted by hybridoma cell line No. LMBP 10376CB deposited at the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection.

5. The (isolated) antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 4. wherein the isolated antibody, antibody-like scaffold or antibody fragment binds to non-phosphorylated tau, phosphoryiated tau and non- phosphorylated tau, and/or phosphory ated tau and non-phosphoryiated aggregates.

6. The antibody, antibody- like scaffold or antibody fragment according to any of claims 1 to 5 wherein the light chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 10, and/or in a CDR2 region an amino acid

32 sequence as set out in SEQ ID NO: 11 and/or in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 12; and/or wherein the heavy chain variable region comprises in a CDR1 region an amino acid sequence as set out in SEQ ID NO: 15, and/or in a CDR2 region an amino acid sequence as set out in SEQ ID NO: 16 and/or in a CDR3 region an amino acid sequence as set out in SEQ ID NO: 17.

7. The antibody, antibody-l ike scaffold or antibody fragment according to any of claims 1 or 4 to 6. wherein bioconj ligation is on a magnetic surface.

8. The antibody, antibody-l ike scaffold or antibody fragment according to any of claims 1 to 7. wherein tau is rPeptide441.

9. An (isolated ) antibody produced by hybridoma cell line No. LMBP 10376CB

deposited at the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection.

10. A(n isolated ) nucleic acid comprising a polynucleotide encoding the antibody,

antibody-like scaffold or antibody fragment according to any of claims 1 to 9.

1 1 . A(n isolated ) cell line producing the antibody, antibody-like scaffold or antibody fragment according to any of claims I to 8.

12. A hybridoma from cell line No. LMBP 10376CB deposited at the Belgian

Coordinated Collections of Microorganisms BCCM™/LMBP Collection.

13. Use of the antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 9 in the detection of tau or in the in vitro diagnosis of a tauopathy.

14. A method for detecting tau in a sample or for the in vitro diagnosis or monitoring of a tauopathy in a subject, comprising the following steps:

- contacting an antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 9 with a sample under conditions suitable for producing an antigen- antibody complex; and

- detecting the formation of said antigen-antibody complex.

15. A kit for the detection of tau or for the in vitro diagnosis or monitoring of a tauopathy in a subject, comprising the antibody, antibody-like scaffold or antibody fragment according to any of claims I to 9.

16. A kit to identify early stage Alzheimer's dementia, in particular to discriminate early stage Alzheimer^'s dementia from other types of dementia, in a subject comprising the antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 9.

33

17. The antibody, antibody-l ike scaffold or antibody fragment according to any of claims 1 to 9 for use in the treatment of a disease.

1 8. The antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 9 for use in the treatment of Alzheimer^' s dementia.

19. A nucleic acid comprising the nucleic acid sequence as set out in SEQ ID NO. 19 and/or SEQ ID NO. 20.

20. A peptide representing an epitope of the tau protein, which epitope is recognized by an antibody, antibody-like scaffold or antibody fragment according to any of claims 1 to 9.

34