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US-12622978-B2 - Binding agents and uses thereof for central nervous system delivery

US12622978B2US 12622978 B2US12622978 B2US 12622978B2US-12622978-B2

Abstract

Disclosed herein are binding agents, conjugates, and extracellular vesicles that enhance penetration of the blood brain barrier. Uses thereof for delivery of agents, e.g., therapeutic agents, to the central nervous system are also provided.

Inventors

  • Steven L. Stice
  • Raymond Swetenburg

Assignees

  • ARUNA BIO, INC.

Dates

Publication Date
20260512
Application Date
20210427

Claims (10)

  1. 1 . An extracellular vesicle (EV) comprising a first exogenous binding agent that specifically binds to Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3).
  2. 2 . The EV of claim 1 , wherein: the first exogenous binding agent enhances transport of the EV across the blood brain barrier; and/or the EV comprises one or more further exogenous binding agents.
  3. 3 . The EV of claim 1 , wherein the EV comprises a second the exogenous binding agent specifically binds to an endothelial cell protein selected from the group consisting of ARHGEF18, ASB12, BAD, DCAF12L1, ECHS1, GORASP2, GPHA2, GRID2IP, HOXD4, KCNT2, LIPJ, MESDC2, MTHFS, OCM, OR4X2, SCLT1, SERACI, SHOC2, SPRYD3, STAG1, TMED10, TRIM67, TTLL7, VLDLR, SFT2D2, CD74, HLA-DOA, ZP2, IFNLR1, HTR6, GPR37L1, MCHR2, CD164, B3GAT1-modified protein, and ST8SIA3-modified protein.
  4. 4 . The EV of claim 1 , wherein the exogenous binding agent is an antibody, or an antigen binding portion thereof, optionally wherein the antibody, or antigen-binding portion thereof, is: an antibody fragment selected from the group consisting of a Fab, a F(ab′)2, an scFv, a tandem scFv, a diabody, a minibody, and a single domain antibody; a humanized antibody, or an antigen binding portion thereof; and/or a fully human antibody, or an antigen binding portion thereof.
  5. 5 . The EV of claim 1 , wherein the exogenous binding agent is a polypeptide ligand or an aptamer.
  6. 6 . The EV of claim 1 , wherein the EV: is about 20 nm to about 250 nm in size; is an exosome; is a microvesicle; is derived from a primary cell, a transformed cell, a stem/progenitor cell, a neural cell, a muscle cell, an immune cell, an adipose cell, or a tumor cell, optionally wherein: the neural cell is an astrocyte, an oligodendrocyte, a neuron, or a glial cell; the immune cell is a microglial cell or a dendritic cell; or the stem/progenitor cell is an embryonic stem cell or an induced pluripotent stem cell, a neural progenitor cell, a neural stem cell, or a mesenchymal stem cell; is derived from a cultured cell line, optionally wherein the cultured cell line is a CHO cell line, a HEK293 cell line, or a Vero cell line; and/or is derived from cells that recombinantly express the exogenous binding agent.
  7. 7 . The EV of claim 1 , wherein the EV further comprises a small molecule, an exogenous nucleic acid, and/or an exogenous polypeptide, optionally wherein the exogenous nucleic acid is a siRNA, a shRNA, an antisense RNA, a miRNA, or a combination thereof.
  8. 8 . A pharmaceutical composition comprising a therapeutically effective amount of the EV of claim 1 , and a pharmaceutically acceptable carrier.
  9. 9 . A method of delivering an EV across the blood brain barrier of a subject, comprising administering to the subject a composition comprising the EV of claim 1 , or a pharmaceutical composition comprising a therapeutically effective amount of the EV of claim 1 .
  10. 10 . The method of claim 9 , wherein: the composition is administered intravenously, intraarterially, intranasally, orally, intramuscularly, intrathecally, intraocularly, intradermally, intracranially