Field of the Invention

The present invention relates to novel fragments of Αβ peptide found in Αβ aggregates, to molecules binding to said fragments, as well as to uses thereof as therapeutics and/or detection tools.

Background of the Invention

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is manifested as a gradual decline in cognitive function. AD is generally characterized by amyloid deposition in senile plaques that are mainly composed of a 40- and 42- residue long amyloid β-protein (Αβ40 and Αβ42). These peptides are cleaved from the amyloid precursor protein (APP) by the sequential enzymatic activity of two proteases, β- and γ- secretases. Αβ42 plays a more important role in the pathogenesis of AD than Αβ40 because of its higher aggregation propensity and neurotoxicity {Haas et al, 2007, Nat. Rev. Mol. Cell. Biol. 8, 101-112). Several in vitro and in vivo studies have provided strong evidence that Αβ peptide-induced loss in synaptic plasticity can be associated with Αβ peptide oligomerization.

In AD, aggregation of Αβ peptide is considered a spontaneous property of this protein. However, recent reports have raised the speculation that aggregation of this protein may be strongly accelerated in the presence of tissue transglutaminase (TGase) (Schmid et al, 2011, J. Biol. Chem. 286, 12172-88). Increased TGase activity appears to coincide with AD pathology and cerebrospinal fluid (CSF) analysis of TGase levels, as well as changes in Αβ concentration have been suggested to serve as useful bio markers in AD. Moreover, CSF analyses indicate that the presence of Αβ oligomers and the decreased levels of Αβ1-42 found in AD patients strongly correlates with AD pathology.

Soluble Αβ oligomers correspond to a heterogeneous protein assembly of different morphology and size and are typically referred to as low molecular weight (LMW, 8- 17kDa range) oligomers of Αβ dimers, trimers and tetramers or high molecular weight (HMW, >20kDa) oligomers such as the dodecameric (56 kDa) and protofibrillar (>150 kDa) forms of Αβ. Αβ dimers and trimers have been reported to form an initial morphological entity during the early events of peptide aggregation and are therefore believed to form part of a molecular seed during oligomerization. Although the pathological loss in synaptic plasticity appears to strongly correlate with the accumulation of Αβ oligomers, the presence of Αβ dimers or trimers per se do not solely account for the pathological decline in neuronal plasticity. The distinct oligomeric morphology responsible for this cognitive loss and the precise mechanisms associated with this neurodegenerative process remain to be determined.

A major reason for this controversy in Αβ oligomer morphology associated neurotoxicity lies in the observation that, soluble Αβ oligomers of different morphologies may be metastable intermediate assemblies on the pathway of insoluble peptide fibrils. On the other hand, certain Αβ oligomeric assemblies may not enter a fibrillogenesis process and are therefore referred to as "off-pathway" oligomers. Nevertheless, soluble Αβ oligomers have raised the most scientific attention during the last decade, mainly because these morphologies are believed to display relatively higher neurotoxicity as compared to either monomeric Αβ or mature amyloid fibrils. Collectively, soluble Αβ oligomers meet to a certain extent the scientific criteria of being a valuable pathogenic target for the early diagnosis and treatment of AD.

The complex profile of different soluble Αβ fragments found in APP-transfected cells (Wang et al., 1996, Biol. Chem. 271: 31894-31902) or human CSF (Portelius et al., 2005, Neurosci. Lett. 409, 215-219) has been reported earlier. However, the presence of these fragments is believed to be the result of an unknown activity associated with β- and γ-secretases or, due to an undefined, concerted activity found with carboxypeptidase and metalloproteases.

Immunization against Αβ peptide is considered to be a promising approach for AD therapy because vaccination of transgenic mouse models of AD with Αβ1-42 aggregates resulted in a reduction of Αβ deposition and the prevention of cognitive impairment (Morgan et al, 2000, Nature 408, 982-985). However, clinical trials (AN1792) of immunization of AD patients against Αβ42 were interrupted because of severe adverse effects of excessive immune activation (Orgogozo et al, 2003, Neurology 61, 46-54). Recently, a follow-up study has shown that immunization against Αβ1-42 suppressed Αβ depositions in AD patients but not the progressive cognitive impairment (Holmes et al, Lancet 372, 216-223). One of the reasons for these problems might be the unintended elimination of both toxic and nontoxic forms of Αβ1-42, whose role in physiological function is controversial at present.

Antibodies binding Αβ peptide fragments are known in the art, they include, for instance, 6E10 antibody that is a mouse monoclonal antibody reactive to amino acid residues 1-16 of human Αβ peptide, the epitope lying within amino acids 3-8 of Αβ peptide (i.e. EFRHDS) (Perchiacca et al, 2012, PNAS, 109(1): 84-9). Another antibody of the prior art, 4G8 that is a mouse monoclonal antibody reactive to amino acid residues 17-24 of human Αβ peptide. The epitope recognized by 4G8 antibody lies within amino acids 18-22 of Αβ peptide (i.e. VFFAE) {Perchiacca et al, 2012, supra). Both 6E10 and 4G8 antibodies react to the enzymatically processed peptides Αβ40, Αβ42 and Αβ43 as well as the APP precursor form. The antibodies of the prior art are for use in research, in particular in immunoblotting (WB), immunohistochemistry (IHC), immunoprecipitation (IP), and ELISA.

Other prior art antibodies against Αβ peptide include Al 1 and NAB61. Antibody Al 1 is a conformational specific antibody which binds to a general oligomeric morphology found in different proteins such as alpha-synuclein protein, prion protein and Αβ peptide {Kayed etal, 2010, Mol Neurodegener. 5: 57). This antibody recognizes a generic epitope that is formed by many different amyloidogenic sequences, which is believed to be on the basis of the similar organization found among these proteins. Similarly, NAB61 is a monoclonal antibody that preferentially recognizes a conformational epitope present in small oligomeric and higher order Αβ structures {Lee et al, 2005, J. Bio. Chem., 281(7): 4292-9). Both Al l and NAB61 antibodies do not bind the soluble monomeric form of Αβ peptide, which reflects the antibody's selectivity for oligomeric morphologies of unknown sequence epitope. However, a major limitation associated with these morphology specific antibodies is the lack of specificity and sensitivity found in the initial events of Αβ aggregation, also known as the "lag-phase".

Therefore, there remains a need for the development of novel agents which show a high affinity and specificity for Αβ peptide in a form associated with presymptomatic stages of Alzheimer's disease or other beta-amyloid related disorders and exhibit a weak or limited affinity and/or specificity to the naturally occurring full-length monomeric form of Αβ peptide that is also present in healthy individuals, which rend them particularly suitable for preventive/therapeutic and prognosis/diagnostic applications in humans.

Summary of the Invention

The present invention is directed towards Αβ-binding molecules as described herewith, in particular antibodies binding specifically to particular fragments of Αβ peptide, as well as methods for detecting the level of fragments of Αβ peptide associated with early stages of Αβ peptide aggregation and/or amyloid plaque formation, methods for prognosis/diagnosis and/or prevention/treatment of beta-amyloid aggregates related disorders including Alzheimer's disease, and methods for screening compounds useful in the treatment of beta-amyloid aggregates related disorders including Alzheimer's disease.

A first aspect of the invention provides an Αβ-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide as described herewith but not to full-length Αβ peptides of 38, 40, 42 and/or 43 amino acids (Αβ1-38, Αβ1-40, Αβ1-42, Αβ1-43, respectively).

Another aspect of the invention relates to an isolated nucleic acid molecule as described herewith.

Other aspects of the invention relate to a recombinant expression vector and to a host cell as described herewith.

Another aspect of the invention relates to a process for producing an Αβ-binding molecule as described herewith.

Another aspect of the invention provides a pharmaceutical composition comprising one or more of (i) an Αβ-binding molecule, (ii) a fusion protein, (iii) a nucleic acid, (iv) a recombinant expression vector, and/or (v) a host cell, as described herewith, and at least one pharmaceutically acceptable carrier.

Another aspect of the invention provides an immunogenic composition comprising one or more fragments of Αβ peptides as described herewith.

Another aspect of the invention relates to an imaging composition or a diagnosis composition comprising at least one Αβ-binding molecule as described herewith.

Another aspect of the invention is a kit comprising at least one Αβ-binding molecule as described herewith. Another aspect of the invention relates to Αβ-binding molecules according to the invention or pharmaceutical compositions thereof for use in the prevention and/or treatment of beta-amyloid aggregation related disorders.

Another aspect relates to a method of preventing and/or treating beta-amyloid aggregation related disorders comprising administering in a subject in need thereof a therapeutically effective amount of said Αβ-binding molecule or said pharmaceutical composition.

Another aspect of the invention relates to an ex vivo method for detecting the level of Αβ peptide fragments associated with Αβ oligomerization and/or amyloid plaque formation, in particular starting from early stages of the process, in a biological sample as described herewith.

Another aspect of the invention provides a method of identifying a compound capable of inhibiting or delaying the process of Αβ peptide aggregation and/or amyloid plaque formation as described herewith.

Another aspect of the invention provides novel isolated fragments of Αβ peptides as described herewith.

Other features and advantages of the invention will be apparent from the following detailed description.

Description of the figures

Figure 1 shows the epitope binding specificity of the neo-C- and neo-N-terminal antibodies using dotblotting. Epitope binding specificity was tested using a repertoire of synthetic peptide standards of different C- or N-termini (indicated in each lane). Binding specificity of some antibodies according to the invention (N-5s, N-3s, N-5ns, N-4, D-4s, D-6ns) was compared with two commercially available anti-Αβ antibodies 6E10 and 4G8. A: Antibodies tested: N-5ns, N-5s, N-3s, 6E10. B: Antibodies tested: N- 4, 6E10 and 4G8. In A and B blots A' and B' were the same as blots A and B but were re-blotted with antibody N-5ns (blot A') or 6E10 (blot B'), respectively. C: Antibodies tested: D-4s and D-6ns. D: Antibodies tested 6E10 and 4G8. E: Summary of antibody specificities. F: Proof of principle of binding of N-5ns antibody during Αβ 1-40 aggregation in the presence and absence of tissue transglutaminase (Tgase). (NH ₂ ) indicates an amidated (CONH ₂ ) form of the peptide at the C-terminal end, whereas no specific indication refers to the form of the peptide with a carboxylic acid (COOH) at the C-terminal end. G: Binding of D-6ns antibody to the neo-N-terminal epitope comprising L-Serine residue at the position 26 (L-S26-40; lane 5) but not to the epitope comprising Serine stereoisomer D-Serine at the position 26 (D-S26-40; lane 6).

Figure 2 shows the screening of Αβ oligomer formation using N-5ns antibody. A: Time dependent screening of oligomer formation and size-exclusion chromatography (SEC) of Αβ oligomers following 20 hours aggregation. B: Electron microscopy imaging of aggregated oligomers (ADDL's) prior to SEC. C: SEC purified oligomers eluted at a column volume of 8-9 ml. D: Zoom of purified oligomers.

Figure 3 shows mass spectrometry (MALDI-TOF/TOF) analysis of Αβ oligomers and typical peptide fragment fingerprint found with oligomers. A: MALDI- TOF/TOF analysis of oligomers. B: Selected reaction monitoring (SRM) mass spectrometry analysis of gel (migration height 14 kDa) extracted and Lys-N digested oligomers.

Figure 4 shows analysis of Αβ oligomers using the neo-N-terminal D-4s antibody and/or neo-C-terminal N-5ns/N-3s antibody. A: Analysis of the amyloid derived diffusible ligands (ADDL's) aggregation kinetics using N-5ns or D-4s antibody. B: SEC analysis and dotblot analysis of oligomers using N-3s or D-4s antibody. C: Increased detection sensitivity with a combination of two complementary antibodies according to the invention (N-5ns and D-4s).

Figure 5 shows analysis of human control and AD patient post-mortem brain tissue extracts using immunprecipitation (IP) with N-5ns and immunoblotting. A:

Immunoblot (4G8) of human control and AD brain samples. B-C: Example of an analysed brain sample (IP with N-5ns followed by Lys-N digestion) using SRM, showing the identification of the diagnostic neo C-terminal fragment (16-25). D: SRM based quantitation of Αβ fragments in an AD brain sample.

Figure 6 shows identification of the diagnostic neo C-terminal fragment 1-25 in human CSF sample of an AD patient. A: IP-MS (MALDI-TOF/TOF) analysis of a control and AD CSF sample using the combination of the two commercially available antibodies 6E10 & 4G8. B: IP-MS analysis of an AD CSF sample using the N-5ns antibody showing the presence of the diagnostic fragment. C: Collision induced dissociation (CID) of the diagnostic fragment showing the identification of the peptide fragment. Detailed Description of the invention

The term « beta-amyloid peptide », « β-amyloid peptide », or « Αβ peptide », designates herewith peptides of 36 to 43 amino acids that are typically involved in Alzheimer's disease as the main component of the amyloid plaques found in the brains of Alzheimer patients. The Αβ peptides result from a 770-residue long amyloid precursor protein (APP, e.g. human APP, Uniprot Accession No. P05067), murine APP (Uniprot Accession No. P12023), which is cleaved before residue 672 and after residue 711, 713, or 714, by the concerted activity of β- and γ-secretases to yield Αβ1-40 (corresponding to residues 672-711 of APP), Αβ1-42 (corresponding to residues 672-713 of APP), Αβ1-43 (corresponding to residues 672-714 of APP) peptides. Αβ peptides can aggregate to form insoluble fibrils or soluble oligomers which may exist in different morphologies generally defined as low molecular weight (LMW) oligomers of Αβ dimers, trimers and tetramers or high molecular weight (HMW) oligomers such as the dodecameric (56 kDa), spherical and protofibrillar (>150 kDa) forms of Αβ. As used herewith, the term Αβ peptide includes the enzymatically cleaved peptide as defined above from the human APP and/or murine APP. For instance, the term Αβ peptide includes human Αβ peptide of amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 3 corresponding to Αβ1-42, Αβ1-40, and Αβ1-43, respectively. Human and guinea pig Αβ peptide sequences are identical. It also includes murine Αβ1-43 peptide of amino acid sequence SEQ ID NO: 26, as well as murine Αβ1-40 and murine Αβ1-42, which correspond to SEQ ID NO: 26 with the last 3 amino acids at the C-terminal end of SEQ ID NO: 26 deleted, or the last amino acid at the C-terminal end of SEQ ID NO: 26 deleted, respectively. Numbers next to "Αβ" refers to the amino acid positions on the Αβ amino acid sequence.

The terms "neo-C-terminal or novo-C-terminal fragment" and "neo-N-terminal or novo- C-terminal fragment" of Αβ peptide designate herewith the two complementary fragments resulting from the intramolecular cleavage of the original Αβ peptide. Said intramolecular cleavage of Αβ peptide generates one type of peptide fragment, wherein the N-term of the original peptide remains unchanged and its C-terminal end differs from that of the original Αβ peptide ("neo-C-terminal fragment"), and a second type of peptide fragment which N-terminal end differs from that of the original Αβ peptide, while the C-terminus remain unchanged as compared to the original Αβ peptide ("neo- N-terminal fragment"). Depending on the exact position of the intramolecular cleavage, different couples of complementary fragments can be formed. Typically, the neo-C- terminal fragments and neo -N-terminal fragments of the invention result from a cleavage of the Αβ peptide between positions 20 and 26 on said Αβ peptide. The terms "neo-C-terminal fragment" and "neo -N-terminal fragment", or equal terms "novo-C- terminal fragment" and "novo -N-terminal fragment" are commonly used in the field of the invention and clear to skilled in the field of peptide chemistry in general.

