Humanized antibodies specific to the protofibrillar form of the beta-amyloid
peptide
The present invention relates to humanized antibodies specific for the
protofibrillar form of p-amyloid peptide. The present invention also relates to the
therapeutic, diagnostic and/or preventive use of these antibodies, in particular
associated with the induction and with the progression of neurodegenerative disorders
and/or with diseases associated with the deposition of amyloid plaques, and notably
Alzheimer's disease.
Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects a
high proportion of the older population. This disease is characterized clinically by
memory loss and a decline in cognitive functions, and neuropathologically by the
presence, in the brain, of intracellular neurofibrillar deposits and of extracellular
deposits of the P-amyloid peptide (A-β) forming amyloid plaques. (Yanker et al. Nature
Med. Vol. 2 No. 8 (1996)). As well as these signs, there are many other abnormal
changes including a deterioration of the immune and inflammatory systems as well as
a deterioration of mitochondrial function, which can lead to an increase in oxidative
stress, activation of the mechanisms of apoptosis and ultimately to cell death.
Amyloid plaques are predominantly composed of A-β peptides with 40 or 42 residues,
which are generated during the proteolytic process of the β-amyloid peptide precursor
(APP) protein. The extracellular deposits of A-β peptides represent the invariable early
characteristic feature of all forms of AD, including the familial forms (FAD). FADs
appear relatively early (between 40 and 60 years) and are due to mutations in the
gene of APP in 5% of cases of FAD (> 20 families) with six single or double missense
mutations; in the gene of presenilin 1 (PS 1) in 50 to 70% of cases of FAD (> 200
families) with more than 80 different mutations identified to date; and in the gene of
presenilin 2 (PS 2) in fewer cases of FAD with 2 missense mutations described in 8
families. Mutations in these three genes have been shown to induce changes in the
proteolysis of APP, which lead to overproduction of A-β and to the early appearance
of the pathology and of symptoms that are similar to those of the sporadic forms of
AD.
The neuronal toxicity of the amyloid plaques might reside in the high molecular weight
fibrils that are formed by aggregation of soluble A-β peptides in fibrillar forms that are
soluble initially (also called protofibrillar form) and are then converted to insoluble

forms incorporated in the amyloid plaques. In fact, it was shown in vitro that the
soluble A-β peptide aggregates progressively to a fibrillar form (i.e. which can be
labelled with agents such as Congo Red or thioflavin S which recognize the beta-
sheet tertiary structures of the peptides/proteins), of high molecular weight (>200 kDa)
but still soluble. Because this form is soluble, it is often called the protofibrillar form,
whereas the fibrils result from even greater aggregation, leading to loss of solubility.
The protofibrillar transitional forms are generally regarded as the precursors of the
amyloid fibres and might be responsible for the cellular dysfunction and the neuronal
loss in Alzheimer's disease and in other diseases associated with the aggregation of
proteins.
It has been shown that the senile amyloid plaques (i.e. aggregated, also called mature
plaques) are correlated with the cognitive status of Alzheimer's patients in contrast to
the diffuse deposits of A-β peptide which are also widely present in unaffected
patients. (Duyckaerts et al., Neurobiol. Aging 1997; 18: 33-42 and Jellinger et al.,
1998; 54:77-95). By targeting these senile amyloid plaques in particular, it is therefore
possible to treat Alzheimer's disease more specifically and effectively.
A great many treatments have been tried for preventing the formation of the A-β
peptides, for example inhibitors of the proteolytic process of APP.
Immunotherapeutic strategies such as the administration of anti-A-β antibody (to
reduce the amyloid deposits) or immunization with antigens of the A-β peptides (to
promote a humoral response) have been tested in order to reduce the size and
density of the plaques.
For example, a method of treatment against Alzheimer's disease has been described
(US 7 179 463), consisting of administering an antibody directed against a protofibril
presenting an Arctic mutation in the region coding for the A-β peptide.
No example of antibody has really been described. Moreover, no comparison of the
affinity of the antibodies for the peptides as a function of the molecular weight of these
peptides has been performed. Other patents (US 6 761 888 and US 6 750 324) have
referred to antibodies recognizing various epitopes along the amino acid sequence of
the peptide A-β42. An international application (WO2007/108756) has been filed
concerning antibodies specific for the protofibrils but the antibodies described
recognize both the high molecular weight A-β peptides and the medium-weight
oligomers. Furthermore, there is no mention of the affinity of the antibodies for the
mature plaques relative to their affinity for the diffuse plaques.

