The present invention relates to humanized antibodies specific for the protofibrillar form of the β-amyloid peptide. The present invention also relates to the therapeutic, diagnostic and / or preventive of such antibodies, in particular associated with initiation and progression of neurodegenerative disorders and / or diseases related to the deposition of amyloid plaques, and in particular of the disease Alzheimer.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects a large proportion of the elderly population. This disease is characterized clinically by loss of memory and cognitive decline, on neuropathologically by the presence in the brain of intracellular neurofibrillary deposits and extracellular deposits of β-amyloid peptide (A-β) forming amyloid plaques. (Yanker et al. Nature Med. Vol 2 No 8 (1996)). To these signs add a significant number of other abnormal changes including an impairment of immune and inflammatory systems and altered mitochondrial function may lead to increased oxidative stress, activation of apoptosis mechanisms and ultimate way cell death.

The amyloid plaques are mainly composed of the peptides A-β for 40 or 42 residues which are generated during the proteolytic processing of the precursor protein of the β-amyloid protein (APP). The extracellular deposits of β-peptides A represent the early and invariable characteristic of all forms of I 1 AD including familial forms (FAD). ADF appear relatively early (between 40 and 60 years) and are due to mutations in the APP gene in 5% of cases of FAD (> 20 families) with six single or double missense mutations; in the gene for presenilin 1 (PS 1) 50 to 70% in cases of FAD (> 200 families) with more than 80 different mutations identified to date; and the gene for presenilin 2 (PS2) in fewer cases of FAD with two missense mutations described in 8 families. Mutations in these three genes have been demonstrated to induce changes in the proteolysis of APP that lead to an A-β overproduction and early appearance of the pathology and symptoms that are similar to those of the sporadic forms of I 1 AD.

Neuron toxicity of amyloid plaques could reside in the high molecular weight fibrils which are formed by aggregation of peptides A-β in soluble forms fibrillar first solubles (also known as protofibrillar form) which are then converted into insoluble forms incorporated into the plates amyloid. Indeed in vitro it was shown that the A-β soluble peptide gradually agrégeait in fibrillar form (ie which may be labeled by agents such as Congo red or thioflavin S which recognize the tertiary structures of the beta-sheet peptide / protein), high molecular weight (> 200 kDa) but also soluble. Because this form is soluble, it is often called protofibrillar form while fibrils result from an even greater aggregation leading to the loss of solubility. transitional forms protofibrillar are generally regarded as the precursors of amyloid fibers and could be responsible for cell dysfunction and neuronal loss in Alzheimer's disease and other diseases related to protein aggregation.

It has been shown that amyloid senile plaques (that is to say, aggregated, they are also called mature plates) are correlated with the cognitive status of Alzheimer's patients in contrast to diffuse deposits of A-β peptide, which are also widely present in non-patients. (Duyckaerts and Al Neurobiol.Aging 1997; 18: 33-42 and Al and Jellinger and 1998; 54. 77-95). Targeting in particular those senile amyloid plaques thus can treat Alzheimer pathology in a more specific and effective.

Many treatments have been tried to prevent the formation of β-peptides A, such as inhibitors of the proteolytic processing of APP.

Immunotherapy strategies such as the administration of anti-A β antibodies (to decrease amyloid deposition) or immunization with antigen peptides A-β (to promote a humoral response) were tested to reduce size and density of the plates. Were described, for example (US 7,179,463) a method of treatment against Alzheimer's disease comprising administering an antibody directed against a protofibril having a Arctic mutation within the region encoding the peptide A-β. No such antibodies is actually described. Furthermore, no antibody affinity comparison of the peptides according to the molecular weight of these peptides was performed. Other patents (US 6,761,888 and US 6,750,324) suggested some antibodies recognizing different epitopes along the amino acid sequence of peptide A-β 42 . An international application (WO2007 / 108756) was submitted for specific antibody protofibrils but the described antibodies recognize both the A-β peptides of high molecular weight and the average weight oligomers. Moreover antibody affinity for mature plates from their affinity for diffuse plaques is not mentioned.

Despite the current evolution of knowledge about Alzheimer's disease, there remains a need for compositions and methods of treatment and / or prevention of this disease minimizing side effects. Antibodies as described herein, humanized and specific form of protofibrillar peptides A-β are intended to solve this problem. Allowing recognition of senile amyloid plaques and not diffuse plaques, the subject antibodies of the invention recognize pathological plates so much more effective than antibodies recognizing all forms of Abeta, which will largely be fixed on the diffuse deposits or attached to soluble forms of A peptide-β monomeric or low molecular weight. Moreover, the fact only recognize shapes protofibrillar peptides A-β and not other forms protofibrillar not related to Alzheimer's disease protein prevents unnecessary connections may decrease the concentration of antibodies effective on the disease.