The term "diagnostic peptide fragment" refers to peptide fragment of the invention that may be used for the purpose of diagnosing AD, in particular the early onsets of the disease.

An "Αβ fragment-binding molecule" as used herein relates to antibodies (including polyclonal antibodies, monoclonal antibodies, antibody fragments, antibody variants), as well as other non-antibody molecules that bind to Αβ peptide fragments as described herewith, including but not limited to antibody mimetics, portions of antibodies that mimic the structure and/or function of an antibody (Qiu et al, 2007, Nature Biotechnology 25, 921-929), chimeric proteins or fusion proteins, aptamers (including peptide aptamers, DNA and RNA aptamers), small molecules. Unless specified otherwise, an Αβ fragment-binding molecule according to the invention can bind at least one neo-C-terminal fragment of Αβ or at least one neo -N-terminal fragment of Αβ peptide as described herewith, but cannot bind full-length forms of Αβ including Αβΐ- 40 peptide, Αβ1-42 peptide, and Αβ1-43 peptide. According to a particular aspect of the invention, an Αβ fragment-binding molecule according to the invention can bind an Αβ peptide fragment even when incorporated within low molecular weight aggregates, such as dimeric, trimeric and tetrameric Αβ peptide species, high molecular weight oligomers, such as dodecamers, Αβ spheres, Αβ peptide protofibrils, and/or insoluble Αβ peptide fibrils, diffuse Αβ deposits, and Αβ plaques. The Αβ fragment-binding molecule according to the invention is capable of specifically binding to and, optionally, neutralizing and/or inhibiting Αβ peptide aggregation, and/or inhibiting or reducing amyloid plaque formation, and/or increasing amyloid plaque dissociation.

The term "antibody" as referred to herein designates a polypeptide that binds to an antigen. This includes whole antibodies and any antigen binding fragments. The term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and further engineered antibodies as long as the characteristic properties of the invention are retained, in particular the ability of binding to the target antigen, more specifically to the same epitope of Αβ peptide fragment as the one recognized by the antibodies of the invention. Examples of antibodies and fragments thereof include a variable domain fragment ("Fv", consisting of the VH and VL domains of a single arm of an antibody), Fab fragment (monovalent fragment consisting of the VH, VL, CHI and CL domains), Fab ₂ fragment (bivalent), Fab ₃ fragment (trivalent), Fab' fragment (Fab with hinge region), F(ab') ₂ fragment (bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region), Fd fragment (consisting of the VH and CHI domains), rlgG (reduced IgG or half-IgG), diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAb), nanobodies, monovalent antibodies, divalent or multivalent antibodies comprising a fragment of more than one antibody, single chain variable fragment (ScFv), bis-scFv (bispecific), and derivatives of antibodies such as disulfide stabilized Fv fragments, CDR-comprising peptides, as well as epitope-binding fragments of any of the above (Holliger and Hudson, 2005, Nature Biotechnology, 23(9): 1126-1136). An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen- binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). In mammalians, the heavy chain can either be alpha (a), delta (δ), epsilon (ε), gamma (γ) or mu (μ), which defines the class of antibody IgA, IgD, IgE, IgG and IgM, respectively. In mammalians, the light chain can either be lambda (λ) or kappa (κ). In mammalians, depending on the class of antibody, the heavy chain constant region comprises three immunoglobulin domains, CHI, CH2, and CH3 (for IgA, IgD, IgG) or four immunoglobulin domains, CHI, CH2, CH3, and CH4 (for IgE and IgM). The light chain constant region comprises one immunoglobulin domain, CL. An antibody can have the structure of an IgA, IgG, IgE, IgD and IgM as well as any subtype thereof. Antibodies may be from any source including in particular primate (human and non-human primate) and primatized sources. The term "variable domain" (variable domain of a light chain (VL), variable domain of a heavy chain (VH)) as used herein refers to each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen. The variable light and heavy chain domains have the same general structure and each domain comprises four framework ("FR") regions whose sequences are widely conserved, connected by three "hypervariable regions" called "complementary determining regions" or "CDRs". The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The term "antigen-binding portion of an antibody" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus: the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The residues of the CDR and FR regions are conventionally numbered according to the standard definition of Kabat et al (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), Publication No. 91-3242). The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR- HI), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. The term "polyclonal antibody" as used herein refers to a composition comprising different antibody molecules which are capable of binding to or reacting with several different specific antigenic determinants (also referred to herein as "epitopes") on the same or on different antigens. The variability in antigen specificity of a polyclonal antibody is located in the variable regions of the individual antibody molecules constituting the polyclonal antibody and in the particular mixture of antibody molecules that constitute the polyclonal antibody. Preferably, compositions comprising the polyclonal antibody of the invention are prepared by immunization of an animal with the target antigen or immunogenic portions thereof as specified below and are derived from the blood, milk, colostrum or eggs obtained from the immunized animal. Typically, the immunized animal is a mouse, a rabbit, or a goat.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The term "chimeric antibody" generally refers to an antibody comprising a variable region from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. A typical example of chimeric antibodies includes those comprising a murine variable region and a human constant region. As defined herewith this term also includes an antibody comprising at least one of the CDRs of a first human antibody and at least a portion of a constant region of a second human antibody. It also includes an antibody comprising heavy chain CDR1, CDR2, and CDR3 of a first human antibody and light chain CDR1, CDR2, and CDR3 of a second human antibody.

The term "humanized antibody" designates antibodies from a non-human species having one or more complementarity determining regions (CDRs) from said non-human species and a framework region from a human immunoglobulin molecule. Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

The term "human antibody" or "fully human antibody" refers to antibodies in which the variable regions and the constant regions of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.

The term "isolated antibody" refers to an antibody that has been separated from a component of its natural environment. For instance, an isolated antibody has been purified to greater than 95% or 99% purity as determined by methods in the art (see e.g. Flatman et al, 2007, J Chromatogr B Analyt Technol Biomed Life Sci, 848: 79-87) including electrophoretic (e.g. SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (e.g. ion exchange or reverse phase HPLC (high performance liquid chromatography) methods.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant includes chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. By generalization, an epitope also designates herewith a region of an antigen that is bound by an antigen binding molecule such as an Αβ fragment-binding molecule as defined herewith.

The terms "immunogenic portion" or "immunogenic fragment" of an antigen or immunogen is any portion of the antigen or immunogen that is capable of inducing an immune response in the host animal being immunized with the antigen or immunogen and that preferably causes the animal to generate polyclonal antibodies against the target antigen. In a preferred embodiment the immunogen is a neo-N-terminal or neo-C- terminal fragment of Αβ peptide as described herewith or an immunogenic fragment thereof. According to a particular aspect of the invention, the immunogenic fragment is a neo-N-terminal or neo-C-terminal fragment of Αβ peptide of synthetic origin as described herewith or an immunogenic fragment thereof.

The term "aptamer" covers peptide aptamers and nucleic acid aptamers, including polypeptidic molecules and non-coding nucleic acid molecules which bind with high specificity and affinity to the neo-N- or neo-C-termini fragments of Αβ peptides as described herewith, by adopting a specific secondary and tertiary structure. Nucleic acid aptamers may be selected from a group of sequences identified using SELEX™ (Systematic Evolution of Ligands by Exponential enrichment) method or a similar process (Cox et ah, 1998, Biotechnol. Prog., 14, 845-850; Berezovski et ah, 2006, J. Am. Chem. Soc, 128:, 1410-1411). For example, aptamers are generated via an iterative process of binding, partitioning, and amplification. Single-stranded DNA primers and templates are amplified into double-stranded transcribable templates by PCR. The library of sequences is either directly used for the selection of DNA aptamers, or transcribed to an RNA library for the selection of RNA aptamers. RNA molecules so generated are then screened for their ability to interact with neo-N- or neo-C-termini fragments of human and/or murine Αβ peptides, for example by affinity chromatography, magnetic bids, or filtration. Alternative rounds of selection and amplification are repeated under increasing stringency in order to generate a limited subset of highly specific, high-affinity candidates to the target molecule. Selected candidates may then be truncated and chemically modified to improve resistance to nucleases and pharmacological properties. Aptamers may comprise biostable aptamers, such as spiegelmers™ (Klussmann et ah, 1996, Nat. Biotechnol, 14, 1112-1115; Vater and Klussmann, 2003, Curr. Opin. Drug. Discov. Devel, 6, 253-261). Peptide aptamers can be selected via in vitro methods such as phage display, ribosome display, and mRNA display, utilizing several rounds of peptide enrichment through in vitro binding to the protein target. Selected binders stay attached to the phage particle, ribosome, or mRNA molecule correspondingly, permitting the recovery of sequence information. Alternatively, peptide aptamers can also be selected via in vivo methods such as the Yeast two Hybrid and similar techniques. The aptamers described herein are capable of specifically binding to and, optionally, neutralizing and/or inhibiting Αβ peptide aggregation, and/or inhibiting or reducing amyloid plaque formation, and/or increasing amyloid plaque dissociation.

The terms "polynucleotide" or "nucleic acid molecule" refers to a polymer comprising nucleotides. Examples of nucleic acid molecules include DNA, RNA, locked nucleic acid (LNA), complementary DNA (cDNA). "Polypeptide" is understood as a peptide, an oligopeptide, an oligomer or a protein comprising at least two amino acids joined to each other by a normal or modified peptide bond, such as in the cases of the isosteric peptides, for example. A polypeptide can be composed of amino acids other than the 20 amino acids defined by the genetic code. A polypeptide can equally be composed of amino acids modified by natural processes, such as post-translational maturation processes or by chemical processes, which are well known to a person skilled in the art. Such modifications are fully detailed in the literature. These modifications can appear anywhere in the polypeptide: in the peptide skeleton, in the lateral chain or even at the carboxy- or amino- terminal ends. For example, polypeptide modifications is understood to include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of flavine, covalent fixation of heme, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation including pegylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination. Such modifications are fully detailed in the literature {Proteins Structure and Molecular Properties (1993) 2 ^nd Ed., T. E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B. C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62).

"Isolated polynucleotide" or "isolated polypeptide" is understood as a polynucleotide or a polypeptide such as previously defined which is isolated from the human body or otherwise produced by a technical process.

The term "variant" can apply to a polynucleotide and/or a polypeptide. For instance, a variant of a peptide or polypeptide, as referred to herein means a peptide or polypeptide substantially homologous to the referenced peptide sequence, but which has an amino acid sequence different from that of the referenced sequence because of one or more amino acid deletions, insertions and/or substitutions. Substantially homologous means a variant amino acid sequence which is identical to the referenced peptide sequence except for the deletion, insertion and/or substitution of a few amino acids, e.g. 1, 2, 3, 4, 5, or 6 amino acids. Substantially homologous means a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the referenced amino acid sequence. A variant nucleic acid sequence can be at least 80%>, at least 85%, at least 90%>, at least 95%, at least 96%o, at least 97%, at least 98%> or at least 99% identical to the referenced nucleic acid sequence. The identity of two amino acid sequences or of two nucleic acid sequences can be determined by visual inspection and/or mathematical calculation, or more easily by comparing sequence information using known computer program used for sequence comparison such as Clustal package version 1.83. A variant may comprise a sequence having at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Generally, substitutions for one or more amino acids present in the original polypeptide should be made conservatively. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known (Kyte, et al, 1982, J. Mol. Biol, 157: 105- 131). For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. The term "variant" also includes a peptide or polypeptide substantially homologous to the referenced peptide sequence, but which has an amino acid sequence different from that of the referenced sequence because one or more amino acids have been chemically modified or substituted by amino acids analogs. This term also includes glycosylated polypeptides. As used herewith the term "bind" or "binding" of an antibody, or generally of an Αβ fragment-binding molecule, to a target antigen means an at least temporary interaction or association of said antibody or molecule with, or to, said target antigen (such as a neo-C-terminal fragment of Αβ peptide or a neo-N-terminal fragment of Αβ peptide as described herewith) or with, or to, fragments of said target antigen comprising an epitope recognized by said antibody or molecule.

The terms "selectively binds", "specifically binds", "specific for", when applied to an antibody or generally to an Αβ fragment-binding molecule, indicate that the antibody or molecule, preferentially recognizes and/or binds the target polypeptide or epitope, i.e. with a higher affinity than to any other antigen or epitope, i.e. the binding to the target polypeptide can be discriminated from non-specific binding to other antigens. The binding affinity of an antibody or Αβ fragment-binding molecule can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard et al, 1949, Ann. N. Y. Acad. 1949. 51, 660-672).

As used herein, "binding affinity" generally refers to the apparent association constant or "Ka". The Ka is the reciprocal of the dissociation constant "Kd". Binding affinity may be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g. using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl ₂ at pH 7.5). These techniques can be used to measure the concentrations of bound and free binding protein as a function of binding protein (or target) concentration. The concentration of bound binding protein ([Bound]) is related to the concentration of free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation: [Bound]=N x([Free])/((l/Ka) + [Free]). Comparison of affinity between two antibodies can be established without actually determining the Ka value for each antibody, but based on a quantitative measurement of affinity (e.g. by ELISA or FACS analysis) that is proportional to Ka or a qualitative measurement of affinity or an inference of affinity (e.g. in functional assay or in vitro or in vivo assay).

The term "blocking" or "neutralizing" activity of an antibody, or generally of an Αβ fragment-binding molecule, refers to its ability to inhibit its target's activity. Applied to an antibody, or generally to an Αβ fragment-binding molecule, binding to as a neo-C- terminal fragment of Αβ peptide or a neo-N-terminal fragment of Αβ peptide as described herewith, this term refers to the antibody's or molecule's ability to generally inhibit or reduce Αβ peptide aggregation and/or amyloid plaque formation and/or increase amyloid plaque dissociation. The neutralizing activity of an antibody, or generally of an Αβ fragment-binding molecule, may be determined by in vitro assays including Thioflavin T or S fluorospectroscopy, Congo red assays, ELISA, immunoblotting, immunohistochemistry or electron microscopy. The "potency" of an antibody, or generally of an Αβ fragment-binding molecule, may be expressed as the concentration of antibody (or molecule)/antigen-binding fragment which produces the half-maximal effect at a given antigen concentration. For example, the "effect" of an antibody may be inhibition or neutralization of its target's activity. In this case, the antibody concentration producing the half-maximal inhibition may be referred to as IC ₅₀ , which is given in mol/1 or M. Potency is usually influenced by affinity until, at a given antigen concentration, an affinity is reached beyond which further improvements in affinity will not enhance binding of the antigen anymore (so-called potency ceiling). The term "efficacy of inhibition" or "efficacy of neutralization", applied to a neutralizing antibody, or generally to an Αβ fragment-binding molecule, is a measure of effectiveness of said antibody or molecule in inhibiting a specific biological or biochemical function. Total inhibition of the biological or biochemical activity is normalized to 100%.

The term "pharmaceutically acceptable" refers to a carrier comprised of a material that is not biologically or otherwise undesirable.

The term "carrier" refers to any components present in a pharmaceutical formulation other than the active agent and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives and the like.

