WO2018111099A1 - Biomarqueurs et traitements de l'angiopathie amyloïde cérébrale (aac) - Google Patents

Biomarqueurs et traitements de l'angiopathie amyloïde cérébrale (aac) Download PDF

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WO2018111099A1
WO2018111099A1 PCT/NL2017/050831 NL2017050831W WO2018111099A1 WO 2018111099 A1 WO2018111099 A1 WO 2018111099A1 NL 2017050831 W NL2017050831 W NL 2017050831W WO 2018111099 A1 WO2018111099 A1 WO 2018111099A1
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caa
biomarker
individual
level
expression
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WO2018111099A8 (fr
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August Benjamin Smit
Jeroen Joseph Maria Hoozemans
David Carolus HONDIUS
Johanna Maria KWAKKEL
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Stichting Vumc
Stichting Vu
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • 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
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a 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
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • CAA cerebral amyloid angiopathy
  • the invention relates to biomarkers and diagnostic methods for determining the presence and/or risk of cerebral amyloid angiopathy (CAA) as well as to methods for screening for treatments for CAA.
  • CAA cerebral amyloid angiopathy
  • AD pathology is characterized by the deposition of amyloid beta ( ⁇ ) in the brain parenchyma as plaques and around the brain vasculature. The latter is referred to as cerebral amyloid angiopathy (CAA). Approximately 80 percent of AD cases have CAA pathology in varying amounts. In AD, patients with CAA present with more vascular pathology and are at higher risk of mortality.
  • ⁇ amyloid beta
  • CAA cerebral amyloid angiopathy
  • CAA type-2 When restricted to the larger blood vessels, including leptomeningeal vessels, cortical arteries and arterioles, this is referred to as CAA type-2.
  • CAA type- 1 In approximately 50% of the AD cases brain capillaries are also affected, which is designated as CAA type- 1 [4,31] .
  • AD the observed plaque pathology and CAA type-1 capillary deposits have an inverse correlation.
  • ⁇ deposition at vessel endothelial cells is known to cause a weakening of the vascular wall structure, which leads to an increase in the occurrence of cerebral infarction, cerebral haemorrhage and microbleeds in CAA [13].
  • ⁇ peptide transport through the blood brain barrier (BBB) is an important mechanism to clear the brain. Disruption of ⁇ clearance is thought to lead to increase in AB deposition in the walls of capillaries and blood vessels, which in turn aggravates drainage capacity and deposition of ⁇ [43,44]
  • CAA amyloid precursor protein
  • Presenilin 1 and Presenilin 2 are associated with CAA.
  • the following mutations in APP are associated with CAA; E693K, E693Q, L705V, A713T, c.*18 OT, c.*331_*332del.
  • Presenilm 1 the G378E mutation is associated with CAA.
  • CAA type- 1 is clinically highly relevant, as it contributes to the symptomatic appearance of AD and, in severe form, CAA type-1 can present itself as the primary cause of rapidly progressive dementia [3, 16] .
  • CAA type-2 is also relevant as, e.g., patients with a familial form of CAA (APP E693Q mutation) generally succumb to bleedings in the brain before dementia sets in.
  • Prion protein cerebral amyloid angiopathy Prion protein cerebral amyloid angiopathy (PrP-CAA) is rare form of hereditary prion protein amyloidosis resulting from stop codon mutations in the gene encoding the prion protein (PRNP) (Jansen et al. Acta Neuropathol 2010 119: 189-197).
  • PRNP prion protein
  • CAA cerebral amyloid angiopathy
  • ICH intracranial hemorrhage
  • microbleeds on T2* MRI may be used as a proxy for diagnosing CAA.
  • definitive diagnosis of CAA can only be determined post- mortem.
  • One aspect of the disclosure provides methods of classifying an individual as having or being at risk of developing cerebral amyloid angiopathy (CAA), comprising
  • an altered level of the biomarker as compared to the reference value indicates that said individual has or is at risk of developing CAA and wherein the reference value is the expression level of said biomarker from one or more individuals not having CAA; or wherein a similar level of the biomarker as compared to the reference value indicates that said individual has or is at risk of developing CAA and wherein the reference value was obtained from one or more individuals having CAA.
  • methods are provided for classifying an individual as having or being at risk of developing CAA type I or CAA type II.
  • methods are provided for classifying an individual as having or being at risk of developing CAA as disclosed herein, wherein said individual is not afflicted with ICH.
  • the biomarker is selected from Table 1A.
  • the expression level of biomarker is determined by detecting the biomarker in vivo.
  • the method comprises administering to said individual a positron emission tomography (PET) compatible biomarker binding tracer and determining whether the brain or the brain vasculature of said individual has an increased level of the biomarker as compared to a reference value.
  • PET positron emission tomography
  • the level of the biomarker is determined from a biological sample from said individual, preferably from blood, serum, or cerebrospinal fluid.
  • One aspect of the disclosure provides methods for determining a treatment schedule for an individual, comprising determining the expression level of at least one biomarker selected from Table 1, comparing the level of the biomarker to a reference value to determine whether said individual has or at risk of developing CAA, and determining a treatment schedule based on the CAA status of said individual.
  • One aspect of the disclosure provides methods of monitoring the treatment and/or progression of CAA in an individual, the method comprising
  • said individual receives a treatment for CAA and/or AD between the first and second time point.
  • One aspect of the disclosure provides methods for the screening and identification of compounds for the treatment of a CAA, the method comprising: (a) administering one or more candidate compounds to a preclinical animal model of CAA; (b) determining the expression level of a biomarker selected from Table 1 in the animal model; (c) comparing the expression level of biomarker to a reference value; and (d) identifying a compound that treats CAA.
  • One aspect of the disclosure provides methods for treating a thromboembolic disorder in an individual, comprising determining as disclosed herein whether said patient has or is at risk of developing CAA and treating such a patient with anti-platelet therapy.
  • the individual is determined to have or be at risk of developing CAA by a method as disclosed herein.
  • One aspect of the disclosure provides methods for treating CAA in an individual, comprising administering to an individual in need thereof a modifier of Norrin expression of activity.
  • the modifier is an inhibitor of Norrin expression or activity.
  • the inhibitor is an anti-Norrin antibody or antigen binding fragment thereof or a nucleic acid molecule that binds Norrin mRNA or pre-mRNA, preferably said inhibitor is an antisense oligonucleotide, miRNA, or siRNA.
  • a further aspect provides methods comprising determining the level of a biomarker selected from Table 1 in an individual.
  • the methods comprise contacting an individual or a sample from said individual with a compound that binds said biomarker (e.g., an antibody).
  • a compound that binds said biomarker e.g., an antibody
  • said contacting occurs in vivo.
  • the method comprises, a. administering to the individual a positron emission tomography (PET)-compatible tracer which binds to the biomarker; b.
  • PET positron emission tomography
  • FIG. 1 Workflow used in this study.
  • Fig. la Workflow used in this study.