The murine antibody that has been humanized bear the name of 13C3 antibody throughout this application.

The sequences coding for humanized antibodies or constitute objects of the invention are shown in Table 2.

The present invention relates to a humanized antibody specifically binds to the protofibrillar form of the peptide A-β or a high molecular weight peptide. In a more advantageous embodiment, the antibody binds to the A-β peptide with a molecular weight greater than 200, 300, 400 or 500 kDa. In one implementation mode, the antibody object of the invention binds to aggregated A β peptide-senile plaques and not to diffuse deposits of A-β peptides.

In an advantageous embodiment, the antibody object of the invention binds specifically to protofibrillar forms of the peptide A-β but not to other amyloid structural proteins (such as the IAPP Islet Amyloid Polypeptide).

The present invention also relates to a humanized antibody with reduced effector functions, thereby limiting adverse effects such as the appearance of micro-hemorrhages and edema vasogéniques In one advantageous embodiment, the subject antibodies of the invention has more effector functions.

In one embodiment even more advantageous, the antibody is an immunoglobulin G Fc domain 4 which has undergone mutations reducing the production of half-molecules.

In one embodiment even more advantageous, the antibody is an immunoglobulin G Fc domain 4 which has undergone mutations decreasing effector activity.

The present invention relates to a humanized antibody comprising at least one CDR encoded by a nucleotide sequence having a sequence identical to one of the sequences SEQ ID NO: 9: 1 1, 13, 15, 17 and 19 or by sequences respectively differing by 1, 2, 3, 4 or 5 nucleotides of these sequences. The present invention also relates to a humanized antibody comprising at least one CDR having a sequence identical to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18 and 20.

In another embodiment, the subject antibodies of the invention comprises at least one CDR whose sequence differs from one to two amino acids with respect to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18, 20 and 32, provided that the antibody keeps its binding specificity.

In an advantageous embodiment, the antibody comprises the CDRs encoded by the nucleotide sequences SEQ ID NO: 9, 1 1, 13, 15, 17 and 19 or by sequences respectively differing by 1, 2,3,4 or 5 nucleotides of these sequences. In another advantageous embodiment, the antibody comprises the CDR sequence of SEQ ID NO: 10, 12, 14, 16, 18 and 20.

The antibody object of the invention may also comprise the CDRs encoded by the nucleotide sequences SEQ ID NO: 9, 1 1, 13, 31, 17 and 19 or by sequences respectively differing by 1, 2, 3, 4 or 5 nucleotides of these sequences.

In an advantageous embodiment, the antibody object of the invention comprises the sequence of CDR SEQ ID NO: 10, 12, 14, 32, 18 and 20.

An object of the invention is the humanized antibody comprising the CDRs encoded by the nucleotide sequences SEQ ID NO: 9, 1 1, 29, 31, 17 and 19 or by sequences respectively differing by 1, 2, 3, 4 or 5 nucleotides of these sequences. Is also an object of the invention a humanized antibody comprising the CDR sequence of SEQ ID NO: 10, 12, 30, 32, 18 and 20.

In an advantageous embodiment, the subject antibodies of the invention comprises a variable part of the heavy chain (VH) encoded by a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 5 or SEQ ID NO 27. in an advantageous embodiment, the subject antibodies of the invention comprises a variable part of the heavy chain (VH) comprising a sequence having at least 80% , 85%, 90%, 95% or 99% identity with the sequence SEQ ID NO: 6 or SEQ ID NO 28 sequence.

In an advantageous embodiment, the subject antibodies of the invention comprises a variable portion of its light chain (VL) encoded by a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 7 or SEQ ID NO 23 sequence.

In an advantageous embodiment, the antibody object of the invention comprises a variable part of the light chain (VL) comprising a sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 8 or SEQ ID NO 24 sequence.

In one embodiment even more advantageous, the antibody comprises a heavy chain comprising a variable (VH) encoded by any of nucleotide sequences SEQ ID NO 5 and SEQ ID NO 27. In an embodiment more advantageous, the antibody comprises a heavy chain comprising a variable (VH) polypeptide sequence of SEQ ID NO 6 or SEQ ID NO 28.

In another embodiment, the antibody comprises a light chain comprising a variable (VL) encoded by any of nucleotide sequences SEQ ID NO 7 and SEQ ID NO 23.

In another embodiment, the antibody comprises a light chain comprising a variable (VL) polypeptide sequence of SEQ ID NO 8 or SEQ ID NO 24.

In an advantageous embodiment, the antibody comprises the sequences encoded by the nucleotide sequences SEQ ID NO: 5 and 7. In an advantageous embodiment, the antibody comprises the polypeptide sequences SEQ ID NO: 6 and 8.

