Methods of treating cognitive disorders

Abstract

This invention relates to the treatment of cognitive impairment, such as age-related cognitive impairment. In some aspects, it involves determining the ratio between the levels of any two proteins selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RSP3, VAV3, SIRT3, and SERPINB8 in a sample obtained from a subject. The subject may have or be suspected of having cognitive impairment. Cognitive impairment may be caused by neurodegenerative diseases such as Alzheimer's disease. The subject can be identified as likely or unlikely to respond positively to plasma exchange therapy based on the ratio between the measured protein levels. In some aspects, methods for treating cognitive impairment in a subject include administering plasma exchange therapy comprising full-volume and / or low-volume plasma exchange. Kits suitable for performing such methods are also provided.

Description

[0001] I. Introduction Technical Field

[0002] This invention relates to a method for treating cognitive impairment in subjects. Background Technology

[0003] The following content is provided for background information only and does not constitute prior art of this invention.

[0004] Aging is a significant risk factor for a variety of human diseases, including cognitive impairment, cancer, arthritis, vision loss, osteoporosis, diabetes, cardiovascular disease, and stroke. Age-related neurodegeneration is a major contributing factor to cognitive impairment. Therefore, aging remains the leading single risk factor for dementia-related neurodegenerative diseases such as Alzheimer's disease (AD) (Bishop, NA et al.). Neural mechanisms of aging and cognitive decline Nature 464(7288), 529-535 (2010); Heeden, T. et al. , Insights into the aging mind: a view from cognitive neuroscience Nat. Rev. Neurosci. 5(2), 87-96 (2004); Mattson, MP, et al. Ageing and neuronal vulnerability . Nat. Rev. Neurosci. 7(4), 278-294 (2006)).

[0005] Aging affects all tissues and functions of the body, including the central nervous system, and neurodegenerative changes and declines in functions such as cognitive or motor skills can severely impact quality of life. Treatments for cognitive decline, motor disorders, and neurodegenerative diseases have had limited success in preventing and reversing damage. Furthermore, while some treatments, such as plasma-based therapies or plasma exchange therapy, have shown promise (Boada et al.), Alzheimers Dement. (16:1412-25, 2020), but these treatments are not always successful. In other words, while some patients show positive outcomes after receiving treatment for this type of cognitive impairment, others do not. This results in a waste of valuable time and resources as the disease progresses.

[0006] Therefore, it is desirable to identify patients who may or may not respond positively to cognitive impairment treatment before administering such treatment.

[0007] II. Summary of the Invention

[0008] In some aspects, the present invention provides methods for treating cognitive impairment. Such treatment may include plasma exchange therapy. In some cases, the subject is identified as potentially or potentially unlikely to respond positively to plasma exchange therapy. Determining whether a subject is likely or unlikely to respond positively to plasma exchange therapy can be based on the determination of the ratio between the levels of any two proteins in the subject's sample, selected from: Delta-like protein 1 (UniProtID: O00548, DLL1), SPARC-associated modular calcium-binding protein 1 (UniProtID: Q9H4F8, SMOC1), CD59 glycoprotein (UniProtID: P13987, CD59), thiosulfate:glutathione S-transferase (UniProtID: Q8NFU3, TSTD1), signal transduction and transcription activator 3 (UniProtID: P40763, STAT3), DNA polymerase δ subunit 4 (UniProtID: Q9HCU8, POLD4), protein mono-ADP-ribosyltransferase PARP11 (UniProtID: Q9NR21, PARP11), and left-right determinant 2 (UniPro... tID: O00292, LEFTY2), Netrin receptor UNC5B (UniProtID: Q8IZJ1, UNC5B), complement C5 (UniProtID: P01031, C5), complement C5b-C6 complex (UniProtID: P01031 / P13671, C5.C6), Set1 / Ash2 histone methyltransferase complex subunit ASH2 (UniProtID: Q9UBL3, ASH2L), inhibin βB chain (UniProtID: P09529, INHBB), small ribosomal subunit protein uS3 (UniProtID: P23396, RPS3), guanine nucleotide exchange factor VAV3 (UniProtID: Q9UKW4, VAV3), NAD-dependent protein deacetylase sirtuin-3, mitochondrial type (UniProtID: Q9NTG7, SIRT3), and Serpin B8 (UniProtID: P50452, SERPINB8).

[0009] Any suitable assay can be used to determine the level of the protein being measured and the ratio between the levels of the protein being measured. In some embodiments, aptamer-based multiplex proteomics assays (such as the SomaScan™ assay) are used to determine the level of the protein being measured.

[0010] Therefore, in some aspects, the present invention provides a method for determining the ratio between the levels of any two proteins selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8. The subject may have or be suspected of having cognitive impairment.

[0011] Cognitive impairment can be caused by neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, and vascular dementia.

[0012] Another aspect of the invention provides a kit comprising reagents for determining the levels of any two proteins in a sample, the two proteins being selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8. The ratio between the levels of the protein pair can be determined based on the determination results of the protein pair.

[0013] III. Merging by Reference

[0014] All publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and separately indicated to be incorporated by reference.

[0015] IV. Description of the attached drawings

[0016] Figure 1 A schematic diagram illustrating an example of plasma exchange therapy is shown. Figure 1In the example described, Alzheimer's disease albumin replacement management (AMBAR) treatment includes a 6-week intensive period during which routine therapeutic PE (TPE, also known as FPE - total plasma exchange, treating one plasma volume [≈ 2500-3000 mL]) is performed and replaced with 5% albumin (1 TPE / week), followed by a 12-month maintenance period during which low-volume PE (LVPE: removing approximately 1 / 3 of the plasma volume [≈ 690-880 mL]) is performed and replaced with 20% albumin or IVIG (1 LVPE / month), applicable to all 3 active groups (arm): in one group, 20 g of 20% Albutein is used for replacement during LVPE, while in the other two groups, visits for 20% Albutein replacement (20 or 40 g) are alternated with visits for 5% Flebogamma DIF (10 or 20 g). (1) through (8) indicate the times when biological samples were collected before and after the TPE or LVPE procedure: (1) serum sample before TPE1, (2) CSF sample before TPE1, (3) serum sample after TPE1, (4) serum sample before LVPE1, (5) CSF sample before LVPE1, (6) serum sample after LVPE1, (7) serum sample at the end of the study (EOS), and (8) CSF sample at EOS. IVIG: intravenous immunoglobulin; Albutein and Flebogamma DIF are commercially available products.

[0017] Figure 2 Proteomics analysis of the samples. A brief description of the statistical analyses used to identify protein pairs and ratios between protein pairs.

[0018] Figures 3A to 3C The predictive power of the baseline plasma protein ratio DLL1 / SMOC1 is shown. A. Relationship between baseline plasma protein ratio DLL1 / SMOC1 and improvement in the Clinical Dementia Rating-Sum of Boxes (CDR-sb) at the end of the study (EOS). A higher baseline plasma protein ratio DLL1 / SMOC1 was associated with improvement in CDR-sb (rho = -0.645). B. The ROC of the DLL1 / SMOC1 ratio predicted EOS with a CDR-sb score superior to bl (AUC_better = 80.8% (70.7%, 91.0%)). C. The ROC of DLL1 / SMOC1 was non-inferior to CMC in predicting EOS (clinically significant change, i.e., a CDR-sb score superior to or equal to bl) (AUC_CMC = 83.3% (75.0%, 91.6%)).

[0019] Figures 4A to 4C The predictive power of the baseline plasma protein ratio DLL1 / CD59 is shown. A. Relationship between baseline plasma protein ratio DLL1 / CD59 and improvement in CDR-sb. A higher baseline plasma ratio DLL1 / CD59 was associated with improvement in CDR-sb (rho = -0.627). B. When predicting EOS using ROC, DLL1 / CD59 showed a better CDR-sb score than bl (AUC_better = 80.7% (70.6%, 90.8%)). C. When predicting EOS using ROC, DLL1 / CD59 was not inferior to CMC (better than or equal to bl) (AUC_CMC = 82.0% (73.3%, 90.8%)).

[0020] Figures 5A to 5C The predictive power of the baseline plasma protein ratio DLL1 / LEFTY2 is shown. A. Relationship between baseline plasma protein ratio DLL1 / LEFTY2 and improvement in CDR-sb. Higher baseline plasma ratio DLL1 / LEFTY2 is associated with improvement in CDR-sb (rho = -0.597). B. DLL1 / LEFTY2's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 84.6% (75.6%, 93.6%)). C. DLL1 / LEFTY2's ROC prediction of EOS was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 77.3% (67.5%, 87.0%)).

[0021] Figures 6A to 6C The predictive power of the baseline plasma protein ratio DLL1 / UNC5B is shown. A. Relationship between baseline plasma protein ratio DLL1 / UNC5B and improvement in CDR-sb. A higher baseline plasma ratio DLL1 / UNC5B is associated with improvement in CDR-sb (rho = -0.591). B. DLL1 / UNC5B's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 80.5% (69.4%, 91.6%)). C. DLL1 / UNC5B's ROC prediction of EOS was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 79.2% (69.8%, 88.6%)).

[0022] Figures 7A to 7CThe predictive power of the baseline plasma protein ratio DLL1 / C5 is shown. A. Relationship between baseline plasma protein ratio DLL1 / C5 and improvement in CDR-sb. A higher baseline plasma ratio DLL1 / C5 is associated with improvement in CDR-sb (rho = -0.584). B. When predicting EOS using ROC, DLL1 / C5's CDR-sb score was better than bl's (AUC_better = 80.8% (70.8%, 90.9%)). C. When predicting EOS using ROC, DLL1 / C5 was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 79.2% (70.0%, 88.5%)).

[0023] Figures 8A to 8C The predictive power of the baseline plasma protein ratio DLL1 / C5.C6 is shown. A. Relationship between baseline plasma protein ratio DLL1 / C5.C6 and improvement in CDR-sb. Higher baseline plasma ratio DLL1 / C5.C6 is associated with improvement in CDR-sb (rho = -0.578). B. When predicting EOS using ROC, DLL1 / C5.C6 showed a better CDR-sb score than bl (AUC_better = 80.6% (70.9%, 90.3%)). C. When predicting EOS using ROC, DLL1 / C5.C6 was not inferior to CMC (better than or equal to bl) (AUC_CMC = 81.4% (72.2%, 90.5%)).

[0024] Figures 9A to 9C The predictive power of the baseline plasma protein ratio DLL1 / INHBB is shown. A. Relationship between baseline plasma protein ratio DLL1 / INHBB and improvement in CDR-sb. A higher baseline plasma ratio DLL1 / INHBB is associated with improvement in CDR-sb (rho = -0.564). B. DLL1 / INHBB's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 80.3% (69.5%, 91.0%)). C. DLL1 / INHBB's ROC prediction of EOS was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 81.0% (71.8%, 90.1%)).

[0025] Figures 10A to 10CThe predictive power of the baseline plasma protein ratio TSTD1 / STAT3 is shown. A. Relationship between baseline plasma protein ratio TSTD1 / STAT3 and improvement in CDR-sb. A higher baseline plasma ratio TSTD1 / STAT3 is associated with improvement in CDR-sb (rho = -0.627). B. TSTD1 / STAT3's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 85.1% (75.9%, 94.2%)). C. TSTD1 / STAT3's ROC prediction of EOS was not inferior to CMC (better than or equal to bl) (AUC_CMC = 79.9% (70.5%, 89.2%)).

[0026] Figures 11A to 11C The predictive power of the baseline plasma protein ratio POLD4 / PARP11 is shown. A. Relationship between baseline plasma protein ratio POLD4 / PARP11 and improvement in CDR-sb. A higher baseline plasma ratio POLD4 / PARP11 is associated with improvement in CDR-sb (rho = -0.606). B. POLD4 / PARP11's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 86.9% (78.1%, 95.8%)). C. POLD4 / PARP11's ROC prediction of EOS was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 80.2% (70.6%, 89.8%)).

[0027] Figures 12A to 12C The predictive power of the baseline plasma protein ratio ASH2L / PARP11 is shown. A. Relationship between baseline plasma protein ratio ASH2L / PARP11 and improvement in CDR-sb. A higher baseline plasma ratio ASH2L / PARP11 is associated with improvement in CDR-sb (rho = -0.571). B. ASH2L / PARP11's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 83.7% (73.0%, 94.3%)). C. ASH2L / PARP11's ROC prediction of EOS was not inferior to CMC (better than or equal to bl) (AUC_CMC = 79.5% (70.0%, 89.0%)).

[0028] Figures 13A to 13CThe predictive power of the baseline plasma protein ratio RPS3 / PARP11 is shown. A. Relationship between baseline plasma protein ratio RPS3 / PARP11 and improvement in CDR-sb. A higher baseline plasma ratio RPS3 / PARP11 was associated with improvement in CDR-sb (rho = -0.564). B. RPS3 / PARP11's CDR-sb score is better than bl's when predicting EOS using ROC (AUC_better = 86.5% (76.3%, 96.7%)). C. RPS3 / PARP11's CMC score is not inferior to bl's (better than or equal to bl's) when predicting EOS using ROC (AUC_CMC = 78.4% (68.5%, 88.4%)).

[0029] Figures 14A to 14C The predictive power of the baseline plasma protein ratio VAV3 / SIRT3 is shown. A. Relationship between baseline plasma protein ratio VAV3 / SIRT3 and improvement in CDR-sb. Higher baseline plasma ratio VAV3 / SIRT3 is associated with improvement in CDR-sb (rho = -0.559). B. VAV3 / SIRT3's ROC prediction of EOS showed that CDR-sb outperformed bl (AUC_better = 79.8% (68.9%, 90.7%)). C. VAV3 / SIRT3's ROC prediction of EOS was not inferior to CMC (outperformed or on par with bl) (AUC_CMC = 80.1% (70.4%, 89.8%)).

[0030] Figures 15A to 15C The predictive power of the baseline plasma protein ratio SERPINB8 / PARP11 is shown. A. Relationship between baseline plasma protein ratio SERPINB8 / PARP11 and improvement in CDR-sb. A higher baseline plasma ratio SERPINB8 / PARP11 is associated with improvement in CDR-sb (rho = -0.554). B. SERPINB8 / PARP11's ROC prediction of EOS showed a better CDR-sb score than bl (AUC_better = 82.4% (72.0%, 92.8%)). C. SERPINB8 / PARP11's ROC prediction of EOS was not inferior to CMC (better than or equal to bl's score) (AUC_CMC = 78.0% (68.2%, 87.8%)).

[0031] V. Detailed Implementation

[0032] A. Introduction

[0033] This invention relates to the treatment of cognitive impairment, such as age-related cognitive impairment. In some aspects, the invention provides a method for determining the ratio between the levels of any two proteins in a sample obtained from a subject, the two proteins being selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8.

[0034] In some cases, the subject has or is suspected of having cognitive impairment. In some embodiments, the cognitive impairment is caused by neurodegenerative diseases such as AD. These methods further include identifying the subject as likely or unlikely to respond positively to plasma exchange therapy. Even further, the present invention describes a method for treating cognitive impairment in a subject by plasma exchange therapy, wherein the subject is identified as likely or unlikely to respond positively to plasma exchange therapy based on specific protein expression data. Plasma exchange therapy can be full-volume and / or low-volume plasma exchange. In some cases, plasma exchange includes albumin replacement. Kits suitable for performing the methods of the present invention are also provided.

[0035] Before describing the invention in detail, it should be understood that the invention is not limited to the specific method or composition described, as such methods or compositions may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0036] The publications discussed herein are provided only for inventions made prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to prior publication by virtue of a prior invention. Furthermore, the publication (announcement) dates provided may differ from the actual publication dates, which may require independent verification.

[0037] When a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated or intermediate value within the stated range and any other stated or intermediate value within the stated range is included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range in which any one or both restrictions are included is also included in this invention, subject to any restrictions specifically excluded from the stated range. Where a stated range includes one or both restrictions, this invention also includes ranges that exclude one or both of these included restrictions.

[0038] It will be apparent to those skilled in the art upon reading this invention that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0039] B. Definition

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, some possible and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with those publications. It should be understood that, in the event of conflict, this invention takes precedence over the inventive content of any incorporated publication.

[0041] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, a reference to “sample” includes multiple such samples.

[0042] In describing the methods of the present invention, the terms "subject," "individual," and "patient" are used interchangeably and refer to any mammal that requires such treatment according to the disclosed methods. Such mammals include, for example, humans, sheep, cattle, horses, pigs, canines, felines, non-human primates, mice, and rats. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a farm animal. In other embodiments, the subject is a pet. In some embodiments, the subject is a mammal. In some instances, the subject is a human. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys). Therefore, the subject of the present invention includes, but is not limited to, mammals, such as humans and other primates, such as chimpanzees and other ape and monkey species; etc., wherein, in some embodiments, the subject is a human. The term subject is also intended to include any person or organism of any age, weight, or other physiological characteristic, wherein the subject may be an adult, child, infant, or newborn.

[0043] As used herein, “treatment” means reducing or eliminating cognitive impairment. Treatment can be administered therapeutically, i.e., after the onset of the disease. Effects can include improving the cognitive performance of a subject with cognitive impairment. Therefore, as used herein, the term “treatment” encompasses any treatment of cognitive impairment in mammals and includes: (a) suppressing the disease, i.e., halting its progression; or (b) alleviating the disease, i.e., causing disease regression. Treatment may result in a variety of different physiological manifestations, such as regulation of gene expression, tissue or organ regeneration (recovery), etc. Therapies can be administered during or after the onset of cognitive impairment. Treatment of ongoing cognitive impairment in which the treatment stabilizes or reduces adverse clinical symptoms in the patient is of interest. Such treatment can be administered before complete loss of function of the affected tissue. Subjective therapy can be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0044] “Cognitive impairment” refers to an individual’s cognitive abilities being impaired relative to healthy individuals (e.g., age-matched healthy individuals) or relative to an individual’s abilities at an earlier point in time (e.g., 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years or more).

[0045] "Cognitive ability" or "cognition" refers to the mental processes that include attention and focus, learning complex tasks and concepts, memory (acquiring, retaining, and retrieving new information in the short and / or long term), information processing (processing information gathered by the five senses), visuospatial functions (visual perception, depth perception, using mental images, copying pictures, constructing objects or shapes), language production and comprehension, verbal fluency (word finding), problem-solving, decision-making, and executive functions (planning and prioritization). "Cognitive decline" refers to a progressive decrease in one or more of these abilities, such as a decline in memory, language, thinking, or judgment.

[0046] In some implementations, cognitive impairment is defined as “age-related cognitive impairment.” “Age-related cognitive impairment” means cognitive impairment that is generally associated with aging, including, for example, cognitive impairment associated with the natural aging process, such as mild cognitive impairment (MCI); and cognitive impairment associated with age-related diseases, that is, diseases that increase in frequency with aging, such as neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, vascular dementia, etc.

[0047] In some implementations, the cognitive impairment in the subject is caused by neuroinflammation, for example, an increase in age-related neuroinflammation in the individual. "Neuroinflammation" refers to the biochemical and cellular responses of the nervous system to injury, infection, or neurodegenerative diseases. Such responses aim to reduce triggering factors by defending against potential harm through involvement of the central nervous system's immunity. Neurodegeneration occurs in the central nervous system and is characterized by the loss of neuronal structure and function. Neuroinflammatory diseases or conditions or illnesses associated with neuroinflammation include, but are not limited to, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, etc.

[0048] C. Measurement Method

[0049] Some aspects of the present invention provide a method for analyzing samples obtained from subjects, the method comprising: The ratio between the levels of any two biomarkers (especially protein biomarkers) in a sample is determined. These biomarkers are selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8.

[0050] In some cases, the subjects had or were suspected of having cognitive impairment.

