Stabilized circulating peptides and uses thereof

Abstract

The present invention provides a method for identifying a subject having a stabilized circulating peptide. The method comprises detecting the stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from the subject. The method may further comprise treating a neurodegenerative disease, or monitoring or adjusting a treatment of a neurodegenerative disease. Also provided is a kit. The kit comprises a binding protein that specifically binds a stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from a subject. The kit may further comprise an agent for detecting, treating or monitoring a neurodegenerative disease in the subject. The neurodegenerative disease may be Alzheimer disease.

Description

[0001] 2103739-000019 20250902

[0002] STABILIZED CIRCULATING PEPTIDES AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application No. 63 / 690,409, filed September 4, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0005] REFERENCE TO SEQUENCE LISTING

[0006] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2103739- 000019_SequenceListing.xml, created August 28, 2025, which is 24 KB in size. The information in XML file format of the Sequence Listing is incorporated herein by reference in its entirety.

[0007] FIELD OF THE INVENTION

[0008] The invention relates to stabilized circulating peptides useful for diagnosing, treating or monitoring neurodegenerative diseases, for example, Alzheimer's disease.

[0009] BACKGROUND OF THE INVENTION

[0010] Worldwide, at least 50 million people are believed to be living with Alzheimer's disease (AD) which is the most common type of neurodegenerative disease characterized by the deposition of extracellular amyloid-p (A3) plaques and Tau protein neurofibrillary tangles. A0 has several proposed physiological functions, including roles in synaptic function, neuronal growth and survival, and protection against oxidative stress. The most commonly studied A0 peptides in Alzheimer's are A04O and A042. A042 is particularly important as it is highly hydrophobic and prone to aggregation, forming the core of amyloid plaques. In Alzheimer's, there's an imbalance in A0 production and clearance, leading to accumulation and aggregation into plaques and its neurotoxic effects contribute to the disease's pathology.

[0011] Tau is a protein that stabilizes microtubules in neurons and is expressed predominantly in the central nervous system (CNS) by neurons and glial cells. Under physiological conditions, tau phosphorylation regulates its association with microtubules. In Alzheimer's, tau becomes abnormally phosphorylated and forms aggregates called neurofibrillary tangles. Specific phosphorylation sites on tau, like p-taul81 and p-tau217, are used as biomarkers because they are elevated in the cerebrospinal fluid and blood of Alzheimer's patients. 2103739-000019 20250902

[0012] Amyloid-0 (A0) is a protein fragment derived from a larger protein called "amyloid precursor protein" (APP). APP is an integral membrane protein and is expressed in many tissues, but can be found in high concentrations in neurons, in the area of the synapse. APP has a large extracellular domain, as well as a trans-membrane domain. Also associated with APP are the secretases, which are enzymes that cut proteins at very specific points. APP can be cut by 3 different secretases: alpha, beta and gamma. Each secretase cuts at specific sites along the transmembrane and extracellular domains of APP. The order in which these secretase cuts are key. Depending on which secretases cut the APP sequence first, one can get fragments of varying sizes. A0, which is the major component of the plaques found in Alzheimer's pathology, is one of several "species" of amyloid. In the "amyloidogenic" pathway, the sequence of cuts is beta secretase then gamma secretase and amyloid beta (ranging in size from ~36 to 42 amino acids) is generated in the extracellular space. These amyloid beta monomers are soluble and can aggregate into oligomers, fibrils and eventually plaques. In particular, A042 is thought to be more toxic to neurons than other forms and is also preferentially found in plaques. On the other hand, in the "non-amyloidogenic”, the sequence of cuts is alpha secretase, then gamma secretase. These cuts do not produce amyloid beta and lead to less plaque formation. One potential molecular therapy in AD is to reduce production of A042 by activating alpha secretase and shifting the equilibrium towards the non-amyloidogenic pathway.

[0013] Less frequently used AD biomarkers include both glial fibrillary acidic protein (GFAP) and neurofilament light polypeptide (NfL). NfL is a protein that provides structural support to neurons. When neurons are damaged, NfL is released into the CSF and blood, making it a valuable biomarker for neurodegeneration. GFAP is a protein primarily found in astrocytes, which are a type of glial cell that provides support for neurons. Elevated levels of GFAP in body fluids indicate astrocyte activation and inflammation, a common feature of various neurodegenerative diseases, including Alzheimer's disease.

[0014] Because CSF is in contact with the extracellular space of the brain, biochemical changes in the brain are reflected in the CSF, making it a good source for AD biomarkers. Screening methods involving analysis of cerebrospinal fluid (CSF) or brain imaging via positron emission tomography (PET) have been used to check for toxic protein buildup in the brain. A marked decrease in CSF A042 (due to aggregated A042 staying in the brain) and an increase in both total tau (t-tau) and phosphorylated tau (p-tau) in CSF have been used to identify symptomatic AD patients with a sensitivity and specificity of detection above 80%. However, these methods are limited by high cost, invasiveness, and insufficient accessibility which impede their use as first-line diagnostic strategies. Efforts have been 2103739-000019 20250902 going on in discovering reliable plasma / serum biomarkers which are the gold standard in clinics. A biomarker based on phosphorylated tau at 217 (pTau217) / A(342 plasma ratio has been approved by FDA. The use of plasma ratio of two proteins rather than levels of a single protein highlights the difficulties in measuring very low-level concentrations of single AD biomarkers. Consequently, ultrasensitive and costly ELISA platforms such as Quanterix's SIMOA or Fujirebio's Lumipulse are required to detect them which preclude their widespread use. It should also be pointed out that plasma ratio of pTau217 / A042 ratio instead of serum ratio is used because the levels of pTau217 in the serum are even lower and much harder to quantify. While both plasma and serum are used as sample types in various assays, using plasma instead of serum can present some disadvantages. These include potential interference from anticoagulants, the need for specialized collection and storage, and the possibility of clotting-induced interferences. Additionally, plasma samples may be less stable than serum samples due to the presence of active cellular components. Discovery of new serum-based AD biomarkers that are present in much higher concentrations and can be measured using conventional and inexpensive methods is urgently needed. Also needed are new serum biomarkers for early detection of AD.

[0015] One reason for the extremely low levels of AD blood biomarkers is because only a fraction of brain proteins is released into the bloodstream and once there, they are severely diluted by high concentration of plasma proteins, such as albumin and IgG. In addition to the dilution, brain proteins released into blood are also degraded by proteases, metabolized in the liver, or cleared by the kidneys.

[0016] When dealing with disease biomarkers, whole protein or specific isoforms are frequently used. While the full proteins, like APP and tau, are the precursors, their processing into specific peptide fragments and the subsequent modifications of those fragments are the crucial events in Alzheimer's pathology. Changes in specific peptide levels, like decreased CSF A|342 or increased p-tau, correlate strongly with the amyloid plaques and neurofibrillary tangle pathology characteristic of Alzheimer's disease. Specific peptides, rather than the whole protein, offer several advantages. For example, targeting specific peptide fragments or modified forms (like phosphorylated tau) can provide greater specificity for detecting disease-related changes. Specific peptides may also be present at higher concentrations in the blood during the early stages of the disease, enabling earlier detection of Alzheimer's disease. Biomarkers for Alzheimer's focus on these disease-specific peptide sequences will also aid in diagnosis and disease monitoring. In drug development, peptide biomarkers are important for developing and evaluating new therapies for AD. 2103739-000019 20250902

[0017] There remains a need for new discoveries of specific peptide-based early-stage AD biomarkers that are present in much higher concentrations in serum samples of AD patients that can be measured using conventional methods such as ELISA or lateral flow immunoassay (LFIA) that can be used in point-of-care or in-home settings.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention relates to methods or kits for identifying a subject having certain stabilized circulating peptides and optionally treating a neurodegenerative disease such as Alzheimer's disease in the subject.

