Patent Application
20210109116
The present invention refers to p53 peptides P1, P2, P3 and P5 and to their use as biomarkers in the diagnosis and/or prognosis of Alzheimer's disease (AD) in a biological sample. The invention also provides for a diagnostic method based on a highly accurate mass spectrometry analysis for the diagnosis of Alzheimer's disease at the pre-clinical and prodromal stages of the disease and for the prognosis of cognitive decline in a subject, by quantitating the levels of said p53 peptides specifically in human plasma of patients.
The confirmation of the presence of a large amount of altered conformational p53 isoform as an early risk factor for Alzheimer's disease (shortly ‘AD’) have been demonstrated in different published studies [1-3]. Initially, more than 400 subjects among AD, Mild Cognitive Impairment, Parkinson Disease, other Dementia and healthy subjects were enrolled in different independent studies and tested for Unfolded p53 by using different techniques (immunoprecipitation experiments, FACS analysis, ELISA) with a commercial conformational specific anti-p53 antibody [4-7]. In 2006 for the first time Uberti et al. [8], demonstrated that fibroblasts from sporadic Alzheimer's disease (AD) patients specifically expressed an anomalous and detectable conformational state of p53 that differentiate these cells from fibroblasts of age-matched non-AD subjects. In this conformational altered state, p53 lost its ability to transactivate its target genes, and consequently its biological functions [9-10]. The higher amount of unfolded p53 was also confirmed in blood of AD compared to healthy-non demented subjects or patients affected by other dementia and PD, as well as in MCI converted to AD.
Altogether these data suggested a direct association between Unfolded p53 and AD pathology.
In EP3201234B1, it has been reported the development of a new conformational specific anti-Up53 antibody named 2D3A8, that binds to an epitope (aa 282-297), accessible only when p53 loses its wild type conformation towards an unfolded phenotype. Comparing to the commercial antibody used at the beginning of Unfolded p53 discovering in AD (PAb240, aa214-217), the 2D3A8 antibody showed higher sensitivity and specificity in identifying AD patients compared to healthy elderly in Oviedo cohort.
In particular, said immunodiagnostic method is able to identify immunocomplex in a biological sample that are indicative of AD and to determine the predisposition of a subject affected by Mild Cognitive Impairment (MCI) to develop AD.
There is now the need of identifying new specific biological markers that can be used in the diagnosis and/or prognosis of Alzheimer's disease and of developing an accurate and sensible diagnostic method that can be used for the diagnosis and/or prognosis of AD, in particular at the pre-clinical and prodromal stages of the disease and for the differential analysis of AD from other forms of dementia.
This object has been achieved by identifying 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, in a biological sample.
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 in the prognosis of Alzheimer's disease at different stages.
The characteristics and the advantages of the present invention will become apparent from the following detailed description and the working examples provided for illustrative purposes.
The invention therefore relates to a highly accurate mass spectrometry method for the diagnosis of Alzheimer's disease at the pre-clinical and prodromal stages of the disease. Said method is based on the identification and quantification of the levels of specific p53 peptides, shorty referred to as “P1”, “P2”, “P3”, and “P5” or “p53 peptides”, that have been detected by mass spectrometry analysis in human plasma of patients affected by Alzheimer's disease or patients that have symptoms that can predispose to the development of AD.
In particular, the mass spectrometry analysis is a qualitative and quantitative method that was performed when no information about the exact correlation between these specific sequences of p53 and AD was known.
First, p53 peptides, present in plasma of patients affected by AD, have been identified by deep sequencing protein method in plasma from patient at pre-clinical, prodromal clinical stages of Alzheimer's, MCI stable patients, and cognitive normal subjects. Then the levels of the p53 peptides have been quantitated in plasma with a highly sensitive selective reaction monitoring (SRM) mass spectrometry method carrying out an Area Under the ROC Curve (AUC) (where ‘ROC Curve’ means ‘receiver operating characteristic curve’).
The data obtained therefore demonstrate a strong evidence that the p53 peptides can be considered as biomarkers in the diagnosis and/or prognosis of AD.
Said method is advantageously fast, requires a small volume of plasma sample and quantifies the concentration of the p53 peptides in each sample analysed.
Furthermore, the method and the biomarkers identified can be used also in the diagnosis of Alzheimer's disease in asymptomatic individuals and people suffering from MCI, allowing the access to the diagnostics market.
