WO2014207122A1

WO2014207122A1 - Tomoregulin in the treatment of neurological diseases

Info

Publication number: WO2014207122A1
Application number: PCT/EP2014/063557
Authority: WO
Inventors: Inge Sillaber
Original assignee: Phenoquest Ag
Priority date: 2013-06-26
Filing date: 2014-06-26
Publication date: 2014-12-31

Abstract

Antagonists of TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) for use in the treatment or prevention of a neurodegenerative disease, in particular plaque related diseases like Alzheimer's disease are described.

Description

TOMOREGULIN IN THE TREATMENT OF NEUROLOGICAL DISEASES

Field of the invention

The present invention generally relates to antagonists of TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) for use in the treatment or prevention of a neurodegenerative disease, in particular a plaque related disease such as a disorder associated with Alzheimer's disease. In addition the present invention relates to pharmaceutical compositions comprising a TMEFF2 antagonist and at least one additional agent suitable for the treatment or prevention of a neurodegenerative disease, in particular Alzheimer's disease or of any of the symptoms of such a disorder.

Background of the invention

Neurodegenerative diseases include a range of conditions which primary affect the neurons in the human brain and are associated with the progressive loss of structure or function of the neurons.

The problems associated with the progressive degeneration and/or death of nerve cells are in particular ataxia or dementia observed in diseases such as Parkinson's disease or Alzheimer's disease. Most of the delimitating and largely untreatable conditions associated with neurodegenerative diseases are linked with age.

The most prominent neurodegenerative disease is Alzheimer's disease (AD). This progressive neurodegenerative disorder is characterized by memory and cognitive dysfunction as well as plaques composed of fibrillar aggregates of amyloid beta (AB) in the patient's brain. AB, a cleavage product of an integral membrane protein, i.e. amyloid precursor protein (APP), is present in the brains and tissues of Alzheimer's patients and its presence correlates with the progression of AD.

Alzheimer's disease can be classified into two types (a) sporadic AD (SAD) and (b) familial AD (FAD). SAD accounts for more than 90 % of all cases of AD in patients aged 65 years or older and the ε-4 allele of the apolipoproteine gene has been identified as the major risk factor. In contrast, FAD is associated with an early onset and the APP, presenilin-1 (PS1), and presenilin-2 (PS2).

AD has great medical need since there are still no ideal AD therapeutics for treating the disease. Current drugs improve symptoms but have no profound disease modifying effect; see for example the development of intravenous bapineuzumab, a humanized monoclonal antibody, which binds to and clears Αβ peptide and which development was ended due to lack of efficiency and to-late-stage trials in patients who had mild to moderate Alzheimer's disease.

Thus, there is still a constant need of providing agents suitable for the treatment or prevention of neurodegenerative diseases, in particular Alzheimer's disease or of any of the symptoms of such a disorder.

The solution to the technical problem in accordance with the present invention is provided by the embodiments as characterized in the claims and described further below.

Summary of the invention

The present invention generally relates to therapeutic compounds capable of modulating TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) activity for use in the treatment or prevention of neurodegenerative diseases. Typically, such modulators in accordance with the present invention are TMEFF2 antagonists capable of for example inhibiting the TMEFF2 activity or reducing the level of TMEFF2 in the human body, particularly the brain.

In one embodiment of the present invention, the TMEFF2 antagonist is for use in the treatment or prevention of plaque related diseases such as due to deposition of Αβ peptide in the brain typically observed in Alzheimer's disease and disorders associated therewith.

In a further embodiment, the present invention relates to pharmaceutical compositions comprising a TMEFF2 antagonist and further an agent suitable for use in the treatment of a neurodegenerative disease, preferably Alzheimer's disease. In addition, or alternatively the pharmaceutical composition comprising a TMEFF2 antagonist is designed for administration to the brain, either directly or indirectly, for example by intranasal application. Further embodiments of the present invention will be apparent from the claims, the description and the Examples that follow.

Brief description of the drawings

Fig. 1: Co-localisation of TMEFF2 and AB plaques in brain sections of human Alzheimer's patients. Double immunofluorescence staining using the anti-amyloid beta antibody ab2539 (green signal, A-l and B-l) and the anti-TMEFF2 antibody PQ001 (red signal, A-2 and B-2) revealed a clear increase of the signal of TMEFF2 in and around Amyloid beta plaques (overlay, A- 1/2, B-l/2).

Fig. 2: Co-localisation of TMEFF2 and AB plaques in brain sections of transgenic mice CVN mice (APPSwDI/NOS2-/-) a mouse model for Alzheimer's disease. Double staining using thio flavin (green signal) and the anti-TMEFF2 antibody PQ001 (red signal) showed a clear increase of the signal of TMEFF2 in and around plaques in the cortex (A) and the dentate gyrus (B) of the CVN mouse model.

Fig. 3: PQ001 -induced increase of phosphorylated CREB (pCREB) in primary cultures of hippocampal neurons. Treatment of cultured primary hippocampal neurons with the anti-TMEFF2 antibody PQ001 alone increased the protein level of pCREB and co- incubation of PQ001 and Activin lead to a further increase of pCREB. A) picture of the original Western blot, B) Quantitation of the fold increase of pCREB by the treatment in relation to control by using densitometry values, normalized to GAPDH.

Detailed description of the invention

The present invention generally relates to antagonists of TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) for use in the treatment or prevention of a neurodegenerative disease, in particular diseases due to and/or associated with amyloid beta (Αβ) pathology/amyloidosis. More specifically, the present invention relates to TMEFF2 antagonists for the treatment of Αβ peptide related brain impairments such as of a disorder associated with Alzheimer's disease (AD). As illustrated in the appended Examples, the present invention is based on the surprising finding that an antagonist of TMEFF2, i.e. anti-TMEFF2 antibody PQ001 is capable of reducing Αβ plaque load in the brains of AD animals and reduces the number of activated astrocytes which normally stimulate the expression of the pro -inflammatory cytokines IL1B and TNFa which is typically associated with Αβ plaque formation.