[0020] The present invention provides a method for identifying a subject having a stabilized circulating peptide. The method comprises detecting a stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from a subject. The stabilized circulating peptide is a fragment of a protein. The protein may be involved in one or more aspects of Alzheimer's disease development. The protein may be selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin- interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein. The presence of the stabilized circulating peptide in the body fluid sample indicates that the subject has the stabilized circulating peptide.

[0021] The method may further comprise exposing the body fluid sample to a binding protein. The binding protein may specifically bind the peptide. The binding protein may be an antibody against the stabilized circulating peptide. The antibody may be a monoclonal antibody, polyclonal antibody, or recombinant antibody.

[0022] The method may further comprise quantifying the stabilized circulating peptide in the body fluid sample.

[0023] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), 2103739-000019 20250902

[0024] DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0025] The stabilized circulating peptide may comprise an amino acid sequence GSTQLLSERNPSKNIR (SEQ ID: NO: 1).

[0026] The stabilized circulating peptide may comprise an amino acid sequence DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2).

[0027] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4).

[0028] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7).

[0029] The stabilized circulating peptide may comprise an amino acid sequence KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8).

[0030] The stabilized circulating peptide may comprise an amino acid sequence NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9).

[0031] The stabilized circulating peptide may comprise the amino acid sequence of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11).

[0032] The stabilized circulating peptide may comprise the amino acid sequence of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13).

[0033] The stabilized circulating peptide may comprise an amino acid sequence GITLDLVSRLVYWA (SEQ ID NO: 14).

[0034] The stabilized circulating peptide may comprise an amino acid sequence MTVTDQVNCPK (SEQ ID NO: 15). 2103739-000019 20250902

[0035] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18).

[0036] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20).

[0037] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22).

[0038] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), and EQLTATKTEQNIK (SEQ ID NO: 26).

[0039] The stabilized circulating peptide may comprise an amino acid sequence LALDIEIATYR (SEQ ID NO: 27).

[0040] The subject may have suffered from a neurodegenerative disease. The neurodegenerative disease may be Alzheimer's disease

[0041] The subject may show early-stage or mild symptoms of Alzheimer's disease. The subject may show a middle-stage symptom of Alzheimer's disease. The subject may show a late-stage or severe symptom of Alzheimer's disease.

[0042] The method may further comprise treating or monitoring Alzheimer's disease in the identified subject.

[0043] Where the identified subject has received a treatment of Alzheimer's disease, the method may further comprise adjusting the treatment.

[0044] A kit is also provided. The kit comprises a binding protein and optionally an agent for detecting, treating or monitoring Alzheimer's disease in a subject having a stabilized circulating peptide. The binding protein specifically binds the stabilized circulating peptide in a body fluid sample from the subject. The stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, 2103739-000019 20250902

[0045] S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein. The binding protein may be an antibody.

[0046] The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0047] A biomarker panel is further provided. The biomarker panel comprises a plurality of biomarkers. Each biomarker comprises one, two or more stabilized circulating peptides. Each stabilized circulating peptide is isolated from a body fluid sample from a subject. Each stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein- Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1- related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Fig. 1 shows the cerebrospinal fluid (CSF) protein complex profiles from three different neurodegenerative patients.

[0050] Fig. 2 shows the 102 separated protein complex spots from CSF of a neurodegenerative patient. 2103739-000019 20250902

[0051] Fig. 3 shows the expression of S100A7 in early-stage or mild AD patients (left panel) and advance Stage or severe AD patients (right panel).

[0052] Fig. 4 shows the expression of insulin degrading enzyme (IDE) in early-stage or mild AD patients (left panel) and advance stage or severe AD patients (right panel).

[0053] Fig. 5 shows the expression of Annexin Al in early-stage or mild AD patients (left panel) and advance Stage or severe AD patients (right panel).

[0054] Fig. 6 shows the expression of Synapsin-1 in early-stage or mild AD patients (left panel) and advance stage or severe AD patients (right panel). Left panel, Control (N = 51), AD Mild (N = 34); For the right panel, Control (N = 51), AD Severe (N = 34).

[0055] Fig. 7 shows a standard curve for detection of synapsin-1 with sensitivity of detection at 0.3 ng / mL.

[0056] DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention relates to identifying a subject having a stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from the subject, and optionally treating or monitoring neurodegenerative disease, for example, Alzheimer's disease (AD), in the subject. The present invention is based on the inventors' surprising discovery that peptides derived from stabilized protein complexes from CSF of neurodegenerative patients are circulating in serum or plasma of the AD patients. In particular, the inventors have discovered that these newly discovered stabilized circulating peptides are AD biomarkers in serum or plasma samples of AD patients and are useful for early detection of and for monitoring progression of AD or other neurodegenerative diseases.

[0058] Once AD biomarkers are released from the brain into CSF, they are subjected to degradation by proteolytic enzymes generating peptides, and this has contributed to high variability in identifying CSF biomarkers using mass spectrometry. For example, when CSF samples from six individual patients were analyzed, only 21% of the proteins were found to be common to all individuals. The inventors took advantage of the fact that inside the cell, proteins normally interact with many other proteins to form protein complexes. Aberrant protein-protein interactions are the basis of multiple aggregation-related diseases including Alzheimer's disease. Once protein complexes are formed, they are much more stable and less subject to degradation than individual proteins because their epitopes are masked. A novel strategy to find peptide biomarkers via separation of stable CSF protein complexes into more than one hundred individual protein complexes, followed by analysis of the 2103739-000019 20250902 peptide content in each of the isolated protein complexes is proposed here. Previously, the inventors used a 2-D High performance liquid electrophoresis (2-D HPLE) method for separating protein complexes using serum samples (U.S. Patent No. 7,326,326). The CSF protein complexes have not been separated previously. The inventors have now found that 2-D HPLE is an ideal procedure for separating protein complexes into individual protein complexes in CSF. The 2-D HPLE method separates protein complexes directly on a polyvinylidene difluoride (PVDF) membrane rather than using the conventional 2-D polyacrylamide electrophoresis (2-D PAGE) or other separation matrices. Using PVDF membrane as a separation matrix is contrary to its intended purpose which is to bind proteins tightly after electrophoresis such as 2-D PAGE. By using specific mixtures of water- miscible organic solvents to weaken the binding forces, protein complexes can be separated under high voltage conditions. The separation of protein complexes on PVDF membrane is based on their isoelectric points (pl) rather than molecular weights or other biochemical properties. Specifically, five microliters of the CSF were mixed with 5 pl of caprolactone and spotted at the center of a PVDF blot membrane (7.5 cmx8 cm). The CSF protein complexes were separated in the first dimension at 3.5 kV for 5 minutes using Buffer A (U.S. Patent No. 7,326,326). The pH of the buffer was adjusted to pH 5.0 with formic acid. The direction of migration was horizontal with protein complexes moving either to the right or left depending on their isoelectric points. Upon completion of the first-dimension separation, the membrane was washed two times for several minutes each in deionized water to remove the first-dimension solvents. The second-dimension separation was carried out at 3.5 kV for 5 minutes in Buffer B (U.S. Patent No. 7,326,326). The pH of the buffer was adjusted to pH 8.0 with 0.5 M piperazine dissolved in propylene carbonate. The direction of migration was vertical with protein complexes moving either to the top or bottom depending on their isoelectric points. At the end of the second-dimension separation, the membrane was washed with deionized water and stained with the Reactive Brown dye which allowed visualization of protein complex spots within one minute (U.S. Patent No. 7,575,858). The entire 2-D HPLE process took less than one hour to accomplish.

