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WO2007068105A1 - PROCEDE DE DIAGNOSTIC DE LA SCLEROSE LATERALE AMYOTROPHIQUE <JavaScript:affichage('1','8870396','FRA','','1')> - Google Patents

PROCEDE DE DIAGNOSTIC DE LA SCLEROSE LATERALE AMYOTROPHIQUE <JavaScript:affichage('1','8870396','FRA','','1')> Download PDF

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WO2007068105A1
WO2007068105A1 PCT/CA2006/002023 CA2006002023W WO2007068105A1 WO 2007068105 A1 WO2007068105 A1 WO 2007068105A1 CA 2006002023 W CA2006002023 W CA 2006002023W WO 2007068105 A1 WO2007068105 A1 WO 2007068105A1
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tau
als
antibody
protein
phosphorylated
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PCT/CA2006/002023
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Michael Strong
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Robarts Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to a method of diagnosing amyotrophic lateral sclerosis (ALS).
  • the invention relates to the identification of a novel biomarker for diagnosing ALS, as well as antibodies selective for the biomarker.
  • ALS has been considered an age-dependant neurodegenerative disease state in which the neuropathological process is restricted to the motor system (1 ;2).
  • the contemporary view of ALS is, however, that of a multisystems disorder in which motor neuron degeneration remains the core neuropathological feature, but in which a frontotemporal lobar degeneration (FTLD) can either accompany the motor neuron degeneration, or in some cases, precede its onset (3).
  • FTLD frontotemporal lobar degeneration
  • This process may be reflected in mild cognitive deficits typical of a frontal dysexecutive syndrome, as behavioural impairment with a disinhibition syndrome, or as a frank frontotemporal dementia (FTD) (4).
  • FTD frank frontotemporal dementia
  • the prevalence of FTD in ALS has been estimated to range from 30% to 50% of cases, with most recent studies suggesting that this may approach 70% (5-7).
  • ubiquitin immunoreactive intraneuronal inclusions are frequently observed in the dentate granule cells, the superficial frontal and temporal cortical layers, and in the entorhinal cortex hippocampus of ALS patients.
  • Ubiquitin immunoreactive dystrophic neurites are evident in the extramotor cortices, but with a predominance of involvement in the frontal, temporal and hippocampal cortex.
  • ALSci ubiquitin immunoreactive inclusions
  • the neuropathological correlates of the FTLD in ALS include superficial linear spongiosus, astrogliosis and microglial proliferation, and tau protein aggregation within the frontal cortex (5; 15).
  • tau pathology in ALSci has recently been re-evaluated.
  • Tau immunoreactive aggregates are observed in neurons, astrocytes, as neuritic threads, and in rare instances, as oligodendroglial coiled bodies within cortical layers II and III, deeper cortical layers and subcortical white matter in both cognitively impaired (ALSci) and cognitively intact ALS patients (15).
  • Tau immunoreactive thread-like structures have been described in the neurophil and in glial cells (as coiled bodies) in the hippocampus, parahippocampal gyrus and amygdala of ALS patients (16).
  • Tau protein aggregation was present in the absence of an alteration in the expression of either the 3 R or 4R isoforms of tau, suggesting a potential post-translational alteration in tau expression (15).
  • these pathological changes of tau aggregation are outside that which would be expected as a function of normal aging (17).
  • ALSci has been examined within the current nosology of the FTLDs in which both the neuropathological characteristics and the molecular characterization of the tau protein are critical components (18).
  • the FTLDs thus, can be considered as either reflective of a tauopathy, or not.
  • a "signature" tau neuropathology can be developed based on the relative presence of either the 3R or 4R isoforms of tau (both by Western blotting and by immunohistochemical analysis) and on the presence or absence of specific neuronal or glial inclusions.
  • corticobasal degeneration can be characterized by the deposition of hyperphosphorylated tau as filamentous inclusions in neurons and glia, with 4R-tau as the predominant isoform (19).
  • the sarkosyl -insoluble tau fraction in both the grey and white matter contains predominantly the hyperphosphorylated 4R-tau isoforms, with the isoforms recognized by the monoclonal antibody AT8 (recognizing phosphorylation at Ser-202/Thr-205) specifically increased in the white matter.