In another embodiment, the antibody comprises the sequences encoded by the nucleotide sequences SEQ ID NO: 5 and 23. In another embodiment, the antibody comprises the polypeptide sequences SEQ ID NO: 6 and 24.

In another embodiment, the antibody comprises the sequences encoded by the nucleotide sequences SEQ ID NO: 27 and 23. In another embodiment, the antibody comprises the polypeptide sequences SEQ ID NO: 28 and 24.

The present invention also relates to an antibody comprising a heavy chain encoded by a sequence having at least 80%, 85%, 90%, 95% or 99% identity with one of the nucleotide sequences SEQ ID NO 1 and SEQ ID NO 25.

The present invention also relates to an antibody comprising a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with the polypeptide sequence SEQ ID NO 2 or with the polypeptide sequence SEQ ID NO 26 .

In an advantageous embodiment the antibody comprises a light chain encoded by a sequence having at least 80%, 85%, 90%, 95% or 99% identity with one of the nucleotide sequences SEQ ID NO 3 and SEQ ID NO 21.

In another embodiment the antibody comprises a light chain comprising a sequence having at least 80%, 85%, 90%, 95% or 99% identity with one of the polypeptide sequences SEQ ID NO 4 and SEQ ID NO 22.

An object of the invention is an antibody comprising the sequences encoded by the nucleotide sequences SEQ ID NO: 1 and 3.

Another object of the invention is an antibody whose sequence comprises the polypeptide sequences SEQ ID NO: 2 and 4.

An object of the invention is an antibody comprising the sequences encoded by the nucleotide sequences SEQ ID NO: 1 and 21.

Another object of the invention is an antibody whose sequence comprises the polypeptide sequences SEQ ID NO: 2 and 22.

An object of the invention is an antibody comprising the sequences encoded by the nucleotide sequences SEQ ID NO: 25 and 21.

Another object of the invention is an antibody whose sequence comprises the polypeptide sequences SEQ ID NO: 26 and 22.

Another object of the invention is an anti-peptide Aß humanized antibody having an affinity for the protofibrillar form of Aß peptide of at least 100 times greater than its affinity for other forms of this peptide.

Another object of the invention is an antibody characterized that it induces a reduction in amyloid plaques.

Another object of the invention is the use of a humanized anti-peptide Aß antibodies in the treatment of diseases associated with neuro-degenerative disorders, in particular in the treatment of Alzheimer's disease. Another object of the invention is a pharmaceutical composition comprising a humanized antibody Aß-peptide and anti excipients.

Another object of the invention is an Alzheimer's disease treatment method comprising administering to the patient an anti humanized antibody peptide Aß.

Another object of the invention is a cell or cells producing a humanized anti-Aß peptide, as well as the process for producing the antibody comprising culturing these cells. Such cells are preferably derived from a cell line.

An object of the invention is a medicament comprising a humanized anti-Aß peptide.

An object of the invention is a polynucleotide encoding a polypeptide having at least 80%, 85%, 90%, 95% or 99% identity with one of the sequences SEQ ID

NO: 2, 4, 6, 8, 22, 24, 26 or 28.

Another object of the invention is a polynucleotide having a sequence having at least 80%, 85%, 90%, 95% or 99% identity with one of the sequences SEQ ID

NO: 1, 3, 5, 7, 21, 23, 25, or 27.

Another object of the invention is a recombinant vector comprising a nucleic acid having one of the sequences SEQ ID NO 1, 3, 5, 7, 21, 23, 25, or 27, and a host cell comprising this vector .

Definitions

Specific binding is defined as a difference of a factor of at least about 10, 20, 30, 40, 50, or 100 between the intensity of the receptor binding relative to another, here between the binding to the protofibrillar form of the peptide A-β and binding to other forms of the peptide.

By "epitope" the antigen site means which the antibody binds. If the antigen is a polymer, such as a protein or polysaccharide, the epitope can be formed by contiguous residues or non-contiguous. Here the epitope is conformational, that is to say linked to the three-dimensional structure of the peptide A-β protofibrillar.

By "protofibrillar form" refers to an oligomeric form of peptide A-β, soluble in vitro and may be isolated as a molecular weight entity greater than 200 kDa, 300 kDa, 400 kDa or 500 kDa and which can fix agents such as thioflavin-S or Congo red.

By "senile plaque" refers to a plate consisting of an amyloid core (fixing thioflavin S or Congo red) surrounded by dystrophic neurites and reaction of glial cells. Senile plaques are found particularly in patients with Alzheimer's disease, unlike diffuse amyloid deposits (not fixing the thioflavin S or Congo red) which are much more numerous but are not associated with disease.