[0051] The DLL1 protein is present in a variety of organisms, including humans. An example of the DLL1 protein in humans is described in the Uniprot database, ID: O00548. The sequence of the human DLL1 protein is given below: MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHRYVCECARGYGGPNCQFLLPELPPGPAWDLTEKLEGQGGPFPWVAVCAGVILVLMLLLGCAAVWCVRLRLQKHRPPADPCRGETETMNNLANCQREKDISVSIIGATQIKNTNKKADFHGDHSADKNGFKARYPAVDYNLVQDLKGDDTAVRDAHSKRDTKCQPQGSSGEEKGTPTTLRGGEASERKRPDSGCSTSKDTKYQSVYVISEEKDECVIATEV(SEQ ID NO: 1)。

[0052] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 1 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 1. For example, those skilled in the art can readily identify homologs of the human DLL1 protein having the sequence of SEQ ID NO: 1 in a target organism and then determine the presence of this DLL1 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0053] The SMOC1 protein is also found in a variety of organisms, including humans. An example of the SMOC1 protein in humans is described in the Uniprot database, ID: Q9H4F8. The sequence of the human SMOC1 protein is given below: MLPARCARLLLTPHLLLVLVQLSPARGHRTTGPRFLISDRDPQCNLHCSRTQPKPICASDGRSYESMCEYQRAKCRDPTLGWHRGRCKDAGQSKCRLERAQALEQAKKPQ EAVFVPECGEDGSFTQVQCHTYTGYCWCVTPDGKPISGSSVQNKTPVCSGSVTDKPLSQGNSGRKDDGSKPTPTMETQPVFDGDEITAPTLWIKHLVIKDSKLNNTNIR NSEKVYSCDQERQSALEEAQQNPREGIVIPECAPGGLYKPVQCHQSTGYCWCVLVDTGRPLPGTSTRYVMPSCESDARAKTTEADDPFKDRELPGCPEGKKMEFITSLL DALTTDMVQAINSAAPTGGGRFSEPDPSHTLEERWHWYFSQLDSNSSNDINKREMKPFKRYVKKKAKPKKCARRFTDYCDLNKDKVISLPELKGCLGVSKEGRLV (SEQ ID NO: 2).

[0054] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 2 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 2. For example, those skilled in the art can readily identify homologs of the human SMOC1 protein having the sequence of SEQ ID NO: 2 in a target organism and then determine the presence of this SMOC1 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0055] The CD59 protein is also present in a variety of organisms, including humans. An example of the human CD59 protein is described in the Uniprot database, ID: P13987. The sequence of the human CD59 protein is given below: MGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO: 3).

[0056] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 3 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 3. For example, those skilled in the art can readily identify homologs of the human CD59 protein having the sequence of SEQ ID NO: 3 in a target organism and then determine this CD59 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0057] The TSTD1 protein is also found in a variety of organisms, including humans. An example of the TSTD1 protein in humans is described in the Uniprot database, ID: Q8NFU3. The sequence of the human TSTD1 protein is given below: MAGAPTTVSLPELRSLLASGRARLFDVRSREEAAAGTIPGALNIPVSELESALQMEPAAFQALYSAEKPKKLEDEHLVFFCQMGKRGLQATQLARSLGYTGARNYAGAYREWLEKES (SEQ ID NO: 4).

[0058] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 4 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 4. For example, those skilled in the art can readily identify homologs of the human TSTD1 protein having the sequence of SEQ ID NO: 4 in a target organism and then determine the presence of this TSTD1 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0059] STAT3 proteins are also found in a variety of organisms, including humans. An example of the human STAT3 protein is described in the Uniprot database, ID: P40763. The sequence of the human STAT3 protein is given below: MAQWNQLQQLDTRYLEQLHQLYSDSFPMELRQFLAPWIESQDWAYAASKESHATLVFHNLLGEIDQQYSRFLQESNVLYQHNLRRIKQFLQSRYLEKPMEIARIVARCLWEESRLLQTAATAAQQGGQANHPTAAWTEKQQMLEQHLQDVRKRVQDLEQKMKVVENLQDDFDFNYKTLKSQGDMQDLNGNNQSVTRQKMQQLEQMLTALDQMRRSIVSELAGLLSAMEYVQKTLTDEELADWKRRQQIACIGGPPNICLDRLENWITSLAESQLQTRQQIKKLEELQQKVSYKGDPIVQHRPMLEERIVELFRNLMKSAFWERQPCMPMHPDRPLVIKTGVQFTTKVRLLVKFPELNYQLKIKVCIDKDSGDVAALRGSRKFNILGTNTKVMNMEESNNGSLSAEFKHLTLREQRCGNGGRANCDASLIVTEELHLITFETEVYHQGLKIDLETHSLPVWISNICQMPNAWASILWYNMLTNNPKNVNFFTKPPIGTWDQVAEVLSWQFSSTTKRGLSIEQLTTLAEKLLGPGVNYSGCQITWAKFCKENMAGKGFSFWVWLDNIIDLVKKYILALWNEGYIMGFISKERERAILSTKPPGTFLLRFSESSKEGGVTFTWVEKDISGKTQIQSVEPYTKQQLNNMSFAEIIMGYKIMDATNILVSPLVYLYPDIPKEEAFGKYCRPESQEHPEADPGSAAPYLKTKFICVTPTTCSNTIDLPMSPRTLDSLMQFGNNGEGAEPSAGGQFESLTFDMELTSECATSPM(SEQ ID NO: 5)。

[0060] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 5 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 5. For example, those skilled in the art can readily identify homologs of the human STAT3 protein having the sequence of SEQ ID NO: 5 in a target organism and then determine the presence of this STAT3 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0061] The POLD4 protein is present in a variety of organisms, including humans. An example of the POLD4 protein in humans is described in the Uniprot database, ID: Q9HCU8. The sequence of the human POLD4 protein is given below: MGRKRLITDSYPWKRREGPAHSKGELAPELGEEPQPRDEEEAELELLRQFDLAWQYGPCTGITRLQRWCRAKQMGLEPPPEVWQVLKTHPGDPRFQCSLWHLYPL (SEQ ID NO: 6).

[0062] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 6 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 6. For example, those skilled in the art can readily identify homologs of the human POLD4 protein having the sequence of SEQ ID NO: 6 in a target organism and then determine the POLD4 protein in a sample obtained from that organism. Such embodiments are within the scope of the invention.

[0063] The PARP11 protein is present in a variety of organisms, including humans. An example of the PARP11 protein in humans is described in the Uniprot database, ID: Q9NR21. The sequence of the human PARP11 protein is given below: MWEANPEMFHKAEELFSKTTNNEVDDMDTSDTQWGWFYLAECGKWHMFQPDTNSQCSVSSEDIEKSFKTNPCGSISFTTSKFSYKIDFAEMKQMNLTTGKQRLIKRAPFSISAFSYICENEAIPMPPHWENVNTQVPYQLIPLHNQTHEYNEVANLFGKTMDRNRIKRIQR IQNLDLWEFFCRKKAQLKKKRGVPQINEQMLFHGTSSEFVEAICIHNFDWRINGIHGAVFGKGTYFARDAAYSSRFCKDDIKHGNTFQIHGVSLQQRHLFRTYKSMFLARVLIGDYINGDSKYMRPPSKDGSYVNLYDSCVDDTWNPKIFVVFDANQIYPEYLIDFH (SEQ ID NO: 7) Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 7 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 7. For example, those skilled in the art can readily identify homologs of the human PARP11 protein having the sequence of SEQ ID NO: 7 in a target organism and then determine the presence of this PARP11 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0064] The LEFTY2 protein is present in a variety of organisms, including humans. An example of the LEFTY2 protein in humans is described in the Uniprot database, ID: O00292. The sequence of the human LEFTY2 protein is given below: MWPLWLCWALWVLPLAGPGAALTEEQLLGSLLRQLQLSEVPVLDRADMEKLVIPAHVRAQYWLLRRSHGDRSRGKRFSQSFREVAGRFLASEASTHLLVFGMEQRLPPNSELVQAVLRLFQEPVPKAALHRHGRLSPRSAQARVTVEWLRVRDDGSNRTSLIDSRLVSVHESGWKAFDVTEAVN FWQQLSRPRQPLLLQVSVQREHLGPLASGAHKLVRFASQGAPAGLGEPQLELHTLDLRDYGAQGDCDPEAPMTEGTRCCRQEMYIDLQGMKWAKNWVLEPPGFLAYECVGTCQQPPEALAFNWPLFLGPRQCIASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP (SEQ IDNO: 8).

[0065] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 8 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 8. For example, those skilled in the art can readily identify homologs of the human LEFTY2 protein having the sequence of SEQ ID NO: 8 in a target organism and then determine this LEFTY2 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0066] The UNC5B protein is present in a variety of organisms, including humans. An example of the UNC5B protein in humans is described in the Uniprot database, ID: Q8IZJ1. The sequence of the human UNC5B protein is given below: MGARSGARGALLLALLLCWDPRLSQAGTDSGSEVLPDSFPSAPAEPLPYFLQEPQDAYIVKNKPVELRCRAFPATQIYFKCNGEWVSQNDHVTQEGLDEATGLRVREVQIEVSRQQVEELFGLEDYWCQCVAWSSAGTTKSRRAYVRIAYLRKNFDQEPLGKEVPLDHEVLLQCRPPEGVPVAEVEWLKNEDVIDPTQDTNFLLTIDHNLIIRQARLSDTANYTCVAKNIVAKRRSTTATVIVYVNGGWSSWAEWSPCSNRCGRGWQKRTRTCTNPAPLNGGAFCEGQAFQKTACTTICPVDGAWTEWSKWSACSTECAHWRSRECMAPPPQNGGRDCSGTLLDSKNCTDGLCMQNKKTLSDPNSHLLEASGDAALYAGLWAIFVWAILMAVGVWYRRNCRDFDTDITDSSAALTGGFHPVNFKTARPSNPQLLHPSVPPDLTASAGIYRGPVYALQDSTDKIPMTNSPLLDPLPSLKVKVYSSSTTGSGPGLADGADLLGVLPPGTYPSDFARDTHFLHLRSASLGSQQLLGLPRDPGSSVSGTFGCLGGRLSIPGTGVSLLVPNGAIPQGKFYEMYLLINKAESTLPLSEGTQTVLSPSVTCGPTGLLLCRPVILTMPHCAEVSARDWIFQLKTQAHQGHWEEWTLDEETLNTPCYCQLEPRACHILLDQLGTYVFTGESYSRSAVKRLQLAVFAPALCTSLEYSLRVYCLEDTPVALKEVLELERTLGGYLVEEPKPLMFKDSYHNLRLSLHDLPHAHWRSKLLAKYQEIPFYHIWSGSQKALHCTFTLERHSLASTELTCKICVRQVEGEGQIFQLHTTLAETPAGSLDTLCSAPGSTVTTQLGPYAFKIPLSIRQKICNSLDAPNSRGNDWRMLAQKLSMDRYLNYFATKASPTGVILDLWEALQQDDGDLNSLASALEEMGKSEMLVAVATDGDC(SEQ ID NO: 9)。

[0067] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 9 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 9. For example, those skilled in the art can readily identify homologs of the human UNC5B protein having the sequence of SEQ ID NO: 9 in a target organism and then determine this UNC5B protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0068] The C5 protein is present in a variety of organisms, including humans. An example of the human C5 protein is described in the Uniprot database, ID: P01031. The sequence of the human C5 protein is given below:

[0069] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 10 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 10. For example, those skilled in the art can readily identify homologs of the human C5 protein having the sequence of SEQ ID NO: 10 in a target organism and then determine this C5 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0070] The C6 protein is present in a variety of organisms, including humans. An example of the human C6 protein is described in the UniProt database, ID: P13671. The sequence of the human C6 protein is given below: MARRSVLYFILLNALINKGQACFCDHYAWTQWTSCSKTCNSGTQSRHRQIVVDKYYQENFCEQICSKQETRECNWQRCPINCLLGDFGPWSDCDPCIEKQSKVRSVLRPSQFGGQPCTAPLVAFQPCIPSKLCKIEEADCKNKFRCDSGRCIARKLECNGENDCGDNSDERDCGRTKAVCTRKYNPIPSVQLMGNGFHFLAGEPRGEVLDNSFTGGICKTVKSSRTSNPYRVPANLENVGFEVQTAEDDLKTDFYKDLTSLGHNENQQGSFSSQGGSSFSVPIFYSSKRSENINHNSAFKQAIQASHKKDSSFIRIHKVMKVLNFTTKAKDLHLSDVFLKALNHLPLEYNSALYSRIFDDFGTHYFTSGSLGGVYDLLYQFSSEELKNSGLTEEEAKHCVRIETKKRVLFAKKTKVEHRCTTNKLSEKHEGSFIQGAEKSISLIRGGRSEYGAALAWEKGSSGLEEKTFSEWLESVKENPAVIDFELAPIVDLVRNIPCAVTKRNNLRKALQEYAAKFDPCQCAPCPNNGRPTLSGTECLCVCQSGTYGENCEKQSPDYKSNAVDGQWGCWSSWSTCDATYKRSRTRECNNPAPQRGGKRCEGEKRQEEDCTFSIMENNGQPCINDDEEMKEVDLPEIEADSGCPQPVPPENGFIRNEKQLYLVGEDVEISCLTGFETVGYQYFRCLPDGTWRQGDVECQRTECIKPVVQEVLTITPFQRLYRIGESIELTCPKGFWAGPSRYTCQGNSWTPPISNSLTCEKDTLTKLKGHCQLGQKQSGSECICMSPEEDCSHHSEDLCVFDTDSNDYFTSPACKFLAEKCLNNQQLHFLHIGSCQDGRQLEWGLERTRLSSNSTKKESCGYDTCYDWEKCSASTSKCVCLLPPQCFKGGNQLYCVKMGSSTSEKTLNICEVGTIRCANRKMEILHPGKCLA(SEQ ID NO: 11)。

[0071] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein complex having the sequences SEQ ID NO: 10 and 11, or a protein complex in which one protein has a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence SEQ ID NO: 10, and the other protein has a sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence SEQ ID NO: 11. For example, those skilled in the art can readily identify homologs of human C5 and C6 proteins having the sequences SEQ ID NO: 10 and 11 in a target organism, and then determine complexes of such proteins in samples obtained from such organisms. Such embodiments are within the scope of the invention.

[0072] The ASH2L protein is present in a variety of organisms, including humans. An example of the ASH2L protein in humans is described in the UniProt database, ID: Q9UBL3. The sequence of the human ASH2L protein is given below: (SEQ ID NO: 12).

[0073] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between two protein levels in a sample from a subject, one of which is a protein having the sequence SEQ ID NO: 12 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence SEQ ID NO: 12. For example, those skilled in the art can readily identify homologs of the human ASH2L protein having the sequence SEQ ID NO: 12 in a target organism and then determine this ASH2L protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0074] INHBB proteins are also found in a variety of organisms, including humans. An example of the human INHBB protein is described in the UniProt database, ID: P09529. The sequence of the human INHBB protein is given below: MDGLPGRALGAACLLLLAAGWLGPEAWGSPTPPPTPAAPPPPPPGSPGGSQDTCTSCGGFRRPEELGRVDGDFLEAVKRHILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGADGQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYVLEKGSRRKVRVKV YFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGERRLNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRIRKRGLECDGRTLCC RQQFFIDFRLIGWNDWIIAPTGYYGNYCEGSCPAYLAGVPGSASSFHTAWNQYRMRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGC A (SEQ ID NO: 13).

[0075] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 13 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 13. For example, those skilled in the art can readily identify homologs of the human INHB protein having the sequence of SEQ ID NO: 13 in a target organism and then determine this INHB protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0076] The RPS3 protein is also found in a variety of organisms, including humans. An example of the RPS3 protein in humans is described in the UniProt database, ID: P23396. The sequence of the human RPS3 protein is given below: MAVQISKKRKFVADGIFKAELNEFLTRELAEDGYSGVEVRVTPTRTEIIILATRTQNVLGEKGRRIRELTAWQKRFGFPEGSVELYAEKVATRGLCAIAQAESLRYKLLGGLAVRRACYGVLR FIMESGAKGCEVVVSGKLRGQRAKSMKFVDGLMIHSGDPVNYYVDTAVRHVLLRQGVLGIKVKIMLPWDPTGKIGPKKPLPDHVSIVEPKDEILPTTPISEQKGGKPEPPAMPQPVPTA (SEQ ID NO: 14).

[0077] Therefore, in some embodiments, the methods disclosed herein include determining the ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 14 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 14. For example, those skilled in the art can readily identify homologs of the human RPS3 protein having the sequence of SEQ ID NO: 14 in a target organism and then determine the presence of this RPS3 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0078] The VAV3 protein is also found in a variety of organisms, including humans. An example of the human VAV3 protein is described in the UniProt database under ID Q9UKW4. The sequence of the human VAV3 protein is given below: MEPWKQCAQWLIHCKVLPTNHRVTWDSAQVFDLAQTLRDGVLLCQLLNNLRAHSINLKEINLRPQMSQFLCLKNIRTFLTACCETFGMRKSELFEAFDLFDVRDFGKVIETLSRLSRTPIALATGIRPFPTEESINDEDIYKGLPDLIDETLVEDEEDLYDCVYGEDEGGEVYEDLMKAEEAHQPKCPENDIRSCCLAEIKQTEEKYTETLESIEKYFMAPLKRFLTAAEFDSVFINIPELVKLHRNLMQEIHDSIVNKNDQNLYQVFINYKERLVIYGQYCSGVESAISSLDYISKTKEDVKLKLEECSKRANNGKFTLRDLLVVPMQRVLKYHLLLQELVKHTTDPTEKANLKLALDAMKDLAQYVNEVKRDNETLREIKQFQLSIENLNQPVLLFGRPQGDGEIRITTLDKHTKQERHIFLFDLAVIVCKRKGDNYEMKEIIDLQQYKIANNPTTDKENKKWSYGFYLIHTQGQNGLEFYCKTKDLKKKWLEQFEMALSNIRPDYADSNFHDFKMHTFTRVTSCKVCQMLLRGTFYQGYLCFKCGARAHKECLGRVDNCGRVNSGEQGTLKLPEKRTNGLRRTPKQVDPGLPKMQVIRNYSGTPPPALHEGPPLQLQAGDTVELLKGDAHSLFWQGRNLASGEVGFFPSDAVKPCPCVPKPVDYSCQPWYAGAMERLQAETELINRVNSTYLVRHRTKESGEYAISIKYNNEAKHIKILTRDGFFHIAENRKFKSLMELVEYYKHHSLKEGFRTLDTTLQFPYKEPEHSAGQRGNRAGNSLLSPKVLGIAIARYDFCARDMRELSLLKGDWKIYTKMSANGWWRGEVNGRVGWFPSTYVEEDE(SEQ ID NO: 15)。

[0079] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 15 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 15. For example, those skilled in the art can readily identify homologs of the human VAV3 protein having the sequence of SEQ ID NO: 15 in a target organism and then determine this VAV3 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0080] The SIRT3 protein is also present in a variety of organisms, including humans. An example of the SIRT3 protein in humans is described in the UniProt database, ID: Q9NTG7. The sequence of the human SIRT3 protein is given below: MAFWGWRAAAALRLWGRWERVEAGGGVGPFQACGCRLVLGGRDDVSAGLRGSHGARGEPLDPARPLQRPPRPEVPRAFRRQPRAAAPSFFFSSIKGGRRSISFSVGASSWGSGGSSDKGKLSLQDVAELIRARACQRVWMVGAGISTPSGIPDFRSPGSGLYSNLQQYDLPYPEAIFELPFFFHNPKPFFTLAKELYPG NEW YORK ID NO: 16) Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 16 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 16. For example, those skilled in the art can readily identify homologs of the human SIRT3 protein having the sequence of SEQ ID NO: 16 in a target organism and then determine the presence of this SIRT3 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0081] The SERPINB8 protein is also found in various organisms, including humans. An example of the SERPINB8 protein in humans is described in the UniProt database, ID: P50452. The sequence of the human SERPINB8 protein is given below: MDDLCEANGTFAISLFKILGEEDNSRNVFFSPMSISSALAMVFMGAKGSTAAQMSQALCLYKDGDIHRGFQSLLSEVNRTGTQYLLRTANRLFGEKTCDFLPDFKEYCQKFYQAELEELSFAEDTEECRKHINDWVAEKTEGKISEVLDAGTVDPLTKLVLVNAIYFKGKWNEQFDRKYTRGMLFKTN EEKKTVQMMFKEAKFKMGYADEVHTQVLELPYVEEELSMVILLPDDNTDLAWEKALTYEKFKAWTNSEKLTKSKVQVFLPRLKLEESYDLEPFLRRLGMIDAFDEAKADFSGMSTEKNVPLSKVAHKCFVEVNEEGTEAAAATAWRNSRCSRMEPRFCADHPFLFFIRHHKTNCILFCGRFSSP (SEQ ID NO: 17).