In addition, since said biomarkers can be used in the prognosis of cognitive decline to Alzheimer's Dementia in asymptomatic and MCI subjects, said method allows the use of a p53 peptide expression to select the subjects in clinical trials to enable success of the trial and to differentiate patients affected by AD from other forms of dementia.
It is therefore an embodiment of the present invention a p53 peptide consisting of formula (I) P1: TEEENLR (SEQ ID N. 1). Alternatively, the p53 peptides of the present invention have an amino acid sequence with at least 80-90% of identity to the sequence of formula (I), preferably at least 90-95% of identity to the sequence of formula (I), more preferably at least 96-99% of identity to the sequence of formula (I).
A further embodiment of the present invention is a p53 peptide having formula (II) P2: TEEENLRK[GG]K (SEQ ID N. 2), where [GG] is a moiety of a residue tail of ubiquitin that derives from the ubiquitination at K291 of the p53 protein after post-translational modification. In this regard, for the purposes of the present invention, the term in square brackets “[GG]” denotes that the first-K amino acid of the P2 sequence is branched by said moiety.
An additional embodiment of the present invention is a p53 peptide consisting of formula (II) P2: TEEENLRK[GG]K (SEQ ID N. 2), where [GG] is as above defined. Alternatively, the p53 peptides of the present invention have an amino acid sequence with at least 80-90% of identity to the sequence of formula (II), preferably at least 90-95% of identity to the sequence of formula (II), more preferably at least 96-99% of identity to the sequence of formula (II).
A further embodiment is the use of the p53 peptides P1 and/or P2 according to the present invention as in vitro biomarker for the diagnosis and/or prognosis of Alzheimer's disease. A further embodiment of the present invention is a p53 peptide having formula (III) P3:
An additional embodiment of the present invention is a p53 peptide consisting of formula (III) P3:
A further embodiment of the present invention is a p53 peptide having formula (V) P5:
An additional embodiment of the present invention is a p53 peptide consisting of (V) P5:
A further embodiment is the use of the p53 peptides P3 and/or P5 according to the present invention as in vitro biomarker for the diagnosis of Alzheimer's disease.
A further 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 steps of:
In a preferred embodiment, the p53 peptide in step a) is the p53 peptide P1 or P2.
In a preferred embodiment, the method of the present invention also comprises a step c) of correlating the quantity of the p53 peptide with the diagnosis of Alzheimer's disease in a patient at different stages of the diseases.
Preferably, said biological sample is blood, plasma, serum, saliva, urine, neuronal cells, blood cells or other types of cells.
In a preferred embodiment, in the method according to the present invention, the step a) is performed by immunoprecipitating the p53 peptide with a monoclonal antibody that binds to a p53 peptide.
Preferably, said monoclonal antibody is the antibody 2D3A8.
In a preferred embodiment in the method according to the present invention the step b) is performed through mass spectrometry analysis, preferably by HPLC-mass spectrometry. In a further preferred embodiment in step b) labelled peptides are used as internal controls. According to a preferred embodiment the in vitro or ex vivo method of the present invention comprises the following steps:
Preferably, the antibody of step a) (ii) is 2D3A8.
Preferably, the biological sample of step a)(i) is subjected to protein plasma depletion by HPLC or chromatographic columns, before performing step a)(ii).
According to a preferred embodiment the in vitro or ex vivo method of the present invention is used for the diagnosis of Alzheimer's disease in asymptomatic individuals and people suffering from MCI.
Preferably, in asymptomatic individuals and people suffering from MCI, the quantity of the p53 peptide is of 0.05 fmol/40 μl to 6.70 fmol/40 μl.
According to a further preferred embodiment, the in vitro or ex vivo method of the present invention is used for the prognosis of cognitive decline of Alzheimer's disease in asymptomatic individuals and people suffering from MCI.
Preferably, in asymptomatic and MCI subjects that have the prognosis of cognitive decline of Alzheimer's dementia, the quantity of the p53 peptide is of 0.203 fmol/40 μl to 6.70 fmol/40 μl.
Preferably, in asymptomatic individuals, the AUC is at least 80%.
Preferably, in people suffering from MCI, the AUC is at least 90%.
