# US 2023/0266341 A1 · Method for diagnosis based on circulating extracellular vesicles

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- Number: US 2023/0266341 A1
- Document ID: US20230266341A1
- Status: Published application
- Kind: A1
- Application number: 17/947,168
- Filed: 2022-09-18
- Published: 2023-08-24
- Earliest claimed: 2022-02-20
- Assignees: Wellsim Biomedical Technologies, Inc
- Applicants: WellSIM Biomedical Technologies, Inc.
- Inventors: Yuchao CHEN; Katherine TUCHEZ
- Web page: https://patentgrep.com/patent/US20230266341A1

## CPC

- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- G01N 33/5076: {involving cell organelles, e.g. Golgi complex, endoplasmic reticulum}
- G01N 33/5091: {for testing the pathological state of an organism}
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding}
- G01N 2333/70596: Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
- G01N 2800/2821: Alzheimer
- G01N 2800/50: Determining the risk of developing a disease
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure provides a method for diagnosis based on proteins present on the surface of circulating extracellular vesicles. The method comprises incubating a sample of the subject with a detection antibody linked to a detectable label, contacting the sample with a capture antibody immobilized on a substrate, and detecting the detectable label on the circulating EV immobilized on the substrate. Compared to the method currently known in the art, the method disclosed herein has the advantages of high sensitivity with low cost and rapid procedure, high specificity.

## Claims

### Claim 1

A method for preparing a circulating extracellular vesicle (EV) from a subject useful for analyzing Alzheimer's disease (AD) in the subject, the method comprising:
incubating a sample of the subject with a detection antibody linked to a detectable label, wherein the sample comprises a circulating EV derived from neurons, wherein the detection antibody specifically binds to an AD-specific antigen present on the surface of the circulating EV derived from neurons;
contacting the sample with an anti-CD56 antibody immobilized on a substrate, thus immobilizing the circulating EV derived from neurons on the substrate; and
detecting the detectable label on the circulating EV immobilized on the substrate.

### Claim at position 2

2-4. (canceled)

### Claim 5 (depends on 1)

The method of claim 1, wherein the substrate is a glass slide.

### Claim 6 (depends on 5)

The method of claim 5, wherein the glass slide is coated with epoxy.

### Claim 7 (depends on 6)

The method of claim 6, wherein the anti-CD56 antibody is immobilized on the glass slide by micro-spotting.

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The method of claim 1, wherein the AD-specific antigen is selected from the group consisting of t-tau, p-tau181, p-tau217, Aβ40, and Aβ42.

### Claim 10 (depends on 1)

The method of claim 1, wherein the detectable label is a fluorophore or a fluorescent microsphere.

### Claim 11 (depends on 10)

The method of claim 10, wherein the detectable label is detected through a laser-induced confocal fluorescence scanner or a fluorescence microscope.

### Claim 12 (depends on 1)

The method of claim 1, further comprising
incubating the sample with a control antibody linked to a second detectable label, wherein the control antibody specifically binds to a control antigen present on the surface of the circulating EV; and
detecting the second detectable label on the circulating EV immobilized on the substrate.

### Claim 13 (depends on 12)

The method of claim 12, wherein the control antigen is selected from the group consisting of CD9, CD63, and CD81.

### Claim 14 (depends on 12)

The method of claim 12, further comprising normalizing the abundance of the detectable label on the circulating EV immobilized on the substrate with the abundance of the second detectable label on the circulating EV immobilized on the substrate.

### Claim 15

A method for preparing a circulating EV from a subject useful for analyzing AD in the subject, the method comprising:
incubating a sample of the subject with a first detection antibody linked to a first detectable label and a second detection antibody linked to a second detectable label, wherein the sample comprises a circulating EV derived from neurons, wherein the first detection antibody and the second detection antibody specifically bind to a first AD-specific antigen and a second AD-specific antigen present on the surface of the circulating EV derived from neurons respectively;
contacting the sample with an anti-CD56 antibody immobilized on a substrate, thus immobilizing the circulating EV derived from neurons on the substrate; and
detecting the first detectable label and the second detectable label on the circulating EV immobilized on the substrate.

### Claim 16 (depends on 15)

The method of claim 15, further comprising:
calculating a ratio of the abundance of the first detectable label to the second detectable label on the circulating EV immobilized on the substrate; and
generating a risk score based on the ratio.

### Claim 17 (depends on 16)

The method of claim 16, wherein the abundance of the first and the second detectable label is normalized with abundance of a control antigen present on the surface of the circulating EV.

### Claim 18

(canceled)

### Claim 19 (depends on 17)

The method of claim 17, wherein the first and the second AD-specific antigen is selected respectively from the group consisting of t-tau, p-tau181, p-tau217, Aβ40, and Aβ42.

### Claim 20 (depends on 19)

The method of claim 19, wherein the ratio is selected from the group consisting of ratio of p-Tau181 to t-Tau, ratio of p-Tau181 to Aβ40, ratio of Aβ42 to Aβ40, ratio of Aβ42 to t-Tau, ratio of p-Tau217 to t-Tau, and ratio of p-Tau217 to Aβ40.

### Claim 21 (depends on 20)

The method of claim 20, wherein the risk score is generated from sum of each ratio multiplied with coefficient thereof.

### Claim 22 (depends on 21)

The method of claim 21, wherein the coefficient is determined by using a machine learning algorithm to study a training data set.

### Claim 23 (depends on 22)

The method of claim 22, wherein the machine learning algorithm is support vector machine (SVM).

### Claim 24 (depends on 1)

The method of claim 1, further comprising administering to the subject a drug treating AD selected from the group consisting of an antibody targeting AP, a neuroprotective agent, a hematopoietic stem progenitor cell expressing at least one neuroprotective agent, a drug that inhibits the aggregation of Aβ 42, a compound that degrade microtubule-associated protein tau, a NMDA receptor antagonist, and an Acetylcholinesterase inhibitor.