Despite the current development of knowledge concerning Alzheimer's disease, there
is still a need for compositions and methods of treatment and/or prevention of this
pathology limiting the secondary effects to the maximum extent. Antibodies such as
described in the present application, humanized and specific for the protofibrillar form
of the A-β peptides, aim to solve this problem. Permitting recognition of the senile
amyloid plaques but not the diffuse plaques, the antibodies according to the invention
recognize the pathological plaques much more effectively than antibodies recognizing
all forms of Abeta, which will largely be attached to the diffuse deposits or attached to
the soluble forms of monomeric or low-molecular-weight A-3 peptide.
Moreover, the fact that only the protofibrillar forms of the A-β peptides are recognized
and not the protofibrillar forms of other proteins not linked to Alzheimer's disease
avoids useless binding that may reduce the concentration of antibodies that are
effective against the disease.
The murine antibody that has been humanized will be called antibody 13C3
throughout the present application.
The sequences that can code for or constitute the humanized antibodies according to
the invention are shown in Table 2.
The present invention relates to a humanized antibody that binds specifically to the
protofibrillar form of the A-β peptide, i.e. a high molecular weight peptide.
In a more advantageous embodiment, the antibody binds to the A-β peptide having a
molecular weight greater than 200, 300, 400 or 500 kDa.
According to one embodiment, the antibody according to the invention binds to the A-
P peptides aggregated into senile plaques and not to the diffuse deposits of A-β
peptides.
In an advantageous embodiment, the antibody according to the invention binds
specifically to the protofibrillar form of the A-β peptide but not to the other proteins of
amyloid structure (for example IAPP, Islet Amyloid Polypeptide).
The present invention also relates to a humanized antibody having reduced effector
functions, making it possible to limit adverse effects such as the development of
microhaemorrhages and vasogenic oedemas.
In an advantageous embodiment, the antibody according to the invention no longer
possesses effector functions.

In an even more advantageous embodiment, the antibody is an immunoglobulin G 4
whose Fc domain has undergone mutations reducing the production of half-
molecules.
In an even more advantageous embodiment, the antibody is an immunoglobulin G 4
whose Fc domain has undergone mutations reducing the 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, 11, 13, 15, 17 and 19, or by sequences differing
respectively by 1, 2, 3, 4 or 5 nucleotides from 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 antibody according to the invention comprises at least
one CDR whose sequence differs by one to two amino acids relative to one of the
sequences SEQ ID NO: 10, 12, 14, 16, 18, 20 and 32, inasmuch as the antibody
maintains its binding specificity.
In an advantageous embodiment, the antibody comprises the CDRs encoded by the
nucleotide sequences SEQ ID NO: 9, 11, 13, 15,17 and 19, or by sequences differing
respectively by 1, 2, 3, 4 or 5 nucleotides from these sequences.
In another advantageous embodiment, the antibody comprises the CDRs of sequence
SEQ ID NO: 10, 12, 14,16, 18 and 20.
The antibody according to the invention can also comprise the CDRs encoded by the
nucleotide sequences SEQ ID NO: 9, 11, 13, 31,17 and 19 or by sequences differing
respectively by 1, 2, 3, 4 or 5 nucleotides from these sequences.
In an advantageous embodiment, the antibody according to the invention comprises
the CDRs of sequence SEQ ID NO: 10, 12, 14, 32, 18 and 20.