  • Figlb Workflow further specifying the tissue isolation.
  • Amyloid Beta pathology was visualized in human postmortem occipital lobe tissue. Unaffected grey matter was isolated from healthy control cases. Grey matter with high burden of AB pathology was isolated from the AD and CAA cases thereby isolating tissue with high plaque load or high CAA type-1 burden, respectively.
  • Tissue was lysed and the proteins were separated using SDS-PAGE and subjected to in-gel trypsin digestion. Peptides were analysed using LC-MS-MS. A database search for protein identification and protein quantification was performed using MaxQuant software. ANOVA (Kruskall Wallis) and t-tests were performed to identify
  • FIG. 3a Identification strategies of proteins that are differentially expressed in CAA type-1 compared to control and AD brains. Three approaches were used (Approach A, Approach B, Approach C). Criteria of each of the selection strategies are specified, numbers of resulted proteins indicated, and selected proteins are listed in tables and figures as indicated. First, t-tests were applied contrasting the different groups and revealing significantly differentially expressed proteins. Only those proteins that have an altered abundance in CAA compared to both the control group and the AD group were considered in this study and are listed in Table 3a and plotted in Fig. 3a. Proteins with 1 or zero detection values in the control group or the AD group (indicating low abundance) and a significant difference between the AD or control group respectively and CAA are listed in Table 3b and plotted in Fig. 3b.
  • Proteins with 1 or zero detections values in both the control group and the AD group, but 4 or more values in the CAA group are listed in Table 3c and plotted in Fig. 3c as are those with 4 or more values in both control and AD groups but 1 or zero values in the CAA group.
  • MS Three groups of selected proteins (panels A-C), with altered levels (MS-derived, log2 LFQ intensity values) in CAA type- 1 compared to the control groups and the AD groups. Selection criteria are specified in Fig.2. Gene names are indicated.
  • A Quantitative values of the proteins that significantly differ when comparing the CAA group with both the AD group and the control group (t-tests, p ⁇ 0.05, cf. Table 3a).
  • B Quantitative values of proteins with zero or one quantitative values in the control or AD group and a significant difference in abundance between the other group (AD or control) and CAA groups (t-test, p ⁇ 0.05, cf. Table 3b).
  • FIG. 5 Immunohistochemical analysis of selected proteins. Representative images were taken. ⁇ pathology was visualized.
  • A The control case does not have any AB pathology.
  • B plaque pathology is confirmed in the AD case and
  • C CAA type- 1 pathology is confirmed in the CAA type-1 case.
  • D,E,F Extensive NDP immunore activity is observed in the CAA type- 1 cases whereas absent in both control and AD cases without CAA.
  • G,H,I COL6A2 immunore activity is hardly observed in the control and AD cases, however, extensive immunore activity is observed in the CAA type cases and includes both capillaries and large vessels.
  • Figure 9 Number of proteins detected per individual case. Proteins were quantified based on a minimum of one peptide adhering to an FDR of ⁇ 0.01.
  • FIG. 10 COL6A2 expression. Immunore activity for COL6A2 is equally present in leptomeningeal vessels in Control, AD and CAA tissue.
  • Figure 13 Analysis of house keeping protein expression. Consistency in the measured abundance of the subunits of several protein complexes indicate correct quantification. Measurements were performed using mass spectrometry, providing semi- quantitative LFQ intensity data, and are taken from the discovery data set. DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS
  • CAA occurs in approximately 80% of patients afflicted with Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • CAA on its own right is a highly prevalent condition affecting approximately 50% of individuals over the age of 80 (Pezzine et al. Neurological Sciences 29 S260-3 2008).
  • the blood-brain-barrier is leaky and the vasculature is vulnerable.
  • the use of anti-coagulants in CAA patients can lead to extravasation of blood cells in the brain and induce an inflammatory response leading to further damage in the brain.
  • other therapeutics, such as anti-amyloid antibody treatment for AD in CAA patients may lead to unwanted side-effects.
  • the CAA status of a patient is therefore an important factor when determining a treatment schedule for a patient and for designing and analysing clinical trials on candidate therapeutics.
  • the invention is based, in part, on the identification of biomarkers that correlate with the conclusively confirmed presence of CAA (i.e., confirmed in post-mortem studies).
  • the examples demonstrate that the biomarkers listed in Table 1 are significantly upregulated in patients with confirmed (post-mortem) CAA as compared to individuals that do not have CAA.
  • Example 1 demonstrates that the biomarkers described herein detect CAA- type 1.
  • methods are provided for classifying an individual as having or being at risk of developing CAA type I or CAA type II.
  • Type I CAA often involves an inflammatory response and a rapid progression of disease. Accordingly, distinguishing between type I and type II can alter the choice in therapeutic treatment.
  • methods are provided for classifying an individual as having or being at risk of developing CAA as disclosed herein, wherein said individual is not afflicted with ICH. Such methods allow for the classification of individuals in an earlier state of disease and/or the classification of individuals that present various clinical symptoms.
  • ACTG1 Actin, cytoplasmic 2; Actin, cytoplasmic 2, N-terminally
  • PNP P00491 Purine nucleoside phosphorylase
  • DHX15 Putative pre-mRNA-splicing factor ATP-dependent RNA
  • the biomarker is selected from Table 1A, more preferably the biomarker is NDS (i.e., Norrin). Determining the expression level of more than one biomarker from Table 1 is also encompassed by the disclosure. In preferred embodiments, the expression level of at least two biomarkers is determined.
  • the biomarker combinations are selected from NDP and CLU, NDP and APOE, NDP and HTRAl, NDP and APCS, NDP and COL6A2, NDP and COL6A3, CLU and APOE, CLU and HTRAl, CLU and APCS, CLU and COL6A2, CLU and COL6A3, APOE and HTRAl, APOE and APCS, APOE and COL6A2, APOE and COL6A3, HTRAl and APCS, HTRAl and COL6A2, HTRAland COL6A3, APCS and COL6A2, APCS and COL6A3, COL6A2 and COL6A3.
  • the expression of at least three or more of the biomarkers is determined.
  • any individual may be tested according to the methods disclosed herein.
  • the individual is older than 50 years, more preferably older than 60 years, suffers from dementia or early signs of cognitive impairment, and/or suffers from AD.
  • an individual may be classified as having CAA, as not having CAA, or at being "at risk" of developing CAA. It is also understood to a skilled person that being at risk of developing CAA indicates that the individual has a higher risk of developing CAA than an age matched population of individuals, or rather the individual has an increased risk over the average of developing CAA.
  • an individual is classified as either having CAA or not having CAA.
  • the methods described herein relate to determining the expression level of biomarker.
  • biomarker protein it is not necessary to determine the absolute amount of biomarker protein in the brain, central nervous system or other relevant biological sample. Rather, as understood by one of skill in the art, the level of biomarker protein is compared between an individual and a reference value and therefore a relative expression level may be determined.
