WO2013010993A1

WO2013010993A1 - Methods and kits for the diagnosis of alzheimer's disease

Info

Publication number: WO2013010993A1
Application number: PCT/EP2012/063913
Authority: WO
Inventors: Christiane Rose; Bernadette Allinquant; Stéphanie CHASSEIGNEAUX; Katell PEOC'H
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2011-07-15
Filing date: 2012-07-16
Publication date: 2013-01-24

Abstract

The invention relates to methods and kits for the diagnosis of Alzheimer's disease. More particularly, the present invention relates to a method for determining whether a patient has Alzheimer's disease or is at risk of having Alzheimer's disease comprising determining the level of at least two biomarkers in a cerebrospinal fluid sample of said patient wherein said two biomarkers are sAPPα and Aβ1-42.

Description

METHODS AND KITS FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE

FIELD OF THE INVENTION

The invention relates to methods and kits for the diagnosis of Alzheimer's disease. BACKGROUND OF THE INVENTION

Alzheimer's disease is a progressive neurodegenerative disease characterized by progressive memory deficits, impaired cognitive function, altered and inappropriate behavior, and a progressive decline in language function. It is the most prevalent age- related dementia, affecting an estimated 18 million people worldwide, according to the World Health Organization. As medical advances continue to prolong the human lifespan, it is certain that Alzheimer's disease will affect an increasing proportion of the population. There is no cure for Alzheimer's disease, and current therapies provide only temporary and symptomatic relief, while doing little to counteract disease progression.

Pathologically, Alzheimer's disease patients display cortical atrophy, loss of neurons and synapses, and hallmark extracellular senile plaques and intracellular neurofibrillary tangles. Senile (or neuritic) plaques are composed of aggregated amyloid β-peptide (A β), which results from APP (Amyloid Precursor Protein) cleavage, and are found in large numbers in the limbic and association cortices. It is widely hypothesized that the extracellular accumulation of A β contributes to axonal and dendritic injury and subsequent neuronal death. Neurofibrillary tangles consist of pairs of about 10 nm filaments wound into helices (paired helical filaments or PHF). lmmunohistochemical and biochemical analysis of neurofibrillary tangles revealed that they are composed of a hyperphosphorylated form of the microtubule-associated protein tau. These two classical pathological lesions of Alzheimer's disease can occur independently of each other. However, there is growing evidence that the gradual accumulation of A β and A β -associated molecules leads to the formation of neurofibrillary tangles. As such, much research is directed at inhibiting the generation of the amyloid β -peptide. Amyloid β peptide is formed by the amyloidogenic processing of APP. This processing requires cleavage at two distinct sites by the β-secretase and γ-secretase. BACEl (β-site APP cleavage enzyme 1) was identified as the major β-secretase and cleaves APP to release an ecto-domain: βΑΡΡβ, into the extracellular space. The remaining C-terminal fragment undergoes subsequent cleavage by γ-secretase to release Αβ and the APP intracellular C-terminal domain (AICD).

In non-pathological situation, APP is predominantly cleaved by the a-secretase within the Αβ domain, thereby precluding the generation of Αβ. This cleavage releases a soluble form of APP: sAPP a, which seems to have neuroprotective functions. Subsequent cleavage of the 83-amino acid remaining C-terminal fragment (C83) releases p3, which is non-amyloidgenic, and the AICD. The functions of these fragments are not known.

Diagnosis of Alzheimer's disease is based on clinical, neuropsychological and neurobiological criteria.

Cerebrospinal fluid biomarkers presently validated include reduced level of AJ31-42, increased total Tau and phospho Tau and a decrease of the IATI index (ABl-42/Tau) (Ibach et al., 2005; Lewczuk et al, 2010, Gabelle et al, 2010). In addition, Lewczuk et al. demonstrated a key role of sAPPa and βΑΡΡβ as biomarkers of Alzeihmer's disease.

However, it remains a strong need to further validate new biomarkers to improve the diagnosis for atypical patients or to discriminate between other neurodegenerative diseases.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining whether a patient has Alzheimer's disease or is at risk of having Alzheimer's disease comprising determining the level of at least two biomarkers in a cerebrospinal fluid sample of said patient wherein said two biomarkers are sAPPa and AB1-42.

DETAILED DESCRIPTION OF THE INVENTION

Typically, the patient has been previously diagnosed with amnesic complaint or even dementia.

As used herein, the term "sAPPa" corresponds to a 100-120 kDa protein (1-687 amino acids of APP770, 1-668 of APP751, 1-612 of APP695 ), obtained by cleavage of APP with a-secretase between amino acid Lys(16) and Leu(17) of the Αβ region.