One object of the invention is the humanized antibody comprising the CDRs encoded
by the nucleotide sequences SEQ ID NO: 9, 11, 29, 31, 17 and 19 or by sequences
differing respectively by 1, 2, 3, 4 or 5 nucleotides from these sequences.
The invention also relates to a humanized antibody comprising the CDRs of sequence
SEQ ID NO: 10,12, 30, 32, 18 and 20.
In an advantageous embodiment, the antibody according to the invention comprises a
variable part of its heavy chain (VH) encoded by a sequence having at least 80%,
85%, 90%, 95% or 99% identity with the sequence SEQ ID NO: 5 or the sequence
SEQ ID NO 27.
In an advantageous embodiment, the antibody according to the invention comprises a
variable part of its heavy chain (VH) comprising a sequence having at least 80%,
85%, 90%, 95% or 99% identity with the sequence SEQ ID NO: 6 or the sequence
SEQ ID NO 28.
In an advantageous embodiment, the antibody according to the invention comprises a
variable part of its light chain (VL) encoded by a sequence having at least 80%, 85%,
90%, 95% or 99% identity with the sequence SEQ ID NO: 7 or the sequence SEQ ID
NO 23.
In an advantageous embodiment, the antibody according to the invention comprises a
variable part of its light chain (VL) comprising a sequence having at least 80%, 85%,
90%, 95% or 99% identity with the sequence SEQ ID NO: 8 or the sequence SEQ ID
NO 24.
In an even more advantageous embodiment, the antibody comprises a heavy chain
comprising a variable part (VH) encoded by one of the nucleotide sequences SEQ ID
NO 5 and SEQ ID NO 27.
In an even more advantageous embodiment, the antibody comprises a heavy chain
comprising a variable part (VH) of polypeptide sequence SEQ ID NO 6 or SEQ ID NO
28.
In another embodiment, the antibody comprises a light chain comprising a variable
part (VL) encoded by one of the nucleotide sequences SEQ ID NO 7 and SEQ ID NO
23.
In another embodiment, the antibody comprises a light chain comprising a variable

part (VL) of polypeptide sequence 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.
One 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.
One 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.
One 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 a humanized anti-peptide Ap antibody having an
affinity for the protofibrillar form of peptide A(3 at least 100 times greater than its
affinity for the other forms of this peptide.
Another object of the invention is an antibody, characterized in that it induces a
reduction of amyloid plaques.
Another object of the invention is the use of a humanized anti-peptide Ap antibody in
the treatment of diseases associated with neurodegenerative disorders, and in
particular in the treatment of Alzheimer's disease.
Another object of the invention is a pharmaceutical composition comprising a
humanized anti-peptide Ap antibody and excipients.
Another object of the invention is a method of treatment of Alzheimer's disease
comprising the administration of a humanized anti-peptide-Ap antibody to the patient.
Another object of the invention is a cell or cells producing a humanized anti-peptide-
Ap antibody, as well as the method of production of this antibody comprising the
culturing of these cells. Said cells are derived advantageously from one cell line.
One object of the invention is a medicinal product comprising a humanized anti-
peptide-Ap antibody.

One object of the invention is a polynucleotide coding for 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 with 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, as well as a
host cell comprising this vector.
Definitions
Specific binding is understood as a difference by a factor of at least about 10, 20, 30,
40, 50, or 100 between the strength of binding to one receptor relative to another,
here between binding to the protofibrillar form of the A-β peptide and binding to the
other forms of the peptide.
"Epitope" means the site of the antigen to which the antibody binds. If the antigen is a
polymer, such as a protein or a polysaccharide, the epitope can be formed by
contiguous or non-contiguous residues. Here the epitope is conformational, i.e.
related to the three-dimensional structure of the protofibrillar A-β peptide.
"Protofibrillar form" means an oligomeric form of A-(3 peptides, soluble in vitro and
which can be isolated as an entity of molecular weight greater than 200 kDa, 300 kDa,
400 kDa or 500 kDa and which can fix agents such as thioflavin-S or Congo Red.
"Senile plaque" means a plaque composed of an amyloid core (fixing thioflavin S or
Congo Red) surrounded by dystrophic neurites and a reaction of glial cells. Senile
plaques are found in particular in patients with Alzheimer's disease, in contrast to the
diffuse amyloid deposits (which do not fix thioflavin S or Congo Red), which are far
more numerous but are not associated with the disease.
An antibody, also called immunoglobulin, is composed of two identical heavy chains
("CH") and two identical light chains ("CL"), which are joined by a disulphide bridge.