As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it for example based on familial history; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage. For instance, treatment of Alzheimer disease comprises preventing, decreasing or even eradicating the symptoms of the disease, for instance partial or total alleviation of cognitive decline including short term memory loss, confusion, irritability, aggression, mood swings, trouble with language, and long- term memory loss.

The terms "beta-amyloid aggregation related disorders" or "β-amyloid aggregation related disorders" are generally defined herewith as disorders associated with Αβ peptide oligomerization, aggregation, and/or amyloid plaque deposit. Non- limitative examples of β-amyloid aggregation related disorders include Alzheimer's disease (also known as Alzheimer disease), cerebral amyloid angiopathy (also known as congophilic angiopathy), Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging.

Although the mechanism of Αβ peptide aggregation remain to be elucidated, the process of formation of Αβ peptides and amyloid plaque can be described as a continuous process comprising the formation of the successive following forms of amyloid beta: soluble LMW oligomers of Αβ dimers, trimers and tetramers, soluble HMW oligomers such as the dodecameric Αβ peptide species, soluble Αβ peptide protofibrils, insoluble Αβ peptide fibrils, diffuse Αβ deposits, and Αβ plaques.

"Early stage of aggregation" is defined herewith as comprising the molecular events associated with the gradual appearance of soluble LMW oligomeric Αβ peptide species. The initial molecular mechanisms associated with early stage aggregation can be considered as part of a prodromal phase of AD, because only the gradual accumulation of HMW Αβ oligomers is known to be associated with loss in neuronal plasticity and hence decline in cognitive function. In contrast, "advanced stage of aggregation" is characterized by the appearance of insoluble Αβ peptide fibrils of typical amyloidogenic nature and morphology. The advanced stage of peptide aggregation is typically monitored by fluorescence spectroscopy using molecules such as Thioflavin T and/or Thio flavin S as well as congo red. These molecules typically bind to the beta-sheet rich architecture found in amyloidogenic fibrils or large aggregates. Advanced stage of peptide aggregation can also be monitored by electron microscopy (EM) imaging. In general, fluorescence spectroscopy is not sufficiently sensitive/specific for monitoring the early stage of peptide aggregation since soluble LMW and HMW Αβ oligomers may bear little or no beta-sheet structures. The early stage of aggregation can be monitored by EM imaging or immuoblotting, which may reveal the presence of small SDS-PAGE stable oligomers. In a particular embodiment of the invention, early stage of aggregation describes the situation where soluble LMW oligomers of dimeric, trimeric and tetrameric Αβ species and soluble HMW oligomers of dodecameric, spherical and proto fibrillar Αβ peptide species, as a whole, represent more than 50%, more than 60%>, more than 70%, more than 80%, more than 90%, or more than 95% of the forms comprising Αβ peptide. In a more particular embodiment, "early stage of aggregation" describes the situation where soluble LMW oligomers of dimeric, trimeric and tetrameric species and soluble HMW oligomers of dodecameric, spherical and proto fibrillar Αβ peptide species, as a whole, represent more than 50%>, more than 60%>, more than 70%, more than 80%, more than 90%, or more than 95% of the forms comprising Αβ peptide.

In another particular embodiment of the invention, early stage of aggregation describes the situation where Αβ peptide fibrils, diffuse Αβ deposits, and Αβ plaques, as a whole, represent less than 50%>, less than 60%>, less than 70%>, less than 80%>, less than 90%>, or less than 95% of the forms comprising Αβ peptide.

The term "subject" as used herein refers to mammals. For example, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents and the like.

The term "efficacy" of a treatment or method according to the invention can be measured based on changes in the course of disease or condition in response to a use or a method according to the invention. For example, the efficacy of a treatment or method according to the invention can be measured by its impact on signs or symptoms of illness or disorder. A response is achieved when the patient experiences partial or total alleviation, or reduction of unwanted symptoms of illness or disorder.

The term "effective amount" as used herein refers to an amount of at least one agent

(e.g. Αβ fragment-binding molecule) according to the invention, or a pharmaceutical formulation thereof, that elicits a detectable reduction of the symptoms of the disease in a subject that is being administered said agent, these symptoms can include, for instance: cognitive decline including short term memory loss, confusion, irritability, aggression, mood swings, trouble with language, and long-term memory loss in the case of Alzheimer's disease. When applied to a vaccine composition such as an immunogenic composition, "effective amount" designates an amount of at least one agent (e.g. neo-N-terminal or neo-C-terminal fragment of Αβ peptide from natural or synthetic origin) according to the invention, or an immunogenic formulation thereof, that elicits the production of antibodies specific for the antigen comprised in the immunogenic composition in a subject that is being administered said agent.

Neo-N-terminal and neo-C-terminal fragments of Αβ peptide

In a first aspect, the present invention provides fragments of Αβ peptide which may be generated as a result of aggregation mediated peptide bond hydrolysis (autocleavage), the appearance of said fragments reflecting the initiation and evolution of the Αβ aggregation process.

In a particular aspect, the invention provides fragments of Αβ peptide, the formation of which is associated with the early stages of Αβ aggregation which precede the appearance of the symptoms of β-amyloid aggregation related disorders, in particular Alzheimer's disease.

In one embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments correspond to the neo-N-terminal and neo-C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of Αβ peptide.

In a particular embodiment, the present invention provides fragments of Αβ peptide as defined herewith, wherein, when the amino acid residue at the equivalent position 26 of the full-length Αβ peptide is present in said fragment, said amino acid residue is a L- Ser.

In another particular embodiment, the present invention provides fragments of Αβ peptide as defined herewith, wherein said fragments comprise only L-amino acids. In another further embodiment, the fragments of Αβ peptide as defined herewith do not include racemized Αβ fragments comprising at least one D-amino acid, in particular a D-Ser at equivalent position 26 on the full-length Αβ peptide amino acid sequence (abbreviated [D-Ser26]).

According to a more particularly embodiment, Αβ peptide fragments of sequences [D- Ser26]Ap25-40, [D-Ser26]Ap25-35, [D-Ser26]Ap26-40, [D-Ser26]Ap26-35, are not included. According to a further more particularly embodiment, Αβ peptide fragments of sequences [D-Ser26]Ap25-42, [D-Ser26]Ap25-43, [D-Ser26]Ap26-42, [D- Ser26]Ap26-43 are not included.

In a more particular embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments correspond to the neo-N-terminal and neo-C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 24 and 25, or 25 and 26, on the amino acid sequence of said Αβ peptide.

In another particular embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments correspond to the neo-N-terminal and neo-C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 22 and 23, or 23 and 24, or 25 and 26, on the amino acid sequence of said Αβ peptide.

In another particular embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments correspond to the neo-N-terminal and neo-C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 22 and 23, or 23 and 24, or 24 and 25, on the amino acid sequence of said Αβ peptide.

In one embodiment, said fragments comprise at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 consecutive amino acids from the full-length Αβ peptide.

In one embodiment, said fragments comprise least 24, or at least 25 consecutive amino acids from the full-length Αβ peptide.

In another embodiment, said fragments comprise not more than 30, 25, 24, 23, 22, 21, or 20 consecutive amino acids from the full-length Αβ peptide. In the invention, said fragments may be fragments of a full-length Αβ peptide (typically Αβ1-40, Αβ1-42, or Αβ1-43) from any mammalian species including human, rabbit, rodent (mice, rat).

In one embodiment of the invention, it is provided isolated neo-C-terminal fragments of Αβ peptide, wherein said fragments correspond to Αβ1-25 or a portion thereof, in particular a portion where a few amino acids (e.g. from 1 to 12, from 1 to 5, or 1, 2, or 3) at the C-terminal end of Αβ1-25 are deleted, or a variant thereof.

In another embodiment of the invention, it is provided neo-N-terminal fragments of Αβ peptide, wherein said fragments correspond to Αβ23-42 or a portion thereof, or Αβ23- 40 or a portion thereof, or Αβ23-43 or a portion thereof, in particular a portion where a few amino acids (e.g. from 1 to 5, or 1, 2, or 3) at the N-terminal end of Αβ23-42,Αβ23- 40, and Αβ23-38, respectively, are deleted, or a variant thereof.

In another particular aspect, the invention provides fragments of Αβ peptide, the formation of which is associated with the later stages of Αβ aggregation, e.g. advanced stages, which are concomitant with the development of the symptoms of β-amyloid aggregation related disorders, in particular, Alzheimer's disease.

Thus, in one embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments are obtained by cleavage of the neo-C-terminal fragments of Αβ peptide described above, e.g. Αβ1-22, Αβ1-23, Αβ1-24, Αβ1-25, said cleavage having occurred between two consecutive amino acids located between positions 12 and 16 on the amino acid sequence of said neo-C-terminal fragments.

In a more particular embodiment, the present invention provides fragments of Αβ peptide, wherein said fragments are obtained by cleavage of said neo-C-terminal fragments of Αβ peptide described above, said cleavage having occurred between two consecutive amino acids present at positions 13 and 14, or 14 and 15, or 15 and 16, on the amino acid sequence of said neo-C-terminal fragments.

In a still other embodiment of the invention, it is provided neo-C-terminal fragments of Αβ peptide, wherein said fragments correspond to Αβ1-15 or a portion thereof, in particular a portion where a few amino acids (e.g. from 1 to 5, or 1, 2, or 3) at the C- terminal end of Αβ1-15 are deleted, or a variant thereof.

In a still other embodiment of the invention, it is provided neo-N-terminal fragments of Αβ peptide, wherein said fragments correspond to Αβ 14-25 or a portion thereof, Αβ15- 25 or a portion thereof, Αβ 16-25 or a portion thereof, in particular a portion where a few amino acids (e.g. from 1 to 5, or 1 , 2, or 3) at the C-terminal end of Αβ14-25, Αβ15-25, and Αβ 16-25, respectively, are deleted, or a variant thereof.

In one embodiment of the invention, it is provided isolated fragments of Αβ peptide, wherein said fragments are selected from the group consisting of:

(i) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-22, Αβ1-23, Αβ1-24, Αβ1-25, or a variant thereof,

(ii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-42, Αβ24-42, Αβ25-42, Αβ26-42, or a variant thereof,

(iii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-40, Αβ24-40, Αβ25-40, Αβ26-40, or a variant thereof,

(iv) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-43, Αβ24-43, Αβ25-43, Αβ26-43, or a variant thereof,

(v) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-13, Αβ1-14, Αβ1-15, or a variant thereof,

(vi) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ14-25, Αβ15-25, Αβ16-25, or a variant thereof.

In a particular embodiment, it is provided isolated fragments of Αβ peptide, wherein said fragment is a neo-N-terminal fragment selected from: Αβ25-42, Αβ24-42, Αβ25- 40, Αβ24-40, Αβ25-43, Αβ24-43.

In a particular embodiment, it is provided isolated fragments of Αβ peptide, wherein said fragments are selected from the group consisting of:

(i) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-22 of SEQ ID NO: 4, Αβ1-23 of SEQ ID NO: 5, Αβ1-24 of SEQ ID NO: 6, Αβ1-25 of SEQ ID NO: 7,

(ii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-42 of SEQ ID NO: 11, Αβ24-42 of SEQ ID NO: 12, Αβ25-42 of SEQ ID NO: 13, Αβ26-42 of SEQ ID NO: 14,

(iii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-40 of SEQ ID NO: 15, Αβ24-40 of SEQ ID NO: 16, Αβ25-40 of SEQ ID NO: 17, Αβ26-40 of SEQ ID NO: 18, (iv) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-43 of SEQ ID NO: 19, Αβ24-43 of SEQ ID NO: 20, Αβ25-43 of SEQ ID NO: 21, Αβ26-43 of SEQ ID NO: 22,

(v) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-13 of SEQ ID NO: 8, Αβ1-14 of SEQ ID NO: 9, Αβ1-15 of SEQ ID NO: 10, and

(vi) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ14-25 of SEQ ID NO: 23, Αβ15-25 of SEQ ID NO: 24, Αβ16-25 of SEQ ID NO: 25.

In a particular embodiment of the invention, said fragment is Αβ1-23, more particularly Αβ1-23 of SEQ ID NO: 5.

In a particular embodiment of the invention, said fragment is Αβ1-24, more particularly Αβ1-24 of SEQ ID NO: 6.

In another embodiment of the invention, the last amino acid positioned at the C-terminal end of the amino acid sequence of the fragments as described above has a free -COOH group.

In an alternative embodiment of the invention, said fragments as described above are amidated at their last amino acid positioned at the C-terminal end, i.e. the last amino acid positioned at the C-terminal end of the amino acid sequence of the fragments as described above has a -CONH ₂ group instead of a free -COOH group.

In a particular embodiment, is provided an isolated fragment of Αβ peptide, wherein said fragment is a neo-C-terminal fragment of Αβ peptide selected from: Αβ1-25, Αβΐ- 24, Αβ1-23, Αβ1-22, wherein the last C-terminal amino acid of said fragment is amidated.

In another particular embodiment of the invention, said fragment is Αβ1-24, more particularly Αβ1-24 of SEQ ID NO: 6, wherein amino acid residue at position 24 has a - CONH ₂ group.

Αβ fragment-binding molecules

The invention provides molecules specifically binding to neo-N-terminal and neo-C- terminal fragments of Αβ peptide as described herewith but not binding to Αβ1-40, Αβ 1-42 and/or Αβ1-43. In a particular embodiment, the invention provides molecules specifically binding to neo-C-terminal fragments of Αβ peptide as described herewith but not binding to Αβΐ- 40, Αβ 1-42 and/or Αβ1-43.

In a particular embodiment, the invention provides Αβ fragment-binding molecule characterized in that said molecule specifically binds to a neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide, but does not bind to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43).

In one embodiment, the Αβ fragment-binding molecules according to the invention bind preferentially to neo-N-terminal or neo-C-terminal fragments of Αβ peptide as described herewith and exhibit virtually no binding (i.e. negligible or not detectable binding) to Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides.

In preferred embodiment, the Αβ fragment-binding molecules according to the invention bind preferentially to neo-C-terminal fragments of Αβ peptide as described herewith and exhibit virtually no binding (i.e. negligible or not detectable binding) to Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides.

In preferred embodiment, the Αβ fragment-binding molecule according to the invention, bind preferentially to neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located at positions 24 and 25, or 23 and 24, or 25 and 26, or 22 and 23, on the amino acid sequence of said Αβ peptide. In a particular embodiment, the Αβ fragment-binding molecules according to the invention bind preferentially to neo-N-terminal or neo-C-terminal fragments of Αβ peptide, wherein, when the amino acid residue at the equivalent position 26 of the full- length Αβ peptide is present in said fragment, said amino acid residue is a L-Ser, and wherein said Αβ fragment-binding molecules exhibit virtually no binding (i.e. negligible or not detectable binding) to Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides.

In a particular embodiment, the Αβ fragment-binding molecules according to the invention bind preferentially to neo-N-terminal or neo-C-terminal fragments of Αβ peptide comprising only L-amino acids as described herewith and exhibit virtually no binding (i.e. negligible or not detectable binding) to Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides. In a particular embodiment, the Αβ fragment-binding molecules according to the invention bind preferentially neo-C-terminal fragments of Αβ peptide comprising only L-amino acids as described herewith and exhibit virtually no binding (i.e. negligible or not detectable binding) to Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides.

