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EP-4735886-A1 - COMPOSITIONS AND METHODS FOR DETECTING ANALYTES IN EXOSOMES

EP4735886A1EP 4735886 A1EP4735886 A1EP 4735886A1EP-4735886-A1

Abstract

Provided herein are compositions and methods for detecting analytes in exosomes. In particular, the present invention relates to the capture and lysis of exosomes and detection of analytes in the exosomes using an immunoassay.

Inventors

  • LUKE, Kimberly
  • SCOTT-WEATHERS, Casey

Assignees

  • Intuitive Biosciences, Inc.

Dates

Publication Date
20260506
Application Date
20240626

Claims (20)

  1. 1. A method of detecting the presence of an analyte in a sample from a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more analyte- specific binding agents and/or one or more detection reagents suitable for detecting the analyte; and determining whether the extracellular vesicles comprise the analyte.
  2. 2. The method of claim 1, wherein the analyte is a protein.
  3. 3. The method of claims 1 or 2, wherein the analyte is a cancer antigen or cancer- specific protein.
  4. 4. The method of claim 3, wherein the analyte is prostate specific antigen (PSA).
  5. 5. The method of claim 2, wherein the protein is from an infectious agent and the presence of the analyte in the sample is indicative of infection of the analyte in the subject.
  6. 6. The method of claim 5, wherein the infectious agent is a virus or a bacteria.
  7. 7. The method of claim 1, wherein the analyte is a nucleic acid.
  8. 8. The method of any one of claims 1 to 2, wherein the analyte is HIV protein p24 and, optionally, at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20.
  9. 9. A method for diagnosing human immunodeficiency vims (HIV) infection in a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more HIV protein binding agents and/or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the extracellular vesicles comprise the HIV protein p24 and optionally at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20; wherein a determination of the presence of p24 and the at least one additional HIV protein is indicative of HIV infection in the subject.
  10. 10. The method of claim 9, wherein the at least one additional HIV protein is Nef.
  11. 11. The method of claim 9, wherein the at least one additional HIV protein is Tat.
  12. 12. The method of claim 9, wherein the at least one additional HIV protein is Vpu.
  13. 13. The method of claim 9, wherein the at least one additional HIV protein is gp!20.
  14. 14. The method of claim 9, wherein the subject has been vaccinated against HIV infection.
  15. 15. The method of claim 9, wherein the subject has or is on antiretroviral therapy.
  16. 16. The method of any one of claims 1 to 15, wherein the extracellular vesicles are captured on a chromatographic test strip.
  17. 17. The method of claim 16, wherein the chromatographic strip is in a lateral flow assay device.
  18. 18. The method of any one of claims 1 to 17, wherein the extracellular vesicles are captured with a tctraspanin.
  19. 19. The method of claim 18, wherein the tetraspanin is selected from the group consisting of CD9, CD63, and CD81.
  20. 20. The method of any one of claims 1 to 19, wherein the extracellular vesicles are lysed on a chromatographic test strip.

Description

COMPOSITIONS AND METHODS FOR DETECTING ANALYTES IN EXOSOMES CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application No. 63/524,517, filed June 30, 2023, and U.S. Provisional Application No. 63/571,733, filed March 29, 2024, which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION Provided herein are compositions and methods for detecting analytes in exosomes. In particular, the present invention relates to the capture and lysis of exosomes and detection of analytes in the exosomes using an immunoassay. BACKGOUND OF THE INVENTION At home or point of care testing has become more prevalent after the COVID-19 pandemic. Lateral flow tests in particular are an easy-to-use platform to detect analytes in liquid samples, which makes at home testing possible for vulnerable populations. Generally, lateral flow tests have reduced sensitivity when compared to the more complicated and expensive clinical testing. However, the trade-offs between sensitivity and simplicity can be reduced by concentrating the analyte to be detected on the lateral flow test. Exosomes are secreted by cells and are characterized as vesicles ranging in size from 40- 200 nm and are a type of extracellular vesicle (EV). These EVs are a novel source of biomarkers indicative of disease, from their unique surface proteins to their cell origin- specific cargo. Not only are EVs prevalent in the blood but can be of tissue origin and circulate throughout the body and be detected in many different body fluids. EVs contain certain membrane proteins like integrins and tetraspanins that are key factors in membrane transport and fusion. These membrane proteins have been utilized as EV “markers” to enrich samples for EVs, including exosomes. EVs from body fluids like blood and urine are a non-invasive alternative to many current diagnostic tests. For some infectious diseases, like HIV, proteins from the pathogen are sequestered within EVs. In the example of HIV, the vims shares many properties, like the presence of tetraspanins in the outer membrane, with EVs. See, e.g., Jing et al., (2021) Exosomes in HIV infection, Current Opinion in HIV and AIDS 16(5):p 262-270. In the example of prostate cancer, prostatespecific antigen (PSA) in found both in the blood and within extracellular vesicles. In this example, the levels of PSA in blood are easily measured but have poor correlation with true incidence of disease. However, EV-associated PSA levels not only distinguish prostate cancer from healthy individuals, but can also distinguish prostate cancer from benign prostatic hyperplasia. See, e.g., Salciccia et al., (2023) Exosome Analysis in Prostate Cancer: How They Can Improve Biomarkers’ Performance, Curr Issues Mol Biol. 2023 Jul; 45(7): 6085-6096. Prior work has demonstrated the ability to capture EV s from bodily fluids using antibodies against tetraspanins on a lateral flow test. See, e.g., Oliveira- Rodriguez et al., (2016) Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids, J. Extracellular Vesicles 5(1): 1-10. However, this work only measured total EVs captured and did not further probe surface or cargo proteins of the captured EVs. The invention described here Not only captures EVs from the liquid sample, but also lyses the captured EVs to release cargo and detect that cargo on the test line of the lateral flow assay. SUMMARY OF THE INVENTION Provided herein are compositions and methods for detecting analytes in extracellular vesicles such as exosomes. In particular, the present invention relates to the capture and lysis of exosomes and detection of analytes in the extracellular vesicles using an immunoassay. Experiments described herein describe a sensitive test for an analyte (e.g., Human Immunodeficiency Virus (HIV) or PSA) using enrichment and capture of EVs to probe EV cargo for HIV specific antigens or PSA. Such methods provide needed improved detection of a variety of analytes. For example, in some embodiments, provided herein is a method of detecting the presence of an analyte in a sample from a subject, comprising: obtaining a patient sample comprising exosomes; capturing exosomes from the patient sample; lysing the exosomes to release proteins contained therein; contacting the proteins with one or more analyte- specific binding agents and/or one or more detection reagents suitable for detecting the analyte; and determining whether the exosomes comprise the analyte. Also provided is a method for diagnosing human immunodeficiency vims (HIV) infection in a subject, comprising: obtaining a patient sample comprising exosomes; capturing exosomes from the patient sample; lysing the exosomes to release proteins contained therein; contacting the proteins with one or more HIV protein binding agents and/or one or more detection reagents suitable for detecting one or more HIV prote