Each chain contains a constant region and a variable region. Each variable region
comprises three segments called "complementarity determining regions" ("CDRs") or
"hypervariable regions", which are mainly responsible for binding to the epitope of an
antigen.
The term "VH" refers to the variable regions of a heavy chain of immunoglobulin of an
antibody, including the heavy chains of a fragment Fv, scFv, dsFv, Fab, Fab' or F(ab)'.
The term "VL" refers to the variable regions of a light chain of immunoglobulin of an
antibody, including the light chains of a fragment Fv, scFv, dsFv, Fab, Fab' or F(ab)'.
"Antibody" also means any functional fragment of antibody: Fab (Fragment antigen
binding), Fv, scFv (single chain Fv), Fc (Fragment, crystallizable). Preferably, these
functional fragments will be fragments of type Fv, scFv, Fab, F(ab') 2, Fab', scFv-Fc,
diabodies, multispecific antibodies (notably bispecific), synthetic polypeptides
containing the sequences of one or more CDRs, which generally possess the same
specificity of fixation as the humanized antibody from which they are derived.
According to the present invention, fragments of antibodies of the invention can be
obtained from the humanized antibodies by methods such as digestion by enzymes,
such as pepsin or papain and/or by cleavage of the disulphide bridges by chemical
reduction.
Nanobodies also come under this definition.
"CDR or CDRs" denotes the hypervariable regions of the heavy and light chains of the
immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of
immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH,
1991, and later editions). There are 3 heavy-chain CDRs and 3 light-chain CDRs. The
term CDR or CDRs is used here to denote, as applicable, one or more, or even all, of
these regions that contain the majority of the amino acid residues responsible for the
affine binding of the antibody for the antigen or the epitope that it recognizes. The
most conserved regions of the variable domains are called FR regions or sequences,
for "framework regions".
The present invention relates to humanized antibodies.

"Humanized antibody" means an antibody that contains mainly human
immunoglobulin sequences. This term generally refers to a non-human
immunoglobulin that has been modified by incorporating human sequences or
residues found in human sequences.
In general, humanized antibodies comprise one or typically two variable domains in
which all or part of the CDR regions correspond to parts derived from the non-human
parent sequence and in which all or part of the FR regions are derived from a human
immunoglobulin sequence. The humanized antibody can then comprise at least one
portion of a constant region of immunoglobulin (Fc), in particular that of the selected
reference human immunoglobulin.
We thus try to obtain an antibody that is the least 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 that is less immunogenic for the human host, without substantially reducing the
binding specificity of the antibody to the A-B peptide of high molecular weight.
Furthermore, the residues of the framework regions need not be human and it is
possible that they are not modified, as they do not contribute to the immunogenic
potential of the antibody.
Several methods of humanization are known by a person skilled in the art for
modifying a non-human parent antibody to an antibody that is less immunogenic in
humans. Complete identity of the sequences with a human antibody is not essential.
In fact complete sequence identity is not necessarily a predictive indicator of reduced
immunogenicity and modification of a limited number of residues can lead to
humanized antibodies presenting a very attenuated immunogenic potential in humans
(Molecular Immunology (2007) 44,1986-1998).
Some methods are for example the inclusion of CDRs (grafting) (EPO 0 239 400; WO
91/09967; and U.S. Pat. Nos. 5,530,101 and 5,585,089), the resurfacing (EPO 0 592
106; EPO 0 519 596; 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 chain
mixing (U.S. Pat. No. 5,565,332).

The present invention relates in particular to humanized antibodies whose variable
parts are modified according to the technology explained in international patent
application WO 2009/032661.
This technique notably uses dynamic molecular simulation based on three-
dimensional models of antibodies, said models being constructed by homology.
The present invention also relates to any form of antibody having diminished effector
functions, such as immunoglobulins bearing mutations of the Fc domain reducing its
affinity for the receptors of the immune system or such as nanobodies.
"Effector functions" means any fixation of the Fc domain of the antibody to receptors
or proteins inducing immune responses. Decreasing these effector functions makes it
possible to reduce adverse effects such as the induction of microhaemorrhages
(Racke et al. J Neurosci 2005, 25:629).
Affinity can be measured by any technique known by a person skilled in the art. It is
advantageously measured by the Biostat Speed technique developed on the basis of
the algorithms described by Ratkovsk DA and Reedy TJ (Biometrics, 1986, 42, 575-
82).
In order to permit expression of heavy chains and/or light chains of the antibody
according to the invention, the polynucleotides coding for said chains are inserted in
expression vectors. These expression vectors can be plasmids, YACs, cosmids,
retroviruses, episomes derived from EBV, and all the vectors that a person skilled in
the art may judge to be suitable for expression of said chains.
These vectors can be used for transforming cells advantageously derived from one
cell line. Said cell line is even more advantageously derived from a mammal.
It is advantageously the CHO line or a line derived from this line, or the HEK293 line
or a line derived from this line.
The transformation of the cells can be carried out by any method known by a person
skilled in the art for introducing polynucleotides into a host cell. Said method can be
transformation by means of dextran, precipitation by calcium phosphate, transfection