[0059] The inventors have carried out analysis of "protein complex" profiles using CSF samples from 20 diseased neurodegenerative patients. Despite the fact that they came from different patients, we found that their CSF 2-D protein complex profiles are surprisingly similar, indicating that these protein complexes are rather stable. CSF protein complex patterns from 3 patients are shown in Fig. 1 and unlike other electrophoresis procedures (such as 2-D PAGE), the CSF protein complex spots are highly compact and extremely well separated without diffusion. Protein complexes from one of the neurodegenerative patients 2103739-000019 20250902 revealed the presence of 102 clearly separated protein complexes (see Fig. 2). The inventors chose to analyze the peptide composition of all of the separated protein complex spots. Spots on the PVDF membrane were cut out and subjected to on-membrane digestion with trypsin, and the peptides were identified by liquid chromatography with tandem spectrometry sequencing of individual peptides (LC-MS / MS). Protein identities were determined from database searches of virtual tryptic peptide databases or fragmentation spectra of tryptic peptides. Mass spectral analysis and protein database searching were performed by the Wistar Institute Protein Sequencing Facility (Philadelphia, PA). Twentyseven new AD peptide biomarkers were discovered via analysis of the peptide compositions of the separated protein complexes. The peptides came from proteins, for example, Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein.

[0060] The newly discovered stabilized circulating peptides in a body fluid sample, for example, a serum or plasma sample, from a subject may be used as biomarkers in, for example, a biomarker panel, for a neurodegenerative disease, for example, Alzheimer's disease.

[0061] The term "peptide" as used herein refers to a short chain of amino acids. The peptide may have a chain of about 5-50, 5-40, 5-30, 5-20, 10-50, 10-40, 10-30, 10-20, 20-50, 20- 40 or 20-30 amino acids.

[0062] The term "circulating peptide" as used herein refers to a peptide moving freely in the circulatory system, for example, blood, in a subject.

[0063] The term "stabilized circulating peptide" as used herein refers to a circulating peptide that remains intact in the circulatory system, for example, blood, in a subject. At least about 100%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% of the peptide sequence remains intact in the circulatory system, for example, blood, for a predetermined time, for example, about 1, 2, 3, 4, 5, 6, 7, 14, 21 or 28 days, or 1, 2, 3, 4, 5, 6 or 12 months.

[0064] The term "subject" as used herein refers to a mammal, for example, human, nonhuman primates, dogs, bears and wolverines. For example, the subject is a human. 2103739-000019 20250902

[0065] The term "body fluid" as used herein refers to any biological secretion or fluid, whether physiological or pathological, which is produced in the body of a subject. Such body fluids include, without limitation, blood, plasma, serum, tear, bronchoalveolar washing fluid, urine, nasal secretion, ear secretion, urethral secretion, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, ascites fluid, pericardial liquid, amniotic fluid, gastric juice, lymphatic fluid, interstitial fluid, saliva, sputum, liquid deposition, tears, mucus, sweat, milk, semen, vaginal secretions, fluid coming from ulcer, blisters, abscesses and other surface eruptions.

[0066] The term "serum" as used herein refers to an amber-colored, protein-rich liquid that separates out when blood coagulates.

[0067] The term "plasma" as used herein refers to the colorless fluid part of blood, lymph, or milk, in which corpuscles or fat globules are suspended.

[0068] The term "tear" as used herein refers to the liquid produced by the lacrimal glands that lubricates, protects, and nourishes the cornea and conjunctiva.

[0069] The term "neurodegenerative disease" as used herein refers to a disease or condition that gradually damages or destroys the nervous system, especially areas in the brain, of a subject. The nerve cells, for example, motor neurons, in the brain or peripheral nervous system may lose function over time and ultimately die. Exemplary neurodegenerative diseases include Alzheimer's disease and other memory disorders, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, multiple system atrophy, and progressive supranuclear palsy. For example, the neurodegenerative disease is Alzheimer's disease.

[0070] The term "early-stage or mild symptom of Alzheimer's disease as used herein refers to having difficulties coming up with the right word or name, remembering names when introduced to new people, and / or performing tasks in social or work settings; forgetting material that was just read; losing or misplacing a valuable object; and experiencing increased trouble with planning or organizing.

[0071] The term "mid-stage symptom of Alzheimer's disease" as used herein refers to being forgetful of events or personal history; feeling moody or withdrawn, especially in socially or mentally challenging situations; being unable to recall information about themselves like their address or telephone number, and the high school or college they attended; experiencing confusion about where they are or what day it is; requiring help choosing proper clothing for the season or the occasion; having trouble controlling their bladder and 2103739-000019 20250902 bowels; experiencing changes in sleep patterns, such as sleeping during the day and becoming restless at night; showing an increased tendency to wander and become lost; and demonstrating personality and behavioral changes, including suspiciousness and delusions or compulsive, repetitive behavior like hand-wringing or tissue shredding..

[0072] The term "late-stage or advanced symptom of Alzheimer's disease" as used herein refers to requiring around-the-clock assistance with daily personal care; losing awareness of recent experiences as well as of their surroundings; experience changes in physical abilities, including walking, sitting and, eventually, swallowing; have difficulty communicating; and become vulnerable to infections, especially pneumonia.

[0073] The present invention provides a method for identifying a subject having a stabilized circulating peptide. The identification method comprises detecting a stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from a subject. The stabilized circulating peptide is a fragment of a protein. The protein may be selected from a group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin- interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein. The presence of the stabilized circulating peptide in the body fluid sample indicates that the subject has the stabilized circulating peptide.

[0074] The identification method may further comprise exposing the body fluid sample to a binding protein. The binding protein specifically binds the stabilized circulating peptide. The binding protein may comprise an antigen binding fragment that specifically binds the stabilized circulating peptide. The binding protein may be an antibody against the stabilized circulating peptide. The antibody may be a monoclonal, polyclonal, or recombinant antibody.

[0075] The identification method may further comprise quantifying the stabilized circulating peptide in the body fluid sample. The stabilized circulating peptide may be quantified by techniques known in the art, for example, lateral flow immunoassay (LFIA), sandwich ELISA, competitive ELISA, and mass spectrometry.