[0082] Therefore, in some embodiments, the methods disclosed herein include determining a ratio between the levels of two proteins in a sample from a subject, one of which is a protein having the sequence of SEQ ID NO: 17 or a protein having at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity with the sequence of SEQ ID NO: 17. For example, those skilled in the art can readily identify homologs of the human SERPINB8 protein having the sequence of SEQ ID NO: 17 in a target organism and then determine this SERPINB8 protein in a sample obtained from such an organism. Such embodiments are within the scope of the invention.

[0083] The markers being measured are also referred to as "target markers" in this paper. A pair of markers whose levels are determined is called a "target marker pair".

[0084] The proteins being measured are also referred to as "target proteins" in this paper. The ratio of the levels of a pair of proteins is determined and called a "target protein pair".

[0085] Samples can be obtained from subjects who have or are suspected of having cognitive impairment. Samples can be any readily available biological sample. "Biological sample" refers both to a natural organism or a subset of its tissues, and to homogenates, lysates, or extracts prepared from such organisms or subsets of their tissues, including but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. Biological samples can be any type of biological tissue, including healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In some embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some instances the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger-prick (wherein the blood may or may not be mixed with any reagents such as preservatives, anticoagulants, etc., prior to measurement). Other examples of biological samples are well known to those skilled in the art, and such embodiments are within the scope of this invention. Methods for obtaining such samples from subjects are well known in the art, and such implementations are within the scope of this invention.

[0086] The term "expression" is used in this document to refer to the process of producing polypeptides from DNA. This process involves transcribing a gene into mRNA and translating that mRNA into a polypeptide. Depending on the context, "expression" can refer to the production of RNA, the production of proteins, or both.

[0087] The terms “expression level” or “level of expression” are used interchangeably and refer to the measurable amount of an expression product produced by a gene in a subject sample, wherein the expression product may be a transcription product or a translation product. As will be understood by those skilled in the art, the expression level can be quantified by measuring the level of the gene’s messenger RNA or by measuring the level of the protein encoded by the gene, including all physiologically relevant post-translational chemical modifications of the protein, such as glycosylation, phosphorylation, acetylation, etc., provided that the protein’s function is maintained.

[0088] In some cases, the methods disclosed herein include the quantitative determination of one or more target biomarkers in a sample.

[0089] In some cases, the methods disclosed herein involve the quantification of one or more target proteins in a sample. Some non-limiting examples of the quantification of one or more target proteins include immunoassays, mass spectrometry, and protein detection array analysis.

[0090] Immunoassays typically involve contacting a sample with a binding agent that specifically binds to a target protein, and then detecting the binding between the binding agent and the target protein. Such assays may include detecting a label conjugated to a binding agent or a label conjugated to a second binding agent that specifically binds to the binding agent and / or the binding agent-target protein complex. The binding agent is typically an antibody or an antigen-binding fragment of an antibody. The binding agent may also be an aptamer or a peptide-binding component (peptide-binding element).

[0091] Non-limiting examples of immunoassays include Western blot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), lateral flow immunoassay, particle-based immunoassay, quantum dot-based immunoassay, etc. Details of methods for performing some exemplary immunoassays are available in Rizzo (2022). Chemosensors The review article , 10(8), 326 describes this, and its content is incorporated into this article in its entirety by reference.

[0092] Mass spectrometry analysis for detecting target proteins typically involves digesting a protein-containing sample into peptides, ionizing the peptides, and analyzing the ionized peptides to determine the identity of the digested protein. Due to the unique sequence of the protein, the specific digestion of the target protein produces unique digested and ionized peptides, which are identified in the mass spectrometer. The presence and amount of these unique ionized peptides are beneficial for the identification of the target protein.

[0093] Non-limiting examples of mass spectrometry methods for determining one or more target proteins include quadrupole mass spectrometry, time-of-flight mass spectrometry, magnetic sector mass spectrometry, electrostatic sector mass spectrometry, quadrupole ion trap mass spectrometry, and ion cyclotron resonance mass spectrometry. Details of methods for performing certain exemplary determinations based on mass spectrometry analysis are provided in Ma (2022). Molecules The description is found in the review article of , 27, 6466, and its content is incorporated into this article in its entirety by reference.

[0094] Protein detection array analysis involves detecting the binding of a target protein in a sample to a binding agent (typically an antibody located at a defined site on a carrier). The sample is exposed to such an array, and the binding of the target protein to the corresponding binding agent (such as an antibody in a localized spot on a solid carrier) is visualized, for example, using a detectable label. For instance, the captured target protein can be detected and / or quantified using a labeled secondary antibody (e.g., a fluorescently labeled secondary antibody).

[0095] As an alternative to binders located on solid supports, lysate microarrays involve immobilizing lysates of a sample onto a support, such as a nitrocellulose-coated glass slide. The immobilized proteins are then detected using a solution-bound binder capable of detecting labeled conjugations, such as a fluorescently labeled solution-phase-specific antibody. Differentially labeled binders, such as various fluorescently labeled antibodies against different target proteins, allow for multiplex detection of proteins, thus enabling the simultaneous determination of two or more proteins.

[0096] Non-limiting examples of protein detection array analysis include analytical protein microarrays, functional protein microarrays, and reversed-phase protein microarrays. Those skilled in the art can determine the protein microarrays suitable for use in the methods disclosed herein. Details of methods for performing certain exemplary protein detection array analyses are found in Neagu et al. (2019). World Acad. Sci. J. The description is found in the review article , 1:113-124, and its content is incorporated into this article in its entirety by reference.

[0097] In some cases, the SomaScan™ assay is used to analyze the ratio of protein pair levels described in this article in subject samples. Some details of the SomaScan™ assay can be found in reference Gold et al. (2010). PLoS OneAs described in 5(12):e15004, which is incorporated herein by reference in its entirety. In short, the SomaScan™ assay uses an aptamer called a SOMAmer, which specifically binds to a target protein. The assay involves binding and dissociating the SOMAmer with the target protein in the sample, resulting in a SOMAmer complex mixture containing SOMAmer in proportion to the protein present in the sample. The resulting SOMAmer mixture is quantified using DNA microarray technology to produce relative fluorescence unit (RFU) readings. Thus, the SomaScan™ assay provides the levels of various proteins in a sample as “relative fluorescence units” (RFU) or derivatives of RFUs (such as log2RFU). The ratio between protein levels in a target protein pair can be determined based on RFU values ​​or derivatives of RFU values ​​(such as log2RFU). The ratio data provided below were obtained following this SomaScan™ technique.

[0098] In some cases, the methods disclosed herein involve the quantitative determination of one or more target mRNA biomarkers in a sample. Suitable methods for determining gene expression levels at the mRNA level include, but are not limited to, standard assays for determining mRNA expression levels, such as qPCR, RT-PCR, RNA protection assays, Northern blotting, RNA dot blot, in situ hybridization, microarray techniques, tag-based methods (such as gene expression serial analysis (SAGE), including variants such as LongSAGE and SuperSAGE), microarrays, fluorescence in situ hybridization (FISH) (including variants such as Flow-FISH, qFiSH, and dual fusion FISH (D-FISH)), and so on.

[0099] In some cases, the ratio between biomarkers is determined by: (a) determining the level (i.e. amount or concentration) at which each of the biomarkers (in the form of protein or mRNA) is expressed in the same unit, and (b) determining the ratio of the level (i.e. amount or concentration) of one biomarker to that of another biomarker.

[0100] In some cases, the method involves determining the ratio between the expression levels of any two biomarkers in the sample, selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8.

[0101] In some cases, the method involves determining the ratio between the levels of any two proteins in the sample, selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8.

[0102] In some cases, one of the two proteins in the protein pair being measured is DLL1. When one of the two proteins is DLL1, the other protein can be selected from the following: SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8, particularly selected from: SMOC1, CD59, LEFTY2, UNC5B, C5, C5.C6, and INHBB. In one implementation, when one of the two biomarkers is DLL1, the ratio is calculated as the expression level of DLL1 relative to the expression levels of biomarkers selected from SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8, particularly the expression levels of biomarkers selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5.C6, and INHBB.

[0103] In some implementations, the method includes determining the ratio between the levels of the following marker pairs (particularly protein pairs) in a sample: i. DLL1 and SMOC1 (especially the ratio DLL1:SMOC1). ii. DLL1 and CD59 (especially the ratio DLL1:CD59), iii. DLL1 and LEFTY2 (especially the ratio DLL1:LEFTY2). iv. DLL1 and UNC5B (especially the ratio DLL1:UNC5B), v. DLL1 and C5 (especially the ratio DLL1:C5), vi. DLL1 and C5.C6 (especially the ratio DLL1:C5.C6) vii. DLL1 and INBBB (especially DLL1:INHBB).

[0104] In some cases, one of the two proteins in the protein pair being measured can be PARP11. When one of the two proteins is PARP11, the other protein can be selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8, particularly from: POLD4, ASH2L, RPS3, and SERPINB8. When one of the two markers is PARP11, the ratio is calculated as follows: The expression level of one biomarker selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8, particularly the expression level of one biomarker selected from POLD4, ASH2L, RPS3, and SERPINB8. Divide by PARP11 expression level.

[0105] In some implementations, the method includes determining the ratio between the levels of the following protein pairs in a sample: i. POLD4 and PARP11 (especially the ratio POLD4:PARP11). ii. ASH2L and PARP11 (especially the ratio ASH2L:PARP11). iii. RPS3 and PARP11 (especially the ratio RPS3:PARP11), and iv. SERPINB8 and PARP11 (especially the ratio SERPINB8:PARP11).

[0106] In some cases, the method involves determining the ratio between the levels of the following protein pairs in the sample: i. TSTD1 and STAT3 (especially the ratio TSTD1:STAT3), and ii. VAV3 and SIRT3 (especially the ratio VAV3:SIRT3).

[0107] Therefore, in some implementations, the method includes determining the ratio between the levels of DLL1 and SMOC1 in the sample.

[0108] In some implementations, the method includes determining the ratio between the levels of DLL1 and CD59 in a sample.

[0109] In some cases, the method involves determining the ratio between the levels of DLL1 and LEFTY2 in the sample.

[0110] In a further embodiment, the method includes determining the ratio between the levels of DLL1 and UNC5B in the sample.

[0111] In even further embodiments, the method includes determining the ratio between the levels of DLL1 and C5 in the sample.

[0112] In some cases, the method involves determining the ratio between the levels of DLL1 and C5.C6 in the sample.

[0113] In other cases, the method involves determining the ratio between the levels of DLL1 and INHB in the sample.

[0114] In some cases, the method involves determining the ratio between the levels of TSTD1 and STAT3 in the sample.

[0115] In a further case, the method includes determining the ratio between the levels of POLD4 and PARP11 in the sample.

[0116] In even more advanced cases, the method includes determining the ratio between the levels of ASH2L and PARP11 in the sample.

[0117] In some cases, the method involves determining the ratio between the levels of RPS3 and PARP11 in the sample.

[0118] In some cases, the method involves measuring the ratio between the levels of VAV3 and SIRT3 in the sample.

[0119] In some cases, the method involves determining the ratio between the levels of SERPINB8 and PARP11 in the sample.

[0120] The method for determining the ratio between any two protein levels in a sample, as disclosed herein, may further include comparing the determination results with a reference ratio.

[0121] The reference ratio is the ratio between protein levels in a protein pair. This ratio can be used to distinguish between subjects who are likely to respond positively to plasma exchange therapy for the treatment of cognitive impairment and subjects who are unlikely to respond positively to plasma exchange therapy for the treatment of cognitive impairment.

[0122] The reference ratio can be a predetermined ratio, for example, based on the ratio between expression levels of a pair of biomarkers in individuals known to have responded to cognitive impairment therapy. Specifically, the reference ratio can be a predetermined ratio, for example, based on the ratio between levels of a pair of proteins in individuals known to have responded to cognitive impairment therapy. For example, certain protein pairs can be retrospectively measured in a cohort of subjects who have responded to cognitive impairment therapy. These retrospective measurements can be used to determine a reference ratio that definitively indicates the likelihood of a subject responding to cognitive impairment therapy. In a specific embodiment, the therapy is plasma exchange therapy, for example, as described in detail below.

[0123] Alternatively, the reference ratio can be predetermined, for example, based on the ratio between expression levels of a pair of biomarkers in individuals known to be unresponsive to cognitive impairment therapy. In particular, the reference ratio can be predetermined, for example, based on the ratio between protein levels in a pair of proteins in individuals known to be unresponsive to cognitive impairment therapy. For example, certain protein pairs can be retrospectively measured in a cohort of subjects unresponsive to cognitive impairment therapy. These retrospective measurements can be used to determine reference ratios that definitively indicate the likelihood of a subject's unresponsiveness to cognitive impairment therapy. In a specific embodiment, the therapy is plasma exchange therapy, which is described in detail below.

[0124] Reference samples may contain pairs of target biomarkers whose relative levels are known to indicate a subject’s responsiveness to plasma exchange therapy for the treatment of cognitive impairment.

[0125] Reference samples may contain target protein pairs whose relative levels are known to indicate a subject’s responsiveness to plasma exchange therapy for the treatment of cognitive impairment.

[0126] The samples used to measure one or more target protein pairs in the methods disclosed herein can be obtained from subjects with cognitive impairment. Alternatively, the samples used to measure one or more target protein pairs in the methods disclosed herein can be obtained from subjects suspected of having cognitive impairment.

[0127] As used in this article, “subjects with cognitive impairment” refers to subjects who exhibit symptoms of cognitive impairment.

[0128] As used herein, the term "individual suspected of having cognitive impairment" refers to a subject exhibiting symptoms of other conditions associated with cognitive impairment. For example, and as discussed in detail later, a subject may exhibit some signs of neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, but has not yet developed cognitive impairment. Such subjects are included in the term "subject suspected of having cognitive impairment."

[0129] In some embodiments, cognitive impairment is caused by neurodegenerative diseases. Non-limiting examples of neurodegenerative diseases that may cause cognitive impairment include Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, glaucoma, myotonic dystrophy, and vascular dementia. These diseases are described below, and further information regarding these diseases is well known in the art.

[0130] In a specific implementation, the neurodegenerative disease is AD, that is, the subject suffers from cognitive impairment caused by AD.

[0131] In some implementations, the samples are obtained from subjects who are candidates for plasma exchange therapy for the treatment of cognitive impairment.

[0132] Any suitable sample from a subject can be measured according to the methods disclosed herein. In a specific embodiment, the sample is a blood sample, serum sample, plasma sample, or cerebrospinal fluid sample.

[0133] In some cases, the sample may be aqueous humor, vitreous fluid, bile, chyle, endolymph, perilymph, lymph, mucus (including nasal drainage (nasal secretions) and sputum), pericardial fluid, peritoneal fluid (ascites), pleural fluid (pleural effusion), pus, tears, saliva, sputum, or synovial fluid. Further examples of biological samples are well known to those skilled in the art, and such embodiments are within the scope of this invention. Methods for obtaining such samples from subjects are also well known in the art, and such embodiments are within the scope of this invention.

[0134] D. Diagnostic methods

[0135] In addition to measuring the ratio of levels of one or more biomarkers in a sample obtained from a subject, as provided in any of the foregoing embodiments, certain aspects of the invention provide for the possibility or impossibility of a subject responding positively to a therapy used to treat the subject's cognitive impairment. In some embodiments, the therapy is plasma exchange therapy. Details of plasma exchange therapy that can be administered according to the invention are discussed in detail below and are applicable to the diagnostic methods described herein.

[0136] All implementation methods provided under the "Measurement Methods" section are also implementation methods for any diagnostic methods provided below.

[0137] As used herein, the expressions “positive response to therapy” or “potential positive response to therapy” refer to an improvement in one or more symptoms of a patient’s cognitive impairment or condition. Preferably, this expression refers to at least a statistically significant improvement in cognitive abilities as measured below. The terms “improvement” and “enhancement” are used interchangeably. For example, according to the invention, a favorable response to treatment in a patient with a neurodegenerative disease (such as AD) could be an improvement in cognitive function, such as learning ability, plasticity, and / or long-term memory.

[0138] The inventors have discovered that certain inflammation-related biomarkers (particularly those listed in the foregoing embodiments) are expressed differentially in responders and non-responders, and that when the ratio of expression levels between biomarker pairs is calculated, robust predictive information about the response to PE therapy can be provided.

[0139] Therefore, in some implementations, a subject is identified as potentially or unpromising to respond positively to plasma exchange therapy for the treatment of cognitive impairment based on the ratio between the expression levels of any two markers selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8 in the subject's sample, particularly the determination of the ratio between the levels of any two proteins.

[0140] For example, based on the determination of the ratio of one or more of the following biomarkers (especially proteins) in a subject's sample, a subject can be identified as likely or unlikely to respond positively to plasma exchange therapy for the treatment of cognitive impairment: i. DLL1 and SMOC1 (especially the ratio DLL1 / SMOC1). ii. DLL1 and CD59 (especially the ratio DLL1 / CD59), iii. DLL1 and LEFTY2 (especially the ratio DLL1 / LEFTY2). iv. DLL1 and UNC5B (especially the ratio DLL1 / UNC5B), v. DLL1 and C5 (especially the ratio DLL1 / C5), vi. DLL1 and C5.C6 (especially the ratio DLL1 / C5.C6) vii. DLL1 and INHB (especially the ratio DLL1 / INHBB), viii. TSTD1 and STAT3 (especially the ratio TSTD1 / STAT3). ix. POLD4 and PARP11 (especially the ratio POLD4 / PARP11). x. ASH2L and PARP11 (especially the ratio ASH2L / PARP11). xi. RPS3 and PARP11 (especially the ratio RPS3 / PARP11). xii. VAV3 and SIRT3 (especially the ratio VAV3 / SIRT3), and xiii. SERPINB8 and PARP11 (especially the ratio SERPINB8 / PARP11).

[0141] In some implementations, determining whether a subject is likely or unlikely to respond positively to a therapy used to treat the subject's cognitive impairment based on the measurement results includes comparing the measurement results to a reference ratio.

[0142] As described above, such reference ratios can be predetermined, for example, based on the relative expression levels of one, two, or more measured biomarker pairs (particularly protein pairs) in individuals known to respond to cognitive impairment therapy. For example, one or more ratios between protein pairs can be retrospectively measured in a cohort of subjects responding to cognitive impairment therapy. These retrospective measurements can be used to determine reference ratios that definitively indicate the likelihood of a subject responding to cognitive impairment therapy. In a specific embodiment, the therapy is plasma exchange therapy, which is described in detail below.

[0143] Similarly, as mentioned above, such reference ratios can be predetermined, for example, based on the relative expression levels of one or more measured biomarker pairs (particularly protein pairs) in individuals known to be unresponsive to cognitive impairment therapy. For example, the relative levels of one or more protein pairs can be retrospectively measured in a cohort of subjects unresponsive to cognitive impairment therapy. These retrospective measurements can be used to determine reference ratios that definitively indicate the likelihood of a subject responding to cognitive impairment therapy. In a specific embodiment, the therapy is plasma exchange therapy, which is described in detail below.