TMEFF2 (transmembrane protein with EGF-like and two follistatin-like domains 2) (Chromosome: 2; NC_000002.11 (192,814,743 to 193,059,644; complement) also known as tomoregulin-2 (TR2), hyperplastic polyposis protein 1 (HPPl), TPEF, transmembrane protein TENB2, and cancer/testis antigen family 120, member 2 (CT120.2) as used in accordance with the present invention is a glycosylated integral membrane protein with EGF- and follistatin-like motifs in its extracellular domain (ECD). The amino acid and nucleotide sequence of TMEFF2 is known in the art; see Uchida et al., Biochem. Biophys. Res. Commun. 266 (1999), 593-602; Horie et al, Genomics 67 (2000), 146-152); or Genbank Accession numbers NM_016192, NM_019790, BC034850, BC008973, AY412287, AY412288, AY412289, AB017270, and AB017269. The nucleotide and amino acid sequence of human TMEFF2 as described in Uchida et al. (1999) (Uchida et al, Biochem. Biophys. Res. Commun. 266(2) (1999), 593-602) is also depicted in Figure 16 of international application WO2007/090631, the disclosure content of which is incorporated herein by reference. When referring herein to TMEFF2, the sequences described in Uchida et al. (1999) and in Figure 16 of international application WO2007/090631, which presented in SEQ ID NO.: 1 are preferred as "reference sequences" when, e.g. determining the degree of identity of nucleotide or amino acid sequences which are encompassed by the term "TMEFF2".

Previously, in a single report or two TMEFF2 has been described to be found extensively in AD plaques and the authors speculated that TMEFF2 may participate in amyloid plaque formation and contribute to the pathogenesis; see Siegel et al. in Int. J. Devi. Neuroscience 20 (2002), 373-389 and J. Neurochemistry 98 (2006), 34-44 as well as Siegel's corresponding US patent application US 2009/0181026 Al . However, no experimental proof or working example for this speculation had been provided. In fact, the Western Blot analysis shown in Fig. 6 of Siegel et al. (2006) even casts doubt on a clear nexus between TMEFF2 and Alzheimer's disease since also a normal subject stained positive for TMEFF2, one of the three severe AD patients had a reduced level of TMEFF2 and the one moderate-stage AD patient had a level of TMEFF2 akin to the two sever AD samples.

Furthermore, besides the fact that among AB peptides as the major component also other proteins are found in AD plaques, for example a-synuclein, secretory protein 7B2, tau, prolyl oligopeptidase (PREP) to name a few (Hannula et ah, Neuroscience 242 (2013), 140-150), which significance towards possible therapeutic intervention of the disease are yet unclear, TMEFF2 has been described to be implicated in substantially different disorders such as cancer and disease involving cell hyperproliferation; see the original publication by Uchida et al. (1999), supra, and international applications WO2004/064612 and WO2007/002525. More recently, TMEFF2 modulation for use in the treatment of affective disorders has been described in international application WO2007/090631 without however considering disorders linked to Αβ plaques and amyloidosis.

In contrast, besides the observation increased levels of TMEFF2 protein in and around AB- plaques in brain slices of human AD patients and the CVN mouse model of AD (Examples 1 and 2, Fig. 1 and 2), experiments performed in accordance with the present invention revealed that modulation of TMEFF2 by antagonists such as anti-TMEFF2 antibodies (i) results in the reduction of Αβ plaque load in the brains of AD animals (Example 3), (ii) reduces the number of activated astrocytes (Example 4) which normally stimulate the expression of the proinflammatory cytokines IL1B and TNFa which is typically associated with Αβ plaque formation and (iii) increases the level of phosphorylated cyclic AMP response element binding protein (CREB), involving the activin pathway (Example 5, Fig. 3), which has also been shown to play a role in cognitive deterioration associated with AD.

Without intending to be bound by theory these results and observations let to the conclusion that TMEFF2 is a suitable target for therapeutic intervention in the treatment and prevention of neurodegenerative diseases in which plaque formation, astrocyte activation and/or CREB phosphorylation (pCREB) play a significant role. In particular, it could be shown in accordance with the present invention that incubation of primary cultures of hippocampal neurons with the anti-TMEFF2 antibody PQ001 alone increased the protein level of pCREB and co-incubation of PQ001 and Activin let to a further increase the level pCREB; see Example 5, Fig. 3. Accordingly, it is believed that antagonizing TMEFF2 is a possibility to increase free Activin A levels in the brain thereby facilitating the anti-inflammatory and neurogenesis promoting activities of Activin A and pCREB.

CREB plays an important role as transcription factor which becomes active following phosphorylation. Activin A is a homomer of two activin BA-subunits. The human activin BA gene contains CREB binding sites and an increased expression of the activin BA by cAMP was reported (Tanimoto et al., J. Biol. Chem. 271(51) (1996), 32760-32769). In consequence, it is suggested that the enhancement of pCREB by the TMEFF2 antibody PQ001 observed potentially increases expression of Activin A. There is evidence, that Activin A plays an important role in synaptic plasticity and was shown to facilitate the maintenance of early LTP whereas follistatin, the inhibitor of Activin A, reduces or blocks LTP and long-term memory (Ageta et al, Learn Mem. 17(4) (2010), 176-85). It is hypothesized that TMEFF2 through its follistatin domains also exerts negative effects on cognitive functions not only via its inhibitory effects on the cAMP -pathway but also by lowering the levels of Activin A.

The protein kinase A (PKA) / CREB pathway has also been shown to play a role in cognitive deterioration associated with AD. CREB is an important factor in learning and memory processes, i.e. synaptic plasticity. In the hippocampus of AD patients, impaired phosphorylation of CREB has been detected (Yamamoto-Sasaki et al., Brain Res 824(2) (1999), 300-303). Soluble Αβ, which is elevated in brains of AD patients, is found to block long term potentiation (LTP), a process dependent on synaptic plasticity. Evidence emerged that soluble AB impairs the PKA/CREB pathway and thereby, contributes to synaptotoxic effects (Vitolo, Proc. Natl. Acad. Sci. USA 99(20) (2002), 13217-21). One target gene of CREB is the brain derived neurotrophic factor (BDNF), another important factor involved in synaptic plasticity, learning and memory processes {e.g. Korte et al. Proc. Natl. Acad. Sci. USA 92 (1995), 8856-60, Mizuno et al. J. Neurosci. 20 (2000), 7116-21). The TMEFF2 protein contains besides the two follistatin modules and the EGF-like domain a G-protein activating motif in its short cytoplasmic domain (Uchida et al., 1999) through which TMEFF2 potentially could affect the cAMP pathway. Previous results (international application WO 2013/093122) showed the decreasing effect of TMEFF2 upon CREB expression under basal conditions or following stimulation of the cAMP pathway. These results with PQ001 show that inhibition of TMEFF2 in primary hippocampal neurons, cells naturally expressing the TMEFF2 protein, lead to an activation of the cAMP pathway resulting in an increase of phosphorylated CREB. However, preliminary results obtained in experiments performed in accordance with the present invention suggest that the increase in phosphorylated CREB is due to an activation of the protein kinase C (PKC), which probably interacts with and otherwise is blocked by TMEFF2.