Measures of accuracy of a diagnostic/prognostic method include sensitivity and specificity. Sensitivity is the probability of a positive test result among those having the target condition, and it is measured by the formula: Sensitivity=true positives/(true positive+false negative). Specificity is the probability of a negative test result among those without the target condition, and it is measured by the formula: Specificity=true negatives/(true negative+false positives).
Preferably, in asymptomatic individuals, the sensitivity of the p53 peptide is at least 70%. Preferably, in people suffering from MCI, the sensitivity of the p53 peptide is at least 90%.
Preferably, in asymptomatic individuals, the specificity of the p53 peptide is at least 90%. Preferably, in people suffering from MCI, the specificity of the p53 peptide is at least 90%.
In a further preferred embodiment, the in vitro or ex vivo method of the present invention is used for the differential analysis of Alzheimer's disease from other dementia, said other dementia being selected from fronto temporal dementia (FTD), Lewi's Body, Parkinson's disease and vascular dementia.
Preferably, in patients affected by Alzheimer's disease, the quantity of said p53 peptide is of 2.21 fmol/40 μl to 6.56 fmol/ 40 μl, while in patients affected by other forms of dementia the quantity of said p53 is lower than 3.81 fmol/ 40 μl.
Preferably, in patients affected by Alzheimer's disease, the AUC is at least 85%. Preferably, in patients affected by Alzheimer's disease, the sensitivity of the p53 peptide is at least 75%.
Preferably, in patients affected by Alzheimer's disease, the specificity of the p53 peptide is at least 80%.
A further embodiment of the present invention is a method of detecting a p53 peptide as above described, said method comprising the following steps:
In a further preferred embodiment, the method of the present invention further comprising a step c) of correlating the quantity of the p53 peptide with the diagnosis and/or prognosis of Alzheimer's disease.
According to a preferred embodiment, in the method of the present invention, step c) correlates the quantity of the p53 peptide with the diagnosis of Alzheimer's disease in asymptomatic individuals and people suffering from MCI.
In a preferred embodiment, the p53 peptide in step a) is the p53 peptide P1 or P2.
According to a further preferred embodiment, in the method of the present invention, in step c) the quantity of the p53 peptide is correlated with the prognosis of cognitive decline of Alzheimer's disease in asymptomatic individuals and people suffering from MCI.
More preferably, in the method of the present invention, in step c) the quantity of the p53 peptide is correlated with the differential analysis of Alzheimer's disease from other dementia, said other dementia being selected from fronto temporal dementia (FTD), Lewi's Body, Parkinson's disease and vascular dementia.
It should be also understood that all the combinations of preferred aspects of the peptides of the invention, as well as of the preparation processes, and methods using of the same, as above reported, are to be deemed as hereby disclosed.
All combinations of the preferred aspects of the peptides of the invention, preparation processes, and methods disclosed above are to be understood as herein described.
Below are working examples of the present invention provided for illustrative purposes.
Materials and Methods
Materials
Antibody-based plasma/serum depletion Seppro® IgY14 LC10 column was purchased from Sigma- Aldrich (Merck KGaA, Darmstadt, Germany). Trypsin (Sequencing grade modified porcine) was obtained from Promega. Acetonitrile was purchased from JT Baker, and formic acid was obtained from EMD Millipore (Billerica, Mass., USA). C18 Cartridges for sample preparation, and chromatography columns for bRPLC and online HPLC of Triple Quadrupole mass spectrometer were purchased from Waters (Milford, Mass.). Plasma Dilution Buffer, -PlasmaWashing Buffer and -Plasma Elution Buffer, were prepared following standard protocols for this method. All other reagents were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated.
Plasma Sample Depletion
The most abundant proteins in the plasma were depleted using a Seppro IgY14 column. Plasma samples were diluted 5× in Plasma Dilution buffer, filtered (0.22 μm), then injected into IgY LC10 columns attached to an Agilent 1200 HPLC system. The unretained fraction was collected. High-abundance proteins were eluted Plasma Elution buffer.