  • the reference value may be the biomarker expression level determined in a comparable sample (e.g., from the same type of tissue as the tested tissue, such as blood or serum), from a healthy, i.e., CAA negative, individual.
  • a healthy individual preferably does not suffer symptoms of dementia or AD.
  • the samples or measurements from healthy individuals may also have been collected retrospectively and the absence of CAA confirmed post-mortem.
  • a pool or population of healthy subjects can be used and the reference value can be an average or mean of the measurements from the population. A significant alteration in the expression level in the patient as compared to the "healthy" expression level, indicates that the individual has or is at risk of developing CAA.
  • an increase in biomarker expression level in a patient indicates that the individual has or is at risk of developing CAA.
  • the alteration in expression level may be an increase in biomarker levels or a decrease in biomarker levels.
  • decreased levels of AB 1.42 in the CSF indicate the presence of AD. The decreased levels are thought to be due to deposition of A61.42 in senile plaques (or rather, an increase in the brain).
  • the reference value may be the biomarker expression level determined in an individual or pool of individuals with CAA.
  • retrospective biological samples may be used from patients that were later confirmed post-mortem to have CAA.
  • determining the biomarker expression level further comprises normalizing the expression level of said biomarker.
  • normalizing the expression level of said biomarker Such methods are well-known to a skilled person and will depend upon the method of protein measurement used.
  • the expression of a reference protein is determined in parallel to biomarker expression.
  • the normalized expression level of said biomarker may be determined as the ratio of the biomarker to the reference protein.
  • Preferred reference proteins include housekeeping proteins such as actin, beta-tubulin, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Normalization can also be performed by other known methods such a by normalizing expression level by total cell number.
  • both the expression level from the individual and the reference value are normalized to allow for direct comparison.
  • the biomarkers exhibit negligible expression in the brain of healthy (non-CAA) individuals.
  • the detection of significant expression in the brain indicates the individual has or is at risk or developing CAA.
  • the reference value can be considered to be zero and any significant increase indicates CAA.
  • a "significant" alteration in a value can refer to a difference which is reproducible or statistically significant, as determined using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation.
  • a significant increase is at least 20, at least 40, or at least 50% higher than the reference value. It is well within the purview of a skilled person to determine the amount of increase or similarity that is considered significant.
  • the expression level of more than one biomarker is determined and each biomarker expression level determined is significant.
  • the expression level of biomarker is determined by detecting the biomarker in vivo.
  • positron emission tomography is used to determine the level of biomarker in vivo.
  • a method is provided, the method comprising providing an individual with a PET compatible biomarker binding tracer and scanning said individual with a PET scanner.
  • the level of biomarker is detected in the human cerebral cortex, especially in the occipital and parietal cortex and in severe cases also in the cerebellar cortex.
  • the disclosure provides methods for determining the level of the biomarkers disclosed herein ire vivo. These data acquisition methods may be useful for collecting information to assist in diagnosis.
  • the disclosure provides the use of the biomarkers, binding molecules and tracers disclosed herein for determining the expression of said biomarkers in the brain.
  • PET is a well-known technique to determine the distribution of a tracer in vivo.
  • a radioactive tracer is administered to an individual.
  • the individual is then subjected to a scanning procedure using a PET or PET/CT scanner. Quantification of
  • radiopharmaceutical (radio-tracer) uptake by the target tissue can be performed using methods known in the art (see, e.g., Boellaard R. et al. Journal of Nuclear Medicine, Vol. 45, No. 9, pp 1519-1527, 2004 and U.S. Publications 20100196274 and
  • a suitable tracer binds to the biomarker.
  • Biomarker binding agents are disclosed further herein.
  • the tracer is an antibody or antigen-binding fragment.
  • the tracer can be labelled with a short-lived radioactive tracer isotope, such as carbon- 11, nitrogen- 13, oxygen- 15, or fluorine- 18.
  • the use of PET has the advantage that not only are expression levels determined, but changes in localized expression can also be measured.
  • the overall level of biomarker may be the same between a CAA affected individual and a healthy individual.
  • the CAA affected individual will demonstrate increased localized expression levels, in particular at or around leptomeningeal vessels, cortical arteries, arterioles, and/or brain capillaries. It is clear to a skilled person that the methods encompass determining and comparing such localized expression levels.
  • an increase in biomarker expression at or around brain capillaries in a patient as compared to the expression in healthy controls indicates the presence of CAA. Based on the results in the examples, the level of biomarker in the brains of healthy individuals is expected to be extremely low.
  • an individual having CAA will exhibit at least 50% more biomarker in the brain as compared to an otherwise healthy individual.
  • the amount of biomarker is preferably measured at or around the brain vasculature. Based on the current PET technology, in preferred embodiments the amount of biomarker present at or around the brain vasculature in an individual not afflicted with CAA is expected to be beneath the limits of detection. In such cases, the reference value can be set as zero.
  • the increased levels of biomarker in the brain of CAA affected individuals can also be detected by sampling other biological samples, in particular blood, serum, or cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the level of biomarker is determined in a CSF sample.
  • the level of biomarker is determined ex vivo, or rather, a biological sample is obtained from an individual and the expression level of biomarker is determined ex vivo.
  • the expression level from a sample of an individual having or at risk of developing CAA will be at least 10, 20, 30, 40, or 50% higher as compared to a comparable sample from an otherwise healthy individual.
  • the level of biomarker is determined by mass spectroscopy.
  • Mass spectroscopy allows detection and quantification of an analyte by virtue of its molecular weight.
  • Suitable ionization methods known in the art can be employed, including electron impact (El), chemical ionization (CI), field ionization (FDI), electrospray ionization (ESI), laser desorption ionization (LDI), matrix assisted laser desorption ionization (MALDI) and surface enhanced laser desorption ionization (SELDI).
  • Any suitable mass spectrometry detection method may be employed, for example quadrapole mass spectroscopy (QMS), fourier transform mass spectroscopy (FT-MS) and time-of-fhght mass spectroscopy (TOF-MS). Mass spectrometry methods are well known in the art and have been widely used to quantify proteins and peptides.
  • the level of biomarker is determined using a biomarker binding compound.
  • a binding compound includes a small molecule, a peptide, a protein, aptamer, an antibody, or an antibody mimic.
  • Antibody mimics refers to molecules capable of mimicking an antibody's ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mime tics include, but are not limited to, Adnectins (i.e., fibronectin based binding molecules), Affibodies, DARPins, Anticalins, Avimers, and Versabodies. Antibodies which bind a particular epitope can be generated by methods known in the art.
  • polyclonal antibodies can be made by the conventional method of immunizing a mammal (e.g., rabbits, mice, rats, sheep, goats). Polyclonal antibodies are then contained in the sera of the immunized animals and can be isolated using standard procedures (e.g., affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography). Monoclonal antibodies can be made by the conventional method of immunization of a mammal, followed by isolation of plasma B cells producing the monoclonal antibodies of interest and fusion with a myeloma cell (see, e.g., Mishell, B. B., et al., Selected Methods In Cellular Immunology, (W.H.