Thus, by interference with the interaction of TMEFF2 with protein kinase the TMEFF2 antagonist such as the anti-TMEFF2 antibody PQ001 induces an increase in pCREB (Fig. 3) and therefore potentially contributes positively to CREB associated positive effects on cognition and synaptic plasticity. Since an increased phosphorylation of CREB by PQ001 alone and an additional increase when applied in combination with Activin A to primary hippocampal neurons is observed it is prudent to expect that patients suffering from Alzheimer's disease or form similar neurodegenerative diseases related to/associated with plaque deposition and neurofibrillary tangles, respectively, will benefit from the application of a TMEFF2 antagonist in terms of reduced plaque load, maintaining or restoring brain plasticity and/or neurogenesis.

Therefore, in its broadest aspect the present invention generally relates to therapeutic compounds capable of modulating TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) activity for use in the treatment or prevention of neurodegenerative diseases. Typically, such modulators in accordance with the invention are TMEFF2 antagonists capable, e.g., for inhibiting TMEFF2 activity or reducing the level of TMEFF2. Unless indicated otherwise the terms "substance", "compound" and "agent" are used interchangeably herein and include but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, antibodies, peptidomimetics, small molecules and other drugs and pro-drugs. In accordance with the present invention the term "substance", "compound" and "agent" also relates to means which are not compounds in the classical sense, for example radiation, stress such as heat and chilling, culture conditions, and the like which result directly or indirectly in substantially reducing TMEFF2 activity or the level of TMEFF2 or nullify it altogether.

As explained above, but without being bound by theory, it is assumed that an antagonist of TMEFF2 can prevent or diminish the effect of e.g. Αβ by reconstituting the activin pathway and thereby stimulating the phosphorylation of CREB or preventing the Αβ induced inactivation of the cAMP pathway, i.e. the protein kinase A (PKA), in particular PKAII_a and/or in view of the above-mentioned preliminary results more probably protein kinase C (PKC).

Accordingly, in an embodiment of the present invention, the TMEFF2 antagonist is capable of and used in, respectively, reducing, inhibiting, or counteracting the action of TMEFF2 on the activin pathway and/or the phosphorylation of CREB.

As used herein, the term "neurodegenerative disease" refers to disorders associated with progressive degeneration and/or death of nerve cells, see also the background section, supra, and may be accompanied by ataxia and/or dementia. Neurodegenerative diseases include but are not limited to Parkinson's disease (PD), PD related disorders, Alzheimer's disease (AD), Huntington's disease (HD), dementia, Motor neuron diseases, Spinocerebellar ataxia, Spinal muscular atrophy, plaque related diseases. Within the term neurodegenerative diseases the term "plaque related disease" is a name for a group of diseases associated with an increased plaque load in the brains.

Since TMEFF2 and Αβ plaques are co-localized in brains of patients and animals (Fig. 1 and 2) and antagonizing and/or inhibiting the activity of TMEFF2 leads to a reduction of plaque load in the brain, i.e. a reduced number of AB positive plaques in various brain areas (Example 3), TMEFF2 seems to be a novel mediator of the plaque development and thus as a new target for the therapeutic intervention in the treatment of neurodegenerative diseases, in particular plaque related diseases, such as AD.

Accordingly, in a preferred embodiment of the present invention, the TMEFF2 antagonist is for use in the treatment of plaque related diseases. In a particular preferred embodiment the TMEFF2 antagonist is for use in the treatment and/or prevention of Alzheimer's disease (AD) or amyloidosis which may or may not be associated with symptoms of AD. In addition, the TMEFF2 antagonist of the present invention can be used in preventing dementia and/or improving cognitive function.

As mentioned above, the antagonist for use in accordance with the present invention may be a compound or agent of any kind, which directly or indirectly affects the activity or level of active TMEFF2 in the brain. TMEFF2 antagonists suitable for use in accordance with the present invention including but not limited to siRNA and antibodies are known in the art; see, e.g., international applications WO2004/064612, WO2007/002525 and in particular WO2007/09063, the disclosure content of which is incorporated herein by reference, in particular the disclosure content international application WO2007/09063 concerning possible TMEFF2 antagonist and methods for their identification.

In principle, the TMEFF2 antagonist for use in accordance with the present invention can be any compound or measure such as radiation, heat treatment, compounds or conditions minimizing oxidative stress, which reduces level of TMEFF2 activity, disrupts TMEFF2 signal pathway and/or counteracts TMEFF2 activity; see also the Examples and supra. Unless indicated otherwise the term "antagonist" and "inhibitor" are used interchangeably herein and includes but is not limited to any nucleic acid, formulation, compound or substance that can regulate TMEFF2 activity in such a way that TMEFF2 is decreased or wherein the effects of TMEFF2 are blocked or altered. Examples of TMEFF2 antagonists include but are not limited to antibody, siRNA or shRNA. In one preferred embodiment, the TMEFF2 antagonist directly interacts with or binds to TMEFF2 protein or its encoding DNA/mRNA.

The term antagonist/inhibitor in accordance with the present invention is also meant to encompass any precursor and individual components of the antagonists/inhibitor. For example, if the TMEFF2 antagonist referred to is a peptide, polypeptide or protein such as an antibody, TMEFF2 protein or peptide inhibitor the respective term also includes the polynucleotide encoding such antagonist, the vector, in particular expression vector comprising the coding sequence of the antagonist as well as the host cell comprising the polynucleotide or vector.