In a particular embodiment, the Αβ fragment-binding molecules according to the invention exhibit virtually no binding to racemized fragments of Αβ peptide comprising at least one D-amino acid, in particular a D-Ser at equivalent position 26 relative to the amino acid sequence of full-length Αβ peptide.

In a more particular embodiment, the Αβ fragment-binding molecules according to the invention do not bind to racemized [ϋ-8εΓ26]Αβ25-35, racemized [ϋ-8εΓ26]Αβ25-40, racemized [ϋ-8εΓ26]Αβ26-35, racemized [ϋ-8εΓ26]Αβ26-40. In a further embodiment, Αβ fragment-binding molecules according to the invention do not bind, either, to racemized [D-Ser26^25-42, racemized [D-Ser26]Ap25-43, [D-Ser26]Ap26-42 and/or racemized [D-Ser26]Ap26-43.

For therapeutic uses, it may be advantageous that the Αβ fragment-binding molecules according to the invention do not bind, and thus do not neutralize, Αβ1-40, Αβ1-42 and/or Αβ1-43 peptides so as not to substantially affect normal Αβ function.

In some embodiments, the binding affinity (e.g. inversely correlated to the Kd value) of the Αβ fragment-binding molecules according to the invention for the herewith described Αβ fragments is at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times higher than their binding affinity for Αβ1-40, Αβ1-42 and Αβ1-43 peptides.

In another embodiment, the Αβ fragment-binding molecules according to the invention, not only bind to the indicated fragments of Αβ peptide but also neutralize or inhibit and/or reduce Αβ peptide aggregation and/or amyloid plaque formation and/or increase amyloid plaque dissociation.

In one embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to full-length Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβΐ- 43), wherein said fragment is a neo-N-terminal or neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26, e.g. between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 24 and 25, or 25 and 26, on the amino acid sequence of Αβ peptide.

In one embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to full-length Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβΐ- 43), wherein said fragment is neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26, e.g. between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 24 and 25, or 25 and 26, on the amino acid sequence of Αβ peptide.

In one embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to full-length Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβΐ- 43), wherein said fragment is neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26, e.g. between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 25 and 26, on the amino acid sequence of Αβ peptide.

In one embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to full-length Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβΐ- 43), wherein said fragment is neo-C-terminal fragment of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26, e.g. between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 24 and 25, on the amino acid sequence of Αβ peptide.

In another particular embodiment, it is provided an Αβ fragment-binding molecule as defined herewith wherein said fragments correspond to the neo-N-terminal and/or neo- C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 25 and 26, on the amino acid sequence of said Αβ peptide.

In another particular embodiment, it is provided an Αβ fragment-binding molecule as defined herewith wherein said fragments correspond to the neo-N-terminal and/or neo- C-terminal fragments of Αβ peptide obtained by cleavage of said Αβ peptide between two consecutive amino acids present at positions 22 and 23, or 23 and 24, or 24 and 25, on the amino acid sequence of said Αβ peptide.

In one embodiment of the invention, it is provided an Αβ fragment-binding moleculecharacterized in that said molecule specifically binds to a fragment of Αβ peptide but not to full-length Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said fragment is a neo-C-terminal fragment of Αβ peptide corresponding to Αβ1-25 or a portion thereof, in particular a portion where a few amino acids, e.g. 1, 2, or 3, at the C-terminal end of Αβ1-25 are deleted, or a variant thereof. In another embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβΐ- 43), wherein said fragment is a neo-N-terminal fragment of Αβ peptide corresponding to Αβ23-42 or a portion thereof, or Αβ23-40 or a portion thereof, or Αβ23-43, in particular a portion where a few amino acids, e.g. 1, 2, or 3, at the N-terminal end of Αβ23-42, Αβ23-40, and Αβ23-43, respectively, are deleted, or a variant thereof.

In a still other embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule binds to a fragment of Αβ peptide but not to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said fragment is a neo-C-terminal fragment of Αβ peptide corresponding to Αβ1-15 or a portion thereof, in particular a portion where a few amino acids, e.g. 1 , 2, or 3, at the C- terminal end of Αβ1-15 are deleted, or a variant thereof.

In a still other embodiment of the invention, it is provided an Αβ fragment-binding molecule characterized in that said molecule binds to a fragment of Αβ peptide but not to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said fragment is a neo-N-terminal fragment of Αβ peptide corresponding to Αβ 14-25 or a portion thereof, Αβ15-25 or a portion thereof, or Αβ 16-25 or a portion thereof, in particular a portion where a few amino acids (e.g. from 1 to 5, or 1, 2, or 3) at the C- terminal end of Αβ14-25, Αβ15-25, and Αβ16-25, respectively, are deleted, or a variant thereof.

In a particular embodiment, the invention provides an Αβ fragment-binding molecule characterized in that said molecule specifically binds to a fragment of Αβ peptide but not to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said fragment is:

(i) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-22, Αβ1-23, Αβ1-24, Αβ1-25, or a variant thereof, or

(ii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-42, Αβ24-42, Αβ25-42, Αβ26-42, or a variant thereof, or

(iii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-40, Αβ24-40, Αβ25-40, Αβ26-40, or a variant thereof, or

(iv) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-43, Αβ24-43, Αβ25-43, Αβ26-43, or a variant thereof, or

(v) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-13, Αβ1-14, Αβ1-15, or a variant thereof, or

(vi) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ 14-25, Αβ15-25, Αβ 16-25, or a variant thereof.

In a particular embodiment is provided an Αβ fragment-binding molecule characterized in that it specifically binds to a fragment of Αβ peptide but not to Αβ peptides of 40, 42 or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said fragment is:

(i) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-22 of SEQ ID NO: 4, Αβ1-23 of SEQ ID NO: 5, Αβ1-24 of SEQ ID NO : 6, Αβ 1 -25 of SEQ ID NO : 7, or

(ii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-42 of SEQ ID NO: 11, Αβ24-42 of SEQ ID NO: 12, Αβ25-42 of SEQ ID NO: 13, Αβ26-42 of SEQ ID NO: 14, or

(iii) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-40 of SEQ ID NO: 15, Αβ24-40 of SEQ ID NO: 16, Αβ25-40 of SEQ ID NO: 17, Αβ26-40 of SEQ ID NO: 18, or

(iv) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ23-43 of SEQ ID NO: 19, Αβ24-43 of SEQ ID NO: 20, Αβ25-43 of SEQ ID NO: 21, Αβ26-43 of SEQ ID NO: 22, or

(v) A neo-C-terminal fragment of Αβ peptide selected from the group consisting of:

Αβ1-13 of SEQ ID NO: 8, Αβ1-14 of SEQ ID NO: 9, Αβ1-15 of SEQ ID NO: 10, or (vi) A neo-N-terminal fragment of Αβ peptide selected from the group consisting of: Αβ14-25 of SEQ ID NO: 23, Αβ15-25 of SEQ ID NO: 24, Αβ16-25 of SEQ ID NO: 25.

In a particular embodiment, said Αβ fragment-binding molecule specifically binds to a neo-C-terminal fragment of Αβ peptide selected from: Αβ1-24 and Αβ1-23.

In a particular embodiment, said Αβ fragment-binding molecule specifically binds to a neo-C-terminal fragment of Αβ peptide wherein the C-terminal amino acid is amidated. In a particular embodiment, said Αβ fragment-binding molecule specifically binds to a fragment of Αβ peptide selected from the group consisting of: Αβ1-23 of SEQ ID NO: 5 with amino acid residue at position 23 having either a free -COOH group or is amidated (i.e. a-CONH ₂ group), Αβ1-24 of SEQ ID NO: 6 with amino acid residue at position 24 having a -CONH ₂ group, Αβ1-25 of SEQ ID NO: 7 with amino acid residue at position 25 having a -CONH ₂ group.

In a more particular embodiment, said Αβ fragment-binding molecule specifically binds to a fragment of Αβ peptide that is Αβ1-24, in particular Αβ1-24 of SEQ ID NO: 6. In a further particular embodiment, said Αβ fragment-binding molecule specifically binds to a fragment of Αβ peptide that is Αβ1-24, in particular Αβ1-24 of SEQ ID NO: 6, wherein amino acid residue at position 24 has a -CONH ₂ group.

The Αβ fragment-binding molecules according to the invention can bind to neo-N- terminal or C-terminal fragments of Αβ peptide by interacting with an epitope comprising amino acids located anywhere in said fragments.

In a particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-N-terminal or neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one amino acid, or two to three consecutive amino acids, of the Αβ peptide, selected from: A21, E22, D23, V24, G25, S26, N27, and K28.

In a further particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-N-terminal or neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one amino acid, or two to three consecutive amino acids, of the Αβ peptide, selected from: A21, E22, D23, V24, G25, S26, N27, and K28, wherein each one of said amino acids is a L-amino acid. In a still further particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-N-terminal or neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least L-S26.

In another particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one amino acid, or two to three consecutive amino acids, of the Αβ peptide, selected from: El l, VI 2, H13, HI 4, and Q15.

It is understood that the numbers indicated herewith refer to the amino acid positions on the Αβ peptide sequence.

In a further particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-N-terminal or neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one of the combinations of amino acids of the Αβ peptide selected from: 23DVG25, 22EDV24, 21AED23, 25GSN27, 2 ₆ SNK ₂ 8, 24VGS26, i ₃ HHQ ₁₅ , and/or„EVH ₁₃ .

In a particular embodiment, the Αβ fragment-binding molecules according to the invention specifically bind to said neo-C-terminal fragments of Αβ peptide by interacting with an epitope comprising at least one of the combinations of amino acids of the Αβ peptide selected from: 22EDV24, 21 AED ₂₃ , and/or 23DVG25.

In a particular embodiment, the Αβ fragment-binding molecule according to the invention specifically binds to said neo-C-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one amino acid of the Αβ peptide selected from: A21, E22, D23, V24, G25, or three consecutive amino acids of the Αβ peptide selected from: 22EDV24, 2iAED ₂₃ , and/or ₂₃ DVG25-

In another particular embodiment, the Αβ fragment-binding molecules according to the invention specifically bind to said neo-C-terminal fragments of Αβ peptide by interacting with an epitope comprising at least one of the combinations of amino acids of the Αβ peptide selected from: 23DVG25, where amino acid G has a free -COOH group or a -CONH2 group, 22EDV24, where amino acid V is amidated (i.e. has a -CONH ₂ group), 2iAED ₂₃ where amino acid D has a free -COOH group or a -CONH ₂ group, i ₃ HHQi ₅ where amino acid Q has a free -COOH group, and nEVHn where amino acid H has a free -COOH group. In another particular embodiment, the Αβ fragment-binding molecules according to the invention specifically bind to said neo-N-terminal fragments of Αβ peptide by interacting with an epitope comprising at least one combination of amino acids of the Αβ peptide selected from: ₂ 6SNK ₂ 8 and ₂₄ VGS ₂₆ .

In a more particular embodiment, the Αβ fragment-binding molecules according to the invention bind to said neo-N-terminal or neo-C-terminal fragments of Αβ peptide by interacting with an epitope comprising at least one combination of amino acids of the Αβ peptide selected from: ₂₃ DVG ₂₅ , ₂₂ EDV ₂₄ , ₂ iAED ₂₃ , ₂₅ GSN ₂₇ , ₂₆ SNK ₂₈ , and ₂₄ VGS ₂₆ . The Αβ fragment-binding molecules according to the invention can be of various nature including antibodies or fragments thereof, nucleic acid derived polymers (such as DNA and RNA aptamers), peptidomimetics, fusion proteins, small molecules.

In a particular aspect of the invention, the Αβ fragment-binding molecules according to the invention are antibodies or fragments thereof.

In a particular aspect of the invention, said Αβ fragment-binding molecules are isolated antibodies, or fragments thereof, specifically binding to neo-N-terminal or neo-C- terminal fragments of Αβ peptide as described herewith, which do not bind to Αβ1-40, Αβ 1-42 and/or Αβ1-43.

In a particular aspect of the invention, said Αβ fragment-binding molecules are isolated antibodies, or fragments thereof, specifically binding to neo-C-terminal fragments of Αβ peptide as described herewith, which do not bind to Αβ1-40, Αβ1-42 and/or Αβ1-43. The antibodies according to the invention can be monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and further engineered antibodies as long as the characteristic properties of the invention are retained, in particular the ability of binding to the target antigen, more specifically to the same epitope of Αβ peptide fragment as the one recognized by the antibodies of the invention, and optionally the ability of neutralizing or reducing Αβ peptide aggregation and/or amyloid plaque formation and/or the ability of increasing amyloid plaque dissociation.

In one embodiment of the invention, the isolated antibody according to the invention, or fragment thereof, is a polyclonal antibody.

Examples of polyclonal antibodies according to the invention include antibodies N-3s, N-4, N-5ns, N-5s, D-4s, and D-6ns, further characterized in the example section, as well as antibodies exhibiting similar antigen binding specificity and, in particular, recognizing the same epitopes.

In another embodiment, the isolated antibody according to the invention, or fragment thereof, is a monoclonal antibody.

In a further embodiment, the isolated antibody according to the invention, or fragment thereof, is selected from the group consisting of: a murine antibody, a humanized antibody, a human antibody, and a chimeric antibody.

Antibodies can be produced by standard techniques in the field including the recovery of polyclonal antibodies from the serum of laboratory or farm animals which have been injected with the antigen of interest, the recovery of monoclonal antibodies produced by fusing antibody-secreting spleen cells from immunized mice with immortal myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant. Alternatively, recombinant antibodies can be produced in various host cells including mammalian cells, plant cells, bacteria, yeasts, which have been engineered to express said antibodies.

In another particular aspect of the invention, said Αβ fragment-binding molecules are aptamers, in particular nucleic acid aptamers such as DNA aptamers or RNA aptamers, specifically binding to neo-N-terminal or neo-C-terminal fragments of Αβ peptide as described herewith, which do not bind to Αβ1-38, Αβ1-40, Αβ1-42 and/or Αβ1-43. The aptamers according to the invention can also, optionally, neutralize or inhibit Αβ peptides aggregation and/or inhibit or reduce amyloid plaque formation and/or increase amyloid plaque dissociation.

Conjugates comprising an auxiliary molecule

In another aspect of the invention, the Αβ fragment-binding molecules according to the invention, e.g. isolated antibodies or antigen-binding fragment thereof and aptamers, are optionally conjugated to an accessory molecule, and are then also referred to herein as "conjugated molecules, "conjugated antibodies, "conjugated antibody fragments", or "conjugated aptamers", depending on the nature of the Αβ fragment-binding molecule. The accessory molecule may be conjugated to the Αβ fragment-binding molecule (e.g. an antibody or antibody fragment) directly or via a spacer of suitable length for instance as described in Kellogg et al. (2011, Bioconjug Chem, 22: 717-27). In one embodiment, particularly adapted for therapeutic purposes, the accessory molecule can be a therapeutic effector group such as a cytotoxic (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragment thereof), cytostatic, or immunomodulatory agent, including radioactive groups (i.e groups comprising a radionucleide or radioisotope), or small molecules.

In a specific embodiment, the accessory molecule may be, for example, an agent active for the treatment of a β-amyloid aggregation related disorder such as, for the treatment of Alzheihmer's disease including cholinesterase inhibitors such as Galantamine, Aricept™ (donepezil HC1), Excelon™ (rivastigmine tartrate), and NMDA (N-Methyl- D-aspartate) receptor antagonists such as Memantine.