  • Peptides corresponding to the genes of Table 1 may be used for immunization in order to produce antibodies which recognize a particular epitope. Screening for recognition of the epitope can be performed using standard immunoassay methods including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting. See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, Edited by Ausubel, F. M et al., 1992. In vitro methods of antibody selection, such as antibody phage display, may also be used to generate antibodies recognizing the biomarkers of Table 1 (see, e.g., Schirrmann et al. Molecules 2011 16:412-426). In addition, a list of commercially antibodies against the biomarkers of Table la is provided in Table 2.
  • the antibodies are immunoglobulin single variable domains (ISVDs).
  • ISVDs are optimal for use in tracer design, given its flexibility in selection and low costs production of high affinity formats, high tissue penetrance, fast kinetics and the associated low radiation burden as PET tracer to allow repeated administrations, low immunogenicity, and lack of immune-mediating (complement- cellular) capacity.
  • Suitable ISVDs include light chain variable domain sequences or heavy chain variable domain sequences.
  • the ISVD may be a domain antibody, or a single domain antibody, or a "dAB” or dAb, or a Nanobody, , or any suitable fragment thereof. (See, e.g., "Single domain antibodies,” Methods in Molecular Biology, Eds. Saerens and
  • the ISVD comprises a single amino acid chain comprising four 4"framework sequences" and 3 "complementary determining regions" or CDRs.
  • ISVDs may be isolated from any organism such as mouse, rat, rabbit, donkey, human, shark (e.g., IgNARs), or camelid (e.g., VHH domain).
  • phage display libraries have been generated in order to select antigen-specific VHH from the VHH repertoire of immunized camels or llamas.
  • the VHH genes are cloned in phage display vectors, the antigen binders are obtained by panning and selected VHH are expressed in bacteria (see, e.g., WO 2004/044204).
  • the ISVD is a nanobody.
  • nanobodies have an amino acid sequence with the structure FR 1 - CDR 1 -FR2 - CDR2 -FR3- CDRS -FR4, , and in which one or more of the Hallmark residues are as defined in WO 08/020079 (Tables A-3 to A-8) (see also, US 20160333099.A further description of Nanobodies, including humanization and/or camelization of Nanobodies, can be found, e.g., in WO 08/101985 and WO 08/142164. Suitable antibodies can also be obtained from e.g., QVQ Research and ProSci Inc. In preferred embodiments, the expression level of biomarker is determined in an immunoassay. Suitable immunoassays include, e.g., radio-immunoassay, ELISA (enzyme -linked immunosorbant assay), "sandwich" immunoassay,
  • immunoradiometric assay gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein A assay, and immunoelectrophoresis assay.
  • assays using other biomarker binding compounds may be used.
  • a biomarker binding peptide can be immobilized on a solid support such as a chip. A biological sample is passed over the solid support. Bound biomarker is then detected using any suitable method, such as surface plasmon resonance (SPR) (See e.g., WO 90/05305, herein incorporated by reference).
  • SPR surface plasmon resonance
  • the detection of biomarker includes the detection of peptide fragments of said biomarkers.
  • Peptide fragments are understood as being from 10 to 100 amino acids in length, preferably from 10 to 50 amino acids in length.
  • the methods described herein classify an individual as having or being at risk of developing CAA. Preferably, the methods predict the likelihood that an individual is either afflicted with or not afflicted with CAA.
  • CAA afflicted patients are susceptible to complications when treated with anti-coagulants (such as heparin and warfarin). For example, it has been demonstrated that patients with CAA have an increased risk of bleeding when treated with warfarin.
  • Anticoagulants are commonly used as medication for thrombotic disorders. Specific indications for anticoagulant therapy include Atrial fibrillation, Coronary artery disease, Deep vein thrombosis, Ischemic stroke, Hypercoagulable states (e.g., Factor V Leiden), Myocardial infarction,
  • Pulmonary embolism, and Restenosis from stents Such disorders are more prevalent in the elderly population, which is also prone to CAA.
  • a positive diagnosis for CAA may result in a physician recommending an alternative treatment, in particular in the use of anti-coagulants.
  • the disclosure provides method for determining a treatment schedule for an individual, comprising determining, as disclosed herein, whether an individual has or is at risk of developing CAA.
  • the recommended dosage of anticoagulant may be lowered or avoided altogether if other treatments are available.
  • a patient with CAA is treated with antiplatelet drugs (e.g., aspirin) instead of anticoagulant therapy.
  • thromboembolic disorder the method comprising determining, as disclosed herein, whether an individual has or is at risk of developing CAA and treating an individual determined to have or be at risk of developing CAA with antiplatelet therapy.
  • Thromboembolic disorders include venous thromboembolism (e.g., deep vein thrombosis in postoperative patients and acute deep vein thrombosis), pulmonary embolism, Atrial fibrillation, Coronary artery disease, Deep vein thrombosis, Ischemic stroke, Hypercoagulable states (e.g., Factor V Leiden), Myocardial infarction, arterial thrombosis, and Restenosis from stents.
  • venous thromboembolism e.g., deep vein thrombosis in postoperative patients and acute deep vein thrombosis
  • pulmonary embolism pulmonary embolism
  • Atrial fibrillation Coronary artery disease
  • Deep vein thrombosis Deep vein thrombosis
  • Ischemic stroke Ischemic stroke
  • Hypercoagulable states e.g., Factor V Leiden
  • Myocardial infarction e.g., Myocardial infarction
  • arterial thrombosis
  • CAA may lead to side-effects when other medicaments are used as well.
  • the presence of CAA may effect the efficacy or side-effects of AD therapies. This is particularly relevant since a majority of AD patients suffer from CAA. A number of therapies are in development for the treatment of AD. Stratifying AD patients in clinical trials based on their CAA status would aid in determining which therapies are effective for which patient populations.
  • AD therapies Two types of AD therapies currently being investigated are active A6-immunotherapy and passive ⁇ -immunotherapy.
  • Active ⁇ -immunotherapy uses synthetic ⁇ peptides as vaccines to stimulate a patient's B cells to generate specific antibodies for sequestering amyloid from the brain in the peripheral system.
  • Passive ⁇ -immunotherapy uses synthetic ⁇ peptides as vaccines to stimulate a patient's B cells to generate specific antibodies for sequestering amyloid from the brain in the peripheral system.
  • immunotherapy refers to treating patients with monoclonal antibodies that are directed to ⁇ peptides (mAbs).
  • mAbs monoclonal antibodies that are directed to ⁇ peptides
  • Such antibodies that are and have been used in clinical trials include bapineuzumap, ponezumab, solanezumab, gantenerumab, crenezumab, aducamab, BAN2401 and SAR228810.
  • the efficacies and side effects of these drugs differ.
  • bapineuzumab is associated with micro-hemorrhages.