As used herein, the term "AB1-42" refers to the amino acid sequence from amino acid 1 to amino acid 42 of the human amyloid B protein.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to Alzheimer's disease, and can mean a patient's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a patient compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. In a particular embodiment, the index ratio AJ31-42/sAPPa is determined and compared to a reference value wherein a difference between said index ratio and said reference value is indicative whether said patient has Alzheimer's disease or is at risk of having Alzheimer's disease. In one embodiment of the present invention, the reference value is derived from the index ratio ABl-42/sAPPa determined in a control sample derived from one or more patients who are substantially healthy (e.g. not been diagnosed for Alzheimer's disease). Such patients who are substantially healthy lack traditional risk factors for Alzheimer's disease. Furthermore, retrospective measurement of the index ratio AJ31-42/sAPPa in properly banked historical patient samples may be used in establishing the reference value. Typically, the index ratio ABl-42/sAPPa in a patient who has Alzheimer's disease or is at risk for Alzheimer's disease is deemed to be lower than the reference value from healthy patients.

Methods for determining the level of a biomarker protein in a fluid sample, such as cerebrospinal fluid sample are well known in the art.

In a particular embodiment, the methods of the invention comprise contacting the cerebrospinal fluid sample with a binding partner capable of selectively interacting with the biomarker protein present in the cerebrospinal fluid sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal. In another embodiment, the binding partner may be an aptamer.

As used herein, the term "antibody" refers to a protein capable of specifically binding an antigen, typically and preferably by binding an epitope or antigenic determinant or said antigen. The term "antibody" also includes recombinant proteins comprising the binding domains, as well as variants and fragments of antibodies. Examples of fragments of antibodies include Fv, Fab, Fab', F(ab')2, dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed from antibody fragments.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV- hybridoma technique (Cole et al. 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies. Antibodies useful in practicing the present invention also include fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the biomarker protein. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

Said antibody is for example an antibody that binds on the cleavage site of sAPPa. In one embodiment, the antibody binds specifically to sAPPa and not to sAPP . Such an antibody is typically an antibody that binds to a fragment of sAPPa that is absent from sAPP , i.e. an antibody that recognizes an epitope located in the C-terminus of sAPPa, between residues 596 and 612 of APP695. Examples of said antibody include, but are not limited to, the monoclonal antibody 6E10, the monoclonal antibody 2B3 and the rabbit antibody 3329 (PMID 9465092).

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. 1997. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance. Examples of suitable labels for this purpose include a chemiluminescent agent, a colorimetric agent, an energy transfer agent, an enzyme, a substrate of an enzymatic reaction, a fluorescent agent, or a radioisotope. The antibody or aptamer may be labelled with a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)). An antibody or aptamer of the invention may be also labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88. Examples of chemiluminescent agent include an enzyme that produces a chemiluminescent signal in the presence of a substrate(s) that produce chemiluminescent energy when reacted with the enzyme. Examples of such an enzyme include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other examples of a chemiluminescent agent include a non- enzymatic direct chemiluminescent label, such as Acrinidium ester system. Examples of a colorimetric agent include an enzyme such as horseradish peroxidase, alkaline phosphatase, and acetylcholine esterase (AChE). Examples of energy transfer agent include fluorescent lanthanide chelates. Examples of fluorescent agents include fluorescent dyes. Examples of radioisotopes include ¹²⁵I, ¹⁴C and ³H.

The aforementioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

In another embodiment of the invention, the detection or quantification of the biomarkers in the sample may be achieved by a cytometric bead array system wherein the antibodies that bind to the biomarkers are coated directly or indirectly on beads.

The level of biomarker protein may be measured by using standard immuno diagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme- labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said biomarker protein. A cerebrospinal fluid sample containing or suspected of containing said biomarker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Measuring the level of the biomarker protein (with or without immunoassay-based methods) may also include separation of the compounds: centrifugation based on the compound's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, said biomarker protein may be identified based on the known "separation profile" e. g., retention time, for that compound and measured using standard techniques.

Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer.

In another embodiment of the invention, the quantification of the biomarkers in the sample may be achieved by homogeneous time resolved fluorescence (HTRF). For example, a first antibody directed to an epitope in the N-terminal domain of sAPPa is coupled with a donor fluorophore, such as Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate), and a second antibody directed to the cleavage site of sAPPa is coupled with an acceptor such as XL665, a modified allophycocyanin, or D2 which represents a second generation of acceptor characterized by an organic structure 100 times smaller.