The conjugated Αβ fragment-binding molecules according to the invention (e.g. conjugated antibodies and conjugated antibody fragments) can target the drug in vivo to a site of disease (e.g. the cortical brain regions or limbic system of the brain, or cerebral arties affected with cerebral amyloid angiopathy) such that the conjugated auxiliary molecule can have a therapeutic effect on the site of disease.

In an alternative embodiment, particularly adapted for diagnostic purposes, the accessory molecule can be, for example, a labeling group including radioisotopes (e.g. 3H, 14C, 32P, 35S, 1251), chromogenic labels, e.g. enzymes which can be used to convert a substrate to a detectable colored (e.g. horseradish peroxidase, alkaline phosphatase, β-galactosidase) or fluorescent compound (e.g. Green Fluorescent Protein, Red Fluorescent Protein), spectroscopic labels (e.g. fluorescent labels such as fluorescein and its derivatives like FITC, Texas red, cyanine dyes, photocyan, rhodamine, or labels presenting a visible color), luminescent labels including luciferins, affinity labels which may be developed by a further compound specific for the label and allowing easy detection and quantification, or any other label used in standard ELISA.

Nucleic acids

According to another embodiment, it is provided an isolated nucleic acid molecule encoding: (i) a neo-N-terminal or neo-C-terminal fragment of Αβ peptide as described herewith, (ii) an Αβ fragment-binding molecule, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, according to the invention, or (iii) a polypeptidic conjugated molecule comprising any one of (i) or (ii) according to the invention. In a specific embodiment, it is provided an isolated nucleic acid molecule encoding: (i) neo-C-terminal fragment of Αβ peptide as described herewith, (ii) an Αβ fragment- binding molecule, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, according to the invention, or (iii) a polypeptidic conjugated molecule comprising any one of (i) or (ii) according to the invention.

In an alternative embodiment, it is provided an isolated nucleic acid molecule comprising a nucleic acid aptamer according to the invention or encoding a peptide aptamer according to the invention.

The isolated nucleic acid according to the invention may be, for instance, natural DNA or RNA or a recombinant or synthetic DNA, RNA or LNA or a recombinant nucleic acid molecule comprising any of the nucleic acid molecules according to the invention either alone or in combination. In a particular embodiment, the nucleic acid molecules according to the invention are cDNA.

Vectors and Host cells for production and purification of the polypeptides of the invention

In one embodiment, the invention provides a vector comprising a nucleic acid molecule according to the invention, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide, operably linked to said nucleic acid molecule.

Numerous expression systems can be used, including without limitation chromosomes, episomes, and derived viruses. More particularly, the recombinant vectors used can be derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia viruses, adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses. These recombinant vectors can equally be cosmid or phagemid derivatives.

The nucleic acid sequence can be inserted in the recombinant expression vector by methods well known to a person skilled in the art such as, for example, those that are described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al, 4 ^th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. The recombinant vector can include nucleotide sequences that control the regulation of the polynucleotide expression as well as nucleotide sequences permitting the expression and the transcription of a polynucleotide of the invention and the translation of a polypeptide of the invention, these sequences being selected according to the host cells that are used.

Thus, for example, an appropriate secretion signal can be integrated in the recombinant vector so that the polypeptide, encoded by the nucleic acid molecule of the invention, will be directed towards the lumen of the endoplasmic reticulum, towards the periplasmic space, on the membrane or towards the extracellular environment. The choice of an appropriate secretion signal may facilitate subsequent protein purification. In a further embodiment, it is provided a host cell comprising a recombinant vector according to the invention.

The introduction of the recombinant vector in a host cell can be carried out according to methods that are well known to a person skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, 2nd ed., McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, such as transfection by calcium phosphate, transfection by DEAE dextran, transfection, microinjection, transfection by cationic lipids, electroporation, transduction or infection.

The host cell can be, for example, bacterial cells such as E. coli or Streptomyces, cells of fungi such as Aspergillus and yeasts such as Saccharomyces, insect cells, Chinese Hamster Ovary cells (CHO), CI 27 mouse cell line, BHK cell line of Syrian hamster cells, Human Embryonic Kidney 293 (HEK 293) cells. In a particular embodiment, the host cell is a CHO cell or a HEK 293 cell.

The host cells can be used, for example, to express a polypeptide of the invention. After purification by standard methods, the polypeptide of the invention can be used in a method described hereinafter.

For instance, when expression systems that secrete the recombinant protein are employed, the culture medium may first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration matrix. Alternatively, an anion exchange and/or an affinity resin can be employed. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP- HPLC media can be employed to further purify the antibodies or fragments thereof. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide a substantially homogeneous recombinant protein. Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

In another embodiment, the invention provides a process for producing cells capable of expressing a polypeptide according to the invention, comprising genetically engineering cells with a vector or a nucleic acid according to the invention.

In another embodiment, the invention provides a process for producing antibodies or fragments thereof according to the invention comprises culturing a host cell transformed with an expression vector comprising a nucleic sequence that encodes said antibodies or fragments thereof under conditions sufficient to promote expression of said polypeptides. The antibody or fragment thereof according to the invention is then recovered from culture medium or cell extracts, depending upon the expression system employed. As known to the skilled artisan, procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium as described above.

Compositions

The invention provides pharmaceutical or therapeutic agents as compositions and methods for preventing and/or treating a patient, preferably a mammalian patient, and most preferably a human patient who is suffering from, or susceptible of suffering from, a β-amyloid aggregation related disorder, in particular a disorder selected from Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging. Alternatively, the invention provides methods for preventing a medical disorder, and in particular a β-amyloid aggregation related disorder, in particular a disorder selected from Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging.

In one embodiment, is provided a pharmaceutical composition comprising one or more of: (i) a neo-N-terminal and/or neo-C-terminal fragment of Αβ peptide as described herewith, (ii) an Αβ fragment-binding molecule, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, according to the invention, or (iii) a polypeptidic conjugated molecule comprising any one of (i) or (ii) according to the invention, (iv) a nucleic acid according to the invention, (v) a vector according to the invention, and/or (vi) a host cell according to the invention, and at least one pharmaceutically acceptable carrier.

In a particular embodiment is provided a pharmaceutical composition comprising one or more Αβ fragment-binding molecules specifically binding to a neo-C-terminal fragment of Αβ peptide as described herewith in combination with at least one further Αβ fragment-binding molecule specifically binding to a neo-N-terminal fragment obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide, but which does not bind to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43).

In a particular embodiment is provided a pharmaceutical composition comprising at least one further Αβ fragment-binding molecule specifically binding to a neo-N- terminal fragment that is Αβ23-42 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-42 are deleted, or Αβ23-40 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-40 are deleted, or Αβ23-43 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-43 are deleted.

In a particular embodiment is provided a pharmaceutical composition comprising at least one further Αβ fragment-binding molecule, wherein the said further Αβ fragment- binding molecule specifically binds to a neo-N-terminal fragment of Αβ peptide by interacting with an epitope comprising at least one combination of amino acids of the Αβ peptide selected from: ₂ 6SNK ₂ 8 and ₂₄ VGS ₂₆ . In a particular embodiment is provided an immunogenic composition comprising neo- C-terminal fragment of Αβ peptide having a sequence obtainable by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide.

In a particular embodiment is provided an immunogenic composition comprising a neo- N-terminal and/or neo-C-terminal fragment of Αβ peptide as described herewith, and at least one pharmaceutically acceptable carrier.

In a particular embodiment, said immunogenic composition comprises a neo-N-terminal and/or neo-C-terminal fragment of Αβ peptide selected from: Αβ1-22, Αβ1-23, Αβ1-24, Αβ1-25, Αβ23-42, Αβ24-42, Αβ25-42, Αβ26-42, Αβ23-40, Αβ24-40, Αβ25-40, Αβ26- 40, Αβ23-43, Αβ24-43, Αβ25-43, Αβ26-43, Αβ1-13, Αβ1-14, Αβ1-15, Αβ14-25, Αβ15- 25, Αβ 16-25, or a variant thereof.

In a further particular embodiment, said immunogenic composition comprises a neo-N- terminal and/or neo-C-terminal fragment of Αβ peptide selected from: Αβ1-23, Αβ1-24, Αβ24-42, Αβ25-42, Αβ24-40, Αβ25-40, Αβ24-43, Αβ25-43, or a variant thereof.

In a further particular embodiment, is provided an immunogenic composition comprising a neo-C-terminal fragment of Αβ peptide that is Αβ1-25 or a portion thereof where 1 to 3 amino acids at the C-terminal end of Αβ1-25 are deleted.

In a further particular embodiment, is provided an immunogenic composition comprising a neo-C-terminal fragment of Αβ peptide sequence selected from the group of: Αβ 1-24 and Αβ1-23.

In a further particular embodiment, said immunogenic composition comprises fragment of Αβ peptide of the invention as mentioned above, comprising only L-amino acids. In a further particular embodiment, is provided an immunogenic composition comprising neo-C-terminal fragment of Αβ peptide having a sequence obtainable by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide comprising at least one further peptide fragment, wherein said further peptide fragment is a neo N-terminal fragment of Αβ peptide obtainable by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide. In a further particular embodiment, said immunogenic composition comprising at least one further peptide fragment, wherein said further peptide fragment is a neo N-terminal fragment of Αβ peptide that is Αβ23-42 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-42 are deleted, or Αβ23-40 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-40 are deleted, or Αβ23-43 or a portion thereof where 1 to 3 amino acids at the N-terminal end of Αβ23-43 are deleted.

In a further particular embodiment said immunogenic composition comprises at least one neo-C-terminal fragment of Αβ peptide selected from: Αβ1-24 and Αβ1-23 or Αβΐ- 25, Αβ1-24, Αβ1-23, Αβ1-22, wherein C-terminal amino acid of said fragment is amidated.

In a further particular embodiment said immunogenic composition comprises at least one further peptide fragment, wherein said further peptide fragment is a neo -N-terminal fragment of Αβ selected from: Αβ25-42, Αβ24-42, Αβ25-40, Αβ24-40, Αβ25-43, Αβ24- 43.

In a still more particular embodiment, said immunogenic composition comprises a neo- N-terminal fragment of Αβ peptide that is Αβ1-24, more particularly Αβ1-24 where the amino acid residue at position 24 has a -CONH ₂ group.

In a further particular embodiment, said immunogenic composition comprises a neo-C- terminal fragment of Αβ peptide selected from: Αβ1-13, Αβ1-14, Αβ1-15, or a variant thereof.

In a further particular embodiment, said immunogenic composition comprises a neo-N- terminal of Αβ peptide selected from: Αβ14-25, Αβ15-25, Αβ16-25, or a variant thereof. In another embodiment, is provided a pharmaceutical composition comprising one or more of:

(i) an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer,

(ii) a nucleic acid encoding said Αβ fragment-binding molecule according to the invention,

(iii) a recombinant expression vector comprising a nucleic acid encoding said Αβ fragment-binding molecule according to the invention, and/or (iv) a host cell comprising a recombinant expression vector comprising a nucleic acid encoding said Αβ fragment-binding molecule according to the invention,

and at least one pharmaceutically acceptable carrier.

In a particular embodiment, said pharmaceutical composition comprises at least one Αβ fragment-binding molecule specifically binding to a neo-N-terminal or neo-C-terminal fragment of Αβ peptide selected from: Αβ1-22, Αβ1-23, Αβ1-24, Αβ1-25, Αβ23-42, Αβ24-42, Αβ25-42, Αβ26-42, Αβ23-40, Αβ24-40, Αβ25-40, Αβ26-40, Αβ23-43, Αβ24- 43, Αβ25-43, Αβ26-43, Αβ1-13, Αβ1-14, Αβ1-15, Αβ14-25, Αβ15-25, Αβ16-25, or a variant thereof.

In a further particular embodiment, said pharmaceutical composition comprises at least one Αβ fragment-binding molecule specifically binding to a neo-N-terminal or neo-C- terminal fragment of Αβ peptide selected from: Αβ1-23, Αβ1-24, Αβ24-42, Αβ25-42, Αβ24-40, Αβ25-40, Αβ24-43, Αβ25-43, or a variant thereof.

In a still more particular embodiment, said pharmaceutical composition comprises at least one Αβ fragment-binding molecule specifically binding to Αβ1-24, more particularly Αβ1-24 where the amino acid residue at position 24 has a -CONH ₂ group, but not binding to Αβ peptides of 40, 42 or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), and at least one pharmaceutically acceptable carrier

In a further particular embodiment, said pharmaceutical composition comprises at least one Αβ fragment-binding molecule specifically binding to a neo-C-terminal fragment of Αβ peptide selected from: Αβ1-13, Αβ1-14, Αβ1-15, or a variant thereof.

In a further particular embodiment, said pharmaceutical composition comprises at least one Αβ fragment-binding molecule specifically binding to a neo-N-terminal fragment of Αβ peptide selected from: Αβ14-25, Αβ15-25, Αβ16-25, or a variant thereof.

Pharmaceutical compositions of the invention can contain one or more Αβ fragment- binding molecule, in particular antibodies specific for the described Αβ fragments or antigen-binding fragment thereof in any form described herein.

Compositions of this invention may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like. The compounds of the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, freeze-dried forms, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.

Compositions of this invention may be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials as well as processing techniques and the like are set out in Part 5 of Remington 's The Science and Practice of Pharmacy, 22 ^nd Edition, 2012, Pharmaceutical Press and the University of the Sciences, Philadelphia College of Pharmacy, which is incorporated herein by reference.

Solid compositions of this invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art.

Compositions of this invention may also be formulated as transdermal formulations comprising aqueous or non-aqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.

Compositions of this invention may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.

Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).

The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials in Remington 's Pharmaceutical Sciences.

Injectable formulations are particularly appropriate for administering the compositions according to the invention.

In another embodiment, the invention provides an imaging composition or diagnostic composition comprising at least one Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, or a conjugated molecule comprising said Αβ fragment-binding molecule, and optionally a diluent and/or a carrier.

In a particular embodiment, said imaging composition or diagnostic composition comprises at least one Αβ fragment-binding molecule binding specifically to a neo-C- terminal Αβ peptide fragment as defined herewith.

In a particular embodiment, said imaging composition or diagnostic composition comprises at least one Αβ fragment-binding molecule binding specifically to a neo-C- terminal Αβ peptide fragment as defined herewith and at least one Αβ fragment-binding molecule binding specifically to a neo-N-terminal Αβ peptide fragment as defined herewith.

More particularly, said imaging composition or diagnosis composition comprises: (i) an Αβ fragment-binding molecule according to the invention, wherein said neo-C-terminal fragment is Αβ1-25 or a portion thereof where 1 to 3 amino acids at the C-terminal end οίΑβ1-25 are deleted.

More particularly, the imaging composition or diagnostic composition according to the invention comprises at least one Αβ fragment-binding molecule binding specifically to neo-C-terminal fragments Αβ1-22, Αβ1-23, Αβ1-24, and/or Αβ1-25 and at least one Αβ fragment-binding molecule binding specifically to neo-N-terminal fragments Αβ23-42, Αβ24-42, Αβ25-42, and/or Αβ26-42.

More particularly, the imaging composition or diagnostic composition according to the invention comprises at least one Αβ fragment-binding molecule binding specifically to neo-C-terminal fragments Αβ1-22, Αβ1-23, Αβ1-24, and/or Αβ1-25.