[0076] According to the identification method of the present invention, the subject may have a body fluid concentration, for example, a serum or plasma concentration, of the stabilized circulating peptide at about 0.001-100, 0.001-10, 0.001-1, 0.001-0.1, 0.001- 0.01, 0.01-100, 0.01-10, 0.01-1, 0.01-0.1, 0.1-100, 0.1-10, or 0.1-1 ng / mL (nanogram / mL). The half-life (ti / 2) of the stabilized circulating peptide in the subject may be 2103739-000019 20250902 about 0.1-24, 0.1-12, 0.1-6, 0.1-3, 0.1-1, 0. 1-0.5, 0.5-24, 0.5-12, 0.5-6, 0.5-3, 0.5-1, 1- 24, 1-12, 1-6, or 1-3 hours. At least about 100%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% of the peptide sequence of the circulating peptide may remain intact in the serum or plasma sample for about 0.1-24, 0.1-12, 0.1-6, 0.1-3, 0.1-1, 0. 1-0.5, 0.5-24, 0.5- 12, 0.5-6, 0.5-3, 0.5-1, 1-24, 1-12, 1-6, or 1-3 hours, for about 1, 2, 3, 4, 5, 6, 7, 14, 21 or 28 days, or for about 1, 2, 3, 4, 5, 6 or 12 months when the serum or plasma sample is stored at a temperature from about -80 °C to about 40 °C, from about -80 °C to about 37 °C, from about -80 °C to about 25 °C, from about -80 °C to about 4 °C, from about 0 °C to about 40 °C, from about 0 °C to about 37 °C, from about 0 °C to about 25 °C, from about 0 °C to about 4 °C, from about 4 °C to about 40 °C, from about 4 °C to about 40 °C, from about 4 °C to about 37 °C, or from about 4 °C to about 25 °C.

[0077] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0078] According to the identification method of the present invention, the stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS 2103739-000019 20250902

[0079] (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0080] According to the identification method of the present invention, the stabilized circulating stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0081] According to the identification method of the present invention, the stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), 2103739-000019 20250902

[0082] KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0083] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may comprise GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of GSTQLLSERNPSKNIR (SEQ ID : NO: 1).

[0084] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may comprise DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2).

[0085] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may comprise DVSQELDPDLYKQ (SEQ ID : NO: 3). The stabilized circulating peptide may consist of DVSQELDPDLYKQ (SEQ ID: NO: 3). 2103739-000019 20250902

[0086] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may comprise SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).

[0087] According to the identification method of the present invention, the stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).

[0088] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7).

[0089] According to the identification method of the present invention, the stabilized circulating peptide may comprise of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating 2103739-000019 20250902 peptide may comprise KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8).

[0090] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may comprise NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9).

[0091] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11).

[0092] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 2103739-000019 20250902

[0093] 13). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13).

[0094] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may comprise GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may consist of GITLDLVSRLVYWA (SEQ ID NO: 14).

[0095] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may comprise MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of MTVTDQVNCPK (SEQ ID NO: 15).

[0096] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). 2103739-000019 20250902

[0097] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20).

[0098] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22).

[0099] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may comprise an amino acid sequence selected from the group 2103739-000019 20250902 consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26).

[0100] According to the identification method of the present invention, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may comprise LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of LALDIEIATYR (SEQ ID NO: 27).

[0101] The subject may show a symptom of a neurodegenerative disease. The subject may have suffered from a neurodegenerative disease. The subject may be predisposed to a neurodegenerative disease. The neurodegenerative disease may be Alzheimer's disease.

[0102] The subject may show early-stage or mild symptoms of Alzheimer's disease. The subject may show middle-stage symptoms of Alzheimer's disease. The subject may show a late-stage or advanced symptom of Alzheimer's disease.

[0103] The method may further comprise treating a neurodegenerative disease in the identified subject. The neurodegenerative disease may be Alzheimer's disease.

[0104] The method may further comprise monitoring a neurodegenerative disease in the identified subject. The neurodegenerative disease may be Alzheimer's disease.

[0105] Where the identified subject has received a treatment of a neurodegenerative disease, the method may further comprise adjusting the treatment. The neurodegenerative disease may be Alzheimer's disease.

[0106] The present invention also provides a kit. The kit comprises a binding protein that specifically binds a stabilized circulating peptide in a body fluid sample, for example, a serum or plasma sample, from a subject. The stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic 2103739-000019 20250902 protein. The binding protein may be an antibody. The kit may further comprise an agent for detecting, treating or monitoring a neurodegenerative disease in the subject. The neurodegenerative disease may be Alzheimer's disease. The agent may be a chemical compound, a biological molecule or a combination thereof. Examples of the agent include biomarkers such as Neurofilament Light Chain (NfL) and glial fibrillary acidic protein (GFAP); Cholinesterase inhibitors such as Donepezil (Aricept) and Rivastigmine (Exelon).

[0107] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID : NO: 1), DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0108] In the kit, the stabilized circulating peptide may consist an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID : NO: 1), DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ 2103739-000019 20250902

[0109] ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0110] In the kit, the stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID : NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0111] In the kit, the stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID : NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), 2103739-000019 20250902

[0112] YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0113] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may comprise GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of GSTQLLSERNPSKNIR (SEQ ID: NO: 1).

[0114] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may comprise DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO:

[0115] 2).

[0116] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may comprise DVSQELDPDLYKQ (SEQ ID: NO:

[0117] 3). The stabilized circulating peptide may consist of DVSQELDPDLYKQ (SEQ ID: NO: 3).

[0118] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may comprise SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO:

[0119] 4).

[0120] In the kit, the stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to 2103739-000019 20250902

[0121] SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).

[0122] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7).

[0123] In the kit, the stabilized circulating peptide may comprise of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may comprise KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8).

[0124] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may comprise NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9).

[0125] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence 2103739-000019 20250902 selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11).

[0126] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO:

[0127] 13).

[0128] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may comprise GITLDLVSRLVYWA (SEQ ID NO:

[0129] 14). The stabilized circulating peptide may consist of GITLDLVSRLVYWA (SEQ ID NO: 14).

[0130] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 2103739-000019 20250902

[0131] 15). The stabilized circulating peptide may comprise MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of MTVTDQVNCPK (SEQ ID NO: 15).

[0132] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18).

[0133] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO:

[0134] 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO:

[0135] 20). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20).

[0136] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to 2103739-000019 20250902 an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22).

[0137] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26).

[0138] In the kit, the stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may comprise LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of LALDIEIATYR (SEQ ID NO: 27).

[0139] A biomarker panel is further provided. The biomarker panel comprises a plurality of biomarkers. Each biomarker comprises one, two or more stabilized circulating peptides. Each stabilized circulating peptide is isolated from a body fluid sample, for example, a serum or plasma sample, from a subject. Each stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2, Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2, Islet cell 2103739-000019 20250902 autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein.

[0140] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0141] In the biomarker panel, each stabilized circulating peptide may consist an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRN SPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), 2103739-000019 20250902

[0142] LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0143] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

[0144] In the biomarker panel, each stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID : NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27). 2103739-000019 20250902

[0145] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may comprise GSTQLLSERNPSKNIR (SEQ ID: NO: 1). The stabilized circulating peptide may consist of GSTQLLSERNPSKNIR (SEQ ID: NO: 1).

[0146] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may comprise DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2). The stabilized circulating peptide may consist of DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2).

[0147] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may comprise DVSQELDPDLYKQ (SEQ ID: NO: 3). The stabilized circulating peptide may consist of DVSQELDPDLYKQ (SEQ ID: NO: 3).

[0148] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may comprise SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).

[0149] In the biomarker panel, each stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). The stabilized circulating peptide may consist of SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4). 2103739-000019 20250902

[0150] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7).