In said embodiment, said first antibody may be the 22C11 clone (ref MAB348 Chemicon) and said second antibody may be the 6E10 clone (ref MAB 1560 Chemicon) or the 2B3 antibody (IBL). In an alternative embodiment, the antibody directed to an epitope in the N-terminal domain of sAPPa is coupled with an acceptor and the antibody directed to the cleavage site of sAPPa is coupled with a donor fluorophore.

When these two fluorophores are brought together by a biomolecular interaction, i.e. interaction between antibodies anti-sAPPa and sAPPa present in the sample, a portion of the energy captured by the donor fluorophore during excitation is released through fluorescence emission at 620nm, while the remaining energy is transfered to the acceptor. This energy is then released by the acceptor as specific fluorescence at 665 nm.

Typically, the level of sAPPa in the sample may be determined homogeneous time resolved fluorescence according to the methods described in the international patent application WO2010046332.

The method of the present invention is also particularly suitable for differential diagnosis of Alzheimer's disease and other neurodegenerative diseases such as Taupathies, Lewy bodies, or vascular dementia.

Another object of the invention is a method for monitoring Alzheimer's disease in a patient in need thereof, said method comprising the step consisting of determining the level of at least two biomarker in a cerebrospinal fluid sample of said patient wherein said two biomarkers are sAPPa and AB1-42.

Said method is particularly suitable for monitoring a patient having a treatment for Alzheimer's disease.

The present invention also relates to a method for confirming the diagnosis of Alzheimer's disease in a patient having amnesic complaint or even dementia but having high level of AJ31-42 (i.e. atypical level of AJ31-42) in his cerebrospinal fluid, said method comprising determining the level of sAPPa in a cerebrospinal fluid sample of said patient.

Typically, said method includes determination of the index ratio AJ31-42/sAPPa and comparison with a reference value as described above. Another object of the invention is a kit for use in the method of the invention as described here above, said kit comprising, means for determining the level of sAPPa and AB1-42 in a cerebrospinal fluid sample.

Typically, said kit comprises as separate components at least one antibody (or aptamer) that binds to sAPPa and at least one antibody (or aptamer) that binds to AJ31-42. Suitable antibodies are similarly identified here above.

In one embodiment, the antibodies may be coated to a solid support. Suitable examples of solid support are identified here above.

In another embodiment, one or more of the antibodies may be labelled. Suitable examples of labels are similarly identified here above.

The kit may also contain optional additional components for performing the method of the invention. Such optional components are for example containers, mixers, buffers, instructions for assay performance, labels, supports, and reagents necessary to couple the antibody to the support or label.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: description of the antibodies used for determined the level of sAPPa with the HTRF assay.

Figure 2: Correlation between sAPPa and Αβ 1-42. Figure 3: Patients with Atypical Αβ 1-42 levels

EXAMPLES:

Methods: Patients: All patients with a amnesic complaint from consultation of neurology or geriatry (Lariboisiere's hospital, Paris). Patients were classified in Lariboisiere's hospital as Alzheimer's disease (D-AD) and other dementias (D-O) according to clinical, neuropsychological, and neurobiological criteria. The population of patients includes:

- 47 D-O: 27 men and 20 women Age: 71.7 + 1.7

- 50 D-AD: 20 men and 30 women Age: 71.9 + 1.4

Samples: Cerebrospinal fluid (CSF) was collected in polypropylene tubes. Within 2h, samples were centrifuged at 1800 g for 10 min at 4°C, aliquoted and stored at -80°C. CSF AB 42, total tau and phosphotau 181 were measured with Innotest sandwich ELISA. sAPPa assay was performed with a HTRF method as described in the International Patent Application WO2010046332 without knowing the diagnosis. Briefly, the antibody 22C11 is labeled with the donor Cryptate and the antibody 6E10 is labeled with the acceptor D2 (Figure 1). Specific signal at 665nm (i.e energy transfer) is proportional to the concentration of sAPP a.

Results:

In CSFs of D-AD patients, we observe a significant higher sAPP a level as reported by two others using multiplexing method based on electrochemilummescence (Lewczuk et al. 2010 and Gabelle et al. 2010) (Table 1).

Table 1:

Biomarker D-O D-AD

MeaniSEM MeaniSEM

(N=47) (N=50)

SAPPa (ng/ml) 371.0±22.4 501.5±25.7 (+35%)*

AB1-42 (pg/ml) 643±36.3 442.8±29.0 (-32%) ** Tau (pg/ml) 247.4±25.0 565.0±37.2 (+128%)**

TauP (pg/ml) 48.7±3.1 99.2±5.9(+103%)**

*:p<0.001; p<0.0001 sAPPa is equivalent to Αβ 1-42 in term of sensitivity (Table 2).

