p53 peptide as a marker in the diagnosis and prognosis of Alzheimer's disease

Abstract

Disclosed are p53 peptides and their use as biomarkers in the diagnosis and / or prognosis of Alzheimer's disease (AD) in biological samples. The present invention also provides a diagnostic method, which is based on highly accurate mass spectrometry analysis, by specifically quantifying the level of the p53 peptide in the human plasma of the patient, for the diagnosis of Alzheimer's disease in the preclinical and prodromal stages of the disease, and for the prognosis of cognitive decline in the subject.

Description

Field of the Invention

[0001] The present invention relates to p53 peptides P1, P2, P3 and P5, and their use as biomarkers in the diagnosis and / or prognosis of Alzheimer's disease (AD) in biological samples. The present invention also provides a diagnostic method, which is based on highly accurate mass spectrometry analysis, by specifically quantifying the level of the p53 peptide in the human plasma of the patient, for the diagnosis of the disease in the preclinical and prodromal stages of Alzheimer's disease, and for the prognosis of cognitive decline in the subject. Background Art

[0002] The presence of numerous conformationally altered p53 isoforms has been demonstrated in different published studies [1-3] to be an early risk factor for Alzheimer's disease (abbreviated as "AD"). Initially, more than 400 subjects with AD, mild cognitive impairment, Parkinson's disease, other dementias and healthy subjects were enrolled in different independent studies and tested for unfolded p53 by different techniques (immunoprecipitation experiments, FACS analysis, ELISA) using commercial conformation-specific anti-p53 antibodies [4-7]. In 2006, Uberti et al. [8] first demonstrated that fibroblasts from sporadic Alzheimer's disease (AD) patients specifically expressed an abnormal and detectable p53 conformational state that distinguished these cells from fibroblasts of age-matched non-AD subjects. In this conformationally altered state, p53 loses its ability to transactivate its target genes and, therefore, loses its biological function [9-10]. Higher amounts of unfolded p53 have also been demonstrated in the blood of AD and in MCI converting to AD compared to healthy non-demented subjects or patients affected by other dementias and PD.

[0003] Together, these data suggest a direct link between unfolded p53 and AD pathology.

[0004] In EP3201234B1, the development of a new conformation-specific anti-Up53 antibody named 2D3A8 has been reported, which binds to an epitope (aa 282-297) that is only accessible when p53 loses its wild-type conformation towards an unfolded phenotype. Compared to the commercial antibody (PAb240, aa214-217) used when unfolded p53 was first discovered in AD, the 2D3A8 antibody showed high sensitivity and specificity in identifying AD patients compared to healthy elderly people in the Oviedo cohort.

[0005] In particular, the immunodiagnostic method is able to identify immune complexes indicative of AD in biological samples and determine the susceptibility of a subject affected by mild cognitive impairment (MCI) to develop AD.

[0006] There is an ongoing need to identify new specific biomarkers that can be used for the diagnosis and / or prognosis of Alzheimer's disease, and to develop accurate and rational diagnostic methods that can be used for the diagnosis and / or prognosis of AD, particularly in the preclinical and prodromal stages of the disease, and for the differential analysis of AD from other forms of dementia. SUMMARY OF THE INVENTION

[0008] This object was achieved by identifying in biological samples four p53 peptides called P1 (SEQ ID N.1), P2 (SEQ ID N.2), P3 (SEQ ID N.3) and P5 (SEQ ID N.5) having the sequences reported in the claims.

[0009] Another aspect of the present invention relates to a diagnostic method based on the identification and quantification of said peptides for use in the diagnosis and / or prognosis of Alzheimer's disease at different stages.

[0010] Features and advantages of the present invention will become apparent from the following detailed description and working examples provided for illustrative purposes. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention therefore relates to a highly accurate mass spectrometric method for the diagnosis of Alzheimer's disease in the preclinical and prodromal stages of the disease. The method is based on the identification and quantification of levels of specific p53 peptides, referred to as "P1", "P2", "P3" and "P5" or "p53 peptides" for short, which have been detected by mass spectrometric analysis in human plasma of patients affected by Alzheimer's disease or with symptoms that may predispose them to the development of AD.

[0013] In particular, mass spectrometry analysis is a qualitative and quantitative method performed when information about the exact correlation between these specific sequences of p53 and AD is unknown.

[0014] First, p53 peptides present in the plasma of patients affected by AD have been identified by deep sequencing protein methods in plasma from patients in the preclinical, prodromal phases of Alzheimer's disease, stable MCI patients, and cognitively normal subjects. The levels of p53 peptides in plasma were then quantified using a highly sensitive Selected Reaction Monitoring (SRM) mass spectrometry method, which yielded the area under the ROC curve (AUC) (wherein "ROC curve" means "receiver operating characteristic curve").