[0151] In the biomarker panel, each stabilized circulating peptide may comprise of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may comprise KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8). The stabilized circulating peptide may consist of KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8).

[0152] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may comprise NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9). The stabilized circulating peptide may consist of NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9).

[0153] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID 2103739-000019 20250902

[0154] NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11).

[0155] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) and SEDFGVNEDLADSDAR (SEQ ID NO: 13).

[0156] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may comprise GITLDLVSRLVYWA (SEQ ID NO: 14). The stabilized circulating peptide may consist of GITLDLVSRLVYWA (SEQ ID NO: 14).

[0157] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may comprise 2103739-000019 20250902

[0158] MTVTDQVNCPK (SEQ ID NO: 15). The stabilized circulating peptide may consist of MTVTDQVNCPK (SEQ ID NO: 15).

[0159] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18).

[0160] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20).

[0161] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may 2103739-000019 20250902 consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO:

[0162] 22). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22).

[0163] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO:

[0164] 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may comprise an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26). The stabilized circulating peptide may consist of an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25) and EQLTATKTEQNIK (SEQ ID NO: 26).

[0165] In the biomarker panel, each stabilized circulating peptide may comprise an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may comprise LALDIEIATYR (SEQ ID NO: 27). The stabilized circulating peptide may consist of LALDIEIATYR (SEQ ID NO: 27).

[0166] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ± 10%, more preferably ±5%, even more preferably ± 1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate. 2103739-000019 20250902

[0167] Example 1. Stabilized circulating peptides

[0168] Many biochemical pathways in addition to the production of amyloid-p (AP) plaques and tau protein neurofibrillary tangles are involved in the development of AD. The peptide fragments we discovered came from proteins that are associated with production and processing of APP, enzymes that degrades AP, proteins that are involved in synapse impairment pathways, immune response-related, and oxidative stress. Table 1 shows circulating peptides of these proteins and other proteins.

[0169] In the brain, amyloid precursor protein (APP) is produced in the endoplasmic reticulum (ER) and processed through the Golgi apparatus. APP is then either transported or degraded via two main pathways: the non-amyloidogenic pathway (which doesn't produce AP) and the amyloidogenic pathway (which produces AP). We have identified two protein biomarkers, amyloid beta precursor protein binding family A member 2 (APBA2) and amyloid beta precursor protein binding family B member 2 (APBB2), and their stabilized circulating peptides that interact with APP and possibly regulate the production of APP.

[0170] 1. Stabilized circulating peptides from amyloid beta precursor protein binding family A member 2

[0171] A 16-amino acid peptide fragment GSTQLLSERNPSKNIR (SEQ ID: NO: 1) from amyloid beta precursor protein binding family A member 2 (APBA2) was identified from mass spectrometric analysis of one of the CSF protein complexes. It came from amino acid residues 378 to 393 of APBA2 which is a neuronal adapter protein that interacts with the APP. It stabilizes APP and inhibits production of proteolytic APP fragments including the AP peptide that is deposited in the brains of Alzheimer's disease patients (ncbi.nlm.nih.gov., APBA2). Therefore, APBA2 may decrease the production and accumulation of amyloid-beta in the brain.

[0172] 2. Stabilized circulating peptides from amyloid beta precursor protein binding family B member 2

[0173] A 25-amino acid peptide fragment DTLAVFMASSGTTDVTN N SPATPP (SEQ ID: NO: 2) from amyloid beta precursor protein binding family B member 2 (APBB2) was identified from mass spectrometric analysis of one of the CSF protein complexes. It came from amino acid residues 12 to 36 of APBB2 which is a protein that binds to the cytoplasmic domain of APP and is implicated in Alzheimer's disease. APBB2, also known as FE65-like (FE65L1), is an adaptor protein that interacts directly with the cytoplasmic domain of the amyloid precursor protein (APP) via phosphotyrosine-binding motifs. Through these interactions, 2103739-000019 20250902

[0174] APBB2 plays a critical role in regulating APP trafficking and processing, which in turn modulates the balance between amyloidogenic and non-amyloidogenic pathways and ultimately influences 0-amyloid (A0) generation. Genetic studies have linked polymorphisms in the APBB2 gene with altered risk for Alzheimer's disease, demonstrating that variations in APBB2 can affect disease onset and amyloid deposition.

[0175] 3. Stabilized circulating peptides from Islet cell autoantigen of 69 kDa

[0176] A 13-amino acid peptide fragment DVSQELDPDLYKQ (SEQ ID: NO: 3) was identified from mass spectrometric analysis of the CSF protein complexes. It came from amino acid residues 175 to 187 of Islet cell autoantigen of 69 kDa which has been shown to influence amyloid precursor protein (APP) processing by regulating the PICKl-PKCa signaling pathway. Specifically, ICA69 increases the expression of APP, and the enzymes with a- secretase activity such as ADAM 10, and ADAM17. The protein is involved in shifting APP processing towards non-amyloidogenic pathways, thus potentially reducing A(3 aggregation.

[0177] 4. Stabilized circulating peptides from Apolipoprotein E receptor

[0178] A 14-amino acid peptide fragment GITLDLVSRLVYWA (SEQ ID NO: 14), corresponding to amino acids from 371 to 384 of Apolipoprotein E receptor was detected in our mass spectrometric analysis of CSF protein complexes of a neurodegenerative patient. Apolipoprotein E (Apo E) receptors play a crucial role in AD pathogenesis, particularly through their interaction with Apo E isoforms. Apo E4 is the strongest genetic risk factor for late-onset AD and has unique properties that influence receptor binding and subsequent effects on A0 processing, tau pathology, and synaptic function.

[0179] 5. Stabilized circulating peptides from Apolipoprotein D

[0180] A 11-amino acid peptide fragment MTVTDQVNCPK (SEQ ID NO: 15) corresponding to amino acids from 86 to 96 of Apolipoprotein D (Apo D) was detected in our mass spectrometric analysis of CSF protein complexes of a neurodegenerative patient. Studies suggest that Apo D plays a role in modulating amyloid plaque pathology in AD, potentially influencing disease progression. Elevated Apo D levels are also linked to oxidative stress and lipid metabolism, which are implicated in AD pathogenesis. Research indicates that Apo D is a component of compact A[3 plaques in the AD brain, and its levels are increased in AD cortex and vasculature.

[0181] 6. Stabilized circulating peptides from Huntingtin-interacting protein 1-related protein 2103739-000019 20250902

[0182] A 24-amino acid peptide fragment SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4) was identified from mass spectrometric analysis of one of the CSF protein complexes. It came from amino acid residues 970 to 993 of Huntingtin-interacting protein 1-related protein. Huntingtin-associated protein 1 (HAP1) plays a role in Alzheimer's disease by influencing the transport of proteins, particularly the APP which is crucial in the formation of amyloid plaques. Because HAP1 is involved in intracellular trafficking, its dysfunction can lead to disruptions in the proper delivery of proteins to their designated locations within neurons. This can affect the processing of APP, potentially contributing to the accumulation of AP peptides, a key component of amyloid plaques.