[0144] Reference samples may contain markers from target marker pairs, the concentration of which is known to indicate a subject’s responsiveness to plasma exchange therapy for the treatment of cognitive impairment.

[0145] The reference sample may contain proteins from the target protein pair, the concentration of which is known to indicate the subject’s responsiveness to plasma exchange therapy for the treatment of cognitive impairment.

[0146] In some implementations, subjects are identified as potentially or unpromising to respond positively to plasma exchange therapy for the treatment of cognitive impairment based on a comparison of different ratios of expression levels of markers (particularly protein markers) in samples obtained from the subjects with a reference ratio.

[0147] As discussed above, in some cases, the SomaScan™ assay is used to analyze the protein levels described herein in patient samples.

[0148] Table 1 below shows the candidate plasma protein pairs identified based on Spearman correlation.

[0149] Table 1. Various protein pairs and their correlation with CDR-sb changes.

[0150] Thirteen protein pairs were identified, involving 17 proteins.

[0151] Of the 13 protein pairs, 7 (61.5%) involved DLL1, of which 4 (30.8%) involved PARP11, and 4 proteins involved inflammation: CD59, STAT3, C5, and C5.C6.

[0152] Tables 2 and 3 below illustrate the assessment of the predictive power of the identified candidate serum proteins for therapeutic benefit: Table 2: Predictive power of treatment benefit (CDR-sb is better than baseline at EOS).

[0153]

[0154] Table 3: Predictive power of treatment benefit (CDR-sb change is <1 point higher than bl)

[0155] [CMC = Clinically significant change; Disease severity and minimum clinically significant difference in clinical outcome assessment of Alzheimer's disease clinical trials in 2019]

[0156] Overall, Tables 2 and 3 demonstrate the strong predictive power of protein ratios for treatment benefit, as evidenced by the superior CDR-sb score at study end compared to baseline. High AUC values, along with sensitivity and accuracy metrics, highlight the effectiveness of these biomarkers in predicting cognitive improvement.

[0157] Table 4 below shows computer simulation validations of other clinical outcomes, predicting AUCs that are superior to baseline or at least not inferior to CMC.

[0158] Table 4. Validation of another clinical outcome using the AMBAR clinical trial. Changes in ADAS_Cog at EOS (n = 88, CMC: 3 points).

[0159]

[0160] Table 4 presents the predictive power of various protein ratios for treatment benefit, as indicated by the ADAS_Cog score at study end compared to baseline. AUC values, along with sensitivity and accuracy metrics, highlight the effectiveness of these biomarkers in predicting cognitive improvement.

[0161] In some cases, the measured ratio between the expression levels of a biomarker pair can be compared with a reference ratio for the corresponding biomarker pair. As those skilled in the art will recognize, the reference ratio used for comparison will depend on the type of sample (e.g., blood or cerebrospinal fluid).

[0162] In some cases, the measured ratio between the levels of protein pairs can be compared with a reference ratio for the corresponding protein pair. As those skilled in the art will recognize, the reference ratio used for comparison will depend on the type of sample (e.g., blood or cerebrospinal fluid).

[0163] Statistical analysis

[0164] The following are some of the terms used in the statistical analysis of data obtained by analyzing bodily fluid samples from subjects. These statistical analyses can be used to identify whether a subject is likely or unlikely to respond positively to treatment for cognitive impairment based on the ratios between one or more protein pairs in the subject's sample.

[0165] First, treatment benefit was confirmed by comparing changes in clinical outcomes between the treatment and placebo groups. Then, candidate protein pairs were identified by significant Spearman correlation coefficients between the baseline ratio of protein levels in the protein pairs, measured by certain assays (here by SomaScan), and the altered cognitive outcomes at the end of the study (Table 1). The predictive power of these candidate protein pairs in indicating whether subjects would have a better cognitive outcome (treatment benefit) or no cognitive outcome (no treatment benefit) after treatment was assessed using receiver operating characteristic (ROC) curves and measured by the area under the ROC curve (AUC). ROC is a graph of sensitivity versus specificity for multiple thresholds of a protein pair. ROC is used to demonstrate the predictive power of consecutive protein pairs on binary outcomes (e.g., treatment benefit versus no treatment benefit).

[0166] AUC represents the probability of correctly ranking patients who have or have not benefited from treatment based on consecutive protein pairs. Generally, an AUC > 70% is considered acceptable, and an AUC > 80% is considered to have excellent predictive power.

[0167] The high or low protein ratio is then defined using an optimal decision threshold. For example, the best cut-off point or reference value for the baseline protein ratio can be defined based on Youden's index, which maximizes accuracy (the overall ratio of correctly classified samples as shown in Table 5 below).

[0168] Ultimately, the best binary protein pairs were identified based on the predictive power of measurements of accuracy, specificity, and sensitivity (the calculations of which are explained below and shown in Table 5).

[0169] The terms A1, B1, A0, and B0 used in the following paragraphs are shown in Table 5: Table 5

[0170] Accuracy represents the percentage of correctly classified subjects: (Number of subjects who actually benefited from treatment + Number of subjects who actually did not benefit from treatment) / Total number of predictions. ((A1 + B0) / N, N = Total number of predictions.)

[0171] Specificity represents the true negative rate, which is the percentage of subjects who actually receive no treatment benefit out of the total number of subjects predicted to receive no treatment benefit (B0 / (A0 + B0)).

[0172] Sensitivity represents the true positive rate, which is the percentage of subjects who actually benefit from treatment out of the total number of subjects predicted to benefit from treatment (A1 / (A1 + B1)).

[0173] These identified protein pairs are then validated using computer simulations, such as testing in other clinical outcomes, randomized reselected samples, other visits, and / or similar clinical trials.

[0174] According to the methods described herein, at least 60% (such as at least 70%, at least 80%, or at least 90%) of the subjects identified as potentially likely to respond positively to treatment for cognitive impairment actually respond positively to treatment. In some cases, plasma exchange therapy, such as... Figure 1 Or as described in the experimental section below.

[0175] Conversely, according to the methods described herein, at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of patients identified as unlikely to respond positively to treatment for cognitive impairment actually do not respond positively to treatment. In some cases, plasma exchange therapy, such as Figure 1 Or as described in the experimental section below.

[0176] In some cases, subjects identified as unlikely to respond positively to treatments for cognitive impairment (such as plasma exchange therapy) are not given treatment (such as plasma exchange therapy).

[0177] In some cases, the ratios of protein levels of protein pairs that indicate whether a subject may or may not respond positively to plasma therapy for the treatment of cognitive impairment, as described in this article, are provided in Table 6 below: Table 6. Ratios of different protein pairs in the blood indicating whether a subject is likely or unlikely to have a positive (better than baseline) response to plasma therapy for the treatment of cognitive impairment (e.g., plasma therapy described in the Experiment section below).

[0178]

[0179] The ratio used to predict a CDR-sb score better than baseline after treatment: a ratio higher than the ratios mentioned in the table indicates treatment benefit, and a ratio lower than the ratios mentioned in the table indicates a lack of treatment benefit.

[0180] As shown in Table 6, in some cases, the present invention provides a ratio of the levels of different protein pairs in the blood, which is used to indicate the therapeutic benefit of CDR-sb over baseline.

[0181] For example, if a blood sample from a subject shows a DLL1 / SMOC1 ratio higher than the ratio mentioned in Table 6, the subject may experience a CDR-sb benefit that is superior to baseline. If a blood sample from a subject shows a DLL1 / SMOC1 ratio lower than the ratio mentioned in Table 6, the subject is unlikely to experience a CDR-sb benefit that is superior to baseline.

[0182] Based on the corresponding values ​​of the ratios, each protein pair listed in Table 6 can be used to indicate whether there is a similar or absent therapeutic benefit.

[0183] In some embodiments, the method may include providing a report indicating whether a subject is likely or unlikely to respond to plasma therapy for the treatment of cognitive impairment. In some embodiments, this step may involve calculating a score based on the measurement of the ratios of one or more protein pairs, where the score is related to responsiveness and may be a numerical value such as a probability, likelihood value, or a ten-point scale. In these embodiments, the method may include inputting a quantity of each ratio of the protein pairs into one or more algorithms or calculations, executing the algorithms or calculations, and receiving a score based on the calculations. In these embodiments, other measurements from the subject (e.g., whether the subject is male or female, the subject's age, the current degree of cognitive impairment, etc.) may be taken into account in the algorithm or calculation.

[0184] In some implementations, the method may involve creating a report (e.g., in electronic form) and forwarding it to a physician or other healthcare professional to help determine an appropriate course of action (e.g., identifying a subject as suitable for plasma exchange therapy to treat cognitive impairment). The report may be used in conjunction with other indicators to determine an appropriate course of action.

[0185] In some cases, reports may be forwarded to a “remote location,” which means a location other than the location where the report was generated. For example, a remote location could be another location within the same city (e.g., an office, laboratory, etc.), another location in a different city, another location in a different state, another location in a different country, etc. Therefore, when an item is designated as “remote” relative to another item, the two items may be in the same room but separate, or at least in different rooms or different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart. “Communication” information refers to data representing that information transmitted in the form of electrical signals through a suitable communication channel (e.g., a private or public network). “Forwarding” an item refers to any means of moving the item from one location to another, whether by physical transport of the item or other means (where possible), and at least for data, includes the physical transport of media carrying the data or communication data. Examples of communication media include radio or infrared transmission channels, network connections to another computer or networked device, and the Internet, or information including email transmissions and records on websites. In some implementations, reports may be analyzed by an MD or other qualified medical professional, and reports based on image-based analysis results may be forwarded to the subject from whom the sample was taken.

[0186] In computer-related implementations, the system may include a computer comprising a processor, storage components (i.e., memory), a display component, and other components typically found in general-purpose computers. The storage components store information accessible to the processor, including instructions executable by the processor and data that can be retrieved, manipulated, or stored by the processor.

[0187] The storage component includes instructions for determining, using the assay results of one or more proteins as discussed above, whether a subject is likely to respond to plasma exchange therapy for the treatment of cognitive impairment. A computer processor is coupled to the storage component and configured to execute the instructions stored in the storage component to receive patient data and analyze the patient data according to one or more algorithms or calculations. A display component can display information about the patient's responsiveness to plasma therapy for the treatment of cognitive impairment.

[0188] Storage components can be any type capable of storing information accessible to the processor, such as hard drives, memory cards, ROM, RAM, DVDs, CD-ROMs, USB flash drives, writable memory, and read-only memory. The processor can be any well-known processor, such as those from Intel Corporation. Alternatively, the processor can be a dedicated controller, such as an ASIC.

[0189] Instructions can be any set of instructions that are executed directly by the processor (such as machine code) or indirectly (such as scripts). In this regard, the terms "instruction," "step," and "program" are used interchangeably herein. Instructions can be stored as object code for direct processing by the processor, or in any other computer language, including scripts or sets of independent source code modules that are interpreted on demand or pre-compiled.

[0190] Data can be retrieved, stored, or modified by a processor according to instructions. For example, although the system disclosed herein is not limited to any particular data structure, data can be stored in computer registers, in a relational database as a table with multiple different fields and records, an XML document, or a flat file. Data can also be formatted in any computer-readable format, such as, but not limited to, binary values, ASCII, or Unicode. Furthermore, data can include any information sufficient to identify relevant information, such as numbers, descriptive text, proprietary code, pointers, references to data stored in other memory (including other network locations), or information applicable to functions for calculating relevant data.

[0191] E. Treatment methods

[0192] Certain aspects of the invention further provide for treating a subject's cognitive impairment by administering a therapy to the subject after determining whether the subject may or may not respond positively to a cognitive impairment therapy. All embodiments provided under the "Diagnostic Methods" section are also embodiments covered under the "Treatment Methods" section.

[0193] Therefore, certain aspects of the present invention provide a method for treating cognitive impairment in a subject, the method comprising administering plasma exchange therapy to the subject, wherein the subject is identified as possibly or possibly not likely to respond positively to plasma exchange therapy for the treatment of cognitive impairment.

[0194] In some cases, a subject may or may not respond positively to plasma exchange therapy for the treatment of cognitive impairment, based on the determination of the expression levels of any two markers selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8 in the subject's sample, particularly the ratio between any two protein levels.

[0195] Details of identifying a subject as potentially or unpromising to respond positively to plasma exchange therapy for the treatment of cognitive impairment based on the determination of the expression levels of any two markers selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8 in the subject sample, particularly the ratio between any two protein levels, are described above under “Diagnostic Methods”.

[0196] In short, subjects can be identified as potentially or unsuitable for a positive response to plasma exchange therapy for the treatment of cognitive impairment based on the expression levels of any two markers selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8, particularly the ratio between the levels of any two proteins. In some embodiments, one or more protein pairs are selected from the following: i. DLL1 and SMOC1, especially DLL1 / SMOC1; ii. DLL1 and CD59, especially DLL1 / CD59; iii. DLL1 and LEFTY2, especially DLL1 / LEFTY2; iv. DLL1 and UNC5B, especially DLL1 / UNC5B; v. DLL1 and C5, especially DLL1 / C5; vi. DLL1 and C5.C6, especially DLL1 / C5.C6; vii. DLL1 and INBBB, especially DLL1 / INHBB; viii. TSTD1 and STAT3, especially TSTD1 / STAT3; ix. POLD4 and PARP11, especially POLD4 / PARP11; x. ASH2L and PARP11, especially ASH2L / PARP11; xi. RPS3 and PARP11, especially RPS3 / PARP11; xii. VAV3 and SIRT3, especially VAV3 / SIRT3; and xiii. SERPINB8 and PARP11; especially SERPINB8 / PARP11.

[0197] Subjects can be identified as potentially or unpromising to respond positively to plasma exchange therapy for the treatment of cognitive impairment by comparing the ratio of one or more levels of one or more markers (such as proteins) in a subject's sample to a reference ratio.

[0198] "Plasma exchange therapy" refers to replacing all or part of a subject's plasma with a plasma replacement solution. Typically, during plasma exchange therapy, blood is gradually removed from the subject's body, blood components (such as blood cells and platelets) are separated from the plasma, the plasma replacement solution is mixed with the separated blood components, and the resulting mixture is returned to the subject.

[0199] Plasma exchange therapy may include replacing substantially all of a subject's plasma with a plasma exchange solution, referred to herein as "total plasma exchange". In total plasma exchange, substantially all of a subject's plasma (such as at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the subject's plasma) is replaced with a plasma exchange solution. In some cases of total plasma exchange, the plasma exchange solution is an albumin solution containing 3% to 10% w / v albumin, such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w / v albumin solutions. In one embodiment, the plasma exchange solution used for total plasma exchange is a 5% w / v albumin solution.

[0200] Plasma exchange therapy may also involve replacing a portion of the subject's plasma with a plasma exchange solution, referred to herein as "low-volume plasma exchange." In low-volume plasma exchange, 10% to 50% (such as 15% to 45%, 20% to 40%, 25% to 35%, or about 30%) of the subject's plasma is replaced with a plasma exchange solution. In some cases of low-volume plasma exchange, the plasma exchange solution is an albumin solution containing 15% to 25% w / v albumin, such as 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% w / v albumin.

[0201] In some embodiments, plasma exchange therapy includes a course of total plasma exchange. In some cases, a course of total plasma exchange includes weekly total plasma exchange for 1 to 8 weeks, such as once a week for 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some such embodiments, the plasma exchange solution contains 5% w / v albumin.

[0202] In another embodiment, plasma exchange therapy includes a low-volume plasma exchange course using a second albumin solution following a total plasma exchange course. The low-volume plasma exchange course may be performed monthly for at least one month to at least ten months, for example, monthly for 12 to 16 months, such as 12, 13, 14, 15, or 16 months. In some such embodiments, the plasma exchange solution contains 20% w / v albumin.

[0203] In specific embodiments, plasma therapy includes, for example: Figure 1The described therapy involves total plasma exchange once a week for 6 weeks using a plasma exchange solution containing 5% w / v albumin. Following this total plasma exchange course, plasma exchange therapy includes monthly low-volume plasma exchange therapy for 10 to 16 months, such as 10, 11, 12, 13, 14, 15, or 16 months. Low-volume plasma exchange therapy involves replacing 15% to 25%, such as 20%, of the subject's plasma with a plasma exchange solution containing 20 gm to 40 gm albumin (such as 20 g, 30 g, or 40 g albumin). In some cases, low-volume plasma exchange therapy involves replacing 15% to 25%, such as 20%, of the subject's plasma with a plasma exchange solution containing 20 gm to 40 gm albumin (such as 20 g, 30 g, or 40 g albumin) and 10 g to 20 g immunoglobulin (such as 10 g, 15 g, or 20 g immunoglobulin).

[0204] In some cases, plasma exchange therapy involves weekly total plasma exchange for 6 weeks using a plasma exchange solution containing 5% w / v albumin. For example, during total plasma exchange, 2500 to 3000 mL of patient plasma is replaced with an equal volume of 5% albumin solution (e.g., 5% albutein). Following total plasma exchange, plasma exchange therapy includes monthly low-volume plasma exchange for 10 to 16 months, such as 10, 11, 12, 13, 14, 15, or 16 months. During low-volume plasma exchange, 600 to 900 mL (e.g., 650 to 880 mL) of patient plasma is replaced with 100 or 200 mL of 20% albumin (e.g., 20% albutein). In some cases, low-volume plasma exchange therapy includes using 20 g to 40 g (e.g., 20 g, 30 g, and 40 g) of albumin in a 20% albumin solution (e.g., Albutein 20%) as the exchange fluid. In some cases, albumin replacement is alternated with immunoglobulin replacement, for example, by using 10 g to 30 g (such as 10 g, 20 g or 30 g) of immunoglobulin in a 5% immunoglobulin solution (e.g. FlebogammaDIF 5%).

[0205] Plasma replacement solution

[0206] In some cases, plasma exchange therapy may be performed using any of the plasma exchange solutions described below.

[0207] Albumin Plasma Products (“APP”) fall into two main categories: plasma protein fractions (“PPF”) and human albumin solutions (“HAS”). PPF is derived from processes with higher yields than HAS, but its minimum albumin purity is lower (>83% for PPF and >95% for HAS) (Production of human albumin solution: a continually developing colloid, P. Matejtschuk et al., British J. of Anaesthesia 85(6): 887-95, at 888 (2000)). In some instances, PPF has an albumin purity of 83% to 95%, or alternatively 83% to 96%. Albumin purity can be determined by electrophoresis or other quantitative assays, such as mass spectrometry.

[0208] Those skilled in the art will recognize that several commercial sources of PPF (“commercial PPF formulations”) exist or have existed. These include Plasma-Plex™ PPF (Armour Pharmaceutical Co., Asphalt Village, NY), Plasmanate™ PPF (Grifols, Clayton, North Carolina), Plasmatein™ (Alpha Therapeutics, Los Angeles, California), and Protenate™ PPF (Baxter Labs, Deerfield, Illinois).

[0209] Those skilled in the art will also recognize several commercial sources of HAS (“commercial HAS formulations”). These include Albaminar™ (CSL Behring), AlbuRx™ (CSL Behring), Albutein™ (Grindford, Clayton, North Carolina), Buminate™ (Baxatla, Bannockburn, Illinois), Flexbumin™ (Baxatla, Bannockburn, Illinois), and Plasbumin™ (Grindford, Clayton, North Carolina).

[0210] Plasma protein components (human) (PPF)

[0211] According to the U.S. Food and Drug Administration (“FDA”), “Plasma Protein Component (Human)” or PPF is the correct name for a product defined as “a sterile solution of proteins consisting of albumin and globulin derived from human plasma” (CFR 21 CFR 640.90, which is incorporated herein by reference). The source material for PPF is whole blood separated (recycled) plasma prepared in accordance with 21 CFR 640.1–640.5 (incorporated herein by reference), or raw plasma prepared in accordance with 21 CFR 640.60–640.76 (incorporated herein by reference).