An antibody, also called immunoglobulin consists of two identical heavy ( "CH") and two identical light chains ( "CL") that are linked by a disulfide bridge. Each chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity determining regions" ( "CDRs") or "hypervariable regions", which are primarily responsible for binding to the epitope of an antigen.

The term "VH" refers to the variable regions of a heavy chain immunoglobulin of an antibody, including the heavy chain of an Fv, scFv, dsFv, Fab, Fab 'or F (ab)'.

The term "VL" refers to variable regions of an immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, Fab, Fab 'or F (ab)'.

By "antibody" is any functional fragment is also understood antibodies: Fab (fragment antigen binding), Fv, scFv (single chain Fv), Fc (Fragment crystallizable). Preferably, these functional fragments will be fragments of Fv, scFv, Fab, F (ab ') 2, Fab', scFv-Fc, diabodies, multispecific antibodies (including bi-specific), synthetic polypeptides containing sequences one or CDRs, which generally have the same binding specificity as the humanized antibody from which they originated. According to the present invention, antibody fragments of the invention can be obtained from the humanized antibodies by methods such as digestion by enzymes, such as pepsin or papain and / or cleavage of disulfide bonds by chemical reduction . The nanobodies are also included within this definition.

By "CDR or CDR regions" is meant the hypervariable regions of the heavy and light chain immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5t "Ed., US Department of Health and Human Services, NIH, 1991 and later editions). There are three heavy chain CDRs and 3 CDRs of light chain. The term CDR or CDRs is used here to refer the case may be, one of these regions or several, or all, of these regions which contain the majority of the amino acid residues responsible for the affinity binding of the antibody for antigen or epitope it recognizes. the most conserved regions of the variable domains are called regions or FR sequences to "framework" regions or "frames".

The present invention relates to humanized antibodies.

By "humanized antibody" means an antibody that contains mostly human immunoglobulin sequences. This term generally refers to non-human immunoglobulin that has been modified by incorporating human sequences or residues found in human sequences.

In general, the humanized antibody comprises one or typically two variable domains in which all or part of the CDR regions correspond to parts from the non-human parent sequence and in which part or all of the FR regions are derived from a human immunoglobulin sequence. The humanized antibody may then comprise at least a portion of an immunoglobulin constant region (Fc), in particular that of human immunoglobulin selected reference. The aim is to obtain an antibody that is minimally immunogenic in a human. Thus it is possible that one or two amino acids of one or more CDRs are modified by an amino acid less immunogenic to the human host, this without substantially reducing the binding specificity of the antibody to peptide A-β high molecular weight. Similarly, the residues of the framework regions can not be human and they may not be modified because they do not contribute to the immunogenic potential of the antibody.

There are several methods of humanization known to the skilled person to change a non-human parent antibody in an antibody less immunogenic to humans. An overall sequence identity with a human antibody is not necessarily required. Indeed the overall sequence identity is not necessarily a predictor of decreased immunogenicity and modification of a limited number of residues can lead to humanized antibodies having a very reduced immunogenic potential in humans (Molecular Immunology (2007)

44, 1986-1998).

Various methods are for example the inclusion of CDR (grafting) (EPO 0,239,400; WO 91/09967; and US Pat 5,530,101 and 5,585,089 Our..), Resurfacing (EPO 0,592,106; EPO 0519596; Padlan, 1991 Molec Imm 28 (4/5): 489-498; Studnicka et al 1994, Prot Eng 7 (6): 805-814; and Roguska et al, 1994, PNAS 91. 969-973) or. mixing channels (US Pat. No. 5,565,332).

The present invention relates in particular humanized antibodies whose variable regions are modified according to the technology explained in international patent application WO 2009/032661.

This technique uses a particular molecular dynamics simulation from three-dimensional antibody models, said models being built by homology.

The present invention also relates to all forms of antibodies with reduced effector functions, such as immunoglobulins bearing Fc domain mutations reducing its affinity for the receptors of the immune system or as nanobodies.

By "effector functions" means any fixation of the Fc domain of the antibody to receptors or proteins that induce immune responses. The reduction of these effector functions reduces adverse effects such as the triggering of blankness (Racke et al J Neurosci 2005, 25:. 629).

The affinity can be measured by any known to those skilled in the art. It is advantageously measured according to the Biostat Speed ​​technique developed from the algorithms described by Ratkovsk DA and Reedy TJ (Biometrics, 1986, 42, 575-82).

To allow expression of heavy and / or light chains of the antibody object of the invention, the polynucleotides encoding said chains are inserted into expression vectors. These expression vectors may be plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the vectors that the skilled person may find appropriate to expression of said chains.

These vectors can be used to transform advantageously derived from a cell line cells. Such a cell line is more preferably derived from a mammal.

It is advantageously the CHO line or a line derived from this cell line, or the HEK293 cell line or a line derived from this lineage.