  • Stratification of patients according to CAA status during clinical trials would demonstrate whether such side effects are over-represented in other the CAA positive or CAA negative groups. Such results would indicate that the AD therapies in development may be more beneficial than previously thought, if administered to a particular patient group.
  • a further embodiment of the disclosure provides for a method for monitoring the treatment and/or progression of CAA in an individual, the method comprising a) determining, as disclosed herein, the level of at least one biomarker selected from Table 1 at a first time point,
  • the individual receives a treatment for CAA and/or AD between the first and second time point.
  • a treatment for CAA and/or AD may be a therapy in development, for example being studied in a clinical trial.
  • the treatment is a candidate treatment for CAA and/or AD.
  • the treatment may be administered once or over a several day, week, month, or year period.
  • the second time point may occur during a treatment regime or after the conclusion of the treatment.
  • a decrease in the level of biomarker between the first and second time point, or rather, after receiving a therapy for CAA and/or AD, indicates that the therapy is useful for treating CAA.
  • the method monitors the progression of CAA in an individual. For example, biomarker expression may be determined in a patient when the first signs of dementia or cognitive decline appear. Biomarker expression may be monitored in the individual, e.g., monthly, every 6 months, or yearly, in order to determine the progression of CAA in the individual. An increase in biomarker level expression at the second time point as compared to the first time point indicates that the disease is progressing.
  • the biomarkers disclosed herein are used in methods related to the sporadic form of CAA.
  • the biomarkers are also useful in methods related to the familial forms of CAA.
  • such methods include monitoring the treatment or progression of CAA in a patient afflicted with the familial form of the disease.
  • a method is provided for screening and identifying compounds for the treatment of a CAA by administering one or more candidate compounds to a preclinical animal model of CAA and using the expression level of a biomarker selected from Table 1 as a readout to determine whether the compound treats CAA in the animal model.
  • the method comprises: (a) administering one or more candidate compounds to a preclinical animal model of
  • CAA CAA
  • CAA animal models are well-known to a skilled person, see e.g., Herzig et al. Brain Pathol. 2006 Jan; 16(l):40-54. and Dommtz et al. J. Neuropathl Exp Neurol 2005 64:588-594 for a description of suitable mouse models of CAA which may be used in the methods.
  • Candidate compounds to be tested include passive AB-immunotherapy treatments as well as PDE3 inhibitors.
  • PDE3 inhibitors are just one class of compounds that is also in development for the treatment of CAA.
  • the expression level of biomarker is compared to the biomarker level in a CAA+ animal or population of CAA+ animals (i.e., the reference value).
  • the reduction of biomarker in the treated animal as compared to the reference value (CAA+ animal) indicates that the treatment is successful.
  • the expression level of biomarker is compared in the same animal before and after treatment.
  • the expression level of biomarker is determined in the test animal at day 0 (i.e., baseline level). Treatment with a candidate compound begins on day 1 and may occur once or over the course of several days, weeks, months. The level of biomarker may be tested during the course of treatment as well as after treatment has stopped.
  • the application further provides COL6A2 as a general small vessel disease marker. While not wishing to be bound by theory, COL6A2 may be acting as a stress marker in, e.g., endothelial cells. COL6A2 is particularly useful for detecting and diagnosing CAA, CADASIL, CARASAL, and hypertension related SVD. In a further aspect of the disclosure, methods are provided for distinguishing CAA from other small vessel diseases.
  • NDP, APOE, and APCS demonstrate significantly higher reactivity in CAA type 1 and Prp-CAA over other small vessel diseases, such as, cerebral autosomal dominant arteriopathy, with sub cortical infarcts and leukoencephalopathy (CADASIL), hypertension related small vessel disease, and Cathepsin A-related arteriopathy with strokes and
  • small vessel diseases such as, cerebral autosomal dominant arteriopathy, with sub cortical infarcts and leukoencephalopathy (CADASIL), hypertension related small vessel disease, and Cathepsin A-related arteriopathy with strokes and
  • the disclosure further provides a method of classifying CAA distinguishing from other small vessel diseases comprising:
  • a method for treating CAA in an individual in need thereof comprising altering the activity or expression of Norrin (NDP) (collectively referred to herein as Norrin modifiers) in said individual.
  • NDP Norrin
  • the present disclosure demonstrates that NDP is highly upregulated in CAA and localizes around the vasculature. NDP is a small secreted protein with a molecular weight of approximately 15kl). It has important function in the formation of the brain vasculature during development and maintaining a proper functioning BBB
  • NDP is primarily expressed by astrocytes (Ye et al. 2011).
  • Norrin activates the canonical Wnt/ b -catenin signaling pathway via frizzled (Fzd)4 low- density lipoprotein receptor-related protein (Lrp)5/6 receptor complex (Xu et al. 2004).
  • Fzd frizzled
  • Lrp low- density lipoprotein receptor-related protein
  • Mice overexpressing NDP had significantly less vascular loss following oxygen exposure.
  • NDP neurodegenerative disease
  • Norrie disease which is primarily an eye disease that leads to blindness. It is characterized by iris atrophy, corneal clouding, cataract, and retinal dysplasia with early vascular proliferation (pseudoglioma) followed by bulbar atrophy.
  • symptoms are also regularly related to the brain and include developmental delay, psychotic features and mental retardation (Sims 1993; Braunger et al. 2012).
  • NDP has been shown to protect neurons against excitotoxicity induced by NMDA (Seitz et al. 2010).
  • NDP seems to be protective protein for both endothelial and neurons.
  • NDP signaling might be impaired, and restoring or fostering NDP signaling might be beneficial in CAA patient.
  • methods are provided for increasing the activity or expression of Norrin in an individual in need thereof.
  • the Norrin is provided as a gene therapy, for example for localized delivery to the brain.
  • a further aspect of the present disclosure is the provision of a vector comprising a nucleic acid molecule encoding Norrin for use in gene therapy. Such therapy is useful in the treatment of CAA.
  • nucleic acid molecule encoding Norrin.
  • the nucleic acid molecule is preferably provided in a viral vector suitable for gene therapy.
  • Appropriate vectors and delivery methods are known to a skilled person and are described, e.g., in Schlachetzki et al. Neurology, Gene therapy of the brain. (2004) 62: 1275-1281 and Richardson et al. Neurosurg Clin N Am. 2009 20(2):205-10.
  • Norrin inhibitors are provided for decreasing the activity or expression of Norrin in an individual in need thereof (herein referred to as Norrin inhibitors).
  • the term "inhibitor” is used in the broadest sense, and includes, e.g., any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of Norrin or reduces expression of Norrin mRNA or Norrin functional protein.
  • Inhibitors of Norrin include polypeptides, small molecules, and nucleic acid based inhibitors.
  • the inhibitor is a nucleic acid molecule, in particular a molecule that causes the degradation of or inhibits the function, transcription, or translation of Norrin in a sequence-specific manner.
  • Exemplary nucleic acid molecules include aptamers, siRNA, artificial microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense oligonucleotides, and DNA expression cassettes encoding said nucleic acid molecules.