[0212] The PPF is tested to determine whether it conforms to the following criteria in 21 CFR 640.92 (incorporated herein by reference): (a) The final product should be a protein solution of 5.0 + / - 0.30%; and (b) The total protein in the final product shall consist of at least 83% albumin and no more than 17% globulin. No more than 1% of the total protein shall be gamma globulin. The protein composition shall be determined by a method approved for each manufacturer by the Director of the Center for Biologics Evaluation and Research, U.S. Food and Drug Administration.

[0213] As used herein, “plasma protein fraction” or “PPF” refers to a sterile protein solution derived from human plasma, consisting of albumin and globulins, which, as determined by electrophoresis, contains at least 83% albumin, no more than 17% globulins (including α1, α2, β, and γ globulins) and other plasma proteins, and no more than 1% γ globulins. (Hink, JH, Jr. et al., Preparation and Properties of a Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957)). PPF can also refer to a solid form having a similar composition when suspended in a solvent. Total globulin fraction can be determined by subtracting albumin from total protein. (Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY, PHYSICAL, ANDLABORATORY EXAMINATIONS, Chapter 10, Walker HK, Hall WD, Hurst JD, eds. (1990)).

[0214] Albumin (human) (HAS)

[0215] According to the FDA, “albumin (human)” (also referred to herein as “HAS”) is the correct name for a product defined as “a sterile solution of albumin derived from human plasma.” (Compilation of Federal Regulations, CFR 21 CFR 640.80, which is incorporated herein by reference.) The source material for albumin (human) is whole blood plasma separated in accordance with 21 CFR 640.1–640.5 (incorporated herein by reference), or raw plasma prepared in accordance with 21 CFR 640.60–640.76 (incorporated herein by reference). Other requirements for albumin (human) are set forth in 21 CFR 640.80–640.84 (incorporated herein by reference).

[0216] Albumin (human) is tested to determine whether it meets the following criteria in 21 CFR 640.82: (a) Protein concentration. The final product shall meet one of the following concentrations: 4.0 + / - 0.25 percent; 5.0 + / - 0.30 percent; 20.0 + / - 1.2 percent; and 25.0 + / - 1.5 percent protein solution.

[0217] (b) Protein composition. As determined by a method approved by the Director of the Biological Products Evaluation and Research Center of the Food and Drug Administration for each manufacturer, at least 96% of the total protein in the final product should be albumin.

[0218] As used herein, “albumin (human)” or “HAS” refers to a sterile protein solution derived from human plasma, consisting of albumin and globulins, with an albumin content of at least 95% and globulins (including α1, α2, β, and γ globulins) and other plasma proteins not exceeding 5%. HAS can also refer to a solid form with a similar composition when suspended in a solvent. The total globulin component can be determined by subtracting albumin from the total protein content.

[0219] As will be appreciated by those skilled in the art, the PPF and HAS components may also be lyophilized or in other solid forms. For example, such formulations, together with suitable additives, can be used to manufacture tablets, powders, granules, or capsules. Solid forms can be formulated into injectable formulations by dissolving, suspending, or emulsifying them in aqueous solutions; and, if desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives can be used.

[0220] F. Indications

[0221] The methods and compounds in question can be used to treat cognitive impairments, such as those caused by neuroinflammation. In some cases, cognitive impairment is age-related, such as age-related decline in individual cognitive abilities.

[0222] Non-limiting examples of such cognitive impairments include age-related dementia, immune conditions, and physical or functional decline. Individuals who have or are suspected of having cognitive impairment and who would benefit from the treatments disclosed herein include those who are approximately 50 years of age or older (e.g., 60 years or older, 70 years or older, 80 years or older, 90 years or older, and 100 years or older, i.e., those between approximately 50 and 100 years of age, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or approximately 100 years old) and who have a cognitive impairment associated with the natural aging process. Individuals with cognitive impairment (such as mild cognitive impairment (MCI)); and individuals approximately 50 years of age or older (e.g., 60 years or older, 70 years or older, 80 years or older, 90 years or older, and generally not exceeding 100 years of age, i.e., between approximately 50 and 90 years of age, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or approximately 100 years old) who have not yet begun to exhibit symptoms of cognitive impairment. Examples of cognitive impairment / indications that may be caused by natural aging include the following: 1. Mild cognitive impairment (MCI) Mild cognitive impairment is a mild disturbance of cognitive function, manifested as problems with memory or other mental functions (such as planning, following instructions, or decision-making), which worsen over time, while overall mental function and daily activities remain unaffected. Therefore, although significant neuronal death does not typically occur, neurons in the aging brain are susceptible to age-related sublethal structural changes, altered synaptic integrity, and changes in molecular processing at synapses, all of which can impair cognitive function. Individuals with or suspected of having age-related cognitive impairment will benefit from treatment using the methods disclosed in this article.

[0223] The subjects envisioned in this study also include individuals of any age with cognitive impairment due to age-related diseases; and individuals of any age diagnosed with age-related diseases that are typically accompanied by cognitive impairment. Examples of such age-related diseases include the following: 2. Alzheimer's disease Alzheimer's disease is a progressive, irreversible loss of cognitive function associated with excessive senile plaques in the cerebral cortex and subcortical gray matter. These areas also contain β-amyloid protein and neurofibrillary tangles composed of tau protein. The most common form affects people over 60 years of age, and its incidence increases with age. It accounts for more than 65% of dementia cases in the elderly.

[0224] The cause of Alzheimer's disease (AD) is unclear. The disease is familial in approximately 15% to 20% of cases. The remaining so-called sporadic cases have some genetic determinants. The disease follows an autosomal dominant inheritance pattern in most early-onset cases and some late-onset cases, but with variable penetrance in later life. Environmental factors are a focus of active research.

[0225] During the disease process, synapses and ultimately neurons are lost in the cerebral cortex, hippocampus, and subcortical structures (including selective cell loss in the basal ganglia of Meynert), locus coeruleus, and dorsal raphe nucleus. Glucose utilization and perfusion in the brain are reduced in certain areas (parietal and temporal lobes in early-stage disease, and prefrontal cortex in later-stage disease). Neuroinflammatory or senile plaques (composed of neurites, astrocytes, and glial cells surrounding an amyloid core) and neurofibrillary tangles (composed of paired spiral filaments) play a role in the pathogenesis of Alzheimer's disease (AD). Senile plaques and neurofibrillary tangles can also occur during normal aging, but they are more prevalent in people with AD.

[0226] 3. Parkinson's disease

[0227] Parkinson's disease (PD) is an idiopathic, slowly progressive, degenerative central nervous system disorder characterized by bradykinesia (slowness of movement), rigidity, resting tremor (dystonia), muscle freezing, and postural instability. Initially thought to be primarily a motor disorder, PD is now also recognized for its role in depression and mood changes. PD can also affect cognition, behavior, sleep, autonomic function, and sensory function. The most common cognitive impairments include impairments in attention and concentration, working memory, executive function, language production, and visuospatial function. A characteristic feature of PD is that symptoms associated with motor function decline often precede those associated with cognitive impairment, which aids in diagnosis.

[0228] In primary Parkinson's disease, pigment neurons in the substantia nigra, locus coeruleus, and other brainstem dopaminergic cell populations undergo degeneration. The etiology is unclear. Loss of substantia nigra neurons projecting to the caudate nucleus and putamen leads to depletion of the neurotransmitter dopamine in these areas. Onset typically occurs after age 40, with an increased incidence in older adults.

[0229] Parkinson's disease (PD) is diagnosed in approximately 60,000 new cases each year in the United States and currently affects about one million Americans. Although PD itself is not fatal, its complications are the fourteenth leading cause of death in the United States. Currently, there is no cure for PD, and treatment is usually aimed at controlling symptoms, with surgery being used in later, severe cases.

[0230] Treatment options for Parkinson's disease (PD) include the administration of medications to help manage motor deficits. These options increase or replace the neurotransmitter dopamine, which is present in low concentrations in the brains of PD patients. Such medications include: carbidopa / levodopa (which produces more dopamine in the brain); apomorphine, pramipexole, ropinirole, and rotingotine (dopamine agonists); selegiline and rasagiline (MAO-B inhibitors that prevent dopamine breakdown); entacapone and tocapone (catechol-O-methyltransferase [COMT] inhibitors that make more levodopa available in the brain); bentropine and trihexyphenidyl (anticholinergics); and amantadine (to control tremors and rigidity). Exercise / physical therapy is also frequently prescribed to help maintain physical and mental function.

[0231] However, current treatment options only treat the symptoms of PD, offering neither a cure nor a halt to disease progression. Furthermore, current medications often lose their effectiveness in advanced PD. Levodopa, the most commonly prescribed drug, typically produces adverse effects within 5 to 10 years of starting treatment. These adverse effects can be severe and can lead to motor fluctuations and unpredictable dose-dependent variability in motor control, as well as tics / tices (motor disorders), which are difficult to manage and can even be as disabling as the symptoms of PD itself. Therefore, new therapies with novel mechanisms of action remain needed, which can be used alone or in combination with current PD medications.

[0232] 4. Parkinson's syndrome

[0233] Secondary Parkinsonism (also known as atypical Parkinson's disease or Parkinson's plus syndrome) is caused by the loss or disruption of dopamine function in the basal ganglia due to other idiopathic degenerative diseases, medications, or exogenous toxins. The most common cause of secondary Parkinsonism is the ingestion of antipsychotic drugs or reserpine, which produce Parkinsonism by blocking dopamine receptors. Less common causes include carbon monoxide or manganese poisoning, hydrocephalus, structural lesions (tumors or infarctions affecting the midbrain or basal ganglia), subdural hematoma, and degenerative diseases, including substantia nigra-striatal degeneration. Certain diseases, such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), and Lewy body dementia (DLB), may present with Parkinsonism symptoms before the major symptoms required for a specific diagnosis appear, and may therefore be labeled as "Parkinsonism syndrome."

[0234] 5. Frontotemporal dementia

[0235] Frontotemporal dementia (FTD) is a condition caused by progressive degeneration of the frontal lobe of the brain. Over time, this degeneration may progress to the temporal lobe. FTD is the second most common form of Alzheimer's disease after Alzheimer's disease, accounting for 20% of all Alzheimer's cases. Based on the function of the affected frontal and temporal lobes, symptoms are divided into three groups: Behavioral variant FTD (bvFTD) is characterized by symptoms including somnolence and lack of initiative on one hand, and disinhibition on the other; progressive nonfluent aphasia (PNFA), in which interruptions in speech fluency are observed due to dysphonia, phonological and / or syntactic errors, but word comprehension is preserved; and semantic dementia (SD), in which patients maintain fluency, with normal phonology and syntax, but increasingly difficulty with naming and word comprehension. Other common cognitive symptoms in all FTD patients include impaired executive function and attention. Other cognitive abilities, including perception, spatial skills, memory, and practical abilities, are usually intact. FTD can be diagnosed by observing frontal and / or anterior temporal lobe atrophy on structural MRI scans.

[0236] There are various forms of frontotemporal dementia (FTD), any of which can be treated or prevented using thematic approaches and compositions. For example, one form of frontotemporal dementia is semantic dementia (SD). SD is characterized by loss of semantic memory in both the verbal and nonverbal domains. Patients with SD typically complain of difficulty finding words. Clinical signs include fluent aphasia, naming difficulties, impaired understanding of word meaning, and associative visual agnosia (inability to match semantically relevant pictures or objects). As the disease progresses, behavioral and personality changes often occur, similar to those seen in frontotemporal dementia, although there are cases described as “pure” semantic dementia with few late-stage behavioral symptoms. Structural MRI imaging shows a characteristic atrophy pattern in the temporal lobe (primarily the upper left), with greater involvement in the lower temporal lobe than the upper temporal lobe, and greater atrophy in the anterior temporal lobe than the posterior temporal lobe.

[0237] As another example, another form of frontotemporal dementia is Pick's disease (PiD, also known as PcD). A defining characteristic of this disease is the accumulation of tau protein within neurons, forming silver-stained spherical aggregates called "Pick bodies." Symptoms include loss of speech (aphasia) and dementia. Patients with orbitofrontal dysfunction may become aggressive and socially inept. They may steal or exhibit compulsive or repetitive stereotyped behaviors. Patients with dorsolateral or dorsolateral frontal lobe dysfunction may exhibit apathy, indifference, or reduced initiative. Patients may exhibit a lack of self-monitoring, abnormal self-awareness, and an inability to understand meaning. Patients with bilateral posterolateral orbitofrontal cortex and right anterior insula gray matter loss may exhibit changes in eating behavior, such as pathological cravings for sweets. Patients with more pronounced focal gray matter loss in the anterolateral orbitofrontal cortex may experience hyperphagia. While some symptoms may initially resolve, the disease progresses, and patients typically die within two to ten years.

[0238] 6. Huntington's disease

[0239] Huntington's disease (HD) is a hereditary, progressive neurodegenerative disorder characterized by mood, behavior, and psychiatric abnormalities; loss of intellectual or cognitive function; and motor abnormalities (motor disturbances). Typical signs of HD include the development of chorea—involuntary, rapid, irregular, jerky (tic-like) movements that may affect the face, arms, legs, or trunk—and cognitive decline, including a gradual loss of cognitive processing abilities and acquired intellectual capacity. Impaired memory, abstract thinking, and judgment may be present; disorientation may occur due to a lack of perception of time, place, or identity; increased agitation; and personality changes (depersonalization). While symptoms typically become apparent in the fourth or fifth decade, the age of onset is variable and ranges from early childhood to late adulthood (e.g., in one's 70s or 80s).

[0240] HD is transmitted in families as an autosomal dominant trait. The disease occurs due to an abnormally long sequence or "duplication" of coding instructions in a gene on chromosome 4 (4p16.3). The progressive loss of neurological function associated with HD is due to the loss of neurons in certain areas of the brain, including the basal ganglia and cerebral cortex.

[0241] 7. Amyotrophic Lateral Sclerosis (ALS)

[0242] Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and inevitably fatal neurological disease that attacks motor neurons. Signs of muscle weakness and atrophy, as well as anterior horn cell dysfunction, are initially most common in the hands and less common in the feet. The site of onset is random, and progression is asymmetrical. Spasticity is common and may precede weakness. Rarely, patients can survive for 30 years; 50% die within 3 years of onset, 20% survive 5 years, and 10% survive 10 years.

[0243] Diagnostic features include onset in mid-to-late adulthood and progressive, widespread motor involvement without sensory abnormalities. Neural conduction velocities remain normal until the late stages of the disease. Recent studies have also documented cognitive impairment, particularly declines in immediate verbal memory, visual memory, language, and executive function.

[0244] Reports indicate that even in outwardly normal neurons of ALS patients, reductions in cell body area, synapse number, and total synaptic length have been observed. It has been proposed that persistent synaptic loss may lead to functional impairment when the plasticity of active areas reaches its limits. Promoting the formation of new synapses or preventing synaptic loss may help maintain neuronal function in these patients.

[0245] 8. Multiple sclerosis

[0246] Multiple sclerosis (MS) is characterized by a variety of symptoms and signs of CNS dysfunction, with remissions and recurrent exacerbations. The most common initial symptoms are sensory disturbances in one or more limbs, trunk, or one side of the face; weakness or clumsiness in the legs or hands; or visual disturbances such as partial blindness and monocular pain (retrobulbar optic neuritis), blurred vision, or scotomas. Common cognitive impairments include difficulties with memory (acquiring, retaining, and retrieving new information), attention and concentration (especially distributed attention), information processing, executive function, visuospatial function, and verbal fluency. Common early symptoms include oculomotor palsy leading to diplopia (diplopia), transient weakness in one or more limbs, mild stiffness or unusual easy fatigue of limbs, mild gait disturbances, bladder control difficulties, dizziness, and mild mood disturbances; all of these indicate scattered CNS involvement and often occur months or years before the disease is recognized. Overheating may exacerbate symptoms and signs.

[0247] The course of the disease is highly variable and unpredictable, and in most patients it is remission-prone. Initially, there may be remission periods of several months or years between attacks, especially when the disease begins with retrobulbar optic neuritis. However, some patients experience frequent attacks and rapid disability; in a minority of patients, the course of the disease may progress rapidly.

[0248] 9. glaucoma

[0249] Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs). Evidence supports a compartmentalized degenerative process in synapses and dendrites, including within the RGCs. Recent evidence also suggests a correlation between cognitive impairment and glaucoma in older adults (Yochim BP et al., Prevalence of cognitive impairment, depression, and anxiety symptoms among older adults with glaucoma. J Glaucoma. 2012;21(4):250-254).

[0250] 10. Myotonic dystrophy

[0251] Myotonic dystrophy (DM) is an autosomal dominant multisystem disease characterized by dystrophic muscle weakness and myotonia. The molecular defect is a trinucleotide (CTG) repeat amplification in the 3' untranslated region of the myotonic protein kinase gene on chromosome 19q. Symptoms can occur at any age and range in clinical severity. Myotonia is prominent in the muscles of the hands, and ptosis is common even in mild cases. In severe cases, significant peripheral muscle weakness is present, often accompanied by cataracts, premature alopecia, axe-shaped facies, cardiac arrhythmias, testicular atrophy, and endocrine abnormalities (e.g., diabetes). Intellectual disability is common in the severe congenital form, while age-related decline in frontal and temporal cognitive functions (particularly language and executive functions) is observed in the milder adult form of the disease. Severely affected individuals die in their early 50s.

[0252] 11. dementia

[0253] Dementia describes a class of diseases with symptoms that severely affect thinking and social abilities to the point of interfering with daily functioning. In addition to dementia observed in the later stages of the age-related diseases discussed above, other examples of dementia include vascular dementia and Lewy body dementia, described below.

[0254] In vascular dementia, or "multiple infarct dementia," cognitive impairment is caused by problems with the brain's blood supply, often resulting from a series of small strokes, or sometimes from a large stroke occurring before or after other smaller strokes. Vascular lesions can be diffuse cerebrovascular disease (such as small vessel disease) or focal lesions, or a combination of both. Patients with vascular dementia develop acute or subacute cognitive impairment following an acute cerebrovascular event, followed by progressive cognitive decline. The cognitive impairment is similar to that observed in Alzheimer's disease, including impairments in language, memory, complex visual processing, or executive function, although the relevant changes in the brain are not due to AD pathology but rather to a chronic reduction in blood flow to the brain that ultimately leads to dementia. Single-photon emission computed tomography (SPECT) and positron emission tomography (PET) neuroimaging can be used in conjunction with assessments involving examination of mental status to confirm the diagnosis of multiple infarct dementia.

[0255] Lewy body dementia (DLB, also known by several other names including Lewy body dementia, diffuse Lewy body disease, cortical Lewy body disease, and Lewy type Alzheimer's disease) is a type of dementia characterized anatomically by the presence of Lewy bodies (clumps of α-synuclein and ubiquitin proteins) in neurons, detectable in post-mortem brain histology. Its main characteristic is cognitive decline, particularly in executive function. Alertness and short-term memory fluctuate.

[0256] Persistent or recurrent visual hallucinations with vivid and detailed images are often an early diagnostic symptom. DLB is frequently confused with Alzheimer's disease and / or vascular dementia in its early stages, although Alzheimer's disease typically begins very slowly, while DLB often has rapid or acute onset. DLB symptoms also include motor symptoms similar to those of Parkinson's disease. The difference between DLB and dementia, which sometimes occurs in Parkinson's disease, lies in the time frame of the onset of dementia symptoms relative to Parkinson's symptoms. When a dementia onset occurs more than a year after a Parkinson's disease onset, the diagnosis is Parkinson's disease with dementia (POD). When cognitive symptoms begin simultaneously with or within a year of Parkinson's symptoms, the diagnosis is DLB.