[0183] 7. Stabilized circulating peptides from Synphilin-1

[0184] A 29-amino acid peptide fragment NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) from synphilin-1 was identified from one of the protein complex spots by mass spectrometric analysis. It came from amino acid residues 382-410 of synphilin-1. Synphilin- 1 has been reported to interact with o-synuclein in both neurons and Lewy bodies to inhibit the docking of the vesicles to membranes of the presynaptic terminals. Patients with dementia with Lewy bodies (DLB) frequently exhibit AD pathology, including A[3 plaques and to some degree tau pathology. In vitro studies showed that overexpression synphilin-1 which co-localizes with a-synuclein (a-syn) inhibits proteasomal degradation of a-syn and increases its aggregation. Polyclonal antibody against the peptide sequence TEKLTPAGLAIK (SEQ ID NO: 20) was used to assess the utility of synphilin-1 as an AD serum or plasma biomarker. Synphilin-1 could be a good serum or plasma biomarker for severe AD.

[0185] 8. Stabilized circulating peptides from Nucleoredoxin isoform XI

[0186] Normal nucleoredoxin has 8 exons and this nucleoredoxin splice variant is produced by removing exon 6 and joining exons 5 and 7, eliminating 60 amino acids. Normal nucleoredoxin, a redox-sensitive enzyme, is implicated in various diseases. It plays a role in regulating cellular processes through redox protein-protein interactions and is associated with several pathologies including cancer, liver diseases, obesity, and neurodegenerative conditions. A 17-amino acid peptide sequence LYGIQDSEDDGESEAAK (SEQ ID NO: 21) was detected by mass spectrometry. The first 5 amino acids come from the end of exon 5 and the 12 come from the beginning of exon 7. Using antibodies against LYGIQDSEDDGE (SEQ ID NO: 22), we found that there is a 2-fold increase in nucleoredoxin levels in Alzheimer's patients compared to normal individuals (data not shown).

[0187] 9. Stabilized circulating peptides from Breast cancer gene 2 (BRCA2) 2103739-000019 20250902

[0188] BR.CA2 is a huge protein with 3,418 amino acids. It is unlikely that we are detecting intact protein. In our analysis, we found the following four peptide fragments from this protein: SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), and EQLTATKTEQNIK (SEQ ID NO: 26). Using antibodies against EQLTATKTEQNIK (SEQ ID NO: 26), we found that there is 2-fold increase in BRCA2 levels in Alzheimer's patients compared to normal individuals (data not shown).

[0189] 10. Stabilized circulating peptides from Glial fibrillary acidic protein

[0190] An 11-amino acid peptide fragment LALDIEIATYR (SEQ ID NO: 27) was identified from one of the protein complex spots by mass spectrometric analysis. It came from amino acid residues 146 to 156 of GFAP. GFAP levels in blood and CSF correlate with the severity of AD pathology, including amyloid plaque deposition, tau tangles, and cognitive decline. Elevated GFAP levels in the blood can be detected even in the pre-symptomatic stages of AD, potentially making it a useful biomarker for early detection and monitoring of the disease. Increased GFAP levels, particularly in blood, have been linked to faster cognitive decline and more severe white matter injury in AD.

[0191] 11. Stabilized circulating peptides from S100A7

[0192] A 33-amino acid peptide fragment KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5) was identified from mass spectrometric analysis of the CSF protein complexes. It came from amino acid residues 64 to 96 of S100A7 (which is also called psoriasin) and is a neuroprotective protein known to be involved in immune responses and promotes a- secretase activity in the brain of AD. By promoting a-secretase activity in the brain of AD, S100A7 precludes the generation of amyloidogenic peptides. Increased levels of S100A7 have been found in the cerebrospinal fluid and brain of patients with Alzheimer's disease. To our knowledge, S100A7 has not been reported in serum samples of AD patients. Here, we showed that using polyclonal antibody directed against KNEDKKIDFSEFLS (SEQ ID NO: 6), S100A7 is overexpressed in the serum or plasma samples of AD. Fig. 3 shows that more S100A7 are expressed in mild AD patients when compared to those from severe AD. This would suggest that S100A7 and the peptide fragment could be a good serum or plasma biomarker for early detection of AD.

[0193] 12. Stabilized circulating peptides from S100A15

[0194] A 33-amino acid peptide fragment KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8) was identified from mass spectrometric analysis of one of the CSF protein complexes. It came from amino acid residues 64 to 96 of S100A15 which is a 2103739-000019 20250902 neuroprotective protein known to be involved in immune responses and promotes a- secretase activity in the brain of AD. Its sequence is identical to that of S100A7 except that the amino acid residue 84 is "A" instead of "T". Therefore, polyclonal antibody directed against KNEDKKIDFSEFLS (SEQ ID NO: 6) used for S100A7 could also come from S100A15.

[0195] 13. Stabilized circulating peptides from S100A9

[0196] A 33-amino acid peptide fragment NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9) was identified from mass spectrometric analysis of one of the CSF protein complexes. It came from amino acid residues of 11 to 43 of S100A9 which has been shown to interact with A[3 peptides through co-aggregation with A(3 peptides and promote their aggregation into amyloid plaques.

[0197] 14. Stabilized circulating peptides from Insulin Degrading Enzyme as an AD biomarker

[0198] A 15 amino acid-peptide fragment DREVNAVDSEHEKNV (SEQ ID NO: 10) was discovered from the analysis of CSF protein complex spots. It came from amino acid residues 180-194 of insulin Insulin-degrading enzyme (IDE). IDE is a protease specific to p- structure-forming aggregation-prone peptides and is highly expressed in the brain. Besides insulin, IDE is able to cleave many substrates including AP, making this enzyme a candidate pathophysiological link between Alzheimer's disease (AD) and type 2 diabetes. Using polyclonal antibody against DREVNAVDSEHEK (SEQ ID NO: 11), Fig. 4 shows that IDE levels in mild AD patients are overexpressed, possibly related to IDE playing a crucial role in the clearance of Ap. An "Area Under the Curve" (AUC) number of 0.89 was found when using serum IDE levels for early detection of AD. This indicates that IDE is a good biomarker for detection of early-stage AD. Because IDE activity levels were found to be inversely correlated with brain A[3 burden, we suspect that IDE levels in mild AD patients are overexpressed possibly related to the need for IDE to degrade toxic Ap oligomers. This ability appears to be significantly decreased with the progression of AD to become severe. Since IDE plays a crucial role in the clearance of Alzheimer's amyloid-p (AP), it will be advantageous for early AD patients to overexpress IDE.

[0199] Because IDE is a large protein with 1,019 amino acids, it is unlikely to remain intact when it is released into the blood. Once IDE is fragmented by proteolytic enzymes in CSF and blood, its fragments may not be stable and detectable by commercially available antibodies against a different or an unspecified region of the protein. This is probably the reason for conflicting reports about the levels of IDE in AD patients. Our polyclonal antibodies, on the other hand, were produced against the specific stabilized peptide 2103739-000019 20250902 fragment and would therefore give a more accurate assessment of IDE levels in either serum or plasma sample.

[0200] 15. Stabilized circulating peptides from Annexin Al as a biomarker for AD

[0201] A 24-amino acid peptide fragment SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12) corresponding to amino acids from 189-213 of Annexin Al was detected in our mass spectrometric analysis of CSF protein complexes. Annexin Al is an anti-inflammatory protein, and its level has been shown to increase in the brains of AD patients at early stages. Annexin Al was reported to reduce the levels of A3 by increasing its enzymatic degradation by neprilysin (NEP) and stimulating AP phagocytosis by microglia. NEP is considered to be the most potent Ap-degrading enzyme and can degrade not only monomeric forms of AP but also its more toxic oligomers. NEP dysfunction was reported to elevate endogenous AP levels in the brain. These findings suggest that NEP may have profound effects on AD pathogenesis by promoting AP clearance. Fig. 5 shows that using antibody directed against the sequence SEDFGVNEDLADSDAR (SEQ ID NO: 13), annexin Al is a very good serum or plasma biomarker for early detection of AD with AUC number approaching 0.90.