  • Other suitable Norrin inhibitors include those described in
  • the nucleic acid molecule is an antisense oligonucleotide.
  • Antisense oligonucleotides generally inhibit their target by binding target mRNA and sterically blocking expression by obstructing the ribosome. ASOs can also be used for "exon-skipping". Exon-skipping oligonucleotides bind to pre-mRNA and modulate splicing such that one or more exons are skipped in the resulting mRNA. Exon- skipping may lead to an in frame deletion resulting in a truncated protein or protein lacking internal amino acids or skipping may lead to a premature stop codon resulting in nonsense-mediated decay. The design of such oligonucleotides is well-known in the art (see, e.g., Aartsma-Rus et al Mol Ther 17(3):548 (2009)).
  • ASOs can also inhibit their target by binding target mRNA thus forming a DNA- RNA hybrid that can be a substance for RNase H.
  • ASOs may also be produced as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Methods for designing and modifying such gapmers are described in, for example, U.S. Patent Publication Nos. 20110092572 and 20100234451.
  • Preferred ASOs include Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and morpholinos.
  • the nucleic acid molecule is an RNAi molecule, i.e., RNA interference molecule.
  • RNAi molecules include siRNA, shRNA, and artificial miRNA.
  • siRNA comprises a double stranded structure typically containing 15 to 50 base pairs and preferably 19 to 25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell.
  • An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure.
  • shRNA small hairpin RNA
  • these shRNAs are composed of a short, e.g.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • siRNA molecules The design and production of siRNA molecules is well known to one of skill in the art (Hajeri PB, Singh SK. Drug Discov Today. 2009 14(17- 18):851-8). Methods of administration of therapeutic siRNA is also well-known to one of skill in the art (Manjunath N, and Dykxhoorn DM. Discov Med. 2010 May;9(48):418-30; Quo J et al, Mol Biosyst. 2010 Jul 15;6(7): 1143-61).
  • siRNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding.
  • Artificial miRNA molecules are pre-miRNA or pri-miRNA comprising a stem-loop structure(s) derived from a specific endogenous miRNA in which the stem(s) of the stem-loop structure(s) incorporates a mature strand-star strand duplex where the mature strand sequence is distinct from the endogenous mature strand sequence of the specific referenced endogenous miRNA.
  • stem-loop structure(s) derived from a specific endogenous miRNA in which the stem(s) of the stem-loop structure(s) incorporates a mature strand-star strand duplex where the mature strand sequence is distinct from the endogenous mature strand sequence of the specific referenced endogenous miRNA.
  • RNA interference refers to a decrease in the mRNA level in a cell for a heterologous target gene by at least about 5%, about 10%, about 20%, about 30%), about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the, e.g., miRNA or siRNA interference molecule
  • RNAi molecules may also include chemical analogues such as, e.g., 2'-0-Methyl ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and other chemical analogues of nucleic acid oligonucleotides.
  • the nucleic acid molecule inhibitors may be chemically synthesized and provided directly to cells of interest.
  • the nucleic acid compound may be provided to a cell as part of a gene delivery vehicle.
  • a gene delivery vehicle is preferably a liposome or a viral gene delivery vehicle. Liposomes are well known in the art and many variants are available for gene transfer purposes.
  • Vectors comprising the nucleic acids disclosed herein are also provided.
  • a “vector” is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached.
  • the term “vector” includes both viral and non-viral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.
  • Viral vectors include lentivirus, retrovirus, adeno-associated virus (AAV), pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors.
  • Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • Lentiviruses have been previously described for transgene delivery to the hippocampus (van Hooijdonk BMC Neuroscience 2009, 10:2) There are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205- 210 (1993)). Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided.
  • the method of introduction is largely dictated by the targeted cell type include, e.g., CaPOt precipitation, liposome fusion, lipofectin, electroporation, dextran-mediated transiection, calcium phosphate precipitation, polybrene mediated transiection, protoplast fusion, , viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • the nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). Such cells are useful for producing isolated polypeptides, which may be used in the methods described herein.
  • the Norrin inhibitors described herein are antibodies.
  • the term "antibody” includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Methods of making antibodies are well known in the art and many suitable antibodies are commercially available.
  • the antibodies disclosed herein include antigen binding fragments (e.g., Fab', F(ab')2, Fv or Fab fragments).
  • Preferred Norrin antibodies or antigen-binding fragments thereof are humanized or human antibodies or antigen-binding fragments thereof. Additional Norrin inhibitors are described in EP2334702 (i.e., "Norrin antagonists").
  • the Norrin modifiers are preferably prepared as pharmaceutically acceptable compositions. When administering the pharmaceutical preparations thereof to an individual, it is preferred that the compound is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution.
  • excipients capable of forming complexes, vesicles and/or liposomes that deliver such a compound as defined herein in a vesicle or liposome through a cell membrane.
  • Suitable excipients comprise polyethylenimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT- 18), lipofectmTM, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, to a cell.
  • a Norrin modifier is provided directly to the brain and or central nervous system.
  • the compound may be delivered by way of a catheter or other delivery device having one end implanted in a tissue, e.g., the brain by, for example, intracranial infusion.
  • a tissue e.g., the brain by, for example, intracranial infusion.
  • intracranial infusion Such methods are known in the art and are further described in U.S. Publications 20120116360 and 20120209110, which are hereby incorporated by reference.
  • a Norrin inhibitor as described herein may also be administered into the cerebral spinal fluid.
  • Methods that use a catheter to deliver a therapeutic agent to the brain generally involve inserting the catheter into the brain and delivering the composition to the desired location.
  • surgeons typically use stereotactic apparatus/procedures, (see, e.g., U.S. Pat. No. 4,350, 159)
  • an incision may be made in the scalp to expose the patient's skull.
  • the catheter may be inserted into the brain.
  • Other delivery devices useful with methods disclosed herein include a device providing an access port, which can be implanted subcutaneously on the cranium through which therapeutic agents may be delivered to the brain, such as the model 8506 ICV Access Port and the 8507 Intraspinal Port, developed by Medtronic, Inc. of Minneapolis, Minn.
  • Actual dosage levels of the pharmaceutical preparations described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start with doses of the compounds described herein at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • an element means one element or more than one element.
  • an individual is any mammal including humans; laboratory animals such as rats, mice, simians and guinea pigs; domestic animals such as rabbits, cattle, sheep, goats, cats, dogs, horses, and pigs and the like. Preferably said individual is human.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • NBB Netherlands Brain Bank
  • NIN Netherlands Institute for Neuroscience
  • Brain tissue was selected based on clinical and neuropathological reports. Three groups were composed. Cognitively healthy control cases lacking any pathology, AD cases with severe plaque pathology but devoid of CAA and capillary CAA cases with severe CAA type-1 pathology (also named CapCAA). All cases are listed in Table 4. The Braak stage for tau pathology [8,9], ⁇ phase according ABC criteria [26,36], CERAD score for neuritic plaques [24] are indicated.