[0257] 12. CADASIL

[0258] Autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL) is a genetic disorder associated with mutations in the NOTCH3 gene (Locatelli M et al., Front. Pharmacol. 11:321 (2020)). It typically occurs in middle-aged adults and manifests as cognitive impairment leading to dementia and disability. Same as above. Other manifestations include mood disorders, migraines with aura, and recurrent strokes. Effective treatment remains elusive because the pathogenesis of the disease is still unclear. Same as above. CADASIL is the most common inherited subcortical vascular dementia. (Kalimo H, et al.) , Future Neurology, 3(6) (2008)).

[0259] CADASIL is characterized by four main common symptoms: migraine with aura, recurrent ischemic stroke, mental disorders, and cognitive decline. The first is usually the initial symptom, occurring in 20-40% of affected individuals. The second symptom occurs in 60-85% of symptomatic individuals. Third, mental disorders, in the form of moderate / major depression, bipolar disorder, panic disorder, schizophrenia, and apathy, occur in 25-30% of patients. Cognitive impairment occurs in 60% of patients, becoming clinically detectable between 35 and 50 years of age, and progressively worsens with age. Same as above. In younger patients, attention, memory, and executive function impairments are predominant. (Buffon F, et al.) , J Neurol NeurosurgPsychiatry 77(2): 175-80 (2006)). Visuospatial and reasoning abilities deteriorate with age, primarily after age 60. Dementia occurs in 25% of patients, 75% of whom are over 60 years old. However, the number of ischemic attacks is not associated with dementia. Same as above. )

[0260] CADASIL is a progressive and fatal disease. To date, no disease-modifying treatment has been developed (Locatelli et al., ibid.). Symptomatic treatment is the only recourse for clinicians, based on routine clinical practice, such as acetazolamide or sodium valproate for migraines; daily aspirin to reduce the risk of heart attack or stroke; and supportive care for cognitive loss. It is noteworthy that no medication has yet been clearly shown to be beneficial for cognitive loss associated with CADASIL. Same as above. Interventions that have been studied but failed include donepezil (used to improve cognitive impairment in Alzheimer's disease), galantamine (an acetylcholinesterase inhibitor used to treat cognitive impairment in Alzheimer's disease), and levodopa (used for Alzheimer's disease and Parkinson's disease).

[0261] 13. Progressive supranuclear palsy

[0262] Progressive supranuclear palsy (PSP) is a brain disorder that causes severe and progressive problems with gait and balance control, as well as complex eye movements and thinking. One of the hallmarks of the disease is the inability to properly aim the eyes, caused by lesions in brain regions that coordinate eye movements. Some describe this effect as blurring. Affected individuals often exhibit mood and behavioral changes, including depression and apathy, as well as progressive mild dementia. The long name of the disease indicates that it begins slowly and steadily worsens (progressive) and causes weakness (paralysis) (supranuclear) by damaging parts of the brain above pea-sized structures called nuclei that control eye movements. PSP was first described as a distinct disease in 1964 when three scientists published a paper differentiating it from Parkinson's disease. It is sometimes referred to as the Steele-Richardson-Olszewski syndrome, reflecting the combined name given by the scientists who defined the disease. Despite the progressive nature of PSP, no one dies from PSP itself.

[0263] 14. Ataxia

[0264] People with ataxia have coordination problems because parts of the nervous system that control movement and balance are affected. Ataxia can affect the fingers, hands, arms, legs, body, speech, and eye movements. The term ataxia is often used to describe a symptom of poor coordination that may be associated with infection, injury, other diseases, or degenerative changes in the central nervous system. Ataxia is also used to refer to a specific group of neurodegenerative diseases called hereditary and sporadic ataxia, which are the main focus of the National Ataxia Foundation.

[0265] 15. Multiple system atrophy

[0266] Multiple system atrophy (MSA) is a degenerative neurological disorder. MSA is associated with the degeneration of nerve cells in specific areas of the brain. This cell degeneration leads to problems with movement, balance, and other autonomic bodily functions, such as bladder control or blood pressure regulation.

[0267] The cause of MSA is unknown, and specific risk factors have not been identified. Approximately 55% of cases occur in men, with a typical age of onset in the late 50s to early 60s. MSA often presents with some of the same symptoms as Parkinson's disease. However, MSA patients typically respond very little (if any) to dopamine medications used for Parkinson's disease.

[0268] 16. weak

[0269] Frailty syndrome (“frailty”) is a geriatric syndrome characterized by functional and physical decline, including decreased mobility, muscle weakness, slowness of movement, poor endurance, low physical activity, malnutrition, and involuntary weight loss. This decline is often accompanied by and is a consequence of diseases such as cognitive impairment and cancer. However, frailty can occur even in the absence of disease. Individuals with frailty have an increased risk of negative outcomes such as fractures, accidental falls, disability, comorbidities, and premature death. (C. Buigues et al., Effect of a Prebiotic Formulation on Frailty Syndrome: A Randomized, Double-Blind Clinical Trial, Int. J. Mol. Sci. 2016, 17, 932). Furthermore, individuals with frailty have an increased incidence of higher healthcare expenditures. (Ibid.)

[0270] Common symptoms of frailty can be identified by certain types of tests. For example, involuntary weight loss involves losing at least 10 pounds or more than 5% of body weight in the previous year; muscle weakness can be identified by grip strength at the lowest 20% of baseline (adjusted for sex and BMI); bradykinesia can be identified based on the time required to walk 15 feet; poor endurance can be identified by an individual’s self-reported fatigue; and low physical activity can be measured using standardized questionnaires. (Z. Palace et al., The Frailty Syndrome, Today's Geriatric Medicine 7(1), at 18(2014)).

[0271] 17. Neuromyelitis optica spectrum disorder

[0272] Neuromyelitis optica spectrum disorder (NMOSD), also known as Devic disease, is a rare inflammatory disorder of the central nervous system. It is characterized by optic neuritis (inflammation of the optic nerve) and myelitis (inflammation of the spinal cord). Typically, patients experience recurrent episodes of inflammation interspersed with periods of remission. The disease is believed to be caused by autoantibodies that typically target myelin oligodendrocyte glycoprotein (MOG-IgG) or aquaporin 4 (AQP4-IgG), leading to demyelination and axonal damage in the optic nerve and spinal cord.

[0273] 18. Postoperative cognitive impairment

[0274] Postoperative cognitive decline occurs after anesthesia and surgery. It is common in patients over 60 years of age and is diagnosed through preoperative and postoperative cognitive tests. Patients typically present with memory impairment, delirium, and impaired performance on intellectual tasks.

[0275] 19. Chronic traumatic encephalopathy

[0276] Chronic traumatic encephalopathy (CTE) is a neurodegenerative brain disorder most commonly seen in athletes, veterans, or others with a history of repeated head injuries. It is one of many tau protein disorders characterized by an excess of tau protein in the brain, leading to neuronal loss. Symptoms include memory loss, mood or personality changes, confusion, impaired judgment, impulse control, aggression, and depression.

[0277] 20. Traumatic brain injury

[0278] Traumatic brain injury (TBI) is caused by a severe impact to the head or body. It can also be caused by an object that penetrates brain tissue during the injury. It results in bleeding, tissue tears, and physical damage to brain cells and cell death. Physical symptoms vary, but include loss of consciousness, headache, nausea, extreme fatigue, impaired speech, difficulty sleeping, dizziness, blurred vision, sensitivity to light or sound, memory loss, and attention problems.

[0279] In some embodiments, the subject matter methods and compositions are used to slow the progression of age-related cognitive impairment. In other words, cognitive impairment in an individual will decline more slowly after treatment with the disclosed methods than before treatment with the disclosed methods or without treatment with the disclosed methods. In some such instances, the subject matter treatment methods include measuring the progression of cognitive impairment after treatment and determining a reduction in the progression of decline. In some such instances, this determination is made by comparison with a reference, such as the individual's rate of decline before treatment, for example, by measuring cognitive abilities at two or more time points prior to administration of the subject matter blood product.

[0280] The subject methods and compositions are also used to stabilize the cognitive abilities of individuals, such as those with age-related cognitive impairment or those suspected of having age-related cognitive decline. For example, an individual may exhibit some age-related cognitive impairment, and the progression of cognitive impairment observed prior to treatment with the disclosed methods will cease after treatment with the disclosed methods.

[0281] As another example, an individual may be at risk of developing age-related cognitive decline (e.g., the individual may be 50 years of age or older, or may have been diagnosed with an age-related disease), and the individual's cognitive abilities are essentially unchanged, i.e., cognitive decline cannot be detected after treatment with the disclosed method compared to before treatment with the disclosed method.

[0282] The subject-based approach and composition are also used to reduce cognitive impairment in individuals with age-related disorders. In other words, the affected cognitive abilities are improved in the individual after treatment with the subject-based approach. For example, cognitive abilities in the individual increase by, for instance, 2 to 5 times, 10 to 15 times, 20 to 30 times, or 40 to 50 times, 60 to 70 times, 80 to 90 times, or 100 to 100 times or more, relative to cognitive abilities observed in the individual before treatment with the subject-based approach.

[0283] In some instances, treatments using thematic methods and compositions have restored cognitive abilities in individuals with age-related cognitive impairment, for example, to levels seen when the individual was approximately 40 years old or younger. In other words, cognitive or motor impairments have been eliminated.

[0284] G. Methods for monitoring and improvement

[0285] In some instances, among various methods for monitoring disease progression and improvement in cognitive impairment, the following types of assessments are used alone or in combination with those for subjects with cognitive impairment. These types of methods are presented as examples and are not limited to the listed methods. Any convenient method for monitoring disease can be used in the practice of this invention as needed. These methods are also included in the methods of this invention.

[0286] i. General understanding

[0287] Some embodiments of the methods of the present invention further include methods for monitoring the effects of a drug or treatment on a subject's treatment of cognitive impairment, such as age-related cognitive impairment. Some such methods include comparing cognitive function before and after treatment. Methods for assessing cognitive function are well known in the art. For example, and without limitation, methods may include assessing cognitive function based on medical history, family history, physical and neurological examination by a clinician specializing in cognitive function, laboratory tests, and neuropsychological assessments. Further embodiments of the present invention include: assessment of consciousness, such as using the Glasgow Coma Scale (EMV); examination of mental status, including the Abbreviated Mental Test Score (AMTS) or the Mini-Mental State Examination (MMSE) (Folstein et al., J. Psychiatr. Res 1975; 12: 1289-198); overall assessment of higher functions; and estimation of intracranial pressure, such as by funduscopy. In one implementation, monitoring the effects on cognitive impairment, such as age-related cognitive impairment, includes examining improvements in scores 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 using the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-COG).

[0288] In one implementation, an examination of the peripheral nervous system can be used to assess cognitive function, including any of the following: olfaction, visual field and visual acuity, eye movements and pupils (sympathetic and parasympathetic), facial sensory function, facial and shoulder girdle muscle strength, hearing, taste, pharyngeal movement and reflexes, tongue movement, which can be tested individually (e.g., visual acuity can be tested using the Snellen visual acuity chart; reflex hammers are used to test reflexes, including masseter, biceps and triceps tendons, patellar tendon, ankle reflex, and plantar reflex (i.e., Babinski sign); muscle strength is typically assessed using an MRC scale of 1 to 5; muscle tone and signs of rigidity).

[0289] Some embodiments of the methods of the present invention further include monitoring the effects of cognitive impairment treatment on motor function before and after treatment. Some such methods include assessing the motor function of subjects with PD who are being treated for cognitive impairment according to the methods disclosed herein. Methods for assessing motor function are well known in the art. For example, and without limitation, methods may include assessing motor function based on medical history, family history, physical and neurological examination by a clinician specializing in neurodegenerative diseases and movement disorders, laboratory tests, and neurodegenerative assessments. Further embodiments of the present invention include the use of rating scales discussed below.

[0290] Several rating scales have been used to assess the progression of PD. The most widely used scales include the Unified Parkinson's Disease Rating Scale (UPDRS, introduced in 1987) (J. Rehabil Res. Dev., 2012 49(8): 1269-76) and the Hoehn and Yahr Scale (Neruology, 1967 17(5):427-42). Other scales include the Movement Disorders Association (MDS) updated UPDRS scale (MDS-UPDRS) and the Schwab and England Activities of Daily Living (ADL) Scale.

[0291] The UPDRS scale assesses 31 items, which are divided into three subscales: (1) mental, behavioral, and emotional; (2) activities of daily living; and (3) motor examination. The Hoehn and Yahr scale divides PD into five stages, each with a defined substage: 0 - no signs of disease; 1 - symptoms on one side only; 1.5 - symptoms on one side but also involving the neck and spine; 2 - symptoms on both sides without balance disorder; 2.5 - mild symptoms on both sides, recovering on the “pull” test; 3 - balance disorder, mild to moderate disease; 4 - severe disability, but able to walk or stand independently; and 5 - requires a wheelchair or is bedridden without assistance. The Schwab and England scale divides PD into several percentages (from 100% - completely independent to 10% - completely dependent).

[0292] General motor function can be assessed using widely used scales, including the General Motor Function Scale (GMF). These tests consist of three components: dependence, pain, and insecurity. (Aberg AC et al. (2003) Disabil. Rehabil. 2003 May 6;25(9):462-72.). Motor function can also be assessed using home monitoring or wearable sensors. For example: gait (speed, variability, leg rigidity) can be sensed with an accelerometer; posture (trunk tilt) with a gyroscope; leg movements with an accelerometer; hand movements with an accelerometer and gyroscope; tremor (amplitude, frequency, duration, asymmetry) with an accelerometer; falls with an accelerometer; gait freeze with an accelerometer; movement disorders with an accelerometer, gyroscope, and inertial sensor; bradykinesia (duration and frequency) with an accelerometer and gyroscope; and aphasia (pitch) with a microphone. (Pastorino M et al., Journal of Physics: Conference Series 450 (2013)012055).

[0293] Some embodiments of the methods of the present invention further include monitoring the effect of cognitive impairment treatment on the progression or improvement of neurodegenerative changes. Some such methods include assessing the progression or improvement of neurodegenerative changes in subjects with neurodegenerative diseases (such as MS, HD, ALS, glaucoma, PSP) who have been treated for cognitive impairment according to the methods disclosed herein.

[0294] Methods for monitoring the progression or improvement of neurodegenerative changes are well known to those skilled in the art. By way of example, and not by limitation, monitoring can be performed using techniques such as: cerebrospinal fluid (CSF) monitoring; magnetic resonance imaging (MRI) to detect the development of lesions and demyelinating plaques; evoked potential studies; and gait monitoring.

[0295] CSF analysis can be performed, for example, via lumbar puncture to obtain pressure, appearance, and CSF content. Normal values ​​typically range as follows: pressure (70-180 mm H2O); appearance clear and colorless; total protein (15-60 mg / 100 mL); IgG 3-12% of total protein; glucose 50-80 mg / 100 mL; cell count 0-5 white blood cells and no red blood cells; chloride (110-125 mEq / L). Abnormal results may indicate the presence or progression of MS.

[0296] MRI is another technique that can be performed to monitor disease progression and improvement. Typical criteria for monitoring MS using MRI include patchy abnormal white matter areas in the cerebral hemispheres and periventricular regions, lesions in the cerebellum and / or brainstem, and cervical or thoracic segments of the spinal cord.

[0297] Evoked potentials can be used to monitor the progression and improvement of MS in subjects. Evoked potentials measure the slowing of electrical impulses, such as in visual evoked responses (VER), brainstem auditory evoked responses (BAER), and somatosensory evoked responses (SSER). Abnormal responses help indicate reduced conduction velocity in central sensory pathways.

[0298] Gait monitoring can also be used to monitor disease progression and improvement in MS subjects. MS is often accompanied by impaired mobility and abnormal gait, partly due to fatigue. Monitoring can be performed, for example, by using motion monitoring devices worn by the subject. (Moon, Y. et al. Monitoring gait in multiple sclerosis with novel wearable motion sensors, PLOS One, 12(2):e0171346 (2017)).

[0299] The present invention also considers treating or improving neurogenesis in subjects with decreased or impaired neurogenesis, which may manifest itself, for example, through reduced cognitive or motor function, or through its association with neuroinflammation.

[0300] Embodiments of the invention also contemplate determining the level of neurogenesis before, during, and / or after treatment (e.g., plasma exchange therapy). Non-invasive techniques for assessing neurogenesis have been reported (Tamura Y. et al., J. Neurosci. (2016) 36(31): 8123-31). Positron emission tomography (PET) combined with the tracer [18F]FLT and the BBB transporter inhibitor probenecid allows the tracer to accumulate in neurogenic regions of the brain. This imaging allows for the assessment of neurogenesis in patients undergoing treatment for neurodegenerative diseases.

[0301] H. Exercise

[0302] Exercise can be characterized by aerobic or anaerobic activity and can involve both high-calorie-expenditure and moderate-calorie-expenditure activities. Exercise may involve strength training (e.g., weight training or isometric exercises). Other forms of exercise may include, for example, running, cycling, walking, dancing, marching, swimming, yoga, tai chi, balance exercises, leg bends, skipping rope, surfing, rowing, rotating or bending arms or legs, gardening, cleaning, active games such as bowling, aerobics, Pilates, and martial arts.

[0303] The exercise program may include performing a single exercise at a specific frequency, or performing a combination of exercises at a specific frequency. The frequency may be once, twice, three times, four times, five times, six times, or seven times per week. The frequency may vary weekly. The exercise program may be at the same intensity and / or frequency level as the subject's practice prior to applying the composition of the present invention. The exercise program may also have a higher intensity and / or frequency than the level the subject practiced prior to applying the therapy of the present invention. The exercise program may be advised or prescribed by a health or fitness professional, or the exercise program may be self-initiated by the subject.

[0304] I. Kits, reagents, and devices

[0305] Some aspects of the present invention provide kits comprising reagents for measuring the ratio between the expression levels of any two markers (in protein or mRNA form) selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8 in a sample.

[0306] As discussed above, the determination of the ratio between levels of protein or mRNA pairs can be performed using any suitable method, such as qualitative or quantitative methods. Therefore, the kits disclosed herein can be designed to determine the ratio of levels of one or more target protein pairs.

[0307] Therefore, in some cases, the kits disclosed herein provide reagents and / or devices for determining the ratio of levels of one or more protein or mRNA pairs in a sample. Some non-limiting examples include kits for quantifying the ratio of levels of one or more target protein pairs via immunoassay, mass spectrometry, and protein detection array analysis.

[0308] As discussed above, non-limiting examples of immunoassays include Western blot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), lateral flow immunoassay, particle-based immunoassay, quantum dot-based immunoassay, etc. Therefore, certain embodiments of the present invention provide kits for performing one or more of these assays on one or more target protein pairs disclosed herein.

[0309] In one embodiment, the kit includes a lateral flow immunoassay device. Such devices allow for the quantification of any two proteins selected from the following: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8. The lateral flow immunoassay device may also include an internal control displaying a reference ratio.

[0310] In some cases, lateral flow immunoassay devices allow for the quantification of the ratio between levels of one or more of the following protein pairs: i. DLL1 and SMOC1, especially the ratio DLL1 / SMOC1; ii. DLL1 and CD59, especially the ratio DLL1 / CD59; iii. DLL1 and LEFTY2, especially the ratio DLL1 / LEFTY2; iv. DLL1 and UNC5B, especially the ratio DLL1 / UNC5B; v. DLL1 and C5, especially the ratio DLL1 / C5; vi. DLL1 and C5.C6, especially the ratio DLL1 / C5.C6; vii. DLL1 and INHBB, especially the ratio DLL1 / INHBB; viii. TSTD1 and STAT3, especially the ratio TSTD1 / STAT3; ix. POLD4 and PARP11, especially the ratio POLD4 / PARP11; x. ASH2L and PARP11, especially the ratio ASH2L / PARP11; xi. RPS3 and PARP11, especially the ratio RPS3 / PARP11; xii. VAV3 and SIRT3, especially the ratio VAV3 / SIRT3; and xiii. SERPINB8 and PARP11, especially the ratio SERPINB8 / PARP11.