Immunoprecipitation (IP)
Antibody clone 2D3A8 was used in the IP reactions. Conjugation of antibodies to beads was optimized and performed using with Link buffers. The monoclonal antibody was added directly to Protein G Dynal Magnetic Beads (as obtained from Invitrogen), and the antibody was bound to the beads in a buffer on a rotator at room temperature for 2 h. The antibody-bound beads then were washed by incubation in 50 mL Link-A buffer and collected on a magnet. The antibody was cross-linked to the protein G on the beads by incubation with 50 mL B buffer on a rotator at room temperature for 1 h. The beads then were washed twice with 50 mL C buffer, resuspended in 50 mL C buffer, and rotated at room temperature for 15 min. The beads were resuspended in C buffer and were stored at −20° C. until application. 200 μl depleted or undepleted plasma samples were used for IP reactions, each sample is reconstituted with IP buffer into a 3 ml system and 3 ml antibody conjugated beads (1:1 volume ratio) were added to the system. The IP systems were incubated on a rotator with a speed of 32 rounds per minute at 4° C. for 18 hours. Beads were collected on a magnet and washed by IP-Wash buffer at 4° C. for 3 times, followed by elution of the antigen through IP-Elution buffer. Flow-through fractions were collected for protein sample preparation.
Protein Preparation
Protein samples were processed in compliance with “Filter Assisted Sample Preparation” (FASP) method (Nat Methods. 2009, 6:359-362). Briefly, protein samples in 9M UREA were reduced with 5 mM TCEP at 37° C. for 45 min and reduced cysteines were blocked using 50 mM iodoacetamide at 25° C. for 15 min. Protein samples (100 μg each) were then cleaned using 10 kDa Amicon Filter (UFC501096, Millipore) three times using 9M urea and two times using Buffer 1. Samples were then proteolyzed with trypsin (V5111, Promega) for 12 hrs at 37° C. The peptide solution then was acidified by adding 1% trifluoroacetic acid (TFA) and was incubated at room temperature for 15 min. A Sep-Pak light C18 cartridge (Waters Corporation) was activated by loading 5 mL 100% (vol/vol) acetonitrile and was washed by 3.5 mL 0.1% TFA solution two times.
Acidified digested peptide solution was centrifuged at 1,800×g for 5 min, and the supernatant was loaded into the cartridge. To desalt the peptides bound to the cartridge, 1 mL, 3 mL, and 4 mL of 0.1% TFA were used sequentially. To elute the peptides from the cartridge, 2 mL of 40% (vol/vol) acetonitrile with 0.1% TFA was used, and this elution was repeated two more times (for a total of 6 mL of eluate). It was important to ensure that the cartridge had stopped dripping before each sequential wash and elution solution was applied. The eluted peptides were lyophilized overnight and reconstituted in 100 μL of Buffer 2.
Strong Cation Exchange Chromatography
Peptides were fractionated by strong cation exchange (SCX) chromatography. Briefly, lyophilized peptides mixture was resuspended in 1 ml of SCX solvent A (5 mM KH2PO4 pH 2.7, 30% ACN) and fractionated by SCX chromatography on a PolySULPHOETHYL A (5 μm, 200 Å) column (200×9.4 mm; PolyLC Inc., Columbia, Md.) by employing an increasing gradient of SCX solvent B (5 mM KH2PO4 pH 2.7, 30% ACN, 350 mM KCl) on an Agilent 1290 Infinity II LC system. For each experiment, a total of 96 fractions were initially collected, which were then pooled into 24 fractions, and dried using SpeedVac (Eppendorf). bRPLC and SCX were performed side by side to compare the compatibility of detection of TP53 proteins, and SCX was chosen because a relative stronger yield of TP53 isoforms has been repeatedly observed for this project.
Optimization of Peptide SRM Transitions
4 peptides from targeting TP53 protein were chosen as SRM quantifying targets and 1092 sets of transition parameters and retention times of the 4 peptides were established individually using an Agilent 6495 Triple Quadrapole Mass Spectrometer for 1+, 2+, 3+ and 4+ charged precursor ions. 2 peptides (P1 and P2) were then selected as main correlated to the AD pathology.
Selected Reaction Monitoring (SRM) Analysis
Peptide samples reconstituted in 37 ul Buffer 3 were spiked with SRM Internal Control Mixture composed of a pool of 1 femto mole heavy isotope labeled peptides covering a large hydrophobicity window and a large M/z range (M/z 200˜1300), Peptide samples were eluted through an online Agilent 1290 HPLC system into the Jet Stream ESI source of an Agilent 6495 Triple Quadrupole Mass spectrometer.