by means of polybrene, protoplast fusion, electroporation, encapsulation of the
polynucleotides in liposomes, biolistic injection and direct micro-injection of DNA into
the nucleus.
The antibody according to the invention can be included in pharmaceutical
compositions with a view to administration by the topical, oral, parenteral, intranasal,
intravenous, intramuscular, subcutaneous, intraocular or other routes. Preferably, the
pharmaceutical compositions contain pharmaceutically acceptable vehicles for an
injectable formulation. These can be in particular sterile, isotonic saline solutions
(monosodium or disodium phosphate, sodium, potassium, calcium or magnesium
chloride, etc., or mixtures of said salts), or dry compositions, notably lyophilized,
which, by adding sterilized water or physiological serum as appropriate, permit
injectable solutes to be constituted.
As an example, a pharmaceutical composition comprises (1) a Dulbecco phosphate
buffer (pH ~ 7.4), optionally containing 1 mg/ml to 25 mg/ml of human serum albumin,
(2) 0.9% w/v of sodium chloride (NaCI), and (3) 5% (w/v) of dextrose. It can also
comprise an antioxidant such as tryptamine and a stabilizer such as Tween 20.
The pathologies in question can be any diseases associated with the deposition of
amyloid plaques. In particular, the pathology in question is Alzheimer's disease.
The doses depend on the desired effect, the duration of the treatment and the route of
administration used; they are generally between 5 mg and 1000 mg of antibody per
day for an adult. Generally the doctor will determine the appropriate dosage in relation
to the stage of the disease, the patient's age and weight, or any other patient-related
factor that has to be taken into account.
The present invention is illustrated, but is not limited, by the examples given below.

Brief description of the drawings:
Fig. 1 A: Map of the plasmid pXL4973 permitting expression of the light chain LC1 of
the antiAbeta antibody 13C3-VH1VL1.
Fig. 1B: Map of the plasmid pXL4979 permitting expression of the heavy chain HC1
of the antiAbeta antibody 13C3-VH1VL1.
Fig. 2A and 2B: Separation of the protofibrils and of the low-molecular-weight
oligomers by gei filtration on Superdex 75 (at t=0 and at t=16h respectively).
Fig. 3: Determination of the molecular weight of the protofibrils.
Fig. 4A, 4B and 4C: Determination of the affinities of the humanized antibodies
(antibodies LP09027 (4A), LP09026 (4B) and LP09028 (4C) respectively) for the
protofibrils (mean value from 3 experiments ± sem).
Fig. 5: Specificity of the humanized antibody LP09027 with respect to fibrils of A(3.
Fig. 6A and 6B: Specificity of the humanized antibody (LP09027) for the mature
senile plaques respectively of the frontal cortex (6A) and of the hippocampus (6B) of a
mouse. The arrows indicate the senile plaques.
EXAMPLES:
Example 1: Obtaining humanized antibodies
A murine antibody 13C3 was humanized.
This example describes the sequence and the production of the humanized
anti-peptide AfJ antibody VH1VL1 (LP09027) by production by transient expression in
the mammalian line HEK293 designated FreeStyie 293-F.
The cDNAs coding for the humanized variable chains VL1 and VH1 are fused
with the cDNAs coding for the human constant regions Ckappa and lgG4 respectively.
The sequence of the constant region lgG4 is that of the variant having the
substitutions S241P and L248E in Kabat's nomenclature, for a significant reduction in
the production of half-molecules (Angla et al., 1993, Mol. Immunol., 30: 105-108) and
the effector functions (WO 97/09351).