[0015] The data thus obtained show strong evidence that p53 peptides can be considered as biomarkers in the diagnosis and / or prognosis of AD.

[0016] Advantageously, the method is rapid, requires small volumes of plasma samples, and quantifies the concentration of p53 peptide in each sample analyzed.

[0017] Furthermore, the methods and identified biomarkers may also be used for the diagnosis of Alzheimer's disease in asymptomatic individuals and in people suffering from MCI, allowing entry into the diagnostics market.

[0018] Furthermore, since the biomarker can be used for prognosis of cognitive decline to Alzheimer's Dementia in asymptomatic and MCI subjects, the method allows the use of p53 peptide expression to select subjects in clinical trials to ensure trial success and distinguish patients affected by AD from other forms of dementia.

[0019] Therefore, an embodiment of the present invention is a p53 peptide consisting of formula (I) P1:TEEENLR (SEQ ID N.1). Optionally, the p53 peptide of the present invention has an amino acid sequence that is at least 80%-90% identical to the sequence of formula (I), preferably at least 90%-95% identical to the sequence of formula (I), and more preferably at least 96%-99% identical to the sequence of formula (I).

[0020] Another embodiment of the present invention is a p53 peptide having formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is a moiety derived from the ubiquitin residue tail of the p53 protein after post-translational modification at K291. In this regard, for the purposes of the present invention, the term "[GG]" in square brackets indicates that the first K amino acid of the P2 sequence is branched by said moiety.

[0021] Another embodiment of the present invention is a p53 peptide consisting of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is as defined above. Optionally, the p53 peptide of the present invention has an amino acid sequence that is at least 80%-90% identical to the sequence of formula (II), preferably at least 90%-95% identical to the sequence of formula (II), and more preferably at least 96%-99% identical to the sequence of formula (II).

[0022] Another embodiment is the use of the p53 peptides P1 and / or P2 according to the invention as in vitro biomarkers for the diagnosis and / or prognosis of Alzheimer's disease.

[0023] Another embodiment of the present invention is a p53 peptide having the formula (III) P3: KKPLDGEYFTLQIR (SEQ ID N. 3).

[0024] Another embodiment of the present invention is a p53 peptide consisting of formula (III) P3: KKPLDGEYFTLQIR (SEQ ID N.3).

[0025] Another embodiment of the present invention is a p53 peptide having the formula (V) P5: GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ ID N.5).

[0026] Another embodiment of the present invention is a p53 peptide consisting of (V)P5:GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ IDN.5).

[0027] Another embodiment is the use of the p53 peptides P3 and / or P5 according to the invention as in vitro biomarkers for the diagnosis of Alzheimer's disease.

[0028] Another embodiment of the present invention is an in vitro or ex vivo method for the diagnosis and / or prognosis of Alzheimer's disease, the method comprising the following steps:

[0029] a) determining the presence of p53 peptide in a biological sample, and

[0030] b) Quantification of the peptide by comparing it to a given internal control.

[0031] In a preferred embodiment, the p53 peptide in step a) is p53 peptide P1 or P2.

[0032] In a preferred embodiment, the method of the invention further comprises the step c) correlating the amount of p53 peptide with the diagnosis of Alzheimer's disease in patients at different stages of the disease.

[0033] Preferably, the biological sample is blood, plasma, serum, saliva, urine, neuronal cells, blood cells or other types of cells.

[0034] In a preferred embodiment, in the method according to the invention, step a) is performed by immunoprecipitating the p53 peptide using a monoclonal antibody that binds to the p53 peptide.

[0035] Preferably, the monoclonal antibody is antibody 2D3A8.

[0036] In a preferred embodiment of the method according to the invention, step b) is performed by mass spectrometry, preferably by HPLC-mass spectrometry.

[0037] In another preferred embodiment, in step b), a labeled peptide is used as an internal control.

[0038] According to a preferred embodiment, the in vitro or ex vivo method of the present invention comprises the following steps:

[0039] a) determining the presence of p53 peptide in a biological sample by (i), (ii), (iii), (iv) as follows:

[0040] (i) providing biological samples;

[0041] (ii) protein immunoprecipitation using antibodies that bind to p53 peptide;

[0042] (iii) protein fragmentation by trypsin;

[0043] (iv) performing strong cation exchange chromatography of the peptide;

[0044] and

[0045] b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v)

[0046] (v) Selective reaction monitoring analysis was performed to identify and quantify p53 peptides by comparing the p53 peptides to a given control.

[0047] Preferably, the antibody of step a)(ii) is 2D3A8.

[0048] Preferably, the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatography columns prior to carrying out step a)(ii).

[0049] According to a preferred embodiment, the in vitro or ex vivo method of the invention is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and in humans suffering from MCI.