[0202] 16. Stabilized circulating peptides from Synapsin-1

[0203] Two peptide fragments were detected from the protein complexes of CSF of a neurodegenerative patient. They are AGIPSVNSLHSVYNFCD (SEQ ID NO: 16) (from amino acid residues 206-224) and IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), (from residues 319-350). Synapsin-1 is a neuron-specific phosphoprotein and is localized to the cytoplasmic surface of synaptic vesicles (SVs). Synaptic dysfunction has been reported to be a major contributor to the symptoms of AD and positively related to the severity of dementia. A polyclonal antibody against IGQNYKAYMRTS (SEQ ID NO: 18) was used to quantify serum levels of synapsin-1 from AD patients. As shown in Fig. 6, compared to normal individuals, there is a 3-fold increase in serum synapsin-1 levels in mild AD patients with an area AUC number of 0.84 and a 5-fold increase in severe AD patients with a very high AUC number of 0.97. The correlation between Synapsin-1 levels and AD progression suggests its potential as a biomarker for early AD diagnosis and monitoring its progression.

[0204] Example 2. Production of quantitative AD LFIA kits for point-of-care and inhome use 2103739-000019 20250902

[0205] A highly sensitive "time-resolved fluorescence lateral flow immunoassay" (TRF-LFIA) to quantify the AD peptide biomarkers for both in-home use and under point-of-care settings was developed. In standard fluorometric detection, excitation and emission are simultaneous with the light emitted. By contrast, time-resolved fluorescence (TRF) relies on using specific fluorescent molecules, called lanthanide chelate labels, which have a long fluorescence lifetime and allow detection of the emitted light after excitation has occurred. The most commonly used lanthanide chelate label is the europium ion (Eu3+).

[0206] Besides the long emitting fluorescence, europium displays a large Stokes shift (290 nm) with no overlap between the excitation and emission spectrum. TRF can also eliminate the background signals caused by auto fluorescence in biological samples such as serum or tissue. Each TRF microsphere contains thousands of fluorescence molecules which greatly increased the sensitivity of detection.

[0207] Using the 32-amino acid peptide fragment from synapsin-1 (IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS) (SEQ ID NO: 17) as an example, in the synapsin-1 test kit, the antibodies against the C terminal region (GSAMLEQIAMS) with attached fluorescent microspheres are in the conjugate pad. The test (T) line on the nitrocellulose membrane is spotted with the peptide conjugated with bovine serum albumin (BSA). The control (C) line contains the goat anti-rabbit IgG. After adding diluted serum sample to the well, it will go through the conjugate pad and flow from one end to the other end. If there is peptide antigen in the sample from synapsin-1, it will bind to the antibodies in the conjugate pad and reduce the availability for interaction with synapsin-1 peptide antigen in the T line, resulting in a decreased fluorescence value. On the other hand, if the amount of the synapsin-1 peptide biomarker in the sample is low, the fluorescence value will be higher. After completion of the reaction, the test strip will be analyzed using the fluorescence reader by measuring the volumes of the test line (VT) and the control line (Vc) to calculate the VR (volume ratio). The fluorescence intensity in the test line will be inversely correlated with the levels of biomarker in the sample. The fluorescence intensity at the control line, on the other hand, will remain nearly constant for the entire test. A standard curve is generated using eight serial diluted standards by plotting the VT (y) against the peptide concentration (x). Fig. 7 shows a standard curve for detection of synapsin-1 with sensitivity of detection at about 0.3 ng / mL. Unlike most fluorescence readers which are only semi-quantitative, our fluorescence reader is quantitative because we have a built-in standard curve, and the reader gives out the actual fluorescence values.

[0208] Example 3. Use of tear fluid samples to verify AD biomarker 2103739-000019 20250902

[0209] Some of the peptide biomarkers present in the serum samples could be caused by other pathological conditions. For example, overexpression of S100A7 have been linked to numerous cancers including skin cancer, squamous cell carcinoma, head and neck squamous cell carcinoma, oral squamous cell carcinoma, bladder cancer, breast cancer, lung cancer, cervical cancer, and gastric cancer. A method to confirm that S100A7 overexpression (and other peptide biomarkers discovered in the present application) is caused by AD is provided. Because the cornea is considered to be an extension of the central nervous system (CNS), it is possible to assess AD peptide biomarkers with the use of tear samples. Tear fluid plays a vital role in maintaining ocular surface health and can be analyzed to detect various diseases. Tear fluid can be collected non-invasively using Schirmer strips, which are placed under the lower eyelid to absorb tears. This method is relatively simple, painless, and can be performed without the assistance of a healthcare professional. Elevated levels of total tau and phosphorylated tau (p-tau) have been observed in the tears of AD patients, and A042 levels are also found to be significantly different in AD patients compared to healthy individuals.

[0210] Example 4. Quantification of AD biomarkers via mass spectrometry

[0211] The identification of stable circulating peptides provides an alternative method for quantification using stable isotope-labeled internal standards (SIS) which are chemically identical to the target biomarker, but with a slight mass difference due to the incorporation of stable isotopes (e.g.,13C or15N). By adding a known amount of a stable isotope-labeled version of the target molecule to the sample and comparing the signal intensities of the labeled and unlabeled molecules, absolute quantification can be achieved. Using DREVNAVDSEHEKNV from insulin degrading enzyme as an example, one of the amino acid residues will be labelled with a heavy isotope. To address the issue of existing endogenous peptides in human serum, plasma, tear or other body fluid, a calibration strategy will be adopted in constructing the standard curves by spiking on top of the endogenous peptide to create an internal standard and varying amounts of heavy peptide are spiked to create a set of concentration standards. The samples to be quantified are spiked with the same amounts of heavy peptides as internal standards spiked into the matrix used to create calibration curves in which the light peptides are used as internal standards. Our discovery of many peptide fragments would also increase the sensitivity of peptide analysis for AD detection by analyzing multiple peptide fragments simultaneously. 2103739-000019 20250902

[0212] Example 5. Development of sandwich ELISA based on circulating serum or plasma peptide fragments for both early detection of AD and monitoring AD progression

[0213] Several of the detected peptides have rather long amino acid sequence and can be developed into a sandwich-based ELISA for example by using rabbit polyclonal antibodies directed against the entire sequence to capture the peptide and a mouse monoclonal antibody against the N-terminal amino acids (or C-terminal amino acids) as the detection antibody. Alternatively, we could use antibodies against the N-terminal region to capture the peptide and use antibody against the C-terminal region for detection (or vice versa).

[0214] Our specific serum or plasma-based peptide biomarkers will help move to a new era where AD could be better intervened at an early stage. If the new serum or plasma biomarkers can achieve comparable high performance like CSF or PET testing, suspected AD patients will be easily determined at primary care settings at an early stage, which will greatly improve the situation of early diagnosis of AD. Early diagnosis of AD is also essential to enable appropriate counselling to take place, as well as for planning treatment and care. The possibility of making an early diagnosis is also essential for the clinical evaluation of novel, potentially disease-modifying drugs for the treatment of AD.