The nucleic acid sequences coding for CH1 (SEQ ID NO 1) and for CL1 (SEQ
ID NO 3) were cloned independently in the expression vector to generate the
plasmids pXL4973 (Fig. 1A) and pXL4979 (Fig. 1B), respectively.
A batch of the antibody is produced by production by transient expression in
the line Freestyle 293-F (Invitrogen) after co-transfection of the plasmids pXL4973
and pXL4979 according to the protocol described by Invitrogen (catalogue reference
K9000-01). This batch (LP09027) is then purified by affinity chromatography on a
column of MabSelect gel (Amersham) according to the supplier's recommendations
and then formulated in PBS buffer (reference Dulbecco 14190-094) and submitted to
sterile filtration (0.2 urn). Starting from 1 L of culture, 33 mg of antibody is obtained at
a purity of 97% by SDS-PAGE in denaturing conditions and by steric exclusion
chromatography. The mass obtained by SDS-PAGE in denaturing conditions and by
LC/MS is in agreement with the primary amino acid sequence and the presence of an
N-glycan on the Fc domain, namely a mass of 23969 Da for LC1 and 49650 Da for
HC1 taking into account the N-glycan in the GOF form. The mass obtained by SDS-
PAGE in non-denaturing conditions and by size exclusion chromatography is in
agreement with the hetero-tetrameric structure of the antibody of 150 kDa (Fig. 4A).
According to the same method, batches of humanized antibodies LP09026
and LP09028 were produced starting from the nucleotide sequences SEQ ID NO 25
and SEQ ID NO 21 for LP09026 (Fig. 4B), and SEQ ID NO 1 and SEQ ID NO 21 for
LP09028 (Fig. 4C).
Example 2: Preparation of protofibrils from peptide Ap (1-42)
The protofibrils were prepared from the synthetic peptide Ap (1-42) according to the
method described by Johansson et al. (FEBS, 2006, 2618-2630). The lyophilized
peptide (Anaspec reference 24224) is dissolved in 10 mM NaOH at a concentration of
100uM, then stirred for 1 min and incubated on ice for 10 min. The solution of peptide
is then diluted in buffer of 100 mM sodium phosphate, 200 mM NaCI pH=7.4 to a
concentration of 50 uM, then stirred for 1 min. The preparation is incubated overnight
at 37°C for formation of protofibrils and then centrifuged at 17900g for 15 min at 16°C
to remove the insoluble aggregates. To separate the protofibrils from the oligomeric
forms of Ap of low molecular weight, the supernatant is loaded on a Superdex 75 gel
filtration column equilibrated in 50 mM ammonium acetate buffer pH=8.5. The

fractions corresponding to the protofibrils and to the low-molecular-weight oligomers
are collected and stored at 4°C. Fig. 2 shows a typical profile of separation of the
protofibrils. The molecular weight of the protofibrils is determined by Superdex200 gel
filtration using, as markers of molecular weight, the Biorad calibration kit (reference
150-1901). Fig. 3 shows that the molecular weight of the protofibrils is greater than
200 kDa.
Example 3: Specificity and affinity of the humanized antibodies with respect to
the protofibrils
50ul of protofibrils and low-molecular-weight oligomers at a concentration of 1 ug/ml in
PBS (Gibco, reference 70011) are deposited in the wells of an ELISA plate (Nunc,
reference 442404) and incubated overnight at 4°C. After removing the excess antigen,
200 ul of buffer PBS + 5% milk powder (weight/volume) is deposited in each well to
remove the non-specific adsorptions and incubated for 2h at room temperature. The
wells are then washed 4 times with 300 pi of buffer PBS Tween 0.02%. 50 pi of a
primary antibody solution (dilution of 3 in 3 in PBS Tween starting from a
concentration of 100pg/ml for the oligomers and from 25 ug/ml for the protofibrils) is
added to each well and incubated for 1 h at room temperature. The wells are washed 4
times with 300ul of buffer PBS Tween. The secondary anti-Fc human antibody
coupled to peroxidase (Goat Anti Human IgG (Fc) peroxidase conjugated, Pierce,
reference 31413) diluted to 1/10000 in buffer PBS Tween is added to each well and
incubated for 1 h at room temperature. After 4 washings with 300 pi of PBS Tween,
100 pi of TMB (Interchim, reference UP664782) is added to each well and incubated
for about 10 min, then the reaction is stopped with a solution of 1M HCI (Interchim,
reference UPS29590) and the plates are read at an OD measured at a wavelength of
450nm. The EC50 values are determined by BioStat Speed. The results obtained are
presented in Table 1 and in Fig. 4 and show the very high specificity of the antibody
for the protofibrils relative to the low-molecular-weight oligomers (factor of 184).