  • both CBD and progressive supranuclear palsy can be differentiated from Pick's disease by the observation of 4R-tau immunostaining of intraneuronal aggregates, a feature not observed in Pick's neuronal aggregates (although observed within glial aggregates) (21).
  • a similar process can be adopted for a number of the remaining tauopathies, including argyrophilic grain disease, dementia lacking distinctive histopathology (DLDH), and Alzheimer's disease (22-25).
  • a method of diagnosing ALS in a mammal comprising the step of analyzing a tau-containing sample obtained from the mammal to determine whether the threonine at position 175 of tau is phosphorylated, wherein phosphorylation at position 175 of tau is indicative of ALS.
  • a polypeptide useful to generate an antibody immunospecific to a Tau protein phosphorylated at threonine 175 comprises the sequence RIPAK[pT]PPAPK, wherein [pT] represents a phosphorylated threonine.
  • kits for use in the diagnosis of ALS comprising a pT175 antigenic polypeptide comprising the sequence, RIPAK[pT]PPAPK, or comprising a phosphospecific T175-Tau antibody.
  • Figure 1 illustrates the results of Western blot analysis exhibiting a unique Tau protein expression profile for ALS in comparison with Alzheimer's
  • Figure 2 illustrates the results of Western blot analysis which shows that ALS and ALSci patients have prominent insoluble tau in both frontal grey and white matter tau protein isolates in contrast to corresponding samples in neurologically intact controls;
  • Figure 3 illustrates the determination of extent and stability of tau phosphorylation at amino acids S202 and T205 in AD, ALS and ALSci isolates of frontal grey matter when exposed to dephosphorylation by lambda alkaline phosphatase (A) andbovine alkaline phosphatase (B);
  • Figure 4 is a schematic illustration of soluble tau phosphoepitopes for control, AD, ALS and ALSci;
  • Figure 5 illustrates the amino acid sequence alignment between different isoforms of human Tau protein
  • Figure 6 shows the expression of tau protein phosphorylated solely at Tl 75 or S217, or tau protein simultaneously phosphorylated at S208/S210 in isolates from the white matter of patients suffering from AD or ALS; and
  • Figure 7 illustrates the immunohistochemical analysis of samples from AD or ALS white matter showing the distribution of Tau protein phosphorylated at T175, S217, or S208/S210.
  • An ALS-specific T175-phosphorylated isoform of Tau protein is provided that is useful in a method of diagnosing ALS in a mammal.
  • the method comprises analyzing a tau-containing sample obtained from the mammal to determine whether or not the threonine at position 175 of tau is phosphorylated. Phosphorylation at position 175 of tau is indicative of ALS.
  • Tau protein is a highly soluble microtubule-associated protein (MAP).
  • the major tau protein in the human brain is encoded by 11 exons. Exon 2, 3 and 10 are alternative spliced, allowing six combinations (2-3-10-; 2+3-10-; 2+3+10-; 2-3-10+; 2+3-10+; 2+3+10+).
  • the tau proteins constitute a family of six iso forms having from 352-441 amino acids. They differ in the number of inserts of 29 amino acids at the N-terminal part (exon 2 and 3) including either none, 1 or 2 inserts, and/or the number of repeat-regions at the C-terminal end (exon 10).
  • the longest isoform in the CNS has four repeats (Rl, R2, R3 and R4) and two inserts (441 amino acids total), while the shortest isoform has three repeats (Rl, R3 and R4) and no insert (352 amino acids total).
  • the amino acid sequences of four human Tau isoforms are set out in Figure 5.
  • Tl 75 is used herein to denote the threonine at position 175 of a tau protein, including any tau isoform as described above.
  • Phosphorylated threonine is denoted by pT or [pT]
  • phosphorylated threonine at position 175 of a tau protein is denoted by pT175 or [pT]175.
  • tau-containing sample as used herein is meant to encompass any mammalian biological sample containing at least one tau isoform that exists in the brain. Samples may be obtained from the cerebrum cortex, hypothalamus, amygdala, hippocampus, thalamus and basal ganglia Such a tau-containing sample is obtained from a living mammal via cerebral spinal fluid, or alternatively, serum. Less desirably, the sample may be obtained from tissue obtained by biopsy. [0016]The term “mammal” is used herein to refer to human and non-human mammals, including both domestic and wild animals.