Antisense or siRNA as TMEFF2 antagonist in accordance with the present invention includes corresponding vectors such as plasmids encoding and producing the same. Thus, the term antagonist and inhibitor have to be construed in their broadest sense in that they include any means and methods which the person skilled in the art would consider to bring about the effect of the recited TMEFF2 antagonist. A "binding molecule" as used in the context of the present invention relates primarily to antibodies, and fragments thereof, but may also refer to other non-antibody molecules that bind to TMEFF2 and exhibit the functional properties of the PQ001 antibody illustrated in the Examples including but not limited to hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell- cell adhesion molecules such as members of the cadherin, intergrin, C-type lectin, immunoglobulin (Ig) superfamilies and in particular designed ankyrin repeat proteins (DARPins) which are a promising class of non- immunoglobulin proteins that can offer advantages over antibodies for target binding; see for review, e.g., Stumpp and Amstutz, Curr. Opin. Drug Discov. Devel. 10 (2007), 153-159, and references cited therein. Thus, for the sake of clarity only and without restricting the scope of the present invention most of the following embodiments are discussed with respect to antibodies and antibody-like molecules which represent the preferred binding molecules for the development of therapeutic and diagnostic agents. Antibodies or antigen-binding fragments, immunospecific fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, murine, human, humanized, primatized, murinized or chimeric antibodies, a recombinant full antibody (immunoglobulin), in particular a monoclonal recombinant full antibody (immunoglobulin), single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide- linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies disclosed herein), a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody, a cross-cloned antibody, a fully-human antibody, a humanized antibody, a xenogenic or a chimeric human- murine antibody, nanobodies, diabodies, and the like. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. Immunoglobulin or antibody molecules of the invention can be of any type {e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class {e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Means and methods for the recombinant production of binding molecules, in particular antibodies and mimics thereof as well as methods of screening for competing binding molecules, which may or may not be antibodies, are known in the art and are summarized, for example, in international application WO2006/103116 with respect to antibodies against AB and the treatment/diagnosis of AD, the disclosure content of which is incorporated herein by reference for this purpose of antibody engineering and administration for therapeutic or diagnostic applications.

As shown in the Examples, the novel and inventive concept of the present invention, i.e. antagonizing TMEFF2 has been illustrated with an anti-TMEFF2 antibody. Accordingly, in an embodiment of the present invention, the TMEFF2 antagonist for use in the treatment or prevention of a neurodegenerative disease is an antibody or antigen-binding fragment thereof. In a preferred embodiment the antibody is an anti-TMEFF2 antibody, preferably a monoclonal antibody.

As demonstrated in the Figures and Examples of the present invention an anti-TMEFF2 antibody, designated PQOOl reduced activated astrocytes which normally stimulates the expression of the pro-inflammatory cytokines IL1B and TNFa leading to an increased TMEFF2/AJ3 plaque formation, and additionally reduced the plaque load in the brain. PQOOl is a mouse monoclonal anti-TMEFF2 antibody which is disclosed in applicant's co-pending international application WO 2013/093122, the disclosure content of which is incorporated herein by reference, in particular with respect to the amino acid sequences of the antigen and epitope recognized by the PQOOl antibody, its CDRs and variable region as well as assays useful for testing the biological activity the PQOOl antibody and equivalent antibodies such as human antibodies and TMEFF2 antagonists in general.

In one embodiment, the TMEFF2 antagonists for use in accordance with the present invention has one or more properties described for the PQOOl antibody in international application WO 2013/093122; see, e.g., claim 1 as originally filed.

Accordingly, in one embodiment of the present invention, the TMEFF2 antagonist is capable of binding an epitope bound the PQOOl antibody and comprising the amino acid sequence EDGHYAR. Preferably, the TMEFF2 antagonist is capable of binding a peptide consisting of the amino acid sequence NTTTTTKSEDGHYAR, a peptide consisting of the amino acid sequence TT SEDGHYARTDYA, and/or a peptide consisting of the amino acid sequence EDGHYARTDYAENAN. In this embodiment, the TMEFF2 antagonist for use in accordance with the present invention is typically an the antibody or TMEFF2 binding fragment thereof comprising in its epitope binding domain

(a) at least one complementarity determining region (CDR) of the VH and/or VL variable region amino acid sequences depicted in

(i) Fig. 4 (VH) (SEQ ID NOs: 3, 4, 5); and

(ii) Fig. 4 (VL) (SEQ ID NOs: 6, 7, 8);

(b) an amino acid sequence of the VH and/or VL region as depicted in Fig. 4;

(c) at least one CDR consisting of an amino acid sequence resulted from a partial alteration of any one of the amino acid sequences of (a);

(d) a heavy chain and/or light variable region comprising an amino acid sequence resulted from a partial alteration of the amino acid sequence of (b); or

(e) at least one CDR comprising an amino acid sequence with at least 90 % identity to any one of the amino acid sequences of (a).

Alternatively, the TMEFF2 antagonist for use in accordance with the the present invention is an antibody or antigen-binding fragment thereof, which competes for binding to the TMEFF2 with the antibody having the VH and VL region as depicted in Fig. 4. Those antibodies may be murine, however, humanized, xenogeneic, or chimeric human-murine antibodies being preferred, in particular for therapeutic applications. However, for diagnostic uses and research in general murine antibodies are suitable as well. An antigen-binding fragment of the antibody can be, for example, a single chain Fv fragment (scFv), a F(ab') fragment, a F(ab) fragment, and an F(ab')₂ fragment.

Competition between antibodies is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as TMEFF2. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; see Stahli et al., Methods Enzymol. 9 (1983), 242-253; solid phase direct biotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986), 3614-3619 and Cheung et al, Virology 176 (1990), 546-552; solid phase direct labeled assay, solid phase direct labeled sandwich assay; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); solid phase direct label RIA using 1125 label; see Morel et al, Molec. Immunol. 25 (1988), 7-15, and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the use of purified TMEFF2 or aggregates thereof bound to a solid surface or cells bearing either of these, an unlabelled test immunoglobulin and a labeled reference immunoglobulin, i.e. the monoclonal antibody of the present invention. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Preferably, the competitive binding assay is performed under conditions as described for the ELISA assay in the appended Examples. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50% or 75%. Hence, the present invention is further drawn to the use of an antibody, or antigen-binding fragment, variant or derivative thereof capable of inhibiting a reference antibody PQ001 from binding to TMEFF2 and/or competing with its binding.