[0050] Preferably, the amount of p53 peptide is from 0.05 fmol / 40 μl to 6.70 fmol / 40 μl in asymptomatic individuals and humans suffering from MCI.

[0051] According to another preferred embodiment, the in vitro or ex vivo method of the invention is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and in humans suffering from MCI.

[0052] Preferably, the amount of p53 peptide is from 0.203 fmol / 40 μl to 6.70 fmol / 40 μl in asymptomatic and MCI subjects with a prognosis of cognitive decline of Alzheimer's dementia.

[0053] Preferably, in asymptomatic individuals, the AUC is at least 80%.

[0054] Preferably, in humans suffering from MCI, the AUC is at least 90%.

[0055] Measures of accuracy of diagnostic / prognostic methods include sensitivity and specificity. Sensitivity refers to the probability of a positive test result occurring in those with the target condition, and is measured by the following formula: Sensitivity = True Positive / (True Positive + False Negative). Specificity is the probability of a negative test result occurring in those without the target condition, and is measured by the following formula: Specificity = True Negative / (True Negative + False Positive).

[0056] Preferably, in asymptomatic individuals the sensitivity of the p53 peptide is at least 70%.

[0057] Preferably, the sensitivity of the p53 peptide in humans suffering from MCI is at least 90%.

[0058] Preferably, the specificity of the p53 peptide in asymptomatic individuals is at least 90%.

[0059] Preferably, the specificity of the p53 peptide in humans suffering from MCI is at least 90%.

[0060] In another preferred embodiment, the in vitro or ex vivo method of the present invention is used for differential analysis of Alzheimer's disease from other dementias selected from the group consisting of frontotemporal dementia (FTD), Lewy bodies, Parkinson's disease and vascular dementia.

[0061] Preferably, in patients affected by Alzheimer's disease, the amount of p53 peptide is between 2.21 fmol / 40 μl and 6.56 fmol / 40 μl, whereas in patients affected by other forms of dementia, the amount of p53 is below 3.81 fmol / 40 μl.

[0062] Preferably, in patients affected by Alzheimer's disease, the AUC is at least 85%.

[0063] Preferably, the sensitivity of the p53 peptide in patients affected by Alzheimer's disease is at least 75%.

[0064] Preferably, the specificity of the p53 peptide in patients affected by Alzheimer's disease is at least 80%.

[0065] Another embodiment of the present invention is a method for detecting p53 peptide as described above, said method comprising the following steps:

[0066] a) determining the presence of p53 peptide in a biological sample, and

[0067] b) quantifying the peptide by comparing it to a given control.

[0068] In another preferred embodiment, the method of the present invention further comprises step c): correlating the amount of p53 peptide with the diagnosis and / or prognosis of Alzheimer's disease.

[0069] According to a preferred embodiment, in the method of the invention, step c) correlates the amount of p53 peptide with the diagnosis of Alzheimer's disease in asymptomatic individuals and in humans suffering from MCI.

[0070] In a preferred embodiment, the p53 peptide in step a) is p53 peptide P1 or P2.

[0071] According to another preferred embodiment, in the method of the present invention, the amount of p53 peptide in step c) is correlated with the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and in humans suffering from MCI.

[0072] More preferably, in the method of the present invention, the amount of p53 peptide in step c) is correlated with differential analysis of Alzheimer's disease from other dementias selected from frontotemporal dementia (FTD), Lewy bodies, Parkinson's disease and vascular dementia.

[0073] It should also be understood that all combinations of preferred aspects of the peptides of the invention as reported above, as well as methods for their preparation and methods for their use are to be considered disclosed herein.

[0074] All combinations of preferred aspects of the peptides of the present invention, methods of preparation and processes disclosed above are understood to be described herein.

[0075] The following are working examples of the present invention provided for illustrative purposes. Example

[0076] Materials and methods

[0077] Material

[0078] Antibody-based plasma / serum depletion IgY14 LC10 column was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Trypsin (sequencing grade modified porcine trypsin) was obtained from Promega. Acetonitrile was purchased from JT Baker, and formic acid was obtained from EMD Millipore (Billerica, MA, USA). C18 cartridges for sample preparation, chromatographic columns for bRPLC, and online HPLC for triple quadrupole mass spectrometers were purchased from Waters (Milford, Massachusetts). Plasma dilution buffer, -plasma wash buffer, and -plasma elution buffer were prepared following the standard protocol for the method. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich (St. Louis, Missouri).

[0079] Plasma sample consumption

[0080] Seppro IgY14 columns were used to deplete the most abundant proteins in plasma. Plasma samples were diluted 5X in plasma dilution buffer, filtered (0.22 μm), and then injected into an IgY LC10 column attached to an Agilent 1200HPLC system. Unretained fractions were collected. Highly abundant proteins were eluted with plasma elution buffer.