Cell transformation can be performed by any method known in the art to introduce polynucleotides into a host cell. Such a method may be processing using dextran, precipitation by calcium phosphate, transfection using polybrene, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, the biolistic injection and direct microinjection of DNA into the nucleus.

The subject antibodies of the invention may be included in pharmaceutical compositions for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. Preferably, the pharmaceutical compositions contain pharmaceutically acceptable vehicles for an injectable formulation. It may be in particular saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which , by addition depending on the case of sterilized water or of physiological saline, allow the constitution of injectable solutions.

For example a pharmaceutical composition comprises (1) Dulbecco's phosphate buffer (pH - 7.4), optionally containing 1 mg / ml to 25 mg / ml human serum albumin, (2) 0.9% w / v sodium chloride (NaCl)), and (3) 5% (w / v) dextrose. It may also comprise an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

The pathologies targeted may be all diseases related to the deposition of amyloid plaques. In particular, the condition is referred Alzheimer's disease.

The doses depend on the desired effect, the duration of treatment and the route of administration used; they are generally between 5 mg and 1000 mg of antibodies per day for an adult. Generally the doctor will determine the appropriate dosage depending on the stage of the disease, the patient's age, weight or any other factor to be considered depending on the patient.

The present invention is illustrated, without being limited by the following examples.

BRIEF DESCRIPTION OF FIGURES:

Fig.1 A: Map of the plasmid pXL4973 which allows expression of the light chain of the antibody LC1 antiAbeta 13C3-VH1VL1.

Fig 1B: Map of the plasmid pXL4979 enabling the expression of the heavy chain

HC1 antibody 13C3 antiAbeta-VH1VL1.

Fig 2A and 2B: Separation of protofibrillles and low molecular weight oligomers by gel filtration on Superdex 75 (respectively at t = 0 and t = 16h). Figure 3: Determination of the molecular weight of protofibrils.

Fig 4A, 4B and 4C: Determination of affinities of the humanized antibodies

(Respectively LP09027 antibody (4A), LP09026 (4B) and LP09028 (4C)) for the protofibril (mean of 3 experiments ± sem).

Fig 5: Specificity of the humanized antibody LP09027 overlooked fibrils Aß. Fig 6A and 6B: Specificity of the humanized antibody (LP09027) for mature senile plaques respectively the frontal cortex (6A) and hippocampus (6B) of a mouse. The arrows indicate the senile plaques.

EXAMPLES:

Example 1 Production of humanized antibodies

A murine antibody 13C3 was humanized.

This example describes the sequence and obtaining the anti-peptide Aß VH1VL1 humanized antibody (LP09027) production by transient expression in HEK293 mammalian line named FreeStyle 293-F.

The cDNA coding for humanized variable chains VL1 and VH1 are fused to cDNA encoding human constant regions Ckappa and IgG4 respectively. The sequence of the constant region IgG4 is the variant having the S241 P and L248E substitutions in the nomenclature of Kabat, to significantly reduce the production of half-molecules (Angla et al, 1993, Mol Immunol, 30... 105 -108) and effector functions (WO 97/09351). The nucleic acid sequences encoding CH1 (SEQ ID NO 1) and CL1 (SEQ

ID NO 3) were cloned independently in the expression vector to generate the plasmids pXL4973, respectively (Fig 1A) and pXL4979 (Fig 1 B).

A batch of the antibody is produced by production in transient expression in FreeStyle 293-F line (Invitrogen) after co-transfection of plasmids pXL4973 and pXL4979 according to the protocol described by Invitrogen (catalog number K9000-01). Lot (LP09027) is then purified by affinity chromatography on a gel column MabSelect (Amersham) as recommended by the supplier and then formulated in PBS buffer (Dulbecco reference 14190-094) and sterile filtered (0.2 .mu.m). From 1 liter of culture, 33 mg antibody are obtained with a purity of 97% by SDS-PAGE under denaturing conditions and by steric exclusion chromatography. The mass obtained by SDS-PAGE under denaturing conditions and by LC / MS is consistent with the primary sequence of amino acid and the presence of a N-glycan on the Fc domain is a mass of 23969 Da and 49650 for LC1 da for HC1 taking into account the N-glycan in the GOF form. The mass obtained by SDS-PAGE under non-denaturing conditions and by size exclusion chromatography is in agreement with the hetero-tetrameric structure of antibodies 15O kDa. (Fig 4A).

According to the same method, humanized antibodies lots LP09026 and LP09028 were produced from the nucleotide sequences SEQ ID NO 25 and SEQ ID NO 21 LP09026 (Fig 4B), and SEQ ID NO 1 and SEQ ID NO 21 LP09028 (Fig 4C).