For some applications only the variable regions of the antibodies are required, which can be obtained by treating the antibody with suitable reagents so as to generate Fab', Fab, or F(ab")₂ portions. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of immunoglobulins to detecting reagents such as radioisotopes.

The TMEFF2 antagonist for use in accordance with the present invention can be immunoglobulin or its encoding cDNAs which may be further modified. Thus, in a further embodiment the TMEFF2 antagonists of the present invention comprises chimeric antibody, humanized antibody, single-chain antibody, Fab-fragment, bi-specific antibody, fusion antibody, labeled antibody or an analog of any one of those. Methods producing such antagonists are known to the person skilled in the art and are described, e.g., in Harlow and Lane, Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor (1988). When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to the same epitope as that of any one of the antibodies described herein (Schier, Hum. Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international application WO 89/09622. Methods for the production of humanized antibodies are described in, e.g., European application EP-A1 0 239 400 and international application WO 90/07861. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., international applications WO 91/10741, WO 94/02602, WO 96/34096, and WO 96/33735. As discussed above, the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab, and F(ab)₂, as well as in single chains; see e.g. international application WO 88/09344. Furthermore, diabodies and V-like domain binding molecules are well-known to the person skilled in the art; see, e.g. US patent No. 7,166,697.

The antibodies for use in accordance with the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory N.Y. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment or removal of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the present invention encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g., international application WO 00/30680 for corresponding technical details. In a further embodiment of the present invention, the binding molecule, antibody, immunoglobulin chain or a binding fragment thereof or the antigen for use in accordance with the present invention is detectably labeled. Labeling agents can be coupled either directly or indirectly to the antibodies or antigens of the invention. One example of indirect coupling is by use of a spacer moiety. Furthermore, the antibodies of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art and described above or can be performed by, e.g., chemical cross-linking as described in, e.g., international application WO 94/04686. The additional domain present in the fusion protein comprising the antibody of the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide- bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the antibody of the invention or vice versa. The therapeutically or diagnostically active agent can be coupled to the antibody of the invention or an antigen-binding fragment thereof by various means. This includes, for example, single- chain fusion proteins comprising the variable regions of the antibody of the invention coupled by covalent methods, such as peptide linkages, to the therapeutically or diagnostically active agent. Further examples include molecules which comprise at least an antigen-binding fragment coupled to additional molecules covalent ly or non-covalent ly include those in the following non-limiting illustrative list. Traunecker, Int. J. Cancer Surp. SuDP 7 (1992), 51-52, describe the bispecific reagent janusin in which the Fv region directed to CD3 is coupled to soluble CD4 or to other ligands such as OVCA and IL-7. Similarly, the variable regions of the antibody of the invention can be constructed into Fv molecules and coupled to alternative ligands such as those illustrated in the cited article. Higgins, J. Infect. Dis. 166 (1992), 198- 202, described a hetero-conjugate antibody composed of OKT3 cross-linked to an antibody directed to a specific sequence in the V3 region of GP120. Such hetero-conjugate antibodies can also be constructed using at least the variable regions contained in the antibody of the invention methods. Additional examples of specific antibodies include those described by Fanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev. Immunol. 12 (1992), 101-124. Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The antibodies of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

The above described fusion protein may further comprise a cleavable linker or cleavage site for proteinases. These spacer moieties, in turn, can be either insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to enable drug release from the antibody at the target site. Examples of therapeutic agents which can be coupled to the antibodies of the present invention for immunotherapy are drugs, radioisotopes, lectins, and toxins. The drugs with which can be conjugated to the antibodies and antigens of the present invention include compounds which are classically referred to as drugs such as mitomycin C, daunorubicin, and vinblastine. In using radio isotopically conjugated antibodies or antigens of the invention for, e.g., immunotherapy, certain isotopes may be more preferable than others depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters may be preferable to others. In general, a and β particle emitting radioisotopes are preferred in immunotherapy. Preferred emitters are short range, high energy emitters such as ²¹²Bi. Examples of radioisotopes which can be bound to the antibodies or antigens of the invention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. Most preferably, the radiolabel is ⁶⁴Cu. Other therapeutic agents which can be coupled to the antibody or antigen of the invention, as well as ex vivo and in vivo therapeutic protocols, are known, or can be easily ascertained, by those of ordinary skill in the art. Wherever appropriate the person skilled in the art may use a polynucleotide of the invention encoding any one of the above described antibodies, antigens or the corresponding vectors instead of the proteineous material itself.

The antibody for use in accordance with the present invention can be labeled (e.g., fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal) and used to detect specific targets in vivo or in vitro including "immunochemistry" like assays in vitro.

As will be explained further below, for pharmaceutical use the TMEFF2 antagonist is typically formulated in a composition further comprising a pharmaceutically acceptable carrier. In a further embodiment, the present invention relates to a composition comprising a TMEFF2 antagonist as described above and at least one further therapeutic agent useful in the treatment or prevention of a neurodegenerative disease and/or symptoms associate therewith. The at least one further therapeutic agent can be selected but is not limited to the group of antibodies against Αβ, α-synuclein or tau, antidepressiva, antipsychotika, or other pharmaceuticals for treatment of neurodegenerative diseases in particular AD, as known in the art, i.e. cholinesterase-inhibitors or Levodopa and/or catechol-O-methyltransferase inhibitors in case of Parkinson's disease (PD). Naturally, the compositions of the present invention are particularly useful in treating and/or preventing neurodegenerative disease, especially plaque related diseases, in particular AD.

Selecting an appropriate drug, as described above, depends on the nature of the neurodegenerative disease, age and the individual's overall health status. A combination therapy is particularly preferred during the progress of neurodegenerative diseases. In the context of the present application, "co-administration" of two or more compounds is defined as administration of the two or more compounds to the patient within 24 h, including separate administration of two medicaments each containing one of the compounds as well as simultaneous administration whether or not the two compounds are combined in one formulation or whether they are in two separate formulations. A "synergistic effect" of two compounds is in terms of statistical analysis an effect which is greater than the additive effect which results from the sum of the effects of the two individual compounds.