  • Sections were briefly washed (3x 30sec) and freshly prepared 3,3' diaminobenzidine (DAB) solution was applied and left to incubate for 5 minutes to visualize antibody binding. Sections were thoroughly washed in ultra-pure H2O and incubated with 1% (w/v) toluidine blue in ultrapure H2O for 1 minute as a counterstain. Sections were then washed in ultra-pure H2O twice for 1 minute and twice in 100% ethanol for 1 minute and air dried.
  • DAB 3,3' diaminobenzidine
  • LCM Laser capture microdissection
  • Micro-dissected tissue lysates were incubated at 95 °C for 5 min to denature the proteins, followed by incubation with 50 mM iodoacetamide for 30 min at RT in the dark to alkylate the cysteine residues.
  • To reduce protein complexity samples were size separated on a NuPAGE® 4-12% Bis-Tris acrylamide gel (Invitrogen, Carlsbad, CA, USA) using MOPS SDS running buffer (Invitrogen, Carlsbad, CA, USA) according to the manufacturers' protocol.
  • the peptides were dissolved in 15 ⁇ of 0.1% (v/v) acetic acid of which 10 ⁇ was loaded onto a nano-liquid chromatography (nano-LC) system (Eksigent). The peptides were separated using a capillary reversed phase C18 column that had been equilibrated with 0.1% (v/v) acetic acid at a flow rate of 400 nL/min. The peptides were eluted by increasing the acetonitrile concentration linearly from 5 to 40% in 80 min and to 90% in 10 min, using the same flow rate. Eluted peptides were transferred into the LTQ/Orbitrap MS (Thermo Scientific) by Electro Spray Ionisation (ESI).
  • ESI Electro Spray Ionisation
  • the Orbitrap was operated in the range of m/z 350-2000 at a full width at half maximum resolution of 30,000 after accumulation to 500,000 in the LTQ with one microscan.
  • the five most abundant precursor ions were selected for fragmentation by collision- induced dissociation (CID) with an isolation width of 2 Da.
  • MaxQuant software was used for spectrum annotation, protein inference, and relative protein quantification [14]. Spectra were annotated against the Uniprot human reference proteome database (version 2016_04). Enzyme specificity was set to Trypsin/P, allowing at most two missed cleavages. Carbamido-methylation of cysteine was set as a fixed modification, and N-acetylation and methionine oxidation were set as variable modifications. Mass deviation tolerance was set to 20 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. False -discovery rate cut-offs for peptide and protein identifications were set to 1% for both. The minimum peptide length was seven amino acids.
  • CAA selective proteins Conditions that were set for inclusion of CAA selective proteins comprise of three approaches (A, B and C) that are visualized in figure 2.
  • Approach A T-tests (two- sided, assuming unequal variances, performed using Excel (Microsoft)) were performed contrasting the three experimental groups. When there was a significant difference (p ⁇ 0.05) between both the control group versus CAA, and the AD group versus CAA, a protein was labelled as CAA specific.
  • Approach B If the number of quantitative values in the control group was zero or one while the AD and CAA groups both had two or more quantitative values, than a t-test was performed between the AD and the CAA group.
  • Protein extracts were prepared by lysis of whole occipital lobe tissue in reducing SDS sample buffer using a 1:20 tissue weight to lysis buffer ratio.
  • Proteins were denatured at 95 °C for 5 min and separated by SDS-PAGE using precast Stain Free gradient gels (Bio-Rad) and transferred (40 V overnight at 4°C) onto a 0.45 ⁇ PVDF membrane (Merck Millipore), which was pre-incubated in 100% methanol.
  • the PVDF membrane was incubated in Odyssey blocking buffer for lh and subsequently incubated with the primary antibody overnight. All primary antibodies used are listed in Table 2.
  • Fresh frozen or paraffin embedded human occipital tissue was cut (5 ⁇ ).
  • the sections were placed on a SuperFrost Microscope Slide (VWR, PA, USA) and air-dried overnight at room temperature (RT). Prior to staining, the sections were fixed in 100%) acetone for 10 minutes.
  • the paraffin was removed by washing in xylene. Next the sections were washed in decreasing concentrations (100%, 96% and 70% (v/v)) of ethanol. . Endogenous peroxidase activity was quenched by incubating in methanol with 0.3% H2O2 for 30 minutes at RT. Next, antigen retrieval was performed by submerging the slides in citrate buffer (pH6) and heating in an autoclave.
  • the slides were dehydrated by incubation in increasing concentrations of ethanol consisting of 70% (v/v), 96% (v/v) and 100% (v/v) ethanol.
  • the slides were then incubated in xylene and mounted using Quick-D mounting medium. A negative control was made by omission of the primary antibody. Quantification of the staining was done using Image-J using the threshold colour plugin.
  • case #5 was clustered with the control cases indicating that the protein expression profile of this sample is more similar to the control cases than to other CAA or AD cases.
  • Visualizing the expression profile of case #5 next to the average expression profiles of the three groups confirmed the resemblance of case #5 to the control group, but also showed several proteins that are similar in expression to the AD and or CAA groups.
  • Case #5 was identified as having an expression profile resembling a control case. Case #5 was found positive for Alzheimer type 2 astrocytes, possibly related to high alcohol intake and exhibited relatively low tau pathology. Otherwise, this case showed no pathological abnormalities when compared to the rest of the CAA type- 1 group. However, The expression of several CAA selective markers that we identified was inspected for case #5. The levels of these markers correspond well with the other cases of the CAA group, indicating that these proteins are inseparably linked to the pathology of CAA type- 1.
  • COL6A2 IHC showed some immunoreactivity in control and AD cases which was restricted to leptomeningeal vessels (Fig. 10) and a few large vessels in the brain tissue.
  • CAA type-1 immunoreactivity for COL6A2 was highly increased and includes brain capillaries and larger vessels. Immunoreactivity was mostly associated with the endothelium and / or the adventitia (Fig. 5G- I). Similar results were obtained using two different antibodies for COL6A2 (data not shown).
  • HTRA1 IHC showed clear overlap with ⁇ in both AD and CAA. Compact and diffuse staining was observed related to the vessels in the CAA cases and showed plaque pathology in the AD cases without CAA. Control cases were all negative for HTRA1 (Fig. 5J-L).
  • IHC for APOE resulted in pronounced staining of the vasculature in CAA cases and appeared related to compact deposits as well as more diffuse dysphoric staining. Also immunoreactivity of APOE was observed in the AD cases related to the A6 plaques, although the staining was less intense than that related to the vascular amyloid in the CAA cases (Fig. 5P-R).
  • APCS IHC illustrated the presence of this protein in relation with both diffuse and compact ⁇ pathology in both the CAA and AD group. However, staining related to the plaque pathology was less intense than that related to the vascular AB pathology (Fig. 5M-0).
  • IHC cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
  • IHC was performed on sections that exhibited the relevant pathological characteristics of each disease including an additional CAA type 1 case (Fig. 7A-E).