Table 2:

A strong decrease of the ratio ΑβΙ-42/sAPPa is observed in the D-AD group as for the IATI index (ΑβΙ-42/Tau) (Table 3).

Table 3:

* p<O.O00i

The two indexes display similar accuracy and could be complementary for the diagnosis (Table 4).

Table 4 index AUC±SE (%) Sensitivity Specificity Cutoff

(%) (%)

IATI 0.89±0.03 92 80 0.86

AB1-42 / 0.86±0.03 70 96 0.97 sAPPa

A significant correlation between sAPPa and Αβ1-42 is observed only in the D-0 group (Figure 2). No correlation between sAPPa and Tau or TauP is observed in any group.

Among all our population we secondly categorized 19 "atypical Αβ levels" patients:

8 D-0 : (according to clinical and neuropsychological criteria but with low CSF AB levels ): 4 men and 4 women Age:71.3 + 3.9

1 1 D-AD: (according to clinical and neuropsychological criteria but with high CSF AB levels ): 4 men and 7 women Age: 73.5 +2.4

Among our population, about 20 % of patient in each group (D-0 and D-AD) exhibit a level of Αβ1-42, opposite to its level in clear diagnosed patients (Table 5):

Patients classified D-Oa have a concentration of Αβ1-42 < to the cutoff as for

D-AD patients

Patients classified D-ADa have a concentration of AB 1-42 > to the cutoff as for D-0 patients

Table 5

Biomarker D-Oa D-ADa

MeaniSEM MeaniSEM

(N=8) (N=ll) sAPPa (ng/ml) 225±32.3 (cutoff:<372) 491.2±57.3 (cutoff:>372)

AB1-42 (pg/ml) 298.1±16.0 icutoff:>474) 777.0±49.2 icutoff:<474)

Tau (pg/ml) 211.8±32.7 (cutoff:<329) 542.8±84.5 (cutoff:>329)

TauP (pg/ml) 37.0±5.7 (cutoff:<69) 86.9±10.0 (cutoff:>69) In such a case, sAPPa associated to Tau and TauP could be an interesting biomarker to strengthen the diagnosis.

In such artificially constituted groups, the sole Αβ biomarker would lead to misdiagnosis. However, the new proposed sAPPa biomarker strengthens the biological diagnosis based on Tau and TauP levels.

The shrinkage of patients with atypical levels of Αβ lead to a significant positive correlation between sAPPa and Αβ1-42 in both D-AD and D-0 group (Figure 3). The selection of patients with atypical levels of Αβ lead to a roughly negative correlation between sAPPa and Αβ1-42 in D-ADa group (Figure 3).

Conclusions:

In this work, we compare a selected population of AD patients (based on clinical, neuropsychological and biological criteria) to a non-AD dementia group. We observe a significant higher level of sAPPa in the DAD group, and we propose the Αβ/sAPPa ratio as a new index for the biological diagnosis, complementary to the IATI already used.

In the case of an atypical Αβ 1-42 CSF level, the sAPPa evaluation could strengthen the biological diagnosis.

Even if present biomarkers can discriminate between stable MCI and progressive MCI to AD, it remains uncertainties in diagnosis, particularly to discriminate between AD and other neurodegenerative diseases (Taupathies, Lewy bodies, vascular dementia). The sAPPa and the Αβ/sAPPa ratio should be interesting in these cases.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

1. A method for determining whether a patient has Alzheimer's disease or is at risk of having Alzheimer's disease comprising determining the level of at least two biomarkers in a cerebrospinal fluid sample of said patient wherein said two biomarkers are sAPPa and AB1-42.

2. The method according to claim 1 wherein the index ratio AJ31-42/sAPPa is determined and compared to a reference value wherein a difference between said index ratio and said reference level is indicative whether said patient has Alzheimer's disease or is at risk of having Alzheimer's disease.

3. The method according to claim 1 or 2 wherein the level of biomarkers in the cerebrospinal fluid sample of said patient is determined with at least one antibody that binds to AJ31-42 and with at least one antibody that binds to sAPPa.

4. The method according to any one of the preceding claims wherein the level of sAPPa is determined with a method involving homogeneous time resolved fluorescence (HTRF).

5. A kit comprising as separate components at least one antibody that binds to sAPPa and at least one antibody that binds to AB1-42.