Still more particularly, the imaging composition or diagnostic composition according to the invention comprises at least one Αβ fragment-binding molecule binding specifically to Αβ1-24 and/or Αβ1-25 and at least one Αβ fragment-binding molecule binding specifically to Αβ24-42.

Still more particularly, the imaging composition or diagnostic composition according to the invention comprises at least one Αβ fragment-binding molecule binding specifically to ΑβΙ-24-CONI^ and/or Αβ1-25 and at least one Αβ fragment-binding molecule binding specifically to Αβ24-42, for instance N-5ns antibody and D-4s antibody as described herewith. Still more particularly, the imaging composition or diagnostic composition according to the invention comprises (i) at least one Αβ fragment-binding molecule binding specifically to one of said neo-C-terminal fragments by interacting with an epitope comprising at least one amino acid, or two to three consecutive amino acids, of the Αβ peptide, selected from: A21, E22, D23, V24, and G25, and (ii) at least one Αβ fragment-binding molecule binding specifically to one of said neo-N-terminal fragments by interacting with an epitope comprising at least one amino acid, or two to three consecutive amino acids, of the Αβ peptide, selected from: D23, V24, G25, S26, N27, and K28.

The imaging composition or diagnostic composition according to the invention is useful for detecting and/or monitoring Αβ peptide aggregation and/or amyloid plaque formation in vitro, in particular starting from early stages of the process.

Combination

According to the invention, an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, can be administered alone or in combination with a co-agent useful in the prevention and/or treatment of a β-amyloid aggregation related disorder, for example cholinesterase inhibitors such as Galantamine, Aricept™ (donepezil HC1), Excelon™ (rivastigmine tartrate), and NMDA (N-Methyl-D-aspartate) receptor antagonists such as Memantine.

The invention encompasses the administration of an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, wherein the Αβ fragment-binding molecule is administered to an individual prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful in the prevention and /or treatment of a β- amyloid aggregation related disorder, in a therapeutically effective amount. The Αβ fragment-binding molecules according to the invention that are administered simultaneously with said co-agents can be administered in the same or different compositions and in the same or different routes of administration.

In a particular embodiment, an Αβ fragment-binding molecule according to the invention can be administered in combination with Aricept™ (donepezil HC1) or Memantine for the treatment of subjects suffering from Alzheimer's disease. Mode of administration

Compositions of this invention may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal or intranasal administration or intra bladder, or combinations thereof.

Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intra-thecal, and intra-articular. The compositions of this invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion. In a particular embodiment, an Αβ fragment-binding molecule and/or the immunogenic composition according to the invention is administered systemically or locally.

In a particular embodiment, an Αβ fragment-binding molecule and/or the immunogenic composition according to the invention is administered by intranasal delivery, or by subcutaneous or intravenous route.

The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.

Patients

In an embodiment, patients according to the invention are subjects suffering from a β- amyloid aggregation related disorder, for instance Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging, or subjects who may be predisposed to said disorder but have not yet been diagnosed as having it.

In a particular embodiment, patients according to the invention are subjects suffering from Alzheimer's disease or who may be predisposed to said disease but who have not yet been diagnosed as having it and/or do not exhibit the symptoms of the disease yet.

Uses and methods according to the invention

The Αβ fragment-binding molecules according to the invention, in particular isolated antibodies or antigen-binding fragments thereof or aptamers, the nucleic acids, the recombinant expression vectors, the host cells, the compositions according to the invention useful in the prognosis, diagnosis, prevention and/or treatment of β-amyloid aggregation related disorders, as well as for monitoring Αβ aggregation and/or amyloid plaque formation, in particular starting from early stages of the process.

In one embodiment is provided an Αβ fragment-binding molecule binding specifically to a neo-C-terminal Αβ peptide fragment as defined herewith for use in the prevention and/or treatment of a β-amyloid aggregation related disorder, wherein the said molecule is used in combination with at least one further Αβ fragment-binding molecule specifically binding to a neo-N-terminal fragment obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide, but which does not bind to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43).

In one embodiment is provided an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, for use as a medicament.

Another embodiment provides an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, for use in the prevention and/or treatment of a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging, more particularly Alzheimer's disease.

In one embodiment is provided a use of an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, for the preparation of a pharmaceutical composition for preventing and/or treating a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging.

In a specific embodiment is provided a use of an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, for the preparation of a pharmaceutical composition for preventing and/or treating Alzheimer's disease.

In another embodiment is provided a method for preventing and/or treating a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging, comprising administering a therapeutically effective amount of an Αβ fragment- binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, to a subject in need thereof.

In an alternative embodiment are provided neo-N-terminal and/or neo-C-terminal Αβ fragments according to the invention, for use as a medicament, in particular as immunogenic composition.

According to an embodiment, the Αβ fragments according to the invention are of synthetic origin.

According to an embodiment, the Αβ fragments according to the invention are of natural origin.

Another embodiment provides neo-N-terminal and/or neo-C-terminal Αβ fragments according to the invention for use in the prevention and/or treatment of a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging.

In one embodiment is provided a use of neo-N-terminal and/or neo-C-terminal Αβ fragments according to the invention for the preparation of an immunogenic composition for preventing and/or treating a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging.

In a specific embodiment is provided a use of neo-N-terminal and/or neo-C-terminal Αβ fragments according to the invention for the preparation of an immunogenic composition for preventing and/or treating Alzheimer's disease. In another embodiment is provided a method for preventing and/or treating a β-amyloid aggregation related disorder, in particular Alzheimer's disease, cerebral amyloid angiopathy, Lewy body dementia (DLB), Frontotemporal dementia (FTD), inclusion body myositis, mild cognitive impairment, and β-amyloid deposition associated with aging, comprising administering a therapeutically effective amount of neo-N-terminal and/or neo-C-terminal Αβ fragments according to the invention to a subject in need thereof.

In an alternative embodiment is provided a method of detecting neo-N-terminal and/or neo-C-terminal fragments of Αβ as described herewith associated with Αβ oligomerization and/or amyloid plaque formation, in particular starting from early stages of the process, in a biological sample comprising contacting a biological sample from a subject with an Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer.

As used herewith "biological sample" refers to cells and/or tissue samples obtained from a subject, in particular from a subject suspected of, or suffering from, a β-amyloid aggregation related disorder or at high risk of developing such a disorder.

Examples of biological sample include blood, serum, plasma, cerebrospinal fluid, synovial fluid, urine, faeces, and tissue samples including cells isolated from said tissue. Tissue samples include formalin- fixed or frozen tissue sections.

In a particular embodiment, a biological sample is a blood sample or a cerebrospinal fluid sample.

Any suitable method for detection and analysis of Αβ peptide fragments can be employed, including immunodetection (Western blot, dot blot), confocal or electron microscopy imaging coupled with immunodetection, immunoprecipitation (I), IP coupled with mass spectrometry analysis, Enzyme Linked Immunoabsorbant Assays (ELISA).

In a particular embodiment, a method for detection and analysis of Αβ peptide fragments is Enzyme Linked Immunoabsorbant Assays (ELISA).

In a particular embodiment, the invention provides an ex vivo method for monitoring Αβ aggregation, in particular initiation, evolution and quantification of Αβ aggregates, in a biological sample, comprising the steps of:

(i) Providing a biological sample from a subject, (ii) Reacting said biological sample with at least one Αβ fragment-binding molecule according to the invention, under conditions sufficient for binding at least one neo-N-terminal and/or neo-C-terminal fragment of Αβ as described herewith present in said biological sample to said at least one Αβ fragment- binding molecule; and

(iii) Detecting a signal proportional to the level of complex formed in step (ii) between said Αβ fragment and said Αβ fragment-binding molecule, wherein the intensity of the signal correlates with the level of Αβ aggregation in the biological sample.

In another embodiment, it is provided an ex vivo method of prognosis or diagnosis of a β-amyloid aggregation related disorder from a biological sample of a subject comprising the steps of:

(a) Providing a biological sample from a subject;

(b) Bringing said biological sample into contact with at least one Αβ fragment- binding molecule according to the invention, wherein the contacting is under conditions sufficient for binding at least one neo-N-terminal and/or neo-C-terminal fragment of Αβ as described herewith, present in said biological fluid sample, to said at least one Αβ fragment-binding molecule;

(c) Washing;

(d) Detecting a signal proportional to the level of complex formed in step (b) between said at least one neo-N-terminal and/or neo-C-terminal fragment of Αβ and said at least one Αβ fragment-binding molecule,

(e) Comparing the level of signal detected in step (d) with the level of signal detected in the same conditions with a negative control,

wherein a level of signal detected in the subject's sample that is higher than the level of signal detected in a negative control is indicative of a β-amyloid aggregation related disorder.

Appropriate negative control includes cells from a healthy subject who is not suffering from, or susceptible to, a β-amyloid aggregation related disorder.

In another embodiment, it is provided an ex vivo method of prognosis or diagnosis of a β-amyloid aggregation related disorder from a biological sample wherein the said biological sample is contacted with at least one further Αβ fragment-binding molecule specifically binding to a neo-N-terminal fragment obtained by cleavage of said Αβ peptide between two consecutive amino acids located between positions 20 and 26 on the amino acid sequence of said Αβ peptide, but which does not bind to Αβ peptides of 40, 42 and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), wherein said contacting is under conditions sufficient for binding at least one of said neo-N-terminal fragment of Αβ present in said biological fluid sample to said at least one further Αβ fragment- binding molecule.

More particularly, it is provided an ex vivo method of prognosis or diagnosis of a β- amyloid aggregation related disorder from a biological sample of a subject comprising the steps of:

(a) Providing a biological sample from a subject;

(b) Bringing said biological sample into contact with a solid matrix where at least one antibody or fragment thereof according to the invention is bound to, wherein the contacting is under conditions sufficient for binding at least one neo-N-terminal and/or neo-C-terminal fragment of Αβ as described herewith, present in said biological fluid sample, to said at least one antibody or fragment thereof through antigen-antibody interactions;

(c) Removing any unbound Αβ fragments from the surface of said solid matrix;

(d) Detecting a signal proportional to the level of antigen-antibody complex bound to said solid matrix,

(e) Comparing the level of signal detected in step (d) with the level of signal detected in the same conditions with a negative control,

wherein a level of signal detected in the subject's sample that is higher than the level of signal detected in the negative control is indicative of a β-amyloid aggregation related disorder.

In another embodiment is provided a method of identifying a compound capable of inhibiting or delaying the process of Αβ peptide aggregation and/or amyloid plaque formation comprising the steps of:

a) Providing a sample comprising Αβ peptides of 40, 42, and/or 43 amino acids (Αβ1-40, Αβ1-42, Αβ1-43), and dividing said sample in a first group and a second group;

b) Exposing the sample from the first group to a test compound; c) Determining the level of neo-N-terminal and/or neo-C-terminal Αβ fragments as described herewith, in the samples from the first and second groups;

d) Comparing the level of said Αβ fragments between the first and second groups; wherein a test compound that reduces the level of said Αβ fragments in the sample from the first group compared to the second group is a compound able to inhibit or delay the process of Αβ peptide aggregation and/or amyloid plaque formation.

Appropriate samples for the method of identifying a compound according to the invention include biological samples (e.g. blood, serum, cerebrospinal fluid) from a subject (e.g. a non-human animal, or a human) not suffering from a β-amyloid aggregation related disorder as well as synthetic preparations comprising Αβ peptides of 40, 42, and/or 43 amino acids (Αβ1-40, Αβ 1-42, Αβ1-43).

Test compounds include small molecules, peptidomimetics, chimaeric proteins, natural or unnatural proteins, nucleic acid derived polymers (such as DNA and RNA aptamers, siRNAs, PNAs, or LNAs), fusion proteins, and antibodies.

In a particular embodiment of the method of identifying a compound according to the invention, the step of determining the level of neo-N-terminal and/or neo-C-terminal Αβ fragments (step c) is carried out using at least one Αβ fragment-binding molecule according to the invention or a mixture thereof.

Kit

One aspect of the invention relates to a kit comprising at least one Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen- binding fragment thereof or an aptamer, and/or at least one nucleic acid encoding said Αβ fragment-binding molecule, and/or at least one recombinant expression vector comprising said nucleic acid, and/or at least one host cell according to the invention, and optionally instructional material.

In a particular embodiment, the kit according to the invention comprises at least one Αβ fragment-binding molecule according to the invention, in particular an isolated antibody or antigen-binding fragment thereof or an aptamer, to be coupled or already coupled to a solid matrix.

Examples of solid matrix suitable for the invention include any solid phase support suitable for carrying out an immunoassay or a method according to the invention, such as beads, microparticles, nanoparticles, tubes, fabrics or plates, films, slides, wells, formed from or coated with glass, polystyrene, polypropylene, nitrocellulose, quartz, ceramic, dextran or other materials. For example, the solid matrix is in a form of microtiter wells, such as a 96-well microtiter plate.

The fixation of the Αβ fragment-binding molecule according to the invention, in particular the isolated antibody or antigen-binding fragment thereof, to the solid matrix in a kit according to the invention may be carried out by adsorption or chemical coupling to a solid phase support. Any means known in the art for immobilizing a protein or peptide to a solid support can be used. The isolated antibody or antigen- binding fragment thereof according to the invention can be either covalently or non- covalently bound to the solid matrix by techniques such as covalent bonding via an amide or ester linkage or adsorption. Peptides can be bound using binding pairs such as biotin and avidin or antibody and antigen. After the peptides are affixed to the solid matrix, the solid matrix can be incubated with a blocking solution (containing a blocking protein such as bovine serum albumin) to reduce non-specific adsorption of antibodies in a test sample to the support surface. According to one aspect, the antibodies or fragment thereof according to the invention can be synthesized directly on the solid matrix of the kit of the invention.

According to one embodiment, when the kit comprises at least one antibody or fragment thereof according to the invention or a combination thereof for coupling to a solid matrix as solid phase support, the kit further optionally comprises coupling reagents and/or a solid matrix for performing an immunoassay.

According to another further embodiment, the kit according to the invention further comprises at least one rinsing reagent for washing unbound material before detection in order to avoid background noise detection. Typically rinsing reagents comprise standard buffers known in the art.

References cited herein are hereby incorporated by reference in their entirety. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

The invention having been described, the following examples are presented by way of illustration, and not limitation.

EXAMPLES

Example 1. Identification of typical Αβ peptide fragment fingerprint found in early and advanced stages of Αβ aggregation (in vitro aggregation)

An aggregation associated stacking of Αβ peptides enables a close proximity induced side chain interaction, which may favour intra- or inter-molecular nucleophilic side chain attack. This putative nucleophilic rearrangement may result in spontaneous peptide bond hydrolysis, generating a typical peptide fragment fingerprint highly associated with peptide aggregation.

Methods:

Synthetic Αβ peptides 1-40 and 1-42 were solubilized in phosphate buffered saline (PBS) solution at a final concentration of 0.1 and 0.05 mg/ml, respectively. Samples were incubated at 37°C and left for spontaneous aggregation over a period of 0-20 hours (early aggregation), 1-5 days (intermediate aggregation), or 1-10 weeks (long-term aggregation). Samples were drawn for mass spectrometry (MS) and dotblot analysis at different time points to assess the degree of peptide aggregation.