Example 2: Preparation of protofibrils from Aß peptide (1 -42).

The protofibrils were prepared from the synthetic peptide Aß (1-42) as described by Johansson et al. (FEBS, 2006, 2618-2630). The lyophilized peptide (Anaspec 24224 reference) is dissolved in 1OmM NaOH concentration of 100 .mu.m, then stirred for 1 min and incubated on ice for 10 minutes. The peptide solution was then diluted in 10OmM sodium phosphate buffer, 20OmM NaCl pH 7.4 at a concentration of 50 microM, then stirred 1 min. The preparation is incubated overnight at 37 ° C to obtain the formation of protofibrils and then centrifuged at 17900g for 15 min at 16 ° C to remove insoluble aggregates. To separate protofibrils oligomeric forms of low molecular weight Aß, the supernatant is loaded onto a gel filtration column Superdex 75 equilibrated with buffer 50 mM ammonium acetate pH = 8.5. The fractions corresponding to protofibrils and low molecular weight oligomers is collected and stored at 4 ° C. Figure 2 shows a typical profile of separation protofibrils. The molecular weight of protofibrils is determined by Superdex200 gel filtration using as molecular weight markers of the calibration kit Biorad (150-1901 reference). Figure 3 shows that the molecular weight of protofibrils is greater than 200kDa.

Example 3: Specificity and affinity of humanized antibodies vis-à-vis protofibrils

50 .mu.l of protofibrils and low molecular weight oligomers at a concentration of 1 .mu.g / ml in PBS (Gibco, reference 70011) are deposited in the wells of an ELISA plate (Nunc, 442404 reference) and incubated at 4 ° C for a night. After removal of excess antigen, 200 .mu.l of PBS + 5% milk powder (w / v) are placed in each well to remove nonspecific adsorptions and incubated for 2h at room temperature. The wells are then washed 4 times with 300 .mu.l of PBS Tween 0.02%. 50 .mu.l of a primary antibody solution (3 in 3 dilution in PBS Tween from a concentration of 100 micrograms / ml for the oligomers and from 25 mcg / ml for protofibrils) are added to each well and incubated for 1 h at room temperature. The wells are washed four times with 300μl of PBS Tween buffer. The anti-human Fc secondary antibody coupled to peroxidase (Goat Anti Human IgG (Fc) conjugated peroxidase, Pierce, 31413 reference) diluted 1/10000 in PBS Tween buffer is added to each well and incubated 1 hr at room temperature . After 4 washes with 300μl of PBS Tween, 100 .mu.l of TMB (Interchim, UP664782 reference) were added to each well and incubated for about 10 minutes, then the reaction was quenched with a solution of 1 M HCl (Interchim, UPS29590 reference) and the plates are read at OD measured at the wavelength of 450nm. The EC50 is determined by BioStat Speed. The results obtained are shown in Table 1 and in Figure 4 and demonstrate the high specificity of the antibody for the protofibrils relative to low molecular weight oligomers (factor 184).

Table 1:

The lyophilized peptide Aβ1-42 (Anaspec 24224 reference) is dissolved according to manufacturer's recommendations: 40 .mu.l of 1% NH 4 OH are added to 500 mcg Aβi-42. After complete dissolution, 460 .mu.l of PBS are added to obtain a concentration of 1 mg / ml. Aliquots of 10 .mu.l were prepared and stored at -80 0 C. 50 .mu.l of Aβ1-42 peptide solution at a concentration of 1 .mu.g / ml in carbonate buffer (NaHCO 3 0.025 M (Acros Organics, 217 120 010 reference), Na 2 CO 3 0.025 M (Acros Organics, 207 810 010 reference), pH 9.7 is added to the wells of an ELISA plate and incubated overnight at room temperature. as before the wells are washed with PBS Tween buffer, incubated with of PBS + 5% milk powder (w / v) and washed with PBS tween buffer. the humanized antibody at a concentration of 0.02 ug / ml was incubated for 1 h at room temperature with a concentration range ( from 1 mcg / ml) of Aβ1-28 peptides (Bachem H7865 reference), Aβ1-16 ((Anaspec, reference 24225), Aβ25-35 ((Anaspec, reference 24227), low molecular weight oligomers or protofibrils prepared as previously described. The antibody / antigen mixture is e nsuite deposited in each well and the microtitre plate incubated for 1 h at room temperature. The free uncomplexed antibody is determined using the same ELISA protocol as described above. These competition experiments show that only the protofibrils with a much higher affinity than the low molecular weight oligomers are capable of neutralizing the humanized antibody by preventing it from interacting with the pepdie Aβ1-42, none of the peptides is capable of neutralizing antibody.