As mentioned above, and illustrated in Example 4 treatment of cultured primary hippocampal neurons with a TMEFF2 antagonist PQ001 alone increased the protein level of pCREB and co-incubation of PQ001 and Activin led to a further increase of pCREB while incubation with Activin alone had no effect. Accordingly, in one preferred embodiment of the present invention the composition comprising the TMEFF2 antagonist further comprises Activin or an agonist of Activin. Most conveniently, the effective concentration of activin will be increased through direct administration using either activin itself or an activin prodrug (a form which is cleaved within the body to release activin). It is however also possible to increase activin concentration through administration of either activin agonists (substances which effect a direct increase in production or activity of activin within the body, e.g. FSH, cAMP (protein kinase A activator), 12-0-tetradecanoylphorbol 13-acetate (TP A, a protein kinase A activator), TGF-β, IL-1B and TNF-a); see, e.g. international application WO 99/15192.

The dosage regimen utilizing the TMEFF2 antagonist and composition in accordance with the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient, patient's size, body surface area, age, the particular compound to be administered, general health; the severity of the condition to be treated; the time and route of administration; and the particular compound employed. It will be acknowledged that an attending physician can easily determine and prescribe the effective amount of the compound required to prevent, counter or arrest the progress of the condition.

A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg {e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Progress can be monitored by periodic assessment. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

The term "subject" and "patient" is used interchangeably herein and means an individual in need of a treatment of a metabolic disease. Preferably, the subject is a mammal, particularly preferred a human.

"Treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of a metabolic disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the manifestation of the condition or disorder is to be prevented.

For use as a pharmaceutical composition, the TMEFF2 antagonist according to the invention, optionally combined with other active agents, may be incorporated together with one or more inert conventional carriers and/or diluents. Pharmaceutically acceptable carriers and administration routes can be taken from corresponding literature known to the person skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683- 306472, Vaccine Protocols, 2nd Edition by Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "nanobodies™") etc. are also envisaged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant. Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) and corresponding updates. For a brief review of methods for drug delivery see Langer, Science 249 (1990), 1527-1533. In one embodiment, the TMEFF2 antagonist and composition of the invention is administered to a human patient once daily, each other day, thrice weekly, twice weekly or once weekly, preferably less than once daily. Furthermore, whereas the present invention includes the now standard (though fortunately infrequent) procedure of drilling a small hole in the skull to administer a drug of the present invention, in a preferred aspect, the binding molecule, especially antibody or antibody based drug of the present invention can cross the blood-brain barrier, which allows for intravenous or oral administration.

Further embodiments of the present invention will be apparent from the Examples that follow, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES

Example 1: Co-localisation of TMEFF2 and Αβ plaques in brain sections of human

Alzheimer's patients

To examine the expression of TMEFF2 in combination with AB plaques double immunofluorescence staining of human Alzheimer brain sections was performed. In brief, frozen tissue arrays with brain slices of human patients diagnosed with Alzheimer's disease (Arrays-I, Cat-No. T6236444Alz-BC; TfHAD-Alz: Brain Hippocampus T1236052Alz-BC) were purchased from BioCat GmbH Heidelberg. The tissue sections (5-10μιη thickness, mounted on positively charged glass slides) represented various regions of the brains of different patients. For immunohistochemical staining sections were fixed with 4% paraformaldehyde in lxPBS for 20 min at room temperature. After three washing steps, (10 min each, in lxTBS-T (tris-buffered saline with Triton X-100), sections were incubated for 1 h in blocking solution TBS-T containing 5% goat serum. The primary antibodies were added to the slides and incubated overnight: For staining of the AB plaques the polyclonal beta- amyloid antibody ab2539 (Abeam) was used, diluted 1 :50 with TBS-T buffer containing 5% goat serum. For staining of TMEFF2, the monoclonal PQ001 antibody was used, diluted 1 : 100 in TBS-T buffer containing 5% goat serum. On the next day, the sections were washed 3-times for 10 min in lxTBS. The secondary antibodies Alexa Fluor goat anti-rabbit antibody 488 (Invitrogen) and Alexa fluor goat anti-mouse antibody 555 (Invitrogen) were diluted 1 :500 in lxTBS-BSA buffer were added to the slides, the slides were protected against light and incubated for 1 h. After 3 washing steps (2 -times for 10 min in lxTBS, once for 10 min in Aqua. Dest) and counterstaining with bisBenzimide (Hoechst No. 33258) for 5 min, another washing step (10 min in Aqua dest.) followed before the autofluorescent eliminator reagent (Millipore) was added to the slides for 5 min. Sections were dehydrated in an ascending alcohol series, mounted with ProLong Gold antifade reagent (Molecular Probes) and covered with a cover slip. Immunofluorescence signals were visualized by using a Leica DMI 6000B microscope equipped with a Leica DFC 350 FX camera and the ImageJ software (VI .44). The immunofluorescence staining revealed a clear TMEFF2 signal around AB plaques, indicating that TMEFF2 is involved in this plaque related disease (Fig. 1). Example 2: TMEFF2 co-localize with Αβ plaques in brain sections of transgenic CVN mice.

To examine the expression of TMEFF2 in combination with AB plaques in an appropriate model, double immunofluorescence staining of brain sections of the CVN mouse (APPSwDI/NOS2 ^{~ ~}) was performed. In brief, the CVN mouse model of AD was provided by Charles River Laboratories International, Inc. The CVN mice (APPSwDI/NOS2 ^{~ ~}) which were generated by crossing transgenic mice overexpressing APP with Swedish, Dutch and Iowa mutations with NOS2 knockout animals. Brains of wildtype and APPSwDI/NOS2 ^{~ ~} mice with an age of 11 months were perfused with saline, snap-frozen and sagittal cryosections were prepared. The brain sections (12 μιη thick) were mounted on glass slides (SuperFrost plus, Thermo Scientific) and stored at -80° until use. For the detection of TMEFF2 the PQ001 was used, a mouse monoclonal antibody, as primary antibody. To eliminate the problem of the anti-mouse secondary antibody to distinguish between the mouse primary antibody and endogenous mouse immunoglobulins in the tissue, a Mouse on Mouse (M.O.M.) Basic Kit (Vector Laboratories) was used. All solutions were prepared according to the manufacturer's instruction. For immunohistochemical staining sections were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. After three washes in PBS sections were blocked in working solution of Mouse Ig Blocking Reagent (M.O.M Basic Kit) for 1 h. PQ001 (diluted 1 :250 in M.O.M. Diluent) was incubated for 30 min at room temperature. After short washes the Antibody was detected by a 10 min Incubation of M.O.M. Biotinilated Anti-Mouse IgG Reagent followed by a 30 min incubation of Vectastain R.T.U. ABC Reagent (Vector Laboratories) and 30 min incubation with Texas Red Avidin D (1 :300, Vector) in the dark. After Counterstain with bisBenzimide (Hoechst No. 33258) for 5 min, sections were incubated in a 0.02% aqueous Thioflavin S (Sigma) Solution, to visualize primary and secondary amyloid deposits. The Thioflavin S staining was differentiated in a 2x 1 min rinse in 50% ethanol, dehydrated in an ascending alcohol series and mounted with ProLong Gold antifade reagent (Molecular Probes). Immunofluorescence signals were visualized by using a Leica DMI 6000B microscope equipped with a Leica DFC 350 FX camera and the ImageJ software (VI .44). In accordance with the immunofluorescence staining in human brain sections, the staining in the mouse model revealed a clear TMEFF2 signal around AB plaques (Fig. 2). Example 3: PQ001 reduced plaque load in brains of AD transgenic mice