Matrix-assisted Laser Desorption/Ionization Time-of- flight Mass Spectrometry (MALDI TOF/TOF): Aliquots (2 μΐ) of samples were used for MALDI TOF/TOF MS (ABI 4800 model, Applied Biosystems) measurements. Matrix solution of a-cyano-4- hydroxycinnamic acid (7 mg/ml in ACN/0.1% TFA (1 : 1, v/v)) was used for sample deposition. The sample (1 μΐ) was mixed with 1 μΐ of matrix solution and then 1 μΐ of this mixture was deposited in duplicates on the target plate and allowed to air dry. Samples were analyzed in reflectron positive mode.

Absolute quantitation of beta-amyloid using selected reaction monitoring (SRM): All samples were analysed on a TSQ-Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific). A 0.7-FWHM-resolution window for both Ql and Q3 was set for parent- and product-ion isolation. Fragmentation of parent ions was performed in Q2 at 1.5 mTorr, using collision energies calculated with the Pinpoint software (vl . l). Parent-ion selection was set for fully Lys-N digested peptides on the positive charged parent ions. All CID energies and transition selection were tested manually by infusion on the TSQ using synthetic peptide standards.

A mixture of accurately quantified heavy isotope labelled peptide standards (JPT Peptide Technologies, Germany) comprising of residues: 16-23, 16-23-NH2, 16-25, 16- 25-NH2, 16-27, 28-38, 28-40, 28-42 and 28-43 were spiked into each tube immediately after resuspension of samples in the Lys-N digestion buffer (50 mM ammonium bicarbonate, pH 10). Following digestion, endogenous peptides (brain samples) were extracted either from gel fractions, or in solution digestions, together with the spiked surrogate heavy peptide standards. Samples were then dried using a speed vacuum and stored at -20°C before analysis. Samples were resuspended in a solution containing 20% DMSO and 10% formic acid (FA) and further diluted to 5% DMSO and 2.5% FA prior to analysis by LC-SRM.

Using mass spectrometry analysis of synthetic Αβ1-40 and Αβ1-42 peptides, it was demonstrated that the increase in abundance of fragments of residues 1-22, 1-23, 1-24 and 1-25 represents an important fraction of a typical peptide fragment fingerprint found during the early events of peptide aggregation. The aggregation induced generation of neo C-terminal fragments is consistent with the finding that peptide aggregation in heavy labelled water (H ₂ ¹⁸ 0) results in the incorporation of a heavy oxygen atom ( ¹⁸ 0) at the neo C-terminal of the above mentioned fragments (data not shown). Residual C-terminal amidation was observed in some peptide fragments, of which the relative abundance accounted for approximately 10-20% of the normal carboxy C-terminal population (data not shown). Interestingly, fragment 1-24 was found to be predominantly amidated when compared to other fragments within the close peptide region.

Since Αβ peptide aggregation is a continuous process, changes in peptide fragment fingerprints were also monitored following long-term peptide aggregation according to the protocol described above.

Mass spectrometry analysis indicated that long-term aggregation results in a clear shift in peptide fragment fingerprints, giving rise to newly formed and predominant peptide fragments comprising residues Αβ1-13, Αβ1-14 and Αβ1-15. Based on the concomitant disappearance of the longer fragments of Αβ1-23, Αβ1-24 and Αβ1-25, it was concluded that these shorter peptide fragments represent additional analytical targets for monitoring Αβ peptide aggregation (Table 1, no. 8-10). However, the appearance of these N-terminal fragments may represent a more advanced stage of Αβ peptide aggregation. This conclusion is corroborated by the observation that, long-term incubation studies of a synthetic Αβ peptide fragment 1-25 results in a gradual decrease in signal intensity of the parent peptide Αβ1-25, accompanied by a concomitant increase of the shorter fragments such as fragment 1-13 (data not shown).

The neo-C-terminal and neo -N-terminal fragments resulting from the aggregation associated Αβ peptide fragmentation, as well as the full length Αβ peptide 1-42, are indicated in Table 1.

Table 1

SEQ peptide sequence name

ID

NO:

Full length Αβ peptide 1-42

1 Αβ peptide DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA Αβ1-42 1-42

Neo-C-terminal fragments

7 Αβ peptide DAEFRHDSGYEVHHQKLVFFAEDVG Αβ1-25 1-25

6 Αβ peptide DAEFRHDSGYEVHHQKLVFFAEDV Αβ1-24 1-24

5 Αβ peptide DAEFRHDSGYEVHHQKLVFFAED Αβ1-23 1-23

4 Αβ peptide DAEFRHDSGYEVHHQKLVFFAE Αβ1-22 1-22

10 Αβ peptide DAEFRHDSGYEVHHQ Αβ1-15 1-15

9 Αβ peptide DAEFRHDSGYEVHH Αβ1-14 1-14

8 Αβ peptide DAEFRHDSGYEVH Αβ1-13 1-13

Neo-N-terminal fragments

14 Αβ peptide SNKGAIIGLMVGGWIA Αβ26-42 26-42

13 Αβ peptide GSNKGAIIGLMVGGWIA Αβ25-42 25-42

12 Αβ peptide VGSNKGAIIGLMVGGWIA Αβ24-42 24-42

11 Αβ peptide DVGSNKGAIIGLMVGGWIA Αβ23-42 23-42

25 Αβ peptide KLVFFAEDVG Αβ 16-25 16-25

24 Αβ peptide QKLVFFAEDVG Αβ15-25 15-25

23 Αβ peptide HQKLVFFAEDVG Αβ 14-25 14-25 Example 2. Identification of typical Αβ peptide fragments fingerprint found in a mouse model of Alzheimer disease

IP-MS analysis of transgenic mouse (Tg2576) brain tissue (formic acid extract) was carried out using the N-5ns antibody (see examples 4 and 5 below for description). Mass spectrometry (MALDI-TOF/TOF) analysis revealed a clear identification of the typical neo-C-terminal fragments of residues 1-22, 1-23, 1-24 and 1-25. Fragment 1-24 appeared to be predominantly amidated at its C-terminal, which is an observation also made in vitro with the synthetic form of Αβ1-42 peptide, whereas fragments 1-22, 1-23 and 1-25 showed residual amounts of C-terminal amidation as could be seen by the isotopic distribution and monoisotopic masses measured for these peptides (not shown). Mouse (Tg2576) brain tissue was extracted with formic acid as described above. The total protein extract was purified by gel filtration and the eluted fractions were probed for Αβ peptide using dotblotting. Fraction volumes of 11.5-12.5ml were pooled and immunoprecipitated separately using either 6E10 or 4G8. MS analysis of both immunoprecipitated samples were identical and revealed the presence of the neo-C- terminal fragment Αβ1-25 as well as full-length Αβ1-40, 1-42 and 1-43 within this fraction (not shown).

These findings strongly indicate that the presence of a typical Αβ peptide fragment fingerprint of neo-C-terminal and neo-N-terminal fragments is highly associated with in vivo Αβ peptide aggregation in transgenic mice.

Example 3. Production of antibodies according to the invention

Antibodies were raised in rabbits against a peptide identical to residues 20-25 (N-5ns & N-5s), 18-23 (N-3s), 19-24 (N-4), 24-29 (D-4s) and 26-31 (D-6ns) of human Αβ peptide. All rabbit polyclonal antibodies were affinity-purified against Αβ peptide with either a -COOH or -CONH ₂ C-terminal end.

Antibodies specific for a neo-C-terminal fragment of Αβ peptide as described above have a name beginning with a "N", while antibodies specific for a neo-N-terminal fragment of Αβ peptide as described above have a name beginning with a "D".

Example 4. Characterization of some antibodies according to the invention

Epitope binding specificity of various polyclonal antibodies according to the invention, prepared as described in Example 3, was tested using a repertoire of synthetic peptide standards of different C- or N-termini. Antibody binding specificity was compared with the two commercially available anti-Αβ antibodies 6E10 and 4G8, which are known to have an Αβ N-terminal epitope binding affinity for residues 1-16 (6E10) and 17-24 (4G8), respectively.

As shown in Figure 1A, binding specificity of the neo-C-terminal epitope antibodies N- 5 s (blot A) and N-3s (blot B) according to the invention is significantly different from the binding specificity of prior art antibody 6E10 (blot C), since N-5s and N-3s do not detect full-length Αβ 1-40 peptide (lane 9). N-5s antibody showed high binding specificity for the neo-C-terminal residue Gly25 (blot A, lane 6). The N-3s antibody appears to have a high binding affinity for the neo-C-terminal residue Asp23 (blot B, lane 3) and therefore clearly distinguishes itself from N-5s regarding C-terminal specificity. Binding specificity of N-3s antibody is significantly different from the binding specificity of prior art antibody 6E10 (blot B'). The selectivity of both antibodies N-5s and N-3s were combined in a third antibody referred to as N-5ns, which revealed an epitope binding specificity comprising the C-terminal residues Gly25 and Asp23. N-5s, N-3s, and N5ns antibodies exhibit a low binding affinity for peptide fragment 1-24 with an amidated C-terminal end (blot A', lanes 3-6). As with antibodies N-5s and N-3s, no binding to full-length Αβ1-40 peptide was observed with N-5ns (blot A', lane 9). Antibodies N-5s and N-5ns do not cross-react with other C-terminal glycine residues, such as the C-terminal of Αβ 1-38 (G38) (data not shown).

High specificity was observed for the amidated C-teminal ending of peptide fragment 1-24 using antibody N-4 (Figure IB). No cross-reaction was observed with full-length peptide fragment 1-40 or any other shorter peptide fragments (blot B, lines 1-9).

Binding specificity of the antibodies D-4s and D-6ns according to the invention for the complementary neo-C-terminal fragments were also tested (Figure 1C). Antibody D-4s showed a high affinity for the N-terminal fragment comprising residue V24 (blot A, lane 4) and a low affinity for the shorter fragment Αβ26-40 (blot A, lane 5) but did not bind to fragment Αβ28-40 (blot A, lane 6) or full-length Αβ1-40 (blot A, lane 7). Antibody D-6ns binds the peptide fragment comprising a neo -N-terminal ending at residue S26 (blot B, lane 5) but does not cross-react with the neo -N-terminal ending at residue 24 or 28, or the full-length Αβ1-40 peptide (blot B, lanes 4, 6, 7). The high affinity for full-length Αβ1-40 of prior art antibodies 6E10 and 4G8 was confirmed and shown in Figure ID, lane 9. However the specificity of these prior art antibodies for Αβ fragments had not been studied before. Prior art antibody 4G8 has a higher binding affinity for peptide fragments Αβ1-23 and Αβ1-25, when compared to 6E10 (Figure IB, blot B', B" lanes 3 and 6 and Figure ID, blot A, A', lanes 3 and 6), whereas binding to fragment Αβ1-24 with amidated C-terminal end was clearly increased with 6E10 (Figure ID, blot A, A', lane 4). Although both 6E10 and 4G8 bind fragment Αβ1-25, they do not recognize the amidated form of this fragment (Figure ID, blot A, A', lane 6).

The diagram represented in Figure IE summarizes the neo-C-terminal binding specificity found for antibodies N-5ns and N-3s as well as for the complementary neo- N-terminal fragments targeted by D-6ns and D-4s. Further comparison with putative epitopes of antibodies 6E10 and 4G8 is provided.

Tissue transglutaminase (Tgase) mediated protein cross-linking has been reported to account for the formation of insoluble lesions found in conformational diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Several reports have suggested that Tgase may play a key role in the pathogenesis of AD. Tgase is known to induce rapid beta-amyloid peptide aggregation by forming SDS-stable dimers and neurotoxic oligomers. Αβ1-40 peptide aggregation was monitored in the presence and absence of Tgase using the N-5ns antibody. As shown in Figure IF, the dotblot analysis revealed a positive signal for peptide aggregation as early as 1 hour in presence of Tgase, whereas in the absence of Tgase this level of significance was only attained following lday of peptide aggregation. This data clearly indicates that the aggregation associated appearance of neo C-terminal fragments, such as peptide fragment 1-25, can be monitored using the N-5ns antibody. More importantly, the use of N-5ns allows for monitoring changes in peptide aggregation speed and therefore serves as an indispensable analytical tool for the development of novel chemical compounds aimed at inhibiting Αβ peptide aggregation. Table 2 provides an overview of targeted neo-C- terminal and neo-N-terminal peptide fragments and the names of the tested antibodies according to the invention which bind said fragments, as well as the sequence of the epitope recognized by said antibodies. Table 2

Specificity of the neo -N-terminal antibody binding to epitopes comprising only amino acid L-stereoisomers using dotblotting was shown on Figure 1G. Epitope binding specificity was tested using a repertoire of synthetic peptide standards of different N- termini (indicated in each lane). The antibody D-6ns was specific for the neo-N- terminal epitope comprising L-Serine residue at the position 26 (L-S26-40; lane 5) but not to epitope comprising Serine stereoisomer D-Serine at the same position (D-S26-40; lane 6). No D-6ns antibody binding was observed for shorter Αβ peptide fragments (lanes 1-3) or peptide fragments comprising S26 as a consecutive sequence (lane 4).

Example 5. Screening of beta amyloid oligomers using N-5ns antibody

Amyloid (1-42) derived diffusible ligands (ADDL's) were prepared as follows: a dried Αβ1-42 peptide film was solubilized in DMSO and further diluted in phenol red free culture media to a final concentration of 0.5 mg/ml. Samples were left for spontaneous aggregation during 0-20 hours at 4°C. Analysis of peptide fragment fingerprints was carried out at different time points in order to monitor the gradual appearance of peptide fragments as a result of peptide oligomerization. The degree of peptide aggregation was monitored by either dotblotting or MS. Five microliter aliquots were taken at each time point and used for either MS and/or dotblot analysis. Typically, one microliter of sample was diluted 1 : 10 with double distilled water (final 0.05 mg/ml) and spotted on a nitrocellulose membrane for dotblotting or mixed with alpha-cyano matrix for MS analysis.

Peptide aggregation was monitored at different time points by dotblotting using theN- 5ns and 6E10 antibodies (Figure 2A). The gradual increase in neo-epitope signal with the N-5ns antibody revealed a time dependent peptide aggregation process taking place. A significant strong antibody signal was observed after 20 hours of peptide incubation and this was highly associated with the presence of typical oligomeric peptide morphology as revealed by electron microscopy (EM) (Figures 2B). Following peptide aggregation the sample was centrifuged in order to precipitate large fibrillar species (pellet) and the supernatant containing soluble oligomers (ADDL) was purified and fractionated by size exclusion chromatography (SEC) using a Supderdex 200 column. In order to identify the SEC fractions containing the neo-epitope of interest, SEC fractions (50 μΐ) were dried by speed vacuum and re-suspended in 5 μΐ of distilled water of which 1 μΐ was spotted onto a nitrocellulose membrane for dotbotting. Membranes were incubated with either the N-5ns or 6E10 antibody. Using the N-5ns antibody, a large population of neo-epitope fragments were identified within the oligomeric fractions (volume 8 ml - 10 ml). This fraction contained mainly large aggregates of protofibrillar and oligomeric morphology as revealed by electron microscopy (Figures 2C, D)

This data strongly indicates that aggregation associated generation of neo-epitopes form part of stable oligomers and that these oligomers can be specifically targeted using the N-5ns antibody.