  • Determination of phosphorylation at position 175 of tau may be conducted using any one of a number of techniques. For example, an immunological method of determining site-specific phosphorylation may be employed using an antibody based on a phosphorylated Tl 75 immunogenic fragment or antigen.
  • the raising of antibodies to a desired peptide or immunogenic fragment can be achieved, for polyclonal antibody production, using immunization protocols of conventional design, and any of a variety of mammalian hosts, such as sheep, goats and rabbits.
  • immunocytes such as splenocytes can be recovered from the immunized animal and fused, using hybridoma technology, to myeloma cells.
  • the fusion cell products i.e. hybridoma cells, are then screened by culturing in a selection medium, and cells producing the desired antibody are recovered for continuous growth, and antibody recovery.
  • Selected hybridoma cells may be implanted into the peritoneum of a mouse, for example, and monoclonal antibodies subsequently produced can be collected from the ascites produced by the mouse in response to implantation of the hybridoma.
  • Recovered antibody can then be coupled covalently to a reporter molecule, i.e. a detectable label, such as a radiolabel, enzyme label, luminescent label or the like, optionally using linker technology established for this purpose.
  • a reporter molecule i.e. a detectable label, such as a radiolabel, enzyme label, luminescent label or the like
  • linker technology such as a radiolabel, enzyme label, luminescent label or the like
  • the antibody may be farther modified, for example, to enable its use in humans for therapy.
  • Techniques well-established in the art may be utilized to humanize the antibody, utilizing CDR (complementarity determining region (CDR)) grafting, in which non-human CDR loops are grafted onto human framework regions may be used as well more recent methodology as described by Studnicka et al. (Protein Engineering vol. 7 no. 6 pp. 805-814, 1994 Oxford Journals).
  • CDR complementarity determining region
  • antibodies may be used to diagnose ALS, to monitor the progression of ALS and to determine the effectiveness of therapies designed to treat ALS.
  • detectably labelled pT175 antibody may be used to identify the existence of phosphorylation of Tl 75 of a tau protein in a tau-containing sample obtained from a mammal.
  • the labelled antibody is added to the sample and incubated under conditions suitable for an immunological interaction between antibody and pT175 tau in the sample.
  • the sample is then treated or washed to remove free antibody (e.g. antibody that did not interact).
  • the presence of antibody in the washed sample is then determined using techniques suitable to identify the label on the antibody. Detection of label in the sample is indicative of pT175, and thus, of ALS.
  • the pT175 antibody can be used to monitor the progression of ALS and to determine the effectiveness of therapies designed to treat ALS by determining the level of pT175 over time in subsequent samples obtained from the mammal being diagnosed/treated.
  • an isolated tau polypeptide useful to generate an antibody immunospecific to a pT175 Tau protein comprising the sequence, RIP AK[pT] PPAPK (SEQ ID NO:1) in accordance with the 3-letter amino acid code.
  • Polypeptides which are functionally equivalent to the polypeptide of SEQ ID No:l are also encompassed.
  • isolated is used herein to refer to peptides which are essentially pure and free from extraneous cellular material including other proteins or peptide fragments.
  • polypeptides comprising SEQ ID NO: 1 may include additional peptide sequence at either or both ends thereof as long as pT175 antigenic property is retained.
  • additional peptide sequence is not particularly restricted with respect to composition or length, but will generally yield an antigenic polypeptide of at least about 12 amino acids in length.
  • additional sequence may be added to bestow on the polypeptide a desirable property such as increased stability, or to enhance cellular uptake.
  • Preferred pT175 antigenic polypeptides may range in size from 12 to about 100 amino acids, but will preferably be 12 to about 50 amino acids in length or smaller.
  • Polypeptides which are "functionally equivalent" to polypeptides comprising the amino acid sequence set out in SEQ ID NO: 1 include peptides comprising one or more amino acid deletions, additions or substitutions, but which retain the antigenic property thereof.
  • functionally equivalent variants of a tau polypeptide according to SEQ ID NO: 1 include analogues, fragments and derivatives thereof.
  • Variants in accordance with the present invention are not necessarily restricted by size, as long as they retain pT175 antigenic activity.