[0081] Immunoprecipitation (IP)

[0082] Antibody clone 2D3A8 is used for IP reaction. The conjugation of antibody to beads is optimized and carried out using a connection buffer. Monoclonal antibodies are added directly to protein G Dynal magnetic beads (obtained from Invitrogen), and the antibody is bound to the beads in the buffer on a rotator at room temperature for 2h. The beads bound to the antibody are then washed by incubation in 50mL Link-A buffer and collected on a magnet. The antibody is cross-linked to the protein G on the beads by incubating with 50mL B buffer on a rotator at room temperature for 1h. The beads are then washed twice with 50mC buffer, resuspended in 50mC buffer, and rotated at room temperature for 15min. The beads are resuspended in C buffer and stored at -20°C until application. 200μl of consumed or unconsumed plasma samples are used for IP reactions, each sample is reconstituted into a 3ml system with IP buffer, and 3ml of beads conjugated to the antibody (1:1 volume ratio) are added to the system. The IP system is incubated at 4°C for 18 hours at a speed of 32 rpm on a rotator. The beads were collected on a magnet and washed 3 times with IP-wash buffer at 4° C., and the antigen was then eluted with IP-elution buffer. The flow-through fractions were collected for protein sample preparation.

[0083] Protein preparation

[0084] Protein samples were processed according to the "filter-assisted sample preparation" (FASP) method (Nat Methods. 2009, 6: 359-362). In short, protein samples in 9M urea were reduced with 5mM TCEP at 37°C for 45min, and reduced cysteine ​​was blocked with 50mM iodoacetamide at 25°C for 15min. Protein samples (100 μg each) were then washed three times with 9M urea using a 10kDa Amicon filter (UFC501096, Millipore), and washed twice with buffer 1. Samples were then proteolyzed with trypsin (V5111, Promega) at 37°C for 12 hours. The peptide solution was then acidified by adding 1% trifluoroacetic acid (TFA), and incubated at room temperature for 15min. Sep-Pak light C18 boxes (Waters Corporation) were activated by loading 5mL 100% (vol / vol) acetonitrile, and washed twice by 3.5mL 0.1% TFA solution.

[0085] The acidified digested peptide solution was centrifuged at 1,800 × g for 5 min, and the supernatant was loaded into the cartridge. To desalt the peptide bound to the cartridge, 1 mL, 3 mL, and 4 mL of 0.1% TFA were used in sequence. To elute the peptide from the cartridge, 2 mL of 40% (vol / vol) acetonitrile with 0.1% TFA was used, and the elution was repeated twice more (a total of 6 mL of eluent). It was important to ensure that the cartridge had stopped dripping before applying each sequential wash and elution solution. The eluted peptide was lyophilized overnight and reconstituted in 100 μL of buffer 2.

[0086] Strong cation exchange chromatography

[0087] The peptides were fractionated by strong cation exchange (SCX) chromatography. Briefly, the lyophilized peptide mixture was resuspended in 1 ml of SCX solvent A (5 mM KH 2 PO 4 The precipitate was resuspended in 5% ACN (pH 2.7) and purified by SCX chromatography on an Agilent 1290 Infinity II LC system in PolySULPHOETHYL A (5 μm, ) column (200×9.4 mm; PolyLC Inc., Columbia, MD) by using SCX solvent B (5 mM KH 2 PO 4The fractions were fractionated by increasing gradients of 1% HPLC (pH 2.7, 30% ACN, 350 mM KCl). For each experiment, a total of 96 fractions were initially collected, which were then pooled into 24 fractions and dried using a SpeedVac (Eppendorf). bRPLC and SCX were performed in parallel to compare the compatibility of TP53 protein detection, and SCX was chosen because relatively higher yields of TP53 isoforms were repeatedly observed in this project.

[0088] Optimization of peptide SRM transitions

[0089] Four peptides from the targeted TP53 protein were selected as SRM quantitative targets, and 1092 sets of transition parameters and retention times of the four peptides were determined individually using an Agilent 6495 triple quadrupole mass spectrometer for precursor ions with 1+, 2+, 3+, and 4+ charges. Two peptides (P1 and P2) were then selected as the main peptides associated with AD pathology.

[0090] Selected Reaction Monitoring (SRM) Analysis

[0091] 37ul of peptide samples reconstituted in buffer 3 were spiked with an SRM internal control mixture consisting of a pool of 1 femtomolar heavy isotope-labeled peptides covering a large hydrophobicity window and a large M / z range (M / z 200-1300), and the peptide samples were eluted into the Jet ESI source of an Agilent 6495 triple quadrupole mass spectrometer via an online Agilent 1290 HPLC system.