  • the PrP-CAA tissue was confirmed negative for ⁇ pathology (Fig. 7K) and showed positive for Prp (data not shown).
  • NDP immunore activity was highly increased and localised to the affected vessels. NDP staining was more pronounced around affected capillaries than at affected larger vessels.
  • COL6A2 immunore activity was clearly present around capillaries and larger affected vessels, of which the vascular pathology was clearly observed using haematoxylin. Although co-occurring in largely the same vessels COL6A2 does not generally co-localize with the deposits, but instead localizes more internally as a component of the basal membrane.
  • APOE and APCS were also highly present and co-localized with the Prp deposits.
  • NDP is explicitly suitable to evidently separate CAA from ⁇ plaque pathology (Table 5).
  • CAA cerebrovascular diseases
  • AD cerebrovascular disease
  • LC-MS-MS LC-MS-MS analysis of AD brain tissue exhibiting severe CAA type- 1 pathology, AD brain tissue without apparent involvement of CAA, and control brain tissue without AD related pathology.
  • proteome of CAA type-1 is different from that of parenchymal plaque pathology in AD, which led to the identification of proteins selectively associated with CAA.
  • NDP is found highly upregulated in CAA specifically type 1, including cotton wool ⁇ pathology and Prp-CAA, and localizes around the affected vasculature.
  • NDPNDP immunore activity was only mildly increased in CADASIL and hypertension.
  • these diseases affect different anatomical regions and present a distinct clinical picture, leaving NDP a promising biomarker for CAA.
  • NDP is a small, secreted protein with a molecular weight of approximately 15 kD. It has important function in the formation of the brain vasculature during development and maintenance of a proper functioning BBB [8] blood brain barrier [15] . In the adult brain the NDP gene is primarily expressed by astrocytes [46]. NDP activates the canonical Wnt/ 6-catenin signalling pathway via the frizzled (Fzd)4/low-density lipoprotein receptor-related protein (Lrp)5/6 receptor complex [45].
  • Fzd frizzled
  • Lrp low-density lipoprotein receptor-related protein
  • NPCs neural progenitor cells derived from FAD mutant PSEN1 subjects it was found that NDP mRNA is upregulated, but no increase in mRNA was found in AD human temporal lobe [35]. In the retina NDP was found to promote regrowth of capillaries and formation of intra-retinal vessels after oxygen-induced retinal damage [27]. Mice overexpressing NDP, had significantly less vascular loss following oxygen exposure.
  • NDP neurodegenerative disease
  • Norrie disease which is primarily an eye disease that leads to blindness.
  • 30-50% of these patients display developmental delay, intellectual disability, behavioural abnormalities, or psychotic-like features [10, 33] .
  • NDP has been shown to protect neurons against excitotoxicity induced by NMDA [32].
  • NDP seems to have protective properties for both endothelial cells and neurons, but whether NDP upregulation is beneficial in the context of CAA pathology is unknown.
  • COL6A2 is a non-fibril collagen and COL6 isoforms are present in various tissues including the vasculature [29] .
  • COL6A2 encodes one of the three alpha chains of type VI collagen, which is found in most connective tissues.
  • Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen [21].
  • collagen VI was shown neuroprotective and its expression increased in animal models of AD [11] .
  • HTRAl is a trypsin-like serine protease which was detected in all CAA samples, with a single value in the AD group and zero quantitative values in the control group. Using immunohistochemistry we found a significant difference with the control groups but not with the AD group as HTRAl marks normal plaque pathology as well. HTRAl is relevant in neurodegeneration as this protease is involved in the degradation of APP and AB [17]. In addition, HTRAl was found to degrade APOE4 more efficiently than APOE3 and the presence of APOE4 reduces digestion of MAPT by HTRAl [12] . In addition, mutations in HTRAl are the cause of the hereditary small vessel disease CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) [19, 37].
  • CARASIL hereditary small vessel disease
  • CAA type-1 biomarkers The proteins discussed in this study, that show specific selective association with CAA type-1 pathology can be used as CAA type-1 biomarkers in patients. As larger vessels are also positive for the markers that were assessed using IHC, these markers might also be relevant in CAA type-2. Although in the case of NDP the intensity of immunore activity is less in larger vessels compared to affected capillaries in CAA. CAA selective markers might be used for pathological assessment of the severity of CAA. The association of AB with the vasculature, and in particular capillaries, is not always obvious in thin microscopic sections. Also, the use of these proteins as potential diagnostic markers should be explored.
  • CAA Cervity-associated edemaoedema and cerebral microhemorrhages.
  • anti- amyloid immunotherapies in development may warrant stratification of AD patients with or without CAA because of expected side- effects associated with CAA, including vasogenic edemaoedema and cerebral microhemorrhages.
  • CAA type-1 is not always clearly visible in immunohistochemical sections, since association of vessels, in particular capillaries, with amyloid deposits are not visible in thin microscopic sections.
  • antibodies to CAA marker proteins may be applicable in PET tracers.
  • protein markers identified in this study showing specific localization in CAA type-1 will provide new opportunities for neuropathological diagnosis and contribute to studies investigating the role of CAA in pathology [6, 34] .
  • these markers would help to increase the safety of anticoagulation therapy in the elderly, which are contra-indicated in CAA.
  • AD cerebral amyloid angiopathy
  • M male
  • F female
  • PMD post mortem delay
  • PMD post mortem delay
  • NA used for mass spectrometry analysis
  • MS used for validation: V. (* ⁇ only present as dysphoric CAA)
  • V 21 CAA type- 1 F 94 A3 B3 C3 4:30 43
  • Protein levels of the markers described in table 1 will be measured in CSF and serum using commercially available ELISA tests or custom made (in-house) ELISA tests. These assays will be used to analyze the body fluid concentrations in post-mortem tissue homogenates, serum and CSF of 10 CAA patients included in the discovery cohort (post mortem derived). Next, the levels of proteins will be analyzed in 4 available CAA cases (post mortem confirmed) of whom CSF was collected in vivo/ante -mortem.
  • Lead antibodies will be tested on postmortem derived tissue sections of human organs, and on human brain (AD, AD + CAA, healthy subjects) to confirm the recognition specificity for CAA.
  • Amyloid 20 179-87. doi: 10.3109/13506129.2013.797389

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Abstract

L'invention concerne des biomarqueurs et des méthodes de diagnostic pour déterminer la présence et/ou le risque d'angiopathie amyloïde cérébrale (AAC) ainsi que des méthodes de criblage pour des traitements de l'AAC.
PCT/NL2017/050831 2016-12-12 2017-12-12 Biomarqueurs et traitements de l'angiopathie amyloïde cérébrale (aac) WO2018111099A1 (fr)

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WO2021234607A1 (fr) * 2020-05-20 2021-11-25 St. Jude Children's Research Hospital Détection de la maladie d'alzheimer à l'aide de biomarqueurs spécifiques

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