  • variant peptides range in size from about 10 to about 100 amino acids, preferably from about 10 to about 50 amino acids, and more preferably from about 10 to about 20 amino acids.
  • a functionally equivalent variant of a tau polypeptide may include one or more amino acid substitutions, particularly a conservative amino acid substitution.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glutamine and glutamic acid, between asparagine and aspartic acid, and between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • a functionally equivalent variant may also include a fragment of a tau polypeptide comprising a sequence that corresponds with SEQ ID NO:1 but which is truncated by one or more amino acid residues. Fragments in accordance with the invention include fragments of SEQ ID No:l which retain pT175 antigenic activity. [0028] A functionally equivalent variant may additionally include a derivative of a tau polypeptide in accordance with the present invention comprising a sequence that corresponds with SEQ ID NO:1 in which one or more of the amino acid residues therein is chemically derivatized. Functionally equivalent derivatives also encompass analogues or fragments comprising one or more derivatized amino acid residues.
  • the amino acids may be derivatized at the amino or carboxy groups, or alternatively, at the side "R" groups thereof. Derealization of amino acids within the peptide may render a peptide having more desirable qualitities such as increased stability or activity.
  • Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form, for example, amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form, for example, O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
  • derivatives include those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, for example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • Terminal modification of a peptide to protect against chemical or enzymatic degradation may also include acetylation at the N-terminus and amidation at the C-terminus of the peptide.
  • Antigenic tau polypeptides in accordance with the present invention may be made using well-established techniques of protein synthesis which may include automated methods utilizing a peptide synthesizer, or manual techniques.
  • the present inhibitors may also be made using any one of a number of suitable techniques based on recombinant technology. It will be appreciated that such techniques are well-established by those skilled in the art, and involve the expression of the nucleic acid encoding the selected tau polypeptide in a genetically engineered host cell.
  • nucleic acid including DNA and RNA, encoding a polypeptide comprising the sequence, RIPAK[ ⁇ T]PPAPK (SEQ ID NO.l), is provided. It will be appreciated that more than one nucleic acid sequence will encode each tau polypeptide according to the invention, given the degeneracy that exists in the genetic code. Nucleic acid encoding functionally equivalent variants of a tau polypeptide is also encompassed by the present invention.
  • DNA encoding a tau polypeptide may be synthesized de novo by techniques well-known in the art. Generally, gene synthesis may be conducted by the successive 3' to 5' coupling of appropriately protected nucleotide reagents in an automated synthesizer, followed by recovery of the deprotected polynucleotide. Sequences obtained by de novo synthesis may be amplified using the polymerase chain reaction as described in United States Patent No. 4,683,195.
  • Recombinant techniques for producing a tau polypeptide generally involve insertion of a tau peptide-encoding DNA sequence into a suitable expression vector which is subsequently introduced into an appropriate host cell (such as Chinese hamster ovary cells (CHO cells) or human embryonic kidney cells of the 293 lineage (ATCC CRL 1573)) for expression.
  • a suitable expression vector such as Chinese hamster ovary cells (CHO cells) or human embryonic kidney cells of the 293 lineage (ATCC CRL 1573)
  • Suitable expression vectors are those vectors which will drive expression of the inserted tau-encoding DNA in the selected host.
  • expression vectors are prepared by site-directed insertion of the DNA construct therein. The DNA construct is prepared by replacing a coding region, or a portion thereof, within a gene native to the selected host, or in a gene originating from a virus infectious to the host, with the tau DNA.
  • regions required to control expression of the tau DNA which are recognized by the host, including both a 5' region to drive expression and a 3' region to terminate expression, are inherent in the DNA construct.
  • a selection marker is generally included in the vector which takes the form of a gene conferring some survival advantage on the transformants such as antibiotic resistance.
  • kits prepared using a pT175 antigenic polypeptide are provided in another aspect of the present invention.
  • a kit for use in the diagnosis of ALS is provided.
  • the kit may include one or more pT175 tau protein antigens comprising the sequence, RIPAK[pT]PPAPK (SEQ ID NO: 1), or a functionally equivalent variant thereof as described above.
  • the kit may include an antibody directed to pT175 tau protein prepared using conventional methodology as set out above.