Treatment of AD mice such as the commercially available Tg2576 mice (Charles River) and CVN mice (Charles River) described in Example 2 and their wildtype littermates started when the mice reach an age of which is known that AB plaque formation starts in the respective AD mouse model. PQ001 at a dose of 10(^g is injected i.p. once every second week for a total treatment period of 4 months, resulting in 9 injections of PQ001 in total. At the end of the treatment period, all animals are euthanized and perfused with saline, subsequently the brains are removed, snap-frozen and stored at -80°C until further use. Of course, other established animal models of AD may be used as well; see, e.g., Knobloch et al, Neurobiol. Aging 28(9) (2007), 1297-306), Elder et al, Mt Sinai J Med. 77(1) (2010), 69-81, Wang et al, J. Neurosci. 31(11) (2011), 4124-4136, and as well.

Binding of the antibody to the pathology specific structures in the brain is then evaluated by immunostaining with a labeled anti- human Ig secondary antibody followed by standard immunohistochemical detection. Tissue slices are prepared from frozen tissue using a cryotome. Presence of TMEFF2 on cryostat sections is assayed by staining with the anti- TMEFF2 antibody PQ001, whereas AB is stained with Thio flavin S as described in Example 2. Analysis of fluorescence is performed on an inverted fluorescence microscope (Leica).

As a result the application of the anti-TMEFF2 antibody PQ001 leads to a reduced number as well as reduced area of AB positive plaques in various brain sections.

Typically, the 100 μg dosis represents a dosage in human adults of 3.5 to 4.5 mg/kg body weight. In accordance with the present invention, the half-life period of the antibody in the brain is about 24 days. Therefore, the dosing may be extended to every 3 or 4 weeks.

Example 4: PQ001 reduced activated astrocytes

To identify the mechanism by which TMEFF2 anticipate on AB plaque formation, its role in the activation of astrocytes is examined. In brief, anti-TMEFF2 antibody PQ001 (100 μg) is injected i.p. bi-weekly over 4 months to the transgenic AD mice as described in Example 3, see supra. Double immunostaining utilizing anti-GFAP-antibody shows a decrease in the GFAP-level of mice administered to PQ001 compared to vehicle induced controls. Not only a change of the total area of the GFAP-immuno fluorescent signal is observed, but also a reduced number of GFAP -positive astrocytes is visible.

Example 5: The anti-TMEFF2 specific antibody PQ001 increases phosphorylated

CREB (pCREB)

To investigate the mechanism through which TMEFF2 might act on the prevention or reduction of AD pathology , rat embryo primary hippocampal neuron cell culture preparation were used to determine of pCREB protein levels. In brief, the hippocampi (HC) from the rat embryo brains (embryonic day 17, CD rat from Charles River) were dissected and placed in a 15 ml tube containing 10 ml Hibernate E (Invitrogen) complete medium (containing 10 ml B27 supplement (Invitrogen) and 0.5 mM Glutamax (Invitrogen)) chilled on ice. The tube was placed in a water bath at 30°C for 8 min. The Hibernate E was removed and 6 ml papain (Worthington)/Hibernate E was added and the tube which was then transferred to a water bath at 30°C for 20 min., swirling every 3-5 min. After removal of the papain/Hibernate E, 2 ml of warm Hibernate E complete medium was added for 5 min. The hippocampi were triturated approximately 7 times in 30 sec. with a 9-inch siliconised (Sigmacote, Sigma), fire-polished Pasteur pipette. The mixture was allowed to settle for approximately 2 min. after which it was transferred to a fresh 15 ml tube. Sediment from the first tube was resuspended in 2 ml Hibernate E complete medium and the procedure repeated one more time. The supernatants from each trituration were combined and non-dispersed tissue was allowed to settle for 3 min. The supernatant was then drawn off and transferred to a new 15 ml tube and centrifuged for 2 min. at 200 g. The pellet was resuspended in 1-2 ml of Neurobasal medium (Invitrogen), supplemented with 10 ml B27, and 0.5 mM Glutamax and 25 μΜ glutamate (Sigma). Cells were counted using a hemocytometer and seeded at a density of 50,000 cells/cm² in Neurobasal complete medium containing 25 μΜ glutamate and 5 μg/ml FGFb (Invitrogen). Four days after seeding, half the medium was removed and replaced with fresh Neurobasal complete medium containing 5 μg/ml FGFb. This was repeated every 6-7 days. After 17 days in vitro (DIV) cells were treated with the PQ001 antibody twice over a 24 h period after which time activin (R&D) was added for 30 min. at 100 ng/ml. Afterwards, for preparing whole cell extracts, the medium was removed, cells were washed with PBS (PBS) and scrapped into a 2 ml Eppendorf tube. Cells were centrifuged at 900 rpm for 4 min. and the PBS was removed. The cell pellet was resuspended in 100 ul RIPA buffer supplemented with 1 : 100 protease inhibitors (Sigma), 1 : 100 phosphatase inhibitors (Sigma) and 1 : 100 lOOmM PMSF (Sigma). Samples were incubated on ice for 30 min. before being centrifuged at 14,000 rpm at 4°C for 20 min. The supernatant was transferred into a new 1.5 ml eppendorf tube. The protein concentration from cell extracts was determined by Pierce^® BCA protein assay kit (Thermo Scientific), as per their instructions. 15 μg/ml of protein per sample was loaded into a 15 well, NuPAGE^® Novex^® 4-12% Bis-Tris Gel (Invitrogen). 20X NuPAGE^® MOPS SDS running buffer (Invitrogen) was used at a final concentration of IX. The gels were run at 200V constant for 45 min. Western blot transfer was performed using a nitrocellulose membrane (GE Healthcare) and 20 X NuPAGE^® transfer buffer (Invitrogen) at a final concentration of lx. The transfer was performed at 30 V constant for 1 h. Following the transfer, membranes were soaked in Ponceau S solution (Sigma) for 5 min., to check for complete transfer. The Ponceau S was washed off with deionized water and the membranes were blocked in 5 % non-fat dry milk (Roth) dissolved in TBS, 0.1 % Tween-20 (TBS-T) (BDH Prolabo) for 1 h at room temperature with gentle shaking. Proteins were detected using phospho-CREB antibody (Cell Signaling) at 1 : 5000 and GAPDH antibody (Cell Signaling) at 1 : 10,000. Densitometry was performed using ImageJ and analysis performed using EXCEL. The pCREB value of each sample was normalized to the respective GAPDH value and fold- increase of the treated samples was calculated in relation to the control. The results revealed that treatment with the anti-TMEFF2 antibody PQOOl alone increased the protein level of pCREB significantly (Fig. 3). In addition, the co -administration of Activin A led to a further increase by 1.5 fold (Fig. 3).