The lyophilized peptide A(31-42 (Anaspec reference 24224) is dissolved according to
the supplier's recommendations: 40 |jl of 1% NH40H is added to 500 pg of Af31-42.
After complete dissolution, 460 pi of PBS is added to obtain a concentration of
1 mg/ml. Aliquots of 10 pi are prepared and stored at -80°C.
50pl of a solution of peptide AB1-42 at a concentration of 1 ug/ml in carbonate buffer
(NaHC03 0.025 M (Acros Organics, reference 217120010), Na2C03 0.025 M (Acros
Organics, reference 207810010), pH 9.7 is deposited in the wells of an ELISA plate
and incubated overnight at room temperature. As previously, the wells are washed
with buffer PBS Tween, incubated in the presence of buffer PBS + 5% milk powder
(weight/volume) and washed with buffer PBS Tween. The humanized antibody at a
concentration of 0.02 ug/ml is incubated for 1h at room temperature with a
concentration range (starting from 1 pg/ml) of peptides Ap1-28 (Bachem, reference
H7865), AP1-16 (Anaspec, reference 24225), A025-35 (Anaspec, reference 24227),
low-molecular-weight oligomers or protofibrils prepared as described previously. The
antibody/antigen mixture is then deposited in each well and the microtitration plate is
incubated for 1h at room temperature. The free, uncomplexed antibody is determined
according to the same ELISA protocol as described previously. These competitive
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 peptide A01-42; none of the peptides is capable
of neutralizing the antibody.

Example 4: Specificity of the humanized antibody LP09027 with respect to the
fibrils of AB1-42
The peptide AB1-42 (Anaspec, 20276) is dissolved in 200 pi of 10 mM NaOH to a
concentration of 5 mg/ml. The peptide IAPP (Anaspec, 60804) is diluted in 200 (Jl of
50% DMSO to a concentration of 5 mg/ml. 100 pi of each preparation is diluted in
400 pi of PBS 1.25X. The final concentration of the peptides is 1 mg/ml in 500 ul. The
samples are incubated for 72h at 37°C. After incubation, the samples are centrifuged
at 17900g for 30 minutes at 4°C. The supernatant is removed and the pellet is washed
3 times with PBS 1X. After the last washing, the pellet of fibrils is taken up in 150 ul of
PBS. To check for the presence of fibrils of amyloid type, a thioflavin T fluorescence
test (Anaspec, 88306) is carried out. 20 ul of thioflavin T (20 uM final), 10 ul of the
sample and 70 ul of PBS 1X (final volume 100 ul) are mixed in a well of a black plate
(Corning, 3792). The thioflavin T is excited at 450 nm and, in the presence of a
structure of amyloid type, emits fluorescence at 482 nm. 50 pi of fibrils of AB1-42 at
1 pg/ml and IAPP at 0.5 pg/ml are deposited in each well of a microtitration plate. The
ELISA protocol is applied using serial dilutions of the humanized antibody starting
from 10 pg/ml. Fig. 5 shows that the humanized antibody LP09027 specifically
recognizes the fibrils of AB1-42 but not those of IAPP.
Example S: Specificity of the humanized antibody LP09027 for the mature senile
plagues but not for the diffuse plaques
The humanized antibody (LP09027) conjugated with digoxigenin (digoxigenin-3-O-
methylcarbonyl-£-aminocaproic acid-N-hydroxysuccinimide ester: Roche
11333054001; 11418165001) was used in immunohistochemistry (Ventana Robot) on
brain sections from mice APP PS1 (Alzheimer model described by Schmitz C. et al.,
Am. J. Pathol, 2004, 164, 1495-1502) as well as human brain sections (cerebral
cortex) derived from patients with Alzheimer's disease. The samples had been fixed in
formol and embedded in paraffin beforehand.
The results obtained in the mouse (Figs. 6A and 6B) clearly show that the humanized
antibody recognizes exclusively the dense, mature senile plaques, but not the diffuse
deposits of peptide AB.