Results
There were cumulatively 5 peptides detected in previous projects for p53 protein 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)]. It was established the SRM detection method for 4 peptides. The peptide GEPHHELPPGSTKRALPNNTSSSPQPK is not feasible for SRM-based detection due to its high molecular weight.
Correlation Between p53 Sequence Peptide and AD Diagnosis.
A deep sequence mass spectrometry analysis of human plasma immuno-precipitated was conducted with the conformationally specific Ab 2D3A8. Plasma sample was depleted from abundant proteins according to the method reported above. Samples were then immunoprecipitated by 2D3A8 and analyzed using Orbitrap (deep sequence mass spectrometry analysis). The resulting peptides present uniquely in AD, MCI to AD and asymptomatic to AD and absent in asymptomatic and MCI stable samples were chosen as markers. The detection of the peptide (presence/absence) by this method was correlated with clinical diagnosis of the subjects.
It was 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 post baseline (Normal), a pool of 10 baseline plasma samples from PiB+ve patients with MCI 18 months before they converted to AD symptoms (Prodromal, MCI due to AD), a pool of 10 baseline plasma samples from PiB-ve patients with MCI who did not experience further cognitive decline to AD symptoms (follow up 18-72 months post baseline), (MCI stable) and 3 baseline plasma samples from PiB+ve patients with MCI 18 months before they converted to AD symptoms (Preclinical, Normal to AD converters).
Results
Initial results from a longitudinal four-year study on clinical progression of cognitively normal subjects and people with mild cognitive impairment to probable AD measuring
U-p53 signals measured by direct ELISA, shown very high predictive values for progression. Based on this finding, it was developed a highly accurate mass spectrometry method based on the identification of p53 peptides P1 and/or P2, for the diagnosis of Alzheimer's at the pre-clinical 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. It is believed that both the diagnostic and prognostic performance of the p53 peptides, P1 and/or P2, is the highest that has been reported to date.
A specific mass spec method, which is the SRM (Selected Monitoring Reaction) method (by triple quadrupole mass spectrometer) was used to quantify both P1 and/or P2 in plasma samples from AD, asymptomatic, MCI subjects. By the triple quadrupole mass spectrometer which acts as essentially as a mass filter, it is possible to sequence the peptides by looking at the different transition peaks. Then, using a heavy labelled internal control for peptide 1 and 2, it is possible to quantitate those peptides with the maximum precision by Selected Monitoring Reaction (SRM) method.
Prognostic Performance
It was evaluated the prognostic power of the biomarker in enriching for patients who are PiB+ but also will exhibit cognitive decline in 18 months from MCI subjects and in 18-72 months for asymptomatic subjects at presentation. This was performed, in order to assess the value of the biomarker in significantly enriching for cognitive decline as an endpoint in disease modifying trials in asymptomatic and patients with MCI. It was examined the prognostic power of P1 and P2, using the future conversion of clinical presentation. Statistical parameters able to describe the prognostic power of P1 and P2 are below reported:
Conclusions
P1 and P2 can accurately enrich for patients that will exhibit cognitive decline from either an asymptomatic or MCI clinical presentation with very high specificity and sensitivity and thus enable the successful recruitment of patients for disease modifying drugs targeting the very early stages of the disease.
Differential Diagnosis of Alzheimer's Disease From Other Forms of Dementia
An analysis was also performed to validate previous data on the specificity of the U-P53 biomarker in Alzheimer's dementia. Well characterized Alzheimer's patients which were amyloid positive and had Alzheimer's dementia were compared with patients with clinical presentations of other dementias such as FTD (frontal-temporal dementia), Parkinson's, Lewi's Body and vascular dementia.
Plasma samples from OD (FTD, Vascular, Parkinson's, Lewi's Body) were collected at disease stage, anyway in a differential diagnosis, it is possible to establish that above a certain cutoff both peptide sequences showed a high specificity and sensitivity toward AD compared to OD.
Furthermore, it has been observed that AUC of both peptides is greater than 90%, it shows the high correlation between biomarker concentration and the AD diagnosis.
There is in literature an unmet need in differential diagnosis in Alzheimer's dementia is between: a) advanced FTD and advanced AD b) advanced LBD and AD as those can mimic clinical presentation is some cases. Anything that can exclude either AD or the other dementia has clinical value, thus high sensitivity of either dementia is enough to bring value.
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| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2019/051785 | Mar 2019 | US |
| Child | 17113559 | US |
