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WO-2026096946-A2 - METHODS FOR COMBINATORIAL IMMUNOSEQUENCING

WO2026096946A2WO 2026096946 A2WO2026096946 A2WO 2026096946A2WO-2026096946-A2

Abstract

The disclosure relates to methods, compositions, and kits for detecting and analyzing surface marker displaying agents (SMDAs). The disclosure further relates to methods for detecting, diagnosing, monitoring response to treatment or recurrence, or predicting overall survival, progression-free survival or duration of response in a subject suspected of having colorectal cancer (CRC) comprising performing one or more assays on at least one biological sample from the subject to detect one or more specific populations of EVs.

Inventors

  • ROUTENBERG, David
  • HEBERT, Lucie
  • GIZZIE, Evan
  • NANJAPPA, Deepak

Assignees

  • MESO SCALE TECHNOLOGIES, LLC.

Dates

Publication Date
20260507
Application Date
20251031
Priority Date
20241101

Claims (20)

  1. 1. A method of determining surface markers of a surface marker displaying agent (SMDA) comprising: (A) contacting the SMDA with (i) a capture reagent bound to a surface, (ii) a plurality of binding reagents, and (iii) an oligonucleotide insert, wherein the plurality of binding reagents comprises: a) a first binding reagent comprising a first detection sequence that comprises (i) a first hybridization sequence, wherein at least a portion of the first hybridization sequence is complexed with a first blocker oligonucleotide, (ii) a first primer site, and (iii) a first barcode sequence; b) a second binding reagent comprising a second detection sequence that comprises (i) a second hybridization sequence, (ii) a third hybridization sequence comprising a second barcode sequence, and (iii) a fourth hybridization sequence; and c) a third binding reagent comprising a third detection sequence that comprises (i) a fifth hybridization sequence, wherein at least a portion of the fifth hybridization sequence is complexed with a second blocker oligonucleotide, (ii) a second primer site, and (iii) a third barcode sequence, wherein the first hybridization sequence and the second hybridization sequence are complementary; wherein the fourth hybridization sequence and the fifth hybridization sequence are complementary; wherein the third hybridization sequence is complementary to the oligonucleotide insert sequence; (B) generating an output oligonucleotide by ligating the hybridized first detection sequence to the hybridized oligonucleotide insert, and ligating the hybridized oligonucleotide insert to the hybridized third detection sequence to form a single-strand output oligonucleotide, wherein when at least three binding reagents bind to surface markers of the SMDA, the output oligonucleotide generated comprises the first barcode sequence, the second barcode sequence and the third barcode sequence; and (C) sequencing the output oligonucleotide to identify the surface markers of the SMDA.
  2. 2. The method of claim 1, wherein the third detection sequence further comprises a third primer site.
  3. 3. The method of claim 2, further comprising (i) binding a strand displacement primer complementary to the third primer site and extending the primer along the single-strand output oligonucleotide to form a double-strand output oligonucleotide, and (ii) releasing the double-strand output oligonucleotide.
  4. 4. The method of any one of claims 1 to 3, further comprising amplifying the output oligonucleotide using a first primer that hybridizes to the first primer site, and a second primer that hybridizes to the second primer site.
  5. 5. The method of any one of claims 1 to 4, wherein the method further comprises sequencing the output oligonucleotide and determining the surface markers of the SMDA.
  6. 6. The method of any one of claims 1 to 5, further comprising displacing the first and second blocker oligonucleotides from the first hybridization sequence and the fifth hybridization sequence prior to hybridization to the second detection sequence.
  7. 7. The method of any one of claims 1 to 6, wherein the first detection sequence further comprises a first blocker complement sequence, wherein the first blocker oligonucleotide is complementary to the first hybridization sequence and the first blocker complement sequence.
  8. 8. The method of any one of claims 1 to 7, wherein the third detection sequence further comprises a second blocker complement sequence, wherein the second blocker oligonucleotide is complementary to the fifth hybridization sequence and the second blocker complement sequence.
  9. 9. The method of claim 8, wherein the nucleotide sequences of the first and second complement blocker sequences are the same or substantially the same.
  10. 10. The method of any one of claims 1 to 9, wherein the first and/or second blocker oligonucleotide is about 5 to about 20 nucleotides in length.
  11. 11. The method of any one of claims 1 to 10, wherein the first and/or second blocker oligonucleotide comprises a 3’ overhang.
  12. 12. The method of any one of claims 1 to 11, wherein the capture reagent is releasably bound to the surface.
  13. 13. The method of any one of claims 1 to 12, wherein the capture reagent is releasably bound to the surface by a labile linker.
  14. 14. The method of claim 13, wherein the labile linker comprises UDG1, a restriction site, or both.
  15. 15. The method of any one of claims 1 to 14, wherein the surface further comprises an oligonucleotide, wherein the oligonucleotide and capture reagent are bound to the same surface.
  16. 16. The method of any one of claims 1 to 15, wherein each of the first, second and third detection sequences has a length of about 20 to about 50 nucleotides.
  17. 17. The method of any one of claims 1 to 16, wherein at least one of the first, second, and third detection sequences comprises a unique molecular identifier.
  18. 18. The method of any one of claims 1 to 17, wherein each of the first, second, and third binding reagents binds to a surface marker selected from: CD9, CD73, CD324, CD325, CD326, CD13. CD66a, CD66e, CD10, CD31. CD36, CD141, CD14, CD54, CD26, MHC Class 11 molecules. FLT1, and any combination thereof.
  19. 19. The method of any one of claims 1 to 18, wherein each of the first, second, and third binding reagents comprises an antibody or antigen binding fragment thereof, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimitope, lipid binding protein, carbohydrate binding protein, DNA aptamer or RNA aptamer.
  20. 20. The method of any one of claims 1 to 19, wherein the first, second, and third binding reagents each further comprise a conjugating oligonucleotide and each of the first, second, and third detection sequences comprises a connecting sequence complementary to the conjugating oligonucleotide.

Description

METHODS FOR COMBINATORIAL IMMUNOSEQUENCING [0001] This invention was made with government support under grant number DK133861 awarded by the National Institutes of Health and grant number TR002886 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE DISCLOSURE [0002] The disclosure relates to methods, compositions, and kits for the analysis, specific detection, and isolation of surface marker displaying agents (SMDAs) such as extracellular vesicles (EVs) and/or their contents by targeting at least two surface markers. The disclosure further relates to methods for detecting, diagnosing, monitoring response to treatment or recurrence, or predicting overall survival, progression-free survival or duration of response in a subject suspected of having colorectal cancer (CRC) comprising performing one or more assays on at least one biological sample from the subject to determine surface markers specific to populations of EVs. REFERENCE TO ELECTRONIC SEQUENCE LISTING [0003] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on October 29, 2025, is named “0076-0097WOI.xml” and is 15,256 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety . BACKGROUND [0004] Surface marker displaying agents (SMDAs) include cells, viruses and viral particles, cellular organelles, and vesicles, including extracellular vesicles (EVs) and exosomes. In addition to the potentially biologically relevant information displayed on the surface of SMDAs, SMDAs may encapsulate biologically relevant materials or components. [0005] EVs are a diverse group of cell-secreted membrane vesicles implicated in a wide variety of physiological and pathological processes, many of which are only beginning to be understood. These include immune regulation, antigen presentation, tumor progression and metastasis, modulation of inflammation, stem cell regulation, neuronal development and regeneration, and cell-to-cell transfer of pathogenic proteins and nucleic acids. EVs are secreted from nearly all cell types through multiple mechanisms including the fusion of specific endosomal compartments called multivesicular bodies (MVB) with the plasma membrane and by budding/shedding directly from the plasma membrane. EVs are present in nearly all body fluids including blood, urine, cerebral spinal fluid, and saliva, and are secreted by most in vitro cultured cells as well. Because of the EV formation mechanisms, EVs contain specific lipids, membrane proteins, and internalized proteins, nucleic acids and metabolites derived from their cells of origin and are thus a rich source of potential biomarkers. [0006] It is now accepted that EVs have numerous roles in inter-cellular communication, facilitated by the transfer of EV cargo to recipient cells following uptake, or by the interaction of EV surface proteins with cellular receptors. EVs are also emerging as useful indicators of disease. EV secretion is involved in the maintenance of normal physiological functions and is linked to numerous disease states, including certain cancers, cardiovascular disease, neurological and immunological disorders. Methods for detecting and isolating EVs are described, for example, in international patent application publication numbers WO2019222708, US20210389304, W02020086751, US20210382043A1, WO2022051481, US20230349920A1, and WO2023212315, and US Application No. 18/860,884, each of which is incorporated by reference in its entirety. [0007] Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for almost 10% of all cases, and is the second leading cause of cancer death in the United States. CRC recurrence rate averages 25%, and follow-up after curative treatment remains a complex challenge for the healthcare system, in terms of diagnostics, treatment and monetary costs. In general, the survival of patients with advanced-stage cancers is lower than that of patients with early-stage cancers, although some survival paradox has been reported between stage IIB/C and stage IIIA. Other prognostic markers are used as indicators of the appropriate therapeutic strategy, but are excluded from the classification, including the histological grade, RAS/BRAF mutation, and plasma carcinoembryonic antigen (CEA) levels. [0008] Assessing the composition of EVs generally requires isolating a pure population of EVs and separating it from non-EV associated factors. Some demonstrations of this idea have focused on enriching EVs from plasma or serum based on immunoaffinity capture of specific EV surface proteins and measuring disease-associated proteins within the enriched EV population. [0009] Despite its utility, this method has significant fundamental and technical drawbacks. Fundamentally, the u