Example 4: Specificity of the humanized antibody LP09027 overlooked the fibril Aß 1 -42

The Aβ1-42 peptide (Anaspec, 20276) was dissolved in 200 .mu.l of 10 mM NaOH at a concentration of 5 mg / ml. The IAPP peptide (Anaspec, 60804) was diluted in 200 .mu.l of 50% DMSO at a concentration of 5 mg / ml. 100 .mu.l of each preparation were diluted in 400 .mu.l of PBS 1.25x. The final peptide concentration is 1 mg / ml in 500 .mu.l. The samples were incubated 72h at 37 ° C. After incubation, the samples were centrifuged at 17900g for 30 minutes at 4 ° C. The supernatant is discarded and the pellet is washed 3 times with 1X PBS. After the last wash, the fibril pellet is taken up in 150 .mu.l of PBS. To monitor the presence of amyloid-like fibrils, a fluorescence test thioflavin T (Anaspec, 88306) is directed. 20 .mu.l of thioflavin T (20 .mu.M final), 10 .mu.l of the sample and 70 .mu.l of 1X PBS (final volume 100 .mu.l) were mixed in a well of a black plate (Corning, 3792). Thioflavin T is excited at 450 nm and in the presence of amyloid-like structure, emits fluorescence at 482 nm. 50 .mu.l of Aβ1-42 fibrils to 1 mcg / ml and IAPP at 0.5 mcg / ml are added to each well of a microtiter plate. The ELISA protocol is applied using serial dilutions of the humanized antibodies from 10 mcg / ml. Figure 5 shows that the LP09027 humanized antibody specifically recognizes Aβ1-42 fibrils but not those of IAPP.

Example 5: Specificity of the humanized antibody LP09027 for mature senile plaques and not to diffuse plaques

The humanized antibody (LP09027) conjugated with digoxigenin (digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester: Roche 11333054001; 11418165001) was used Immunohistochemistry (Robot Ventana) on brain sections of PS1 mice APP (Alzheimer model described by Schmitz C. et al., Am. J. Pathol, 2004, 164, 1495-1502)) and the human brain slices (cerebral cortex) from patients with Alzheimer ' Alzheimer. The samples were previously fixed in formalin and embedded in paraffin.

The results obtained in mice (Figures 6A and 6B) clearly show that the humanized antibody recognizes exclusively mature and dense neuritic plaques, but not diffuse deposits of Aß peptide. These data correlate with the properties of this antibody which is specific for the protofibrillar form Abeta and therefore does not recognize the soluble forms, mono-or oligomeric this peptide.

claims

1. A humanized antibody specific for the protofibrillar form of the peptide A-β.

2. Antibody according to claim 1 characterized in that it binds to aggregated A β peptide-senile plaques and not to diffuse deposits of A-β peptides.

3. Antibody according to one of the preceding claims, characterized in that it comprises at least a CDR encoded by a nucleotide sequence having a sequence identical to one of the sequences SEQ ID NO: 9: 1 1, 13, 15, 17 and

19 or by sequences respectively differing by 1, 2, 3, 4 or 5 nucleotides of these sequences.

4. Antibody according to one of the preceding claims, characterized in that it comprises at least a CDR having a sequence identical to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18 and 20.

5. Antibodies according to one of the preceding claims, characterized in that it comprises at least one CDR whose sequence differs from one to two amino acids with respect to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18 and 20, provided that the antibody keeps its binding specificity.

6. Antibodies according to one of the preceding claims, characterized in that it comprises the CDR encoded by the nucleotide sequences SEQ ID NO 9, 1 1, 13, 15, 17 and 19, or sequences differing by one respectively, 2, 3, 4 or

5 nucleotides of these sequences.

7. Antibody according to one of the preceding claims, characterized in that it comprises the CDR sequence of SEQ ID NO: 10, 12, 14, 16, 18 and 20.

8. Antibody according to one of the preceding claims, characterized in that it comprises the CDR encoded by the nucleotide sequences SEQ ID NO 9, 11, 13, 31, 17 and 19, or sequences differing respectively by 1, 2 , 3, 4 or 5 nucleotides of these sequences.

9. Antibody according to one of the preceding claims, characterized in that it comprises the CDR sequence of SEQ ID NO: 10, 12, 14, 32, 18 and 20.

10. Antibody according to one of the preceding claims, characterized in that it comprises the CDR encoded by the nucleotide sequences SEQ ID NO 9: 1 1

29, 31, 17 and 19, or sequences differing respectively by 1, 2, 3, 4 or 5 nucleotides of these sequences.

11. Antibody according to one of the preceding claims, characterized in that it comprises the CDR sequence of SEQ ID NO: 10, 12, 30, 32, 18 and 20.