[0215] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0216] 2103739-000019 20250902

[0217] Table 1. Sequences of stabilized circulating peptides

Claims

2103739-000019 20250902WHAT IS CLAIMED:

1. A method for identifying a subject having a stabilized circulating peptide, comprising detecting a stabilized circulating peptide in a body fluid sample from a subject, wherein the stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 protein-binding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin- interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin-1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein, whereby the presence of the stabilized circulating peptide in the body fluid sample indicates that the subject has the stabilized circulating peptide.

2. The method of claim 1, further comprising exposing the body fluid sample to a binding protein, wherein the binding protein specifically binds the stabilized circulating peptide.

3. The method of claim 2, wherein the binding protein is an antibody against the stabilized circulating peptide.

4. The method of claim 3, wherein the antibody is a monoclonal antibody, polyclonal antibody, or recombinant antibody.

5. The method of claim 1, further comprising quantifying the stabilized circulating peptide in the body fluid sample.

6. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21),2103739-000019 20250902LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

7. The method of claim 1, wherein the stabilized circulating peptide comprises GSTQLLSERNPSKNIR (SEQ ID: NO: 1).

8. The method of claim 1, wherein the stabilized circulating peptide comprises DTLAVFMASSGTTDVTNRNSPATPP (SEQ ID: NO: 2).

9. The method of claim 1, wherein the stabilized circulating peptide comprises DVSQELDPDLYKQ (SEQ ID: NO: 3).

10. The method of claim 1, wherein the stabilized circulating peptide comprises SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4).

11. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), and ATDYHKQSHGAAPC (SEQ ID NO: 7).

12. The method of claim 1, wherein the stabilized circulating peptide comprises of KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8).

13. The method of claim 1, wherein the stabilized circulating peptide comprises NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9).

14. The method of claim 1, wherein the stabilized circulating peptide comprises DREVNAVDSEHEKNV (SEQ ID NO: 10) and DREVNAVDSEHEK (SEQ ID NO: 11).

15. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), and SEDFGVNEDLADSDAR (SEQ ID NO: 13).

16. The method of claim 1, wherein the stabilized circulating peptide comprises of GITLDLVSRLVYWA (SEQ ID NO: 14).

17. The method of claim 1, wherein the stabilized circulating peptide comprises of MTVTDQVNCPK (SEQ ID NO: 15).

18. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of AGIPSVNSLHSVYNFCD (SEQ2103739-000019 20250902ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17) and IGQNYKAYMRTS (SEQ ID NO: 18).

19. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19) and TEKLTPAGLAIK (SEQ ID NO: 20).

20. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of LYGIQDSEDDGESEAAK (SEQ ID NO: 21) and LYGIQDSEDDGE (SEQ ID NO: 22).

21. The method of claim 1, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), and EQLTATKTEQNIK (SEQ ID NO: 26).

22. The method of claim 1, wherein the stabilized circulating peptide comprises of LALDIEIATYR (SEQ ID NO: 27).

23. The method of claim 1, wherein the subject has suffered from a neurodegenerative disease.

24. The method of claim 21, wherein the neurodegenerative disease is Alzheimer's disease.

25. The method of claim 1, wherein the subject shows an early-stage or mild symptom of Alzheimer's disease.

26. The method of claim 1, wherein the subject shows a middle-stage or moderate symptom of Alzheimer's disease.

27. The method of claim 1, wherein the subject shows a late-stage or advanced symptom of Alzheimer's disease.

28. The method of claim 1, further comprising treating or monitoring Alzheimer's disease in the identified subject.

29. The method of claim 1, wherein the identified subject has received a treatment of Alzheimer's disease, further comprising adjusting the treatment.

30. A kit comprising a binding protein for detecting, treating or monitoring Alzheimer's disease in a subject having a stabilized circulating peptide, wherein the binding2103739-000019 20250902 protein specifically binds the stabilized circulating peptide in a body fluid sample from the subject, and wherein the stabilized circulating peptide is a fragment of a protein selected from the group consisting of; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E receptor, Apolipoprotein D, Synapsin-1, Synphilin- 1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein.

31. The kit of claim 30, wherein the binding protein is an antibody.

32. The kit of claim 31, wherein the antibody is a monoclonal antibody, polyclonal antibody, or recombinant antibody.

33. The kit of claim 30, wherein the stabilized circulating peptide comprises an amino acid sequence selected from the group consisting of GSTQLLSERNPSKNIR (SEQ ID: NO: 1), DTLAVFMAS SGTTDVTNRNSPATPP (SEQ ID: NO: 2), DVSQELDPDLYKQ (SEQ ID: NO: 3), SGQEQIEDRDTMDFSGLSLIKLKK (SEQ ID: NO: 4), KNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPC (SEQ ID NO: 5), KNEDKKIDFSEFLS (SEQ ID NO: 6), ATDYHKQSHGAAPC (SEQ ID NO: 7), KNEDKKIDFSEFLSLLGDIAADYHKQSHGAAPC (SEQ ID NO: 8), NIETIINTFHQYSVKLGHPDTLNQGEFKELVRK (SEQ ID NO: 9), DREVNAVDSEHEKNV (SEQ ID NO: 10), DREVNAVDSEHEK (SEQ ID NO: 11), SEDFGVNEDLADSDARALYEAGERR (SEQ ID NO: 12), SEDFGVNEDLADSDAR (SEQ ID NO: 13), GITLDLVSRLVYWA (SEQ ID NO: 14), MTVTDQVNCPK (SEQ ID NO: 15), AGIPSVNSLHSVYNFCD (SEQ ID NO: 16), IGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMS (SEQ ID NO: 17), IGQNYKAYMRTS (SEQ ID NO: 18), NTEKLTPAGLAIKNGQLECVRWMVSETEA (SEQ ID NO: 19), TEKLTPAGLAIK (SEQ ID NO: 20), LYGIQDSEDDGESEAAK (SEQ ID NO: 21), LYGIQDSEDDGE (SEQ ID NO: 22), SKMFFKDIEEQYPT (SEQ ID NO: 23), YILQKSTFEVPENQMTILK (SEQ ID NO: 24), KEDLSDLTFLEVAK (SEQ ID NO: 25), EQLTATKTEQNIK (SEQ ID NO: 26), and LALDIEIATYR (SEQ ID NO: 27).

35. A biomarker panel comprising a plurality of biomarkers, wherein each biomarker comprises one, two or more stabilized circulating peptides, wherein each of the one, two or more stabilized circulating peptides is isolated from a body fluid sample from a subject, and wherein each of the one, two or more stabilized circulating peptide is a fragment of a protein selected from the group consisting of Amyloid beta A4 proteinbinding, family A, member 2; Amyloid Beta (A4) Precursor Protein-Binding, Family B, Member 2; Islet cell autoantigen 69, Huntingtin-interacting protein 1-related protein, S100A7, S100A15, S100A9, Insulin-degrading enzyme, Annexin Al, Apolipoprotein E2103739-000019 20250902 receptor, Apolipoprotein D, Synapsin-1, Synphilin- 1, Nucleoredoxin isoform XI, BRCA2, and Glial fibrillary acidic protein.

Images