This crucial finding allows for specific prognosis/diagnostic analysis and targeted depletion of toxic oligomers in vitro and in vivo, using the specific neo-C-terminal and neo-N-terminal epitope antibodies described herein.

Example 6. Selected Reaction Monitoring (SRM) mass spectrometry of gel extracted low molecular weight (LMW) oligomers

Applying a similar protocol as described in Example 1, MALDI-TOF/TOF analysis of amyloid derived diffusible ligands (ADDL's) showed the presence of the typical peptide fragment fingerprint generated during ADDL formation Figure 3A. The zoom shows the monoisotopic distribution of the peptide fragments typically found during aggregation. The arrows indicate the presence of the monoisotopic peak corresponding to an amidated C-terminal peptide. Fragment Αβ1-25 and Αβ1-23 appeared to have a relative abundance of 20%-30% of C-terminal amidation, whereas fragment Αβ1-24 was predominantly observed with a fully amidated at the C-terminal. Inset: SDS-PAGE (coomassie stained) showing the gel migration pattern typically observed with these ADDL's.

SDS-PAGE gel bands were cut at the migration level of the Αβ tetramer, trimer, dimer and monomer level. Protein bands were subjected to Lys-N in gel digestion and the extracted proteolytic fragments were analyzed by SRM mass spectrometry (Figure 3B, upper panel). The total ion extraction chromatogram of the SRM analysis shows an example of the analysis of the trimer band (gel migration height of 14kDa) (Figure 3B, gel inset: arrow), where the fragment of residues 16-27 represents the abundance of full- length peptide present in the trimer band since Lys-N proteolysis cleaves full-length Αβ1-42 peptide at positions 15 and 28. Fragments 16-23, 16-24-FIN ₂ (amidated C-term) and 16-25 or 16-25-NH ₂ (amidated C-term) represent the typical peptide fragment fingerprint found within a population of soluble oligomers and are therefore representative of the neo-C-terminal fragments associated with Αβ aggregation. A clear chromatographic separation is achieved between normal, carboxy (COOH) and amidated (CONH ₂ ) C-terminal fragments. The window below shows the identification of the SRM peptide fragment transitions monitored for fragment 16-25 (Figure 3B, lower panel).

In summary, this data demonstrates that LMW oligomers, such as Αβ trimers and/or tetramers, as well as larger Αβ oligomeric assemblies, incorporate a substantial amount of neo-C-terminal and possibly neo-N-terminal fragments within their structure. This implies that soluble Αβ oligomers can be specifically targeted using the herein described neo-epitope antibodies such as N-5ns, N-3s, N4 or D-4s and D-6ns respectively.

Example 7. Analysis of Abeta oligomers using the D-4s antibody

Αβ peptide aggregation was monitored using D-4s antibody according to the invention following the protocol described in Example 5.

Analysis of the ADDL's aggregation kinetics shows a similar time dependent increase in D-4s antibody signal as found with N-5ns (Figure 4A). Dotblot (D-4s) analysis of the purified ADDL sample shows a significant signal only for the oligomeric fraction (Figure 4B). These oligomers were also detected using the neo C-terminal specific N-3s antibody, which binds to the complementary peptide fragment 1-23. No signal was observed for the monomeric fraction using either antibody.

Example 8. The use of a combination of two complementary antibodies increases detection sensibility

Since antibodies N-5ns, N-3s and D-4s showed selective binding for fragments found within the oligomer (ADDL) fraction, it was investigated whether the detection sensibility was altered by using a combination of two complementary antibodies. An antibody mixture consisting of N-5ns and D-4s was used to monitor the aggregation kinetics of Αβ1-42 and compared with the signal readout with only one antibody (N- 5ns) alone.

The results clearly demonstrate an amplification of antibody signal using a combination of two antibodies as compared to a single antibody (Figure 4C).

Example 9. Use of N-5ns antibody for the detection of Αβ1-25 fragment and diagnosis of Alzheimer disease in humans

Human brain tissue samples were serially extracted using TBS, SDS and formic acid as described earlier. Briefly, tissue samples were homogenized (20 strokes on ice) in TBS with protease inhibitors (Roche, Switzerland) using a Teflon homogenizer. Samples were then subjected to centrifugation (150,000g) during 45min and supernatant was recovered as the TBS soluble fraction. Protein pellets were subjected to serial extractions using either Triton-X 100 (1%) or SDS (2%) followed by centrifugation. Finally, pellets were extracted with 70% formic acid (FA) overnight at 4°C followed by centrifugation. SDS fractions were diluted to 0.1% SDS final concentration and FA fractions were neutralized to pH 7.5 with 5M NaOH solution prior to IP. All samples were initially depleted of endogenous IgG's using a mixture of protein A&G agarose (Roche, Switzerland). Typically, 3-5ug/ml rabbit polyclonal antibody (N-3s, N-5s or N- 5ns) or 3-5ug/ml of 6E10 or 4G8 mouse monoclonal was used for overnight IP under continues rotation (4rpm/min) at 4°C. Following IP, samples were either directly analysed by WB, by MALDI-TOF/TOF or further digested overnight using Lys-N proteolysis prior to SRM analysis as outlined above. Human CSF samples (500 μΐ) were immunoprecipitated with either N-5ns, or a mixture of the two commercial antibodies 6E10 and 4G8. Human CSF samples were pre-cleared of endogenous IgG antibodies during 4hr at 4°C. Antibody N-5ns was bound to protein A agarose beads during 2hr at 4°C without crosslinking. CSF samples were IP'ed over night at 4°C under constant rotation. Following IP, samples were washed twice with IP buffer and eluted with 40% ACN /H ₂ 0 & 0.1% TFA and dried in a speed vacuum. Samples were re-suspended in 50 μΐ digestion buffer (50 mM ammonium bicarbonate pH 9-10) and digested overnight at 37°C with Lys-N (70 ng). Following digestion, samples were dried and re-suspended in 20-30 μΐ of 10% DMSO & 5% FA. prior to LC-MS analysis. Where indicated, samples were spiked with a heavy labelled (lysine, K) surrogate peptide corresponding to the Lys-N cleavage product of residues 16-27, 16-25, 16-24 and 16-23.

Immunoprecipitation (IP) using N-5ns antibody, of human control and Alzheimer's disease (AD) post-mortem brain samples resulted in a pull-down of low molecular weight oligomers in AD samples (Figure 5 A, lanes 6-14). No oligomers or monomers were detected in human control brain samples (Figure 5 A, lanes 2-4). These findings highlight the specificity of the N-5ns antibody for the detection of diagnostic peptide fragments with neo C-termini such as peptide fragment 1-25 found in a complex sample matrix. Moreover, IP with N-5ns allows for an identification of oligomeric Αβ species, since neo C-terminal fragments form a stable entity with HMW oligomers. SRM mass spectrometry was used for the targeted analysis of diagnostic fragments (post Lys-N digestion) and the resulting data is shown in Figure 5B. The total ion chromatogram shows the liquid chromatography (LC) elution profile and MS identification of fragments 16-27 (representative of full-length Αβ digest), 16-25 (representative of fragment 1-25 digest) and the complementary C-terminal fragments of 28-40 and 28-42. The SRM findings corroborate the observation made by WB shown in Figure 5A, that IP of brain samples results in a pull-down of the target peptide fragment together with full-length Αβ 1-40/42. The spiking with heavy labelled peptides standards allows for a comprehensive identification and quantitation of endogenous peptide fragments, since these surrogate peptides have identical physico-chemical properties as the light, endogenous peptides (Figure 5C). Determination of absolute values for fragment 1-25 was achieved using SRM mass spectrometry and the quantitative results are shown in Figure 5D. These findings indicate that IP of human brain tissue using N-5ns targets a small population (>5 pg) of the diagnostic peptide fragment 1-25 and that these fragments form part of larger aggregates as seen by the presence of full-length Αβ peptide (16-27) (<150 pg) (Figure 5D).

These findings further show the specificity of the N-5ns antibody for the detection of a small population of neo-C-terminus peptide fragments 1-25 found in larger peptide aggregates found in Alzheimer's disease post-mortem brain samples.

Example 10. Immunoprecipitation (IP) of human cerebrospinal fluid (CSF) derived from a clinically diagnosed AD patient

IP-MS analysis of a human AD and non-demented (ND) control CSF sample using a combination of two commercially available antibodies (6E10 & 4G8). MS analysis (MALDI-TOF/TOF) showed no significant difference in detection of Αβ peptide fragments between these two samples (Figure 6A).

In order to reveal the presence of any diagnostic peptide fragments, CSF samples were immunoprecipitated with N-5ns antibody and subjected to by Lys-N digestion (N- terminal cleavage at residue K16) prior to targeted LC-MS analysis. Samples were spiked with a known concentration of the heavy (lysine, K) labelled surrogate peptide which represents the lys-N cleavage product of residues 16-25. Figure 6B shows the LC-MS analysis of a human CSF sample derived from a clinically diagnosed AD patient subjected to IP with N-5ns and LC-MS analysis, revealing the presence of the diagnostic peptide fragment 16-25 ([M+2H] ²⁺ m/z= 562.8022) (post Lys-N digest) as well as the heavy labelled surrogate (spiked reference) peptide ([M+2H] ²⁺ m/z= 566.8097). Collision induced dissociation (CID) allowed for comprehensive identification of the (endogenous) diagnostic peptide fragment of residues 16-25 and the fragment ions are shown in Figure 6C.

These findings show the specificity and efficiency of the N-5ns antibody for the detection of peptide fragments of the invention within CSF derived from a clinically diagnosed AD patient.

Example 11. Measurement of oligomeric Αβ in human CSF using sandwich ELISA approach Antibodies of the invention, in particular, N-5ns antibody can be used for the detection of peptide fragments of the invention within CSF derived from a patient, as illustrated below.

CSF is known to closely reflect the AD pathology associated changes in Αβ peptide (see Example 10). Changes in CSF levels of Αβ x-42, total and phosphorylated tau, as well as the combination of total tau and Αβ x-42 levels allows to differentiate AD from normal aging and from other forms of dementia such as frontotemporal dementia (FTD). Today, CSF biomarkers for AD have reached a scientific consensus regarding the use of these specific biomarkers for differentiation of early and incipient AD from normal aging subjects. Αβ oligomers are collectively defined as a group of neuro-toxic Αβ aggregates of pre-fibrillar morphology and reflect the initial, early events of Αβ misfolding associated with AD pathology. Therefore, the presence of toxic Αβ oligomeric species found in CSF or blood of AD patients may highlight the onset of AD pathology. Targeted and quantitative MS analysis of Αβ oligomers indicates that aggregation associated auto-cleavage at positions D23, V24 or G25 results in the generation of N-terminal fragments with high aggregation properties, which in turn gives rise to the formation of large Αβ oligomers, consisting of full-length Αβ1-42 as well as novo C-terminal and N-terminal fragments (Figure 2 and 4). Antibodies of the invention (N-3s, N-4, N-5s, N-5ns, optionally in combination with D-4s and D-6ns) therefore serve as an indispensable analytical and diagnostic tool for the use of capture antibodies in diagnostic ELISAs, because these antibodies target specific epitopes exposed on the surfaces both LMW and HMW Αβ oligomers.

Enzyme-linked immunosorbend assays (ELISA) are typically used for the detection of oligomeric Αβ in CSF using a "sandwich" ELISA approach. The sandwich ELISA approach involves the use of a capture anti-Αβ antibody allowing for immobilization of Αβ oligomers on the plate surface, whereas the application of a second, typically biotinylated anti-Αβ antibody (of identical or similar binding epitope) allows for the detection of any free binding surfaces present in Αβ oligomers. Hence, the larger the surface of the oligomer the stronger the signal readout of the second antibody.

Two independent standard curves are generated using synthetic, aggregated (t=3d) Αβ peptide fragment 1-25 alone, or as a peptide mixture of aggregated (t=3d) fragments: 1- 23, 1-24 and 1-25 at a total peptide concentration range of 5 to 50 pg/ml in PBS. The antibody of invention N-5ns is used as a capture antibody whereas the commercially available mouse monoclonal, biotinylated form of 6E10 is used as a detection antibody. Capture antibody (N-5ns, 50 μΐ, 20 μ§/ηι1) is bound to a PVC, 96 well plate following over-night incubation at 4°C to allow complete binding. Following binding, the wells are washed twice with PBS. Blocking of unbound well surfaces is done by using 3% BSA/PBS with 0.02% sodium azide and left for 2h at room temperature followed by two washing steps. CSF samples from clinically diagnosed AD patients and non- demented controls (ND) are thawed on ice. Samples and controls are added in duplicates at 75 μΐ per assay well. The plate is incubated overnight on a shaker at 4°C. The following day, the plate is washed three times with PBS, 0.05%> Tween. The detection antibody (6E10) is added to the wells and left for incubation during 2h room temperature and washed several times with PBS prior to plate reading.

This protocol presents the method of use of the N-5ns antibody for the detection of peptide fragments within CSF derived from a patient.

LIST OF SEQUENCES

SEQ ID NO: 1 : human Αβ1-42

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA SEQ ID NO: 2: human Αβ1-40

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV SEQ ID NO: 3: human Αβ1-43

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

SEQ ID NO: 4: Αβ1-22

D AEFRHD S G YE VHHQKL VFF AE

SEQ ID NO: 5: Αβ1-23

D AEFRHD S G YE VHHQKL VFF AED

SEQ ID NO: 6: Αβ1-24

D AEFRHD S G YE VHHQKL VFF AED V

SEQ ID NO: 7: Αβ1-25

D AEFRHD S G YE VHHQKL VFF AED VG

SEQ ID NO: 8: Αβ1-13

DAEFRHDSGYEVH

SEQ ID NO: 9: Αβ1-14

DAEFRHDSGYEVHH

SEQ ID NO: 10: Αβ1-15

DAEFRHDSGYEVHHQ SEQ ID NO: 11 : Αβ23-42

DVGSNKGAIIGLMVGGVV

SEQ ID NO: 12: Αβ24-42

VGSNKGAIIGLMVGGVVIA

SEQ ID NO: 13: Αβ25-42

GSNKGAIIGLMVGGVVIA

SEQ ID NO: 14: Αβ26-42

SNKGAIIGLMVGGVVIA

SEQ ID NO: 15: Αβ23-40

DVGSNKGAIIGLMVGGVV

SEQ ID NO: 16: Αβ 24-40

VGSNKGAIIGLMVGGVV

SEQ ID NO: 17: Αβ25-40

GSNKGAIIGLMVGGVV

SEQ ID NO: 18: Αβ26-40

SNKGAIIGLMVGGVV

SEQ ID NO: 19: Αβ23-43

DVGSNKGAIIGLMVGGVVIAT

SEQ ID NO: 20: Αβ24-43

VGSNKGAIIGLMVGGVVIAT

SEQ ID NO: 21 : Αβ25-43

GSNKGAIIGLMVGGVVIAT

SEQ ID NO: 22: Αβ26-43

SNKGAIIGLMVGGVVIAT

SEQ ID NO : 23: Αβ 14-25

HQKLVFFAEDVG

SEQ ID NO: 24: Αβ15-25

QKLVFFAEDVG

SEQ ID NO : 25: Αβ 16-25

KLVFFAEDVG

SEQ ID NO: 26: Mouse Αβ1-43:

DAEFGHDSGFEVRHQKLVFFAED VGSNKGAIIGLMVGGVVIAT