[0092] result

[0093] In a previous project, five peptides of p53 protein were cumulatively detected in plasma [P1: TEEENLR (SEQ ID N.1); P2: TEEENLRK[GG]K (SEQ ID N.2), P3: KKPLDGEYFTLQIR (SEQ ID N.3), P4: EPGGSRAHSSHLK (SEQ ID N.4) and P5: GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ ID N.5)]. SRM detection methods for four peptides were determined. The peptide GEPHHELPPGSTKRALPNNTSSSPQPK was not suitable for SRM-based detection due to its high molecular weight.

[0094] Correlation between p53 sequence peptides and AD diagnosis.

[0095] Example 1. Detection of p53 peptide markers of Alzheimer's disease in asymptomatic (cognitively normal) and MCI stages by deep sequencing protein approach.

[0096] Human plasma immunoprecipitated with conformation-specific Ab 2D3A8 was subjected to deep sequence mass spectrometry analysis. Plasma samples were depleted of abundant proteins according to the method reported above. The samples were then immunoprecipitated by 2D3A8 and analyzed using Orbitrap (deep sequence mass spectrometry). The resulting peptides that are uniquely present in AD, MCI to AD, and asymptomatic to AD and are absent in asymptomatic and MCI stable samples were selected as markers. The detection (presence / absence) of the peptides by this method is related to the clinical diagnosis of the subject.

[0097] The following were analyzed: a pool of 10 samples from PiB (Pittsburgh compound B) +ve AD (PiB+ve) patients (AD), a pool of 10 baseline plasma samples from asymptomatic PiB-negative individuals who remained both asymptomatic and PiB-ve for at least 108 months after baseline (normal), a pool of 10 baseline plasma samples from PiB+ve patients with MCI 18 months before their conversion to AD symptoms (prodromal, MCI due to AD), 10 baseline plasma samples from PiB-ve patients with MCI (follow-up 18-72 months after baseline) who did not experience further cognitive decline to AD symptoms (MCI stable) and a pool of 3 baseline plasma samples from PiB+ve patients with MCI (preclinical, normal to AD converters) 18 months before their conversion to AD symptoms.

[0098] Table 1. Inclusion criteria for baseline characteristics

[0099] Minimum age f / mmin % Prodromal (MCI to AD) 70 0.40 Preclinical (normal cognition to AD conversion) 70 0.4 MCI stable 70 0.40 Normal cognition 66 0.5 Alzheimer's disease with dementia 67 0.5

[0100] result

[0101] Table 2. Validation of the performance of the p53 marker in the diagnosis of AD in asymptomatic and MCI stages: results by deep sequencing mass spectrometry.

[0102] A transition of N=1 can be interpreted as "possibly negative", a transition of N=2 is possibly positive; a transition of N=0 is "negative", a transition of N=3 is positive.

[0103]

[0104] Example 2 Validation of a human plasma biomarker of p53 conformation specific for Alzheimer's disease in the preclinical and prodromal stages of the disease

[0105] Preliminary results of a four-year longitudinal study investigating the clinical progression of cognitively normal subjects and people with mild cognitive impairment to possible AD by measuring U-p53 signals by direct ELISA showed a very high predictive value for progression. Based on this finding, a highly accurate mass spectrometry method based on the identification of p53 peptides P1 and / or P2 was developed for the diagnosis of Alzheimer's disease in the preclinical and prodromal stages of the disease and for the prognosis of cognitive decline. The maximum prognostic PPV for cognitive decline to Alzheimer's dementia was achieved in both subjects with MCI and asymptomatic subjects. It is believed that both the diagnostic and prognostic performance of p53 peptides P1 and / or P2 are the highest among those reported so far.

[0106] A specific mass spectrometry method, the SRM (selective reaction monitoring) method (by triple quadrupole mass spectrometer) is used to quantify both P1 and / or P2 in plasma samples from AD, asymptomatic, MCI subjects. By means of a triple quadrupole mass spectrometer, which essentially acts as a mass filter, peptides can be sequenced by observing distinct transition peaks. Then, using heavily labeled internal controls of peptide 1 and peptide 2, these peptides can be quantified with maximum accuracy by the selective reaction monitoring (SRM) method.

[0107] Prognosis

[0108] The prognostic ability of the biomarkers was evaluated in enriched patients who were PiB+ and presented cognitive decline within 18 months from MCI subjects and subjects who presented cognitive decline within 18-72 months from asymptomatic at presentation. This was to evaluate the value of the biomarkers in significantly enriched cognitive decline as an endpoint in disease modifying trials in patients who were asymptomatic and had MCI. The prognostic ability of P1 and P2 was examined using future conversion of clinical presentation. The statistical parameters that can describe the prognostic ability of P1 and P2 are reported as follows:

[0109] Table 3. Prognostic ability of asymptomatic subjects to AD diagnosis

[0110] Sequence peptide 1 Sequence peptide 2 Sensitivity 72.2％ 89％ Specificity 95.5％ 100％ PPV (Positive Predictive Value) 70.2％ 100％ NPV (Negative Predictive Value) 99.7％ 99.8％ AUC 83％ 90.7％ P-value 0.0022 0.0020