  • Tau protein was isolated from 1.0 gm. of either grey or white matter, using the technique of Hanger et al with minor modifications (26).
  • tissue was homogenized in 1.0 ml of MES buffer, pH 6.5, centrifuged at 27,000 X g for 60 minutes at 4 0 C and the pellet discarded. The supernatant was further centrifuged at 95,000 X g for 60 minutes at 4 0 C and the supernatant saved (containing the soluble tau isoforms).
  • the pellet (containing insoluble tau) was solubilized in 150 ⁇ l 4 M guanidine HCl with a brief sonication (1 hours, room temperature) and dialyzed against 50 raM Tris-HCl, pH 7.5, 1 mg/ml PMSF overnight at 4 0 C.
  • the dialysate was centrifuged at 15,000 X g for 60 minutes at 4 0 C and the supernatant retained (containing insoluble tau). The pellet was discarded.
  • the supernatant containing soluble tau isoforms was boiled at 100 0 C for 10 minutes and then centrifuged at 15,000 X g for 30 minutes at 4 0 C.
  • the supernatant was brought to approximately 3.0 ml in 50 mM Tris-HCl, 1.35 gm ammonium sulphate added, and then cooled on ice for 15 minutes. Precipitated proteins were collected following centrifugation at 15,000 X g for 30 minutes at 4 0 C and resuspended in 150 ⁇ l 50 mM Tris-HCl, pH 7.5. The suspension was dialyzed against 50 MM Tris-HCl, pH 7.5, 1 mg/ml PMSF overnight at 4°C and then dialysate clarified by centrifugation at 15,000 X g for 30 minutes at 4 0 C.
  • Protein solutions were dried in the Speed Vac (Savant) and protein residue (200 ⁇ g protein for soluble tau and 100 ⁇ g for insoluble tau from control, ALS and ALSci) were taken up in 100 ⁇ l 8M urea/0.4M NH 4 HCO 3 for reduction, alkylation, tryptic digestion, SPE clean up, immobilized metal affinity chromatography (IMAC) and LC-MS/MS analysis as previously described (27).
  • AD soluble tau protein 200 ⁇ g was also digested with subtilisin (10 ug, Boehringer Mannheim) and after acidification with 12ul TFA, pepsin (10 ⁇ g, Boehringer Mannheim) as described above for trypsin digestion.
  • AD samples were analyzed by LC/MS/MS utilizing an extended HPLC gradient of 2-50% acetonitrile (0.1% HCOOH) over 240 min. MS/MS spectra were searched against a database consisting of the human tau proteins utilizing the SEQUEST program (Thermo) for identification of phosphopeptides and specific phosphorylated residues as previously described.
  • Soluble ALS-de ⁇ ved tau protein is hyperphosphorylated with unique Tl 75, S208 and S210 phosphoepitopes - Because a difference in the extent of dephosphorylation between soluble tau from ALS, ALSci, control and AD was observed, the phosphoepitopes of soluble tau protein in ALS were characterized. Tau protein from frontal cortex of ALS, ALSci, AD and normal control brains was digested with trypsin and the resulting digests subjected to immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment and LC-MS/MS analysis for the global identification of phosphopeptides and their phosphorylated residues (constituting a phosphoproteomics analysis).
  • IMAC immobilized metal affinity chromatography
  • AD samples were analyzed utilizing an extended HPLC gradient of 2-50% acetonitrile (0.1% HCOOH) over 4 hr which is designed to maximize phosphopeptide detection (32).
  • Phosphopeptides thus identified are listed in Table 1 and are seen to exhibit excellent agreement between the observed and calculated molecular weights of the identified phosphopeptides. Also listed are methylated phosphopeptides that were identified from a set of sample digests that were methylated prior to IMAC in order to improve the efficiency of the phosphopeptide enrichment step (33).
  • Table 1 lists non-tryptic AD phosphopeptides that were identified from soluble AD tau protein digested with two essentially non-specific enzymes (subtilisin and pepsin) in order to increase phosphorylation coverage (34).
  • Table 1 LC-MS/MS identification of phosphopep tides from tau fractions isolated from frontal cortex of ALS, ALSci and AD brains.
  • Phosphorylated residues or pairs of phosphorylated residues are listed in order of decreasing probability as listed in the SEQUEST output report. (First residue or pair is most probable.) e Partially methylated phosphopeptide. f From both IMAC materials (see Methods section).