[0311] Optionally, follow the instructions used to calculate the ratio.

[0312] In addition to the ratio of levels of one or more target protein pairs, the lateral flow immunoassay device can also allow for the quantification of levels of one or more control protein pairs.

[0313] Some kits allow the detection of the ratio between levels of one or more target protein pairs via a protein detection array. Some of these protein detection arrays may include specific binding agents, such as antibodies that specifically bind to any two proteins selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8. The antibody is localized to a specific spot on a solid support. The kit may further include reagents that facilitate visualization of the specific binding between the target protein pair in the sample and the binding agent on the array. Such reagents include labeled secondary antibodies, for example, fluorescently labeled secondary antibodies.

[0314] In some implementations, the kit allows for the detection of one or more target protein pairs via lysate microarray analysis. Such kits allow for the preparation of lysates of a sample and the immobilization of the lysates onto a support, such as a nitrocellulose-coated slide. The target protein pairs are then detected using a labeled solution-phase specific binder for one or more target protein pairs, such as a fluorescently labeled antibody that specifically binds to the protein in the target protein pair. Therefore, the kit includes a labeled binder for one or more target protein pairs, particularly a fluorescently labeled antibody that specifically binds to the protein in one or more target protein pairs. In some cases, antibodies against different proteins in the target protein pair are differentially labeled, thereby allowing for multiplex detection and / or quantification of two or more target protein pairs.

[0315] In some cases, kits are designed to allow the determination of the ratio between the expression levels of only one of the following biomarker pairs (particularly protein pairs) in a sample: i. DLL1 and SMOC1, especially the ratio DLL1 / SMOC1; ii. DLL1 and CD59, especially the ratio DLL1 / CD59; iii. DLL1 and LEFTY2, especially the ratio DLL1 / LEFTY2; iv. DLL1 and UNC5B, especially the ratio DLL1 / UNCB5; v. DLL1 and C5, especially the ratio DLL1 / C5; vi. DLL1 and C5.C6, especially the ratio DLL1 / C5.C6; vii. DLL1 and INHBB, especially the ratio DLL1 / INHBB; viii. TSTD1 and STAT3, especially the ratio TSTD1 / STAT3; ix. POLD4 and PARP11, especially the ratio POLD4 / PARP11; x. ASH2L and PARP11, especially the ratio ASH2L / PARP11; xi. RPS3 and PARP11, especially the ratio RPS3 / PARP11; xii. VAV3 and SIRT3, especially the ratio VAV3 / SIRT3; and xiii. SERPINB8 and PARP11; especially the ratio SERPINB8 / PARP11.

[0316] In some cases, the kit is designed to allow the determination of the ratio between the levels of any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, or all thirteen protein pairs listed in the preceding paragraph in a sample.

[0317] In some cases, each of one or more specific binding members in the kits described herein independently comprises an antibody or its antigen-binding fragment, aptamer, or peptide-binding component. The specific binding member is labeled with a detectable portion, such as an optically detectable portion.

[0318] In some cases, the kits disclosed herein are used in the methods disclosed herein, namely, to identify whether a subject is likely to respond positively to plasma exchange therapy for the treatment of cognitive impairment in the subject.

[0319] In addition to the components described above, the kit may further include instructions for use in practicing the methods described herein. These instructions may be present in the kit in various forms, including one or more. One form of these instructions may be as information printed on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, in the kit packaging, in a packaging insert, etc. Another form would be computer-readable media, such as a disk, CD, portable flash drive, etc., on which the information is recorded. Yet another possible form is a website address that can be used via the Internet to access information at a remote site. In some cases, the kit may provide information about a smartphone application, which will then provide the relevant information. Any other convenient means of communication may be present in the kit.

[0320] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0321] Those skilled in the art will understand that, generally, the terms used herein, especially those used in the appended claims (e.g., the body of the appended claims), are intended to be “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” the term “comprising” should be interpreted as “including but not limited to,” etc.). Those skilled in the art will further understand that if the intent is a specific number of introduced claim statements, such intent will be explicitly stated in the claims, and without such a statement, such intent does not exist. For example, to aid understanding, the appended claims below may contain the use of the introductory phrases “at least one” and “one or more” to introduce claim statements. However, the use of such phrases should not be construed as implying that introducing a claim statement with the indefinite article “a (a)” or “an (an)” limits any particular claim containing such an introduced claim statement to an implementation containing only one such statement, even when the same claim includes the introductory phrase “one or more” or “at least one” and an indefinite article (such as “a (a)” or “an (an)” (e.g., “a (a)” and / or “an (an)” should be interpreted as meaning “at least one” or “one or more”); this also applies to the use of definite articles used to introduce a claim statement. Furthermore, even if a specific number of introduced claim statements are explicitly stated, those skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (e.g., the basic statement “two statements” without other modifiers means at least two statements, or two or more statements). Moreover, in those cases where conventions such as “at least one of A, B, and C” are used, such a construction is generally intended to… This is in the sense that a person skilled in the art would understand the agreement to (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where an agreement similar to "at least one of A, B, or C, etc." is used, generally, such a construction is intended to be in the sense that a person skilled in the art would understand the agreement to (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). A person skilled in the art will further understand that virtually any transitional words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to imply the possibility of including one, any, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B".

[0322] Furthermore, when features or aspects of the invention are described in the Markush group, those skilled in the art will recognize that the invention is therefore also described in the name of any single member or subgroup of the Markush group.

[0323] As those skilled in the art will understand, for any and all purposes, such as in providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive and such that the scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As those skilled in the art will also understand, all language such as “up to,” “at least,” “greater than,” “less than,” etc., includes the listed numbers and refers to a scope that can subsequently be decomposed into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 items means a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0324] VI. Examples

[0325] This embodiment describes an exemplary method for identifying protein pairs to predict a subject's response to plasma exchange (PE) therapy for the treatment of cognitive impairment. First, treatment benefit is confirmed by group comparisons of altered clinical outcomes between the treatment group and the placebo group, based on appropriate statistical tests such as t-tests, ANOVA, MMRM, linear mixed models, chi-square tests, Fisher's exact test, logistic regression, etc.

[0326] In this embodiment, the Clinical Dementia Rating Scale Total Score (CDR-sb) was used for clinical outcomes. This is a holistic assessment of AD patients and a key secondary endpoint in the AMBAR clinical trial. Six categories were assessed: memory, orientation, judgment and problem-solving, community affairs, family and hobbies, and personal care. A higher CDR-sb indicates more severe dementia.

[0327] A significant improvement in EOS score of 1.1 points was observed in the treatment group after PE treatment. p = 0.002). Similar improvement was observed in moderate AD (1.5 points). p = 0.01), where both the PE group and the control group worsened, but PE treatment reduced the worsening (Boada et al., (2020)). Alzheimer's & Dementia , 16: 10, pp. 1412-1425. (2020)).

[0328] Based on CDR-sb, predictive protein pairs were identified for predicting responsiveness to PE-Alb treatment in terms of clinical improvement. Spearman correlation analysis was used to identify the ratios of plasma levels of protein pairs associated with changes in CDR-sb at EOS. Such analyses indicate the strength of the ratios of protein levels associated with continuous clinical changes.

[0329] The report included Spearman correlation coefficients or Spearman rho and related data. p Values. Positive rho indicates a positive correlation, while negative rho indicates a negative correlation.

[0330] An absolute value of rho (abs(rho)) > 0.3 is considered a true correlation; rho of 0.3-0.5 is considered a low correlation; rho of 0.5-0.7 is considered a moderate correlation; rho of 0.7-0.9 is considered a high correlation; and rho > 0.9 is considered an extremely high correlation. (See Mukaka (2012)) MalawiMed J. , 24(3): 69-71).

[0331] Based on the concept of diagnostic testing, the CDR-sb changes at EOS were transformed into two binary variables compared to baseline: better than baseline or not better than baseline (including equal to baseline and worse than baseline), and not worse than clinically meaningful changes (CDR.sb increases by 1 point). ROC was generated, and the AUC of the diagnostic / predictive ability of candidate protein pairs was calculated and compared.

[0332] The high or low baseline ratios for different protein pairs were then defined based on the optimal decision threshold (reference value) of the Youden index. The binary ratios (higher or lower than the reference ratio) were then assessed based on their predictive power as measured by accuracy, sensitivity, and specificity. The estimated mean change in CDR-sb for each new group (high biomarker group and low biomarker group) was then calculated using a linear mixture model adjusted for age, sex, and baseline CDR-sb scores.

[0333] These identified candidate biomarkers were validated through computer simulations, including testing in other clinical outcomes (ADAS_COg from the AMBAR study) and in randomly reselected samples (1001 resampled datasets (80% of the original sample size)).

[0334] The final list of candidate protein pairs based on the above results is... Figure 2 The flowchart shown is prepared.

[0335] Materials and methods

[0336] Research Design

[0337] This study was conducted on serum samples from patients participating in the AMBAR study (EudraCT#: 2011-001598-25; ClinicalTrials.govID: NCT01561053), which recruited 322 individuals diagnosed with mild to moderate Alzheimer's disease (Mini Mental State Examination [MMSE] scores ranging from 18 to 26) [Boada M et al. (2019) Alzheimers Dement 26, 5: 61-69]. The patient received a 14-month treatment plan (treatment procedure) of plasma exchange combined with albumin replacement (PE-Alb) for AD.

[0338] Treatment group

[0339] In the AMBAR trial, patients were randomly assigned in a 1:1:1:1 ratio to one of three PE-Alb treatment groups or the control group (sham PE). Figure 1 The control (placebo) group received simulated PE treatment via a non-invasive procedure (sham) that simulated PE but without any actual fluid replacement.

[0340] The intervention regimen lasted 14 months, including the first baseline visit, a 6-week intensive phase 1 treatment, and routine therapeutic plasma exchange (TPE) with albumin replacement (5% Albutein®, Grifols) once a week in all active groups, followed by an intermediate visit and a 12-month maintenance phase 2 treatment with monthly low-volume plasma exchange (LVPE) using albumin replacement (20% Albutein®, Grifols) and with or without IVIG (Flebogamma® 5% DIF, Grifols) depending on the three PE-Alb treatment modalities: - (LA) Low-dose albumin (20g albumin per PE procedure). - Alternating infusions of low-dose albumin (LAF) (20g albumin per PE regimen) and IVIG (F) (10g IVIG per PE regimen); and - Alternate infusions of high-dose albumin (HAF) (40 g albumin per PE procedure) with IVIG (F) (20 g IVIG per PE procedure).

[0341] The study concluded with a final follow-up visit at month 14. Summary of the treatment period and treatment group in [the study / group / etc.] is as follows: Figure 1 middle.

[0342] Each TPE removes and replaces approximately one plasma volume, depending on the patient's sex, height, weight, and hematocrit (approximately 35 to 45 mL / kg, corresponding to a volume of approximately 2500 to 3000 mL). This volume is calculated automatically by the device or manually by the operator depending on the device used. During the procedure, the removed plasma volume is replaced with the same volume of 5% albumin (50 g / L, approximately 125 to 150 g albumin). This procedure is a routine plasma exchange, and each site performs TPE using its standard plasma separation device.

[0343] Therapeutic plasma exchange is performed using a commercially available continuous flow cell separator employing centrifugation or filtration-based techniques. Depending on the patient's individual characteristics, a peripheral access route (e.g., radial / elbow vein) or a central access route (e.g., subclavian / jugular vein) is used.

[0344] During each plasma ablation procedure during LVPE, the plasma volume removed ranges from 650 mL to 880 mL (depending on patient weight). Following LVPE, randomization is performed on the treatment arm (LA or HA), and a 20% albumin infusion is administered, approximately 20 g to 40 g of albumin. Additionally, the HAF treatment group and one LAF group receive IVIG (F) infusions as directed above.

[0345] For analytical purposes, two comparison groups were considered: control / placebo patients and patients receiving combined PE-Alb treatment (three treatment groups).

[0346] sampling

[0347] The AMBAR study recruited patients (and thus collected serum samples) between 2012 and 2017.

[0348] To perform biomarker analysis, serum samples were considered at three time points: PRE-TPE 1 (week 1); PRE-LVPE 1 (month 3); and the final visit (month 14).

[0349] Biomarker assay

[0350] The 7K SomaScan™ panel (SomaLogic, Boulder, Colorado, USA) utilizes aptamer-based proteomics technology for the determination of protein levels in serum. This technology uses modified DNA aptamers called SOMAmers to specifically bind to proteins in a sample. This binding is then quantified using a DNA microarray, enabling the simultaneous measurement of thousands of proteins with high precision and sensitivity. Therefore, the SomaScan™ assay provides the levels of various proteins in a sample as “relative fluorescence units” (RFUs) or derivatives of RFUs (such as log2RFUs).

[0351] Clinical assessment

[0352] The following clinical and neuropsychological measurements were performed: the Clinical Dementia Rating Scale-Total Score (CDR-sb) and the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) as cognitive scales.

[0353] The Clinical Dementia Rating Scale (CDR) is a numerical scale used to quantify the severity of dementia symptoms, producing an overall score and a total score (SOB). The CDR is obtained through semi-structured interviews with patients and informed parties. Cognitive function is scored across six functional domains: memory, orientation, judgment and problem-solving, community affairs, family and hobbies, and personal care. Each domain is scored on a 5-point scale: 0 for no impairment; 0.5 for suspected impairment; 1 for mild impairment; 2 for moderate impairment; and 3 for severe impairment (personal care is scored on a 4-point scale, with no 0.5 score available). The CDR has demonstrated good reliability and has been validated against neuropathological findings. Scoring was performed according to the methodology established in Hughes CP et al., “A new clinical scale for the staging of dementia,” Br J Psychiatry, 1982, 140:566-572.

[0354] The Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog; Rosen WG et al., “A new ratingscale for Alzheimer's disease”, Am. J. Psychiatry, 1984;141:1356-1364) is a short neuropsychological assessment used to evaluate the severity of cognitive symptoms of dementia, and it is considered the “gold standard” for evaluating antidementia treatment. Importantly, the assessors / rating subjects in the trial have no access to any information that would allow them to assign a patient to treatment. When the assessor signs a certification document, it confirms the assessor's blinding of the patient's treatment.

[0355] Data analysis and statistics

[0356] First, treatment benefit is confirmed by group comparison of altered clinical outcomes between the treatment group and the placebo group, based on appropriate statistical tests (such as t-test, ANOVA, MMRM, linear mixed model, chi-square test, Fisher's exact test, logistic regression, etc.).

[0357] A significant improvement in EOS was observed in the treatment group after PE treatment, with a decrease of 1.1 points. p = 0.002) (Boada et al., "A randomized, controlled clinical trial of plasma exchange with albuminreplacement for Alzheimer's disease: Primary results of the AMBAR Study", Alzheimer's & Dementia, 2020, 16:10, pp. 1412-1425).

[0358] Based on clinical improvement in CDR.sb, baseline predictive biomarker ratios for response to PE-Alb treatment were determined. Spearman correlation analysis was used to determine baseline plasma protein ratios associated with changes in CDR.sb at EOS. This analysis indicated the strength of proteomic biomarker ratios associated with continuous clinical changes.

[0359] The Spearman correlation coefficient, or Spearman rho, and the associated p-values ​​are reported.

[0360] Positive rho indicates a positive correlation, while negative rho indicates a negative correlation.

[0361] An absolute value of rho (abs(rho)) > 0.3 is considered a true correlation, rho of 0.3-0.5 is considered a low correlation, rho of 0.5-0.7 is considered a moderate correlation, rho of 0.7-0.9 is considered a high correlation, and rho > 0.9 is considered an extremely high correlation. (Mukaka (2012)) MalawiMed J. , 24(3): 69-71).

[0362] Based on the concept of diagnostic testing, the CDR.sb changes at EOS were transformed into two binary variables and compared to baseline: better than baseline or worse than baseline, and no worse than clinically meaningful changes (CDR.sb increased by 1 point). ROC was generated and AUC was calculated, and the diagnostic / predictive power of the ratios of candidate proteomic biomarkers was compared.

[0363] The ratio of high or low baseline protein levels was then defined using an optimal decision threshold (reference) based on the Youden index. The binary biomarker ratio (higher or lower than the reference) was then assessed based on its predictive power as measured by accuracy, sensitivity, and specificity. The estimated mean change in CDR.sb for each new group (high biomarker group and low biomarker group) was then calculated using a linear mixture model adjusted for age, sex, and baseline CDR.sb scores.

[0364] These identified candidate biomarkers were validated through computer simulations, including testing in other clinical outcomes (ADAS_Cog from the AMBAR study) and in randomly reselected samples (1001 resampled datasets (80% of the original sample size)).

[0365] All statistical analyses were performed using R version 4.1 (https: / / cran.r-project.org / ).

[0366] result

[0367] The final list of identified candidate protein pairs is shown in Table 1. This table displays the Spearman correlation index and the p-value associated with each protein pair. It can be observed that the Spearman correlation index ranges from -0.6452961 for the DLL1 / SMOC1 protein pair to -0.5536106 for the SERPINB8 / PARP11 protein pair. These correlations can be further expressed in... Figure 3A and Figure 15A The results are observed graphically. All evaluated protein pairs showed very high statistical significance.

[0368] When assessing the predictive power parameters of therapeutic benefit (CDR-sb superior to baseline at EOS) for the identified candidate protein pairs, all protein pairs demonstrated very good performance, with AUC values ​​ranging from 86.9% for the POLD4 / PARP11 protein pair to 79.8% for the VAV3 / SIRT3 protein pair (Table 2). These ROC curves can be plotted separately on... Figure 11B and Figure 14B It is observed graphically.

[0369] Another way to assess treatment effectiveness, especially important in neurodegenerative diseases, is not only to assess whether patients show improvement at EOS, but also whether these patients do not worsen at the end of treatment. This has been assessed by calculating whether the increase in CDR-sb at the end of treatment is less than 1 point compared to baseline (Table 3). In this table, the predictive power values ​​of the identified candidate protein pairs are again shown, and the DLL1 / SMOC1 protein pair achieved the best AUC value (83.3%). The ROC curve can be obtained from... Figure 3C It is observed graphically.

[0370] Computer simulation validation of candidate protein pairs was evaluated in another clinical outcome: ADAS_Cog. Table 4 shows the Spearman correlation index, p-values ​​associated with each protein pair, and predictive power parameters. Rho values ​​ranged from -0.488 for the DLL1 / C5.C6 protein pair to -0.343 for the POLD4 / PARP11 protein pair. Again, all evaluated protein pairs showed very high statistical significance. Regarding the predictive power of treatment benefit, the DLL1 / SMOC1 protein pair achieved the best AUC value for ADAS_Cog improvement at EOS (AUC = 76.0%), while the DLL1 / C5.C6 protein pair achieved the best AUC-CMC (the value for patient non-worsening at EOS) (AUC = 71.3%).

[0371] Table 6 shows the ratios of different protein pairs in serum, which indicate whether subjects may or may not respond positively (better than baseline) to plasma therapy for the treatment of cognitive impairment (such as the plasma therapy described previously).

[0372] The results provided in Table 1-6 above are also supported by the graph in Figure 3-15.

[0373] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity, it will be readily apparent to those skilled in the art, based on the teachings of the present invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0374] Therefore, the foregoing has only described the principles of the invention. It will be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown herein, that implement the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language cited herein are primarily intended to help the reader understand the principles of the invention and the concepts contributed by the inventors to advance the technology, and should be interpreted as not being limited to these specifically cited examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover both their structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and future development equivalents, i.e., any element developed to perform the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such invention is explicitly stated in the claims.