[0111] Table 4. Prognostic ability of MCI subjects to AD diagnosis

[0112] Sequence peptide 1 Sequence peptide 2 Sensitivity 95％ 100％ Specificity 95％ 100％ PPV (Positive Predictive Value) 88.2％ 100％ NPV (Negative Predictive Value) 98％ 100％ AUC 96.3％ 100％ P-value 0.0082 0.4724

[0113] in conclusion

[0114] P1 and P2 are able to accurately enrich patients who will exhibit cognitive decline from those who are asymptomatic or clinically manifesting MCI with very high specificity and sensitivity, and thereby enable successful recruitment of patients for disease-modifying drugs that target very early stages of the disease.

[0115] Example 3.

[0116] Differential diagnosis of Alzheimer's disease from other forms of dementia

[0117] An analysis was also performed to validate previous data on the specificity of the U-P53 biomarker in Alzheimer's dementia. Amyloid-positive Alzheimer's patients with well-characterized Alzheimer's dementia were compared with patients with clinical manifestations of other dementias such as FTD (frontotemporal dementia), Parkinson's, Lewy bodies, and vascular dementia.

[0118] Plasma samples from OD (FTD, vascular dementia, Parkinson's, Lewy bodies) were collected during the disease phase. In any case, in differential diagnosis, it was possible to determine that above a certain cutoff, both peptide sequences showed high specificity and sensitivity for AD (compared to OD).

[0119] Furthermore, AUCs greater than 90% have been observed for both peptides, showing a high correlation between biomarker concentrations and AD diagnosis.

[0120] In the literature, there is an unmet need in the differential diagnosis of Alzheimer's dementia to differentiate between: a) advanced FTD and advanced AD; b) advanced LBD and AD, as these can mimic the clinical presentation in some cases. Anything that can rule out AD or another dementia is of clinical value, so high sensitivity for either dementia is of value.

[0121] Table 5. Performance measures of p53 peptides PI and P2 in distinguishing Alzheimer's disease from other dementias in patients with dementia.

[0122] Sequence peptide 1 Sequence peptide 2 Sensitivity 100％ 80％ Specificity 85.7％ 85.7％ PPV (Positive Predictive Value) 87.5％ 84.8％ NPV (Negative Predictive Value) 100％ 81.1％ AUC 94.3％ 90.7％ P-value 0.0510 0.0171

[0123] References

[0124] 1. Stanga, S. et al., 2010. Unfolded p53 in the pathogenesis of Alzheimer's disease: Is HIPK2 thelink? Aging, 2(9), pp.545-554.

[0125] 2.Lanni,C.et al.,2007Unfolded p53:A potential biomarker forAlzheimer’s disease.In Journal of Alzheimer’s Disease.pp.93-99.

[0126] 3.Uberti,D.et al.,2008.Conformationally altered p53:a putativeperipheral marker for Alzheimer’s disease.Neuro-degenerative diseases,5(3-4),pp.209-11.

[0127] 4.Lanni,C.et al.,2008.Conformationally altered p53:a novel Alzheimer’s disease marker?Molecular psychiatry,13(6),pp.641-7.

[0128] 5.Lanni,C.,Racchi,M.,et al.,2010.Unfolded p53 in blood as apredictive signature signature of the transition from mild cognitiveimpairment to Alzheimer’s disease.Journal ofAlzheimer’s disease:JAD,20(1),pp.97-104.

[0129] 6.Buizza,L.et al.,2012.Conformational altered p53 as an early markerof oxidative stress in Alzheimer’s disease.PloS one,7(1),p.e29789

[0130] 7.Arce-Varas N, et al.Comparison of extracellular and intracellularblood compartments highlights redox alterations in Alzheimer's and MildCognitive Impairment patients.Current Alzheimer Research2017;14(1):112-122.

[0131] 8. Uberti, D. et al., 2006. Identification of a mutant-like conformation ofp53in fibroblasts from sporadic Alzheimer's disease patients. Neurobiology ofaging, 27(9), pp.1193-201.

[0132] 9.Lanni, C., Nardinocchi, L., et al., 2010. Homeodomain interacting proteinkinase2: a target for Alzheimer's beta amyloid leading to misfolded p53andinappropriate cell survival. PloS one, 5(4), p.e10171.