These data correlate with the properties of this antibody, which is specific for the
protofibrillar Abeta form and so does not recognize the soluble, mono- or oligomeric
forms of this peptide.

Claims
1. Humanized antibody specific for the protofibrillar form of the A-β peptide.
2. Antibody according to Claim 1, characterized in that it binds to the A-β
peptides aggregated in senile plaques and not to the diffuse deposits of A-β
peptides.
3. Antibody according to one of the preceding claims, characterized in that it
comprises at least one CDR encoded by a nucleotide sequence having a
sequence identical to one of the sequences SEQ ID NO: 9, 11, 13, 15, 17 and
19, or by sequences differing respectively by 1, 2, 3, 4 or 5 nucleotides from
these sequences.
4. Antibody according to one of the preceding claims, characterized in that it
comprises at least one CDR having a sequence identical to one of the
sequences SEQ ID NO: 10, 12,14, 16,18 and 20.
5. Antibody according to one of the preceding claims, characterized in that it
comprises at least one CDR whose sequence differs by one to two amino
acids relative to one of the sequences SEQ ID NO: 10, 12, 14, 16, 18 and 20,
inasmuch as the antibody maintains its binding specificity.
6. Antibody according to one of the preceding claims, characterized in that it
comprises the CDRs encoded by the nucleotide sequences SEQ ID NO 9, 11,
13, 15, 17 and 19, or by sequences differing respectively by 1, 2, 3, 4 or 5
nucleotides from these sequences.
7. Antibody according to one of the preceding claims, characterized in that it
comprises the CDRs of sequence 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 CDRs encoded by the nucleotide sequences SEQ ID NO 9, 11,

13, 31, 17 and 19, or by sequences differing respectively by 1, 2, 3, 4 or 5
nucleotides from these sequences.
9. Antibody according to one of the preceding claims, characterized in that it
comprises the CDRs of sequence 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 CDRs encoded by the nucleotide sequences SEQ ID NO 9, 11,
29, 31, 17 and 19, or by sequences differing respectively by 1, 2, 3, 4 or 5
nucleotides from these sequences.
11. Antibody according to one of the preceding claims, characterized in that it
comprises the CDRs of sequence 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 its 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 its 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 its 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 its 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 encoded 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 encoded 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 a 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
and 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. Antibody according to any one of the preceding claims, characterized in that it
induces a reduction of amyloid plaques.
33. Antibody according to any one of the preceding claims, characterized in that its
affinity for the protofibrillar form of peptide A(3 is at least 100 times greater than
its affinity for the 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 neurodegenerative disorders.
35. Use of an antibody according to any one of the preceding claims in the
treatment of Alzheimer's disease.

36. Pharmaceutical composition comprising an antibody according to any one of
Claims 1 to 34 and excipients.
37. Method of treatment of Alzheimer's disease, characterized in that it comprises
the administration of an antibody according to any one of Claims 1 to 34 to a
patient.
38. Cell producing antibodies according to Claim 1 to 34.
39. Method of production of an antibody according to any one of Claims 1 to 34,
characterized in that it comprises the culturing of cells according to Claim 38.
40. Medicinal product comprising an antibody according to any one of Claims 1 to
34.
41. Polynucleotide coding for a polypeptide having at least 80% identity with one
of the sequences SEQ ID NO: 2, 4, 6, 8, 22, 24, 26 or 28.
42. Polynucleotide, characterized in that it has a sequence having at least 80%
identity with one of the sequences SEQ ID NO: 1, 3, 5, 7, 21, 23, 25, or 27.
43. Recombinant vector comprising a nucleic acid according to either one of
Claims 41 and 42.
44. Host cell comprising a vector according to Claim 43.

The present application relates to humanized antibodies
specific to the protofibrillar form of the beta-amyloid peptide, and to the
use of said antibodies in the field of Alzheimer's disease,