In response to injuries or degenerative processes, the brain also possesses a wide repertoire to maintain its plasticity and counteract with e.g. neuroregeneration as well as neurogenesis. Emerging evidence suggests that especially neurogenesis is impaired in chronic disease like AD. Besides many others, Activin A - a member of the transforming growth factor beta (TGFB) superfamily - is supposed to play a key role in the response to neurodegenerative processes (Abdipranoto-Cowley, Stem Cells 27(6) (2009), 1330-46). The neuronal expression of Activin A is increased and reduces gliosis, a characteristic of central inflammation, in the hippocampus injured by an infusion of an excitotoxic agent. In the same experiment, follistatin, a very potent antagonist of Activin A, aggravated gliosis in the injured hippocampus. It has also been shown that Activin A has potent anti- inflammatory effects in the brain by suppressing microglial activation and pro -inflammatory cytokine release. Additionally, Activin A increases the number of proliferating neural stem cells and neural precursors in the hippocampus, key events of neurogenesis, whereas the application of follistatin impaired neurogenesis. Activin acts mainly via the smad 2/3 and smad 4 pathway and it has been shown that TGFB signaling effectors smad 4, smad 1/5/8 and smad 2/3 play a role in neurogenesis. On the other hand, recent studies have also reported that TGFB signaling is deficient in neurodegenerative disorders like AD.

The TMEFF2 protein contains two follistatin- like domains and therefore, it is proposed that an increase of TMEFF2 in AD prevents Activin A to exert its anti-inflammatory and neurogenesis promoting role by binding of Activin A. In consequence, TMEFF2 indirectly, via inhibition of Activin A effects, would increase pro -inflammatory events most likely within neuritic plaques and in neurons around plaques. The unfolded protein response (UPR) induced TMEFF2 translation in neurons, triggered by AB peptides, and could therefore contribute to the formation of AB plaques. Application of PQ001, leading to inhibiton of TMEFF2 effects, could consequently reduce formation of AB plaques, resulting in a considerable lowering of the neurotoxic and inflammatory events in the brain.

Thus, inhibiton of TMEFF2 for example by PQ001, a monoclonal anti-TMEFF2 antibody, is one possibility to increase free Activin A levels and thereby facilitate the anti-inflammatory and neurogenesis promoting activities of Activin A.

Claims

1. A TMEFF2 (Transmembrane protein with EGF-like and two follistatin-like domains 2) antagonist for use in the treatment or prevention of a neurodegenerative disease.

2. The TMEFF2 antagonist for use according to claim 1, wherein the neurodegenerative disease is a plaque related disease.

3. The TMEFF2 antagonist for use according to claim 1 or 2, wherein the disease is a disorder associated with Alzheimer's disease.

4. The TMEFF2 antagonist for use according to any one of claims 1 to 3, which is an antibody or antigen-binding fragment thereof.

5. The TMEFF2 antagonist for use according to claim 4, wherein the antibody is an anti- TMEFF2 antibody.

6. The TMEFF2 antagonist for use according to claim 4 or 5, wherein the antibody is a monoclonal antibody.

7. The TMEFF2 antagonist for use according to any one of claims 1 to 6, which is capable of binding an epitope comprising the amino acid sequence EDGHYAR.

8. The TMEFF2 antagonist for use according to any one of claims 1 to 7, which is capable of binding a peptide consisting of the amino acid sequence NTTTTTKSEDGHYAR, a peptide consisting of the amino acid sequence TTKSEDGHYARTDYA, and/or a peptide consisting of EDGHYARTDYAENAN.

9. The TMEFF2 antagonist for use according to any one of claims 1 to 8, which is an antibody or antigen-binding fragment comprising in its variable region

(i) Fig. 4 (VH) (SEQ ID NOs: 3, 4, 5); and (ii) Fig. 4 (VL) (SEQ ID NOs: 6, 7, 8);

(b) an amino acid sequence of the VH and/or VL region as depicted in Fig. 4;

10. The TMEFF2 antagonist for use according to any one of claims 4 to 9, wherein the antibody is a human, humanized, xenogenic or a chimeric human-murine antibody.

11. The TMEFF2 antagonist for use according to any one of claims 4 to 10, wherein the antibody is a Fab or scFv antibody.

12. The TMEFF2 antagonist for use according to any one of claims 1 to 11, which is detectably labeled or attached to a drug, preferably wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore and a heavy metal.

13. The TMEFF2 antagonist for use according to any one of claims 1 to 12, wherein the TMEFF2 antagonist is formulated in a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising an agent for use in the treatment of a neurodegenerative disease, preferably Alzheimer's disease; preferably wherein the agent is activin or an agonist thereof.

15. The pharmaceutical composition of claim 13 to 14, which is designed for nasal administration or injection, preferably for extended release.