12. Antibody according to one of the preceding claims, characterized in that the variable part of the heavy chain is encoded by a sequence having at least 80% identity with one of the sequences SEQ ID NO: 5 and SEQ ID NO 27.

13. Antibody according to one of the preceding claims, characterized in that the variable part of the heavy chain comprises a sequence having at least 80% identity with one of the sequences SEQ ID NO: 6 and SEQ ID NO 28.

14. Antibody according to one of the preceding claims, characterized in that the variable part of the light chain is encoded by a sequence having at least 80% identity with one of the sequences SEQ ID NO: 7 and SEQ ID NO 23.

15. Antibody according to one of the preceding claims, characterized in that the variable part of the light chain comprises a sequence having at least 80% identity with one of the sequences SEQ ID NO: 8 and SEQ ID NO 24.

16. Antibody according to one of the preceding claims, characterized in that it comprises a heavy chain comprising a variable part coded by one of the nucleotide sequences SEQ ID NO 5 and SEQ ID NO 27.

17. Antibody according to one of the preceding claims, characterized in that it comprises a heavy chain comprising a variable part of polypeptide sequence SEQ ID NO 6 or SEQ ID NO 28.

18. Antibody according to one of the preceding claims, characterized in that it comprises a light chain comprising a variable part coded by one of the nucleotide sequences SEQ ID NO 7 and SEQ ID NO 23.

19. Antibody according to one of the preceding claims, characterized in that it comprises a light chain comprising a variable part of polypeptide sequence SEQ ID NO 8 or SEQ ID NO 24.

20. Antibody according to one of the preceding claims, characterized in that it comprises sequences encoded by the nucleotide sequences SEQ ID NO: 5 and 7.

21. Antibody according to one of the preceding claims, characterized in that its sequence comprises the polypeptide sequences SEQ ID NO: 6 and 8.

22. Antibody according to one of the preceding claims, characterized in that it comprises a heavy chain encoded by a sequence having at least 80% identity with one of the nucleotide sequences SEQ ID NO 1 and SEQ ID NO 25.

23. Antibody according to one of the preceding claims, characterized in that it comprises a heavy chain having at least 80% identity with one of the polypeptide sequences SEQ ID NO 2 and SEQ ID NO 26.

24. Antibody according to one of the preceding claims, characterized in that it comprises a light chain encoded by a sequence having at least 80% identity with one of the nucleotide sequences SEQ ID NO 3 and SEQ ID NO 21.

25. Antibody according to one of the preceding claims, characterized in that it comprises a light chain sequence having at least 80% identity with one of the polypeptide sequences SEQ ID NO 4 and SEQ ID NO 22.

26. Antibody according to one of the preceding claims, characterized in that it comprises the sequences encoded by the nucleotide sequences SEQ ID NO: 1 and 3.

27. Antibody according to one of the preceding claims, characterized in that its sequence comprises the polypeptide sequences SEQ ID NO: 2 and 4.

28. Antibody according to one of the preceding claims, characterized in that it comprises sequences encoded by the nucleotide sequences SEQ ID NO: 5 and 23.

29. Antibody according to one of the preceding claims, characterized in that its sequence comprises the polypeptide sequences SEQ ID NO: 6 and 24.

30. Antibody according to one of the preceding claims, characterized in that it comprises sequences encoded by the nucleotide sequences SEQ ID

NO: 27 et 23.

31. Antibody according to one of the preceding claims, characterized in that its sequence comprises the polypeptide sequences SEQ ID NO: 28 and 24.

32. An antibody according to any one of the preceding claims, characterized in that it induces a reduction in amyloid plaques.

33. An antibody according to any preceding claim characterized in that its affinity for the protofibrillar form of Aß peptide is at least 100 times greater than its affinity for other forms of this peptide.

34. Use of an antibody according to any one of the preceding claims in the treatment of diseases associated with neuro-degenerative disorders

35. Use of an antibody according to any one of the preceding claims in the treatment of Alzheimer's disease

36. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 34 and of the excipients.

37. A method of treating Alzheimer's disease characterized in that it comprises administering to the patient an antibody according to any one of claims 1 to 34.

38. A cell producing antibody according to claim 1 to 34.

39. A method of producing an antibody according to any one of claims 1 to 34 characterized in that it comprises culturing cells of claim 38.

40. A medicament comprising an antibody according to any one of claims 1 to 34.

41. A polynucleotide encoding a polypeptide having at least 80% identity with one of SEQ ID NOS: 2, 4, 6, 8, 22, 24, 26 or 28.

42. A polynucleotide characterized in that it has a sequence having at least 80% identity with one of SEQ ID NOS: 1, 3, 5, 7, 21, 23,

25 or 27.

43. A recombinant vector comprising a nucleic acid according to any one of claims 41 and 42.

44. A host cell comprising a vector according to claim 43.