[0375] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, a limitation is expressly defined as a reference to 35 USC § 112(f) or 35 USC § 112(6) only if the limitation in the claim begins with the exact phrase “means for…” or the exact phrase “steps for…”; if the limitation in the claim does not use such an exact phrase, then 35 USC § 112(f) or 35 USC § 112(6) is not referenced.

[0376] Terms and Conditions

[0377] For the sake of completeness, various aspects of the invention are set forth in the following numbered clauses: Clause 1. A method for analyzing samples obtained from a subject, the method comprising: determining the ratio between the levels of any two proteins in the isolated sample, the two proteins being selected from: Delta-like protein 1 (DLL1), SPARC-associated modular calcium-binding protein 1 (SMOC1), CD59 glycoprotein (CD59), thiosulfate:glutathione S-transferase (TSTD1), signal transduction and transcription activator 3 (STAT3), DNA polymerase δ subunit 4 (POLD4), protein mono-ADP-ribosyltransferase PARP11 (PAR). P11), Left-Right Determinant 2 (LEFTY2), Netrin receptor UNC5B (UNC5B), Complement C5 (C5), Complement C5b-C6 complex (C5.C6), Set1 / Ash2 histone methyltransferase complex subunit ASH2 (ASH2L), Inhibin βB chain (INHBB), Small ribosomal subunit protein uS3 (RPS3), Guanine nucleotide exchange factor VAV3 (VAV3), NAD-dependent protein deacetylase sirtuin-3, mitochondrial type (USIRT3), and Serpin B8 (SERPINB8).

[0378] Clause 2. A method for predicting whether a subject will respond positively to a cognitive impairment therapy, the method comprising: determining, in isolated test samples, the ratio between the levels of any two proteins selected from: Delta-like protein 1 (DLL1), SPARC-associated modular calcium-binding protein 1 (SMOC1), CD59 glycoprotein (CD59), thiosulfate:glutathione S-transferase (TSTD1), signal transduction and transcription activator 3 (STAT3), DNA polymerase δ subunit 4 (POLD4), and protein mono-ADP-ribosyltransferase PAR. P11 (PARP11), Left-Right Determinant 2 (LEFTY2), Netrin receptor UNC5B (UNC5B), Complement C5 (C5), Complement C5b-C6 complex (C5.C6), Set1 / Ash2 histone methyltransferase complex subunit ASH2 (ASH2L), Inhibin βB chain (INHBB), Small ribosomal subunit protein uS3 (RPS3), Guanine nucleotide exchange factor VAV3 (VAV3), NAD-dependent protein deacetylase sirtuin-3, mitochondrial type (USIRT3), and SerpinB8 (SERPINB8).

[0379] Clause 3. The method described in Clause 1 or 2, wherein one of the two proteins is DLL1 or PARP11.

[0380] Clause 4. The method according to Clause 3, wherein the other of the two proteins is selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5, C6 and INHB.

[0381] Clause 5. The method according to Clause 1 or 2, wherein the method comprises determining the ratio between levels of one or more of the following proteins in a sample: i. DLL1 and SMOC1, ii. DLL1 and CD59, iii. DLL1 and LEFTY2, iv. DLL1 and UNC5B, v. DLL1 and C5, vi. DLL1 and C5.C6, vii. DLL1 and INHB, viii. TSTD1 and STAT3, ix. POLD4 and PARP11, x. ASH2L and PARP11, xi. RPS3 and PARP11, xii. VAV3 and SIRT3, and xiii. SERPINB8 and PARP11.

[0382] Clause 6. The method according to Clause 1 or 2, the method comprising determining the ratio between the levels of DLL1 and SMOC1 in a sample.

[0383] Clause 7. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of DLL1 and CD59 in the sample.

[0384] Clause 8. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of DLL1 and LEFTY2 in the sample.

[0385] Clause 9. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of DLL1 and UNC5B in the sample.

[0386] Clause 10. The method according to Clause 1 or 2, the method comprising determining the ratio between the levels of DLL1 and C5 in a sample.

[0387] Clause 11. The method according to Clause 1 or 2, the method comprising determining the ratio between the levels of DLL1 and C5, C6 in a sample.

[0388] Clause 12. The method of Clause 1, wherein the method includes determining the ratio between the levels of DLL1 and INHB in a sample.

[0389] Clause 13. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of TSTD1 and STAT3 in a sample.

[0390] Clause 14. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of POLD4 and PARP11 in the sample.

[0391] Clause 15. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of ASH2L and PARP11 in the sample.

[0392] Clause 16. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of ROS3 and PARP11 in the sample.

[0393] Clause 17. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of VAV3 and SIRT3 in a sample.

[0394] Clause 18. The method of Clause 1 or 2, wherein the method includes determining the ratio between the levels of SERPINB8 and PARP11 in a sample.

[0395] Clause 19. The method according to any one of Clauses 2 to 18, wherein the subject has cognitive impairment.

[0396] Clause 20. The method according to any one of Clauses 2 to 18, wherein the subject is suspected of having cognitive impairment.

[0397] Clause 21. The method according to any one of the preceding clauses, wherein the cognitive impairment is caused by a neurodegenerative disease.

[0398] Clause 22. The method described in Clause 21, wherein the neurodegenerative disease is Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, or vascular dementia.

[0399] Clause 23. The method described in accordance with Clause 22, wherein the neurodegenerative disease is AD.

[0400] Clause 24. The method according to any one of the preceding clauses, wherein the subject is a candidate for plasma exchange therapy for the treatment of cognitive impairment.

[0401] Clause 25. The method according to any one of the preceding clauses, wherein the method comprises determining a protein in an immunoassay, mass spectrometry analysis or protein detection array analysis.

[0402] Clause 26. The method according to any one of Clauses 1 to 25, including the determination of proteins in aptamer-based multiplex proteomics assays.

[0403] Clause 27. The method according to any one of the preceding clauses, the method comprising comparing a ratio between measured protein levels with a reference ratio obtained from a reference sample.

[0404] Clause 28. The method according to any one of the preceding clauses, wherein the sample is a blood sample, serum sample, plasma sample or cerebrospinal fluid sample.

[0405] Clause 29. The method according to any one of the preceding clauses, the method further comprising: determining, based on the ratio between measured protein levels, whether the subject is likely to respond positively to plasma exchange therapy for treating the subject's cognitive impairment.

[0406] Clause 30. The method according to Clause 29, further comprising treating the subject’s cognitive impairment by administering plasma exchange therapy to the subject if it is determined that the subject may respond positively to plasma exchange therapy.

[0407] Clause 31. A method for treating cognitive impairment in a subject, the method comprising: administering plasma exchange therapy to the subject, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on a determination of the ratio between the levels of any two proteins selected from DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8 in a sample from the subject.

[0408] Clause 32. The method described in Clause 31, wherein one of the two proteins is DLL1.

[0409] Clause 33. The method according to Clause 32, wherein the other of the two proteins is selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5, C6 and INHB.

[0410] Clause 34. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the results of determining the ratio between levels of one or more of the following proteins in the subject's sample: xiv. DLL1 and SMOC1, xv. DLL1 and CD59, xvi. DLL1 and LEFTY2, xvii.DLL1 and UNC5B, xviii. DLL1 and C5, xix.DLL1 and C5.C6, xx.DLL1 and INHB, xxi. TSTD1 and STAT3, xxii. POLD4 and PARP11, xxiii. ASH2L and PARP11, xxiv. RPS3 and PARP11, xxv. VAV3 and SIRT3, and xxvi. SERPINB8 and PARP11.

[0411] Clause 35. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and SMOC1 in the subject's sample.

[0412] Clause 36. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and CD59 in the subject's sample.

[0413] Clause 37. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and LEFTY2 in the subject's sample.

[0414] Clause 38. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and UNC5B in the subject's sample.

[0415] Clause 39. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and C5 in the subject's sample.

[0416] Clause 40. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and C5,C6 in the subject's sample.

[0417] Clause 41. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of DLL1 and INHB in the subject's sample.

[0418] Clause 42. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of TSTD1 and STAT3 in the subject's sample.

[0419] Clause 43. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of POLD4 and PARP11 in the subject's sample.

[0420] Clause 44. The method according to Clause 31, wherein the subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of ASH2L and PARP11 in the subject's sample.

[0421] Clause 45. The method according to Clause 31, wherein a subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of RPS3 and PARP11 in the subject's sample.

[0422] Clause 46. The method according to Clause 31, wherein a subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of VAV3 and SIRT3 in the subject's sample.

[0423] Clause 47. The method according to Clause 31, wherein a subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on the result of measuring the ratio between the levels of SERPINB8 and PARP11 in the subject's sample.

[0424] Clause 48. The method according to any one of Clauses 31 to 47, wherein a subject is identified as potentially responding positively to plasma exchange therapy for the treatment of cognitive impairment based on a ratio between measured protein levels in a sample obtained from the subject that differs from a reference ratio obtained from a reference sample.

[0425] Clause 49. The method according to any one of Clauses 28 to 48, wherein plasma exchange therapy comprises a course of total plasma exchange.

[0426] Clause 50. The method according to Clause 49, wherein total plasma exchange comprises replacing substantially all of the subject's plasma with a primary albumin solution.

[0427] Clause 51. The method described in accordance with Clause 49 or 50, wherein the course of total plasma exchange comprises performing total plasma exchange once a week for 5 to 8 weeks.

[0428] Clause 52. The method according to any one of Clauses 49 to 51, wherein the course of total plasma exchange comprises performing total plasma exchange once a week for 6 weeks.

[0429] Clause 53. The method according to any one of Clauses 50 to 52, wherein the first albumin solution contains 5% albumin.

[0430] Clause 54. The method according to any one of Clauses 49 to 53, wherein plasma exchange therapy includes a course of low-volume plasma exchange using a second albumin solution following a course of total plasma exchange.

[0431] Clause 55. The method according to Clause 54, wherein low-volume plasma exchange comprises replacing 20% ​​to 40% of the subject's plasma with a second albumin solution.

[0432] Clause 56. The method described in accordance with Clause 54 or 56, wherein the course of low-volume plasma exchange comprises performing low-volume plasma exchange once a month for at least 10 months.

[0433] Clause 57. The method according to any one of Clauses 54 to 55, wherein the course of low-volume plasma exchange comprises monthly low-volume plasma exchange for 12 to 16 months.

[0434] Clause 58. The method according to any one of Clauses 54 to 57, wherein the second albumin solution contains 20% albumin.

[0435] Clause 59. A kit comprising reagents for measuring the ratio between the levels of any two proteins in a sample, the two proteins being selected from: DLL1, SMOC1, CD59, TSTD1, STAT3, POLD4, PARP11, LEFTY2, UNC5B, C5, C5.C6, ASH2L, INHBB, RPS3, VAV3, SIRT3, and SERPINB8.

[0436] Clause 60. The kit according to Clause 59, wherein one of the two proteins is DLL1.

[0437] Clause 61. The kit according to Clause 60, wherein the other of the two proteins is selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5, C6 and INHB.

[0438] Clause 62. The kit according to Clause 59, wherein the kit comprises reagents for measuring the ratio between levels of one or more of the following proteins in a sample: i. DLL1 and SMOC1, ii. DLL1 and CD59, iii. DLL1 and LEFTY2, iv. DLL1 and UNC5B, v. DLL1 and C5, vi. DLL1 and C5.C6, vii. DLL1 and INHB, viii. TSTD1 and STAT3, ix. POLD4 and PARP11, x. ASH2L and PARP11, xi. RPS3 and PARP11, xii. VAV3 and SIRT3, and xiii. SERPINB8 and PARP11.

[0439] Clause 63. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and SMOC1 in a sample.

[0440] Clause 64. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and CD59 in a sample.

[0441] Clause 65. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of TSTD1 and STAT3 in a sample.

[0442] Clause 66. The kit according to Clause 59, wherein the kit contains a reagent for measuring the ratio between the levels of POLD4 and PARP11 in a sample.

[0443] Clause 67. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and LEFTY2 in a sample.

[0444] Clause 68. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and UNC5B in a sample.

[0445] Clause 69. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and C5 in a sample.

[0446] Clause 70. The kit according to Clause 56, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and C5,C6 in a sample.

[0447] Clause 71. The kit according to Clause 59, wherein the kit contains a reagent for measuring the ratio between the levels of ASH2L and PARP11 in a sample.

[0448] Clause 72. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of DLL1 and INHB in a sample.

[0449] Clause 73. The kit according to Clause 59, wherein the kit contains reagents for measuring the ratio between the levels of RPS3 and PARP11 in a sample.

[0450] Clause 74. The kit as described in Clause 59, wherein the kit contains a reagent for measuring the ratio between the levels of VAV3 and SIRT3 in a sample.

[0451] Clause 75. The kit according to Clause 59, wherein the kit contains a reagent for measuring the ratio between the levels of SERPINB8 and PARP11 in a sample.

[0452] Clause 76. The kit according to any one of Clauses 59 to 75, wherein the reagent comprises two or more specifically binding components that specifically bind to the protein being measured.

[0453] Clause 77. The kit according to Clause 76, wherein each of the two or more specific binding components is independently an antibody or its antigen-binding fragment, aptamer or peptide-binding component.

[0454] Clause 78. The kit according to Clause 76 or 77, wherein two or more specific binding components are labeled with a detectable portion.

[0455] Clause 79. The kit according to Clause 78, wherein the detectable portion is an optically detectable portion.

[0456] Clause 80. A kit according to any one of Clauses 59 to 79, wherein the kit is suitable for performing immunoassays, Western blot analysis, mass spectrometry analysis or protein detection array analysis.

[0457] Clause 81. The kit according to any one of Clauses 59 to 80, wherein the kit is suitable for performing aptamer-based multiplex proteomics assays.

[0458] Clause 82. The kit according to any one of Clauses 59 to 81, wherein the kit is used to identify whether a subject is likely to respond positively to plasma exchange therapy for the treatment of cognitive impairment in the subject.

Claims

1. A method for predicting whether a subject with or suspected of having cognitive impairment will respond positively to a therapy, particularly plasma exchange therapy, said method comprising determining a ratio between the expression levels of two inflammation-related biomarkers in isolated test samples, wherein One of the biomarkers is selected from Delta-like protein 1 (DLL1) and protein mono-ADP-ribosyltransferase PARP11 (PARP11), and Another biomarker was selected from SPARC-associated modular calcium-binding protein 1 (SMOC1), CD59 glycoprotein (CD59), thiosulfate:glutathione S-transferase (TSTD1), signal transduction and transcription activator 3 (STAT3), DNA polymerase δ subunit 4 (POLD4), left-right determinant 2 (LEFTY2), Netrin receptor UNC5B (UNC5B), complement C5 (C5), complement C5b-C6 complex (C5.C6), Set1 / Ash2 histone methyltransferase complex subunit ASH2 (ASH2L), inhibin βB chain (INHBB), small ribosomal subunit protein uS3 (RPS3), guanine nucleotide exchange factor VAV3 (VAV3), NAD-dependent protein deacetylase sirtuin-3, mitochondrial type (USIRT3), and Serpin B8 (SERPINB8).

2. The method according to claim 1, wherein one of the markers is DLL1 or PARP11, and the other marker is selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5.C6 and INHB.

3. The method of claim 1, wherein one of the markers is DLL1 or PARP11, and the other marker is selected from ASH2L, RPS3 and SERPINB8.

4. The method according to any one of the preceding claims, the method comprising determining a ratio between the expression levels of one or more of the following biomarkers: i. DLL1 and SMOC1, ii. DLL1 and C5.C6, iii. DLL1 and CD59, iv. DLL1 and LEFTY2, v. DLL1 and UNC5B, vi. DLL1 and C5, vii. DLL1 and INHB, viii. POLD4 and PARP11, ix. ASH2L and PARP11, x. RPS3 and PARP11, and xi. SERPINB8 and PARP11; in particular, the method includes determining one or more of the following ratios: DLL1 / SMOC1, DLL1 / CD59, DLL1 / LEFTY2, DLL1 / UNC5B, DLL1 / C5, DLL1 / C5.C6, DLL1 / INHBB, POLD4 / PARP11, ASH2L / PARP11, RPS3 / PARP11 and SERPINB8 / PARP11.

5. The method according to any one of the preceding claims, wherein the cognitive impairment is caused by a neurodegenerative disease; particularly caused by a neurodegenerative disease selected from Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, and vascular dementia; more particularly, the neurodegenerative disease is AD.

6. The method according to any one of the preceding claims, wherein the expression level corresponds to a protein level or an mRNA level; particularly, it corresponds to a protein level.

7. The method according to any one of the preceding claims, wherein the expression level corresponds to the protein level, and the expression level of each of the biomarkers is determined by immunoassay, mass spectrometry or protein detection array analysis, such as by performing aptamer-based multiplex proteomics assays; particularly by performing aptamer-based multiplex proteomics assays.

8. The method according to any one of the preceding claims, the method comprising the additional step of comparing a ratio determined in the test sample with a reference ratio obtained from a reference sample.

9. The method according to any one of the preceding claims, wherein the sample is a blood sample, serum sample, plasma sample or cerebrospinal fluid (CSF) sample; particularly serum or CSF.

10. A kit comprising reagents for measuring the expression levels of two inflammation-related biomarkers, wherein, One of the biomarkers is selected from Delta-like protein 1 (DLL1) and protein mono-ADP-ribosyltransferase PARP11 (PARP11), and Another biomarker was selected from SPARC-associated modular calcium-binding protein 1 (SMOC1), CD59 glycoprotein (CD59), thiosulfate:glutathione S-transferase (TSTD1), signal transduction and transcription activator 3 (STAT3), DNA polymerase δ subunit 4 (POLD4), left-right determinant 2 (LEFTY2), Netrin receptor UNC5B (UNC5B), complement C5 (C5), complement C5b-C6 complex (C5.C6), Set1 / Ash2 histone methyltransferase complex subunit ASH2 (ASH2L), inhibin βB chain (INHBB), small ribosomal subunit protein uS3 (RPS3), guanine nucleotide exchange factor VAV3 (VAV3), NAD-dependent protein deacetylase sirtuin-3, mitochondrial type (USIRT3), and Serpin B8 (SERPINB8); and Optionally, instructions for use in determining the ratio between the two markers.

11. The kit according to claim 10, wherein one of the two markers is DLL1 or PARP11, and the other marker is selected from SMOC1, CD59, LEFTY2, UNC5B, C5, C5.C6 and INHB.

12. The kit according to claim 10, wherein one of the markers is DLL1 or PARP11, and the other marker is selected from ASH2L, RPS3 and SERPINB8.

13. The kit according to any one of claims 10 to 12, wherein the kit comprises reagents and instructions for use for determining the ratio between the expression levels of one or more of the following biomarkers: xiv. DLL1 and SMOC1, xv. DLL1 and CD59, xvi. DLL1 and LEFTY2, xvii.DLL1 and UNC5B, xviii. DLL1 and C5, xix.DLL1 and C5.C6, xx.DLL1 and INHB, xxi. POLD4 and PARP11, xxii. ASH2L and PARP11, xxiii. RPS3 and PARP11, and VII. SERPINB8 and PARP11; in particular, the method includes determining one or more of the following ratios: DLL1:SMOC1, DLL1:CD59, DLL1:LEFTY2, DLL1:UNC5B, DLL1:C5, DLL1:C5.C6, DLL1:INHBB, POLD4:PARP11, ASH2L:PARP11, RPS3:PARP11 and SERPINB8:PARP11.

14. The kit according to any one of claims 10 to 13, wherein the reagent comprises two or more specific binding components that specifically bind to the marker; particularly wherein each of the two or more specific binding components is independently an antibody or its antigen-binding fragment, aptamer, or peptide-binding component; particularly wherein the two or more specific binding components are labeled with a detectable portion, such as an optically detectable portion.

15. Use of the kit according to any one of claims 10 to 14 in the method defined in any one of claims 1 to 9.

Images