  • soluble tau isolates from ALS, ALSci, AD and control brains exhibit unequivocal phosphorylation at Tl 81, Tl 99, S202, S205 (not observed unequivocally in AD isolate), T217, T231, S396 and S404.
  • the Tl 75 site is observed to be unequivocally phosphorylated in both ALS and ALSci as compared to normal control and AD.
  • the S208 and S210 sites are observed to be unequivocally phosphorylated in the ALS as compared to the control and ALSci.
  • phosphospecific polyclonal antibodies were generated as described below in Example 2. These antibodies were designed to specifically recognize tau protein phosphorylated solely at Tl 75 or S217 or tau protein simultaneously phosphorylated at S208 and S210. These antibodies were used to probe soluble and insoluble tau isolates from the white matter of patients suffering from AD or ALS. This analysis demonstrated that S208/S210 was predominantly found in AD, while S217 appeared to be equally present in both AD and ALS. This analysis also demonstrated that phosphorylation of T 175 was predominantly found in ALS.
  • Antibody 0308 was successfully used to identify the unique pT175 in ALS samples.
  • Wilson CM Wilson GM, Farb GM, Munoz DG, He BP, Strong MJ. Cognitive impairment in sporadic ALS. A pathological continuum underlying a multisystem disorder. Neurology 2001;57:651-657.
  • Garruto RM Amyotrophic lateral sclerosis and Parkinsonism-dementia of Guam: Clinical, Epidemiological and Genetic Patterns. Am J Human Biol 1989;l :367-382.
  • Garruto RM Cellular and Molecular mechanisms of neuronal degeneration: Amyotrophic lateral sclerosis, parkinsonism-dementia, and Alzheimer disease. Am J Human Biol 1989;l:529-543.

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Abstract

La présente invention concerne un procédé de diagnostic d'ALS (sclérose latérale amyotrophique <JavaScript:affichage('1','8870396','FRA','','1')>) chez un mammifère comprenant l'étape consistant à analyser un échantillon contenant la protéine tau obtenu à partir du mammifère pour déterminer si l'échantillon comprend tau pT175, dans lequel la phosphorylation à la position 175 de tau est indicative d'ALS. Des polypeptides de tau pT175 antigéniques peuvent être utilisés dans le procédé, ainsi que des anticorps préparés en utilisant les polypeptides antigéniques.
PCT/CA2006/002023 2005-12-12 2006-12-12 PROCEDE DE DIAGNOSTIC DE LA SCLEROSE LATERALE AMYOTROPHIQUE <JavaScript:affichage('1','8870396','FRA','','1')> WO2007068105A1 (fr)

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Cited By (11)

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WO2010115843A3 (fr) * 2009-04-03 2010-12-02 Ac Immune S.A. Composition pharmaceutique
WO2012049570A1 (fr) * 2010-10-11 2012-04-19 Panima Pharmaceuticals Ag Anticorps anti-tau humain
WO2013036833A1 (fr) * 2011-09-09 2013-03-14 Mayo Foundation For Medical Education And Research Détection de démence fronto-temporale et de sclérose latérale amyotrophique
CN105939722A (zh) * 2014-02-14 2016-09-14 伊皮埃里安股份有限公司 Tau肽，抗Tau抗体，及其使用方法
US9598484B2 (en) 2012-12-21 2017-03-21 Biogen Ma Inc. Human anti-tau antibodies
US9598485B2 (en) 2013-03-15 2017-03-21 Ac Immune S.A. Anti-tau antibodies and methods of use
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US9598485B2 (en) 2013-03-15 2017-03-21 Ac Immune S.A. Anti-tau antibodies and methods of use
EP3104870A4 (fr) * 2014-02-14 2017-09-13 Ipierian, Inc. Peptides tau, anticorps anti-tau, et leurs procédés d'utilisation
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US10400018B2 (en) 2014-02-14 2019-09-03 Ipierian, Inc. Tau peptides, anti-tau antibodies, and methods of use thereof
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US12324855B2 (en) 2019-02-08 2025-06-10 Ac Immune Sa Method of safe administration of phosphorylated Tau peptide vaccine
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