[0133] 10.Lanni, C. et al., 2008. Pharmacogenetics and Pharmagenomics, Trends in Normal and Pathological Aging Studies: Focus on p53. Current PharmaceuticalDesign, 14(26), pp.2665-2671. Sequence Listing <110> Dell Daimo Ltd. <120> p53 peptide as a marker in the diagnosis of Alzheimer's disease <160> 5 <170> BiSSAP 1.2 <210> 1 <211> 7 <212> PRT <213> Homo sapiens <220> <223> P1 <400> 1 Thr Glu Glu Glu Asn Leu Arg 1 5 <210> 2 <211> 11 <212> PRT <213> Homo sapiens <220> <223> P2 <400> 2 Thr Glu Glu Glu Asn Leu Arg Lys Gly Gly Lys 1 5 10 <210> 3 <211> 14 <212> PRT <213> Homo sapiens <220> <223> P3 <400> 3 Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg 1 5 10 <210> 4 <211> 13 <212> PRT <213> Homo sapiens <220> <223> P4 <400> 4 Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys 1 5 10 <210> 5 <211> 27 <212> PRT <213> Homo sapiens <220> <223> P5 <400> 5 Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu 1 5 10 15 Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys 20 25

Claims

1. A p53 peptide, the sequence of which is composed of the formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid in the P2 sequence.

2. A p53 peptide, the sequence of which is composed of formula (V) P5: GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ IDN.5).

3. Use of p53 peptide in the preparation of a product for diagnosis and / or prognosis of Alzheimer's disease, wherein: The sequence of the p53 peptide Composed of formula (I) P1:TEEENLR (SEQ ID N.1); or It is composed of the formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid in the P2 sequence.

4. Use of a p53 peptide in the preparation of a product for the diagnosis of Alzheimer's disease, wherein the sequence of the p53 peptide is Composed of formula (III) P3: KKPLDGEYFTLQIR (SEQ ID N.3); or Composed of formula (V) P5:GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ ID N.5).

5. Use of an agent for detecting a p53 peptide in the preparation of a product for the diagnosis and / or prognosis of Alzheimer's disease, wherein the sequence of the p53 peptide is Composed of formula (I) P1:TEEENLR (SEQ ID N.1); or Composed of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the portion branching from the first K amino acid of the P2 sequence; When using the product, follow these steps: a) determining the presence of said p53 peptide in a biological sample, and b) quantifying the p53 peptide by comparing it to a given control.

6. The use according to claim 5, when using the product, further comprising performing step c) correlating the amount of the p53 peptide with the diagnosis of Alzheimer's disease in patients at different stages of Alzheimer's disease.

7. The use according to claim 5 or 6, wherein the biological sample is blood, plasma, serum, saliva, urine, neuronal cells, blood cells or other types of cells.

8. The use according to any one of claims 5 to 6, wherein step a) is performed by immunoprecipitating the p53 peptide using a monoclonal antibody that binds to the p53 peptide.

9. The use according to claim 7, wherein step a) is performed by immunoprecipitating the p53 peptide using a monoclonal antibody that binds to the p53 peptide.

10. The use according to claim 8, wherein the monoclonal antibody is antibody 2D3A8.

11. The use according to claim 9, wherein the monoclonal antibody is antibody 2D3A8.

12. The use according to any one of claims 5-6 and 9-11, wherein step b) is performed by mass spectrometry.

13. The use according to claim 7, wherein step b) is performed by mass spectrometry.

14. The use according to claim 8, wherein step b) is performed by mass spectrometry.

15. The use according to claim 12, wherein step b) is performed by HPLC-mass spectrometry.

16. Use according to any one of claims 13-14, wherein step b) is performed by HPLC-mass spectrometry.

17. The use according to any one of claims 5-6, 9-11 and 13-15, wherein step b) is performed by using a labeled peptide as an internal control.

18. The use according to claim 7, wherein step b) is performed by using a labeled peptide as an internal control.

19. The use according to claim 8, wherein step b) is performed by using a labeled peptide as an internal control.

20. The use according to claim 12, wherein step b) is performed by using a labeled peptide as an internal control.

21. The use according to claim 16, wherein step b) is performed by using a labeled peptide as an internal control.

22. The use according to any one of claims 5-6, 9-11, 13-15 and 18-21, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

23. The use according to claim 7, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

24. The use according to claim 8, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

25. The use according to claim 12, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

26. The use according to claim 16, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

27. The use according to claim 17, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

28. The use according to claim 22, wherein the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatography columns prior to performing step a)(ii).

29. The use according to any one of claims 23 to 27, wherein the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatography columns prior to performing step a)(ii).

30. The use according to any one of claims 5-6, 9-11, 13-15, 18-21 and 23-28, wherein the product is for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

31. The use according to claim 7, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

32. The use according to claim 8, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

33. The use according to claim 12, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

34. The use according to claim 16, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

35. The use according to claim 17, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

36. The use according to claim 22, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

37. The use according to claim 29, wherein the product is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

38. The use according to any one of claims 5-6, 9-11, 13-15, 18-21 and 23-28, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

39. The use according to claim 7, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

40. The use according to claim 8, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

41. The use according to claim 12, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

42. The use according to claim 16, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

43. The use according to claim 17, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

44. The use according to claim 22, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

45. The use according to claim 29, wherein the product is used for the prognosis of cognitive decline in Alzheimer's disease in asymptomatic individuals and humans suffering from MCI.

46. ​​The use according to any one of claims 5-6, 9-11, 13-15, 18-21 and 23-28, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

47. The use according to claim 7, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

48. The use according to claim 8, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

49. The use according to claim 12, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

50. The use according to claim 16, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

51. The use according to claim 17, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

52. The use according to claim 22, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

53. The use according to claim 29, wherein the product is used for differential analysis of Alzheimer's disease from other dementias, wherein the other dementias are selected from frontotemporal dementia, Parkinson's disease and vascular dementia.

54. The use according to any one of claims 5-6, 9-11, 13-15, 18-21, 23-28, 31-37, 39-45 and 47-53, wherein the sequence of the p53 peptide in step a) is composed of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

55. The use according to claim 7, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

56. The use according to claim 8, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

57. The use according to claim 12, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

58. The use according to claim 16, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

59. The use according to claim 17, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

60. The use according to claim 22, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

61. The use according to claim 29, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

62. The use according to claim 30, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

63. The use according to claim 38, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

64. The use according to claim 46, wherein the sequence of the p53 peptide in step a) consists of formula (II) P2:TEEENLRK[GG]K (SEQ ID N.2), wherein [GG] is the part branching from the first K amino acid of the P2 sequence.

65. Use of an agent for detecting p53 peptide in the preparation of a product for the diagnosis of Alzheimer's disease, wherein the sequence of the p53 peptide is Composed of formula (III) P3: KKPLDGEYFTLQIR (SEQ ID N.3); or Composed of formula (V) P5: GEPHHELPPGSTKRALPNNTSSSPQPK (SEQ ID N.5); When using the product, follow these steps: a) determining the presence of said p53 peptide in a biological sample, and b) quantifying the p53 peptide by comparing it to a given control.

66. The use according to claim 65, when using the product, further comprising performing step c) correlating the amount of the p53 peptide with the diagnosis of Alzheimer's disease in patients at different stages of Alzheimer's disease.

67. The use according to claim 65 or 66, wherein the biological sample is blood, plasma, serum, saliva, urine, neuronal cells, blood cells or other types of cells.

68. The use according to claim 65 or 66, wherein step a) is performed by immunoprecipitating the p53 peptide using a monoclonal antibody that binds to the p53 peptide.

69. The use according to claim 67, wherein step a) is performed by immunoprecipitating the p53 peptide using a monoclonal antibody that binds to the p53 peptide.

70. The use according to claim 68, wherein the monoclonal antibody is antibody 2D3A8.

71. The use according to claim 69, wherein the monoclonal antibody is antibody 2D3A8.

72. The use according to any one of claims 65-66 and 69-71, wherein step b) is performed by mass spectrometry.

73. The use according to claim 67, wherein step b) is performed by mass spectrometry.

74. The use according to claim 68, wherein step b) is performed by mass spectrometry.

75. Use according to claim 72, wherein step b) is performed by HPLC-mass spectrometry.

76. Use according to any one of claims 73-74, wherein step b) is performed by HPLC-mass spectrometry.

77. The use according to any one of claims 65-66, 69-71 and 73-75, wherein step b) is performed by using a labeled peptide as an internal control.

78. The use according to claim 67, wherein step b) is performed by using a labeled peptide as an internal control.

79. The use according to claim 68, wherein step b) is performed by using a labeled peptide as an internal control.

80. The use according to claim 72, wherein step b) is performed by using a labeled peptide as an internal control.

81. The use according to claim 76, wherein step b) is performed by using a labeled peptide as an internal control.

82. The use according to any one of claims 65-66, 69-71, 73-75 and 78-81, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

83. The use according to claim 67, when using the product, performing the following steps: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

84. The use according to claim 68, when using the product, performing the following steps: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

85. The use according to claim 72, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

86. The use according to claim 76, when using the product, the following steps are performed: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

87. The use according to claim 77, when using the product, performing the following steps: a) determining the presence of the p53 peptide in a biological sample by the following (i), (ii), (iii), (iv): (i) providing biological samples; (ii) protein immunoprecipitation using antibodies that bind to p53 peptide; (iii) protein fragmentation by trypsin; (iv) performing strong cation exchange chromatography of the peptide; and b) quantifying the p53 peptide by comparing the p53 peptide to a given internal control by: (v) performing a selected reaction monitoring assay to identify and quantify the p53 peptide by comparing the p53 peptide to a given control.

88. The use according to claim 82, wherein the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatography columns prior to performing step a)(ii).

89. The use according to any one of claims 83-87, wherein the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatography columns prior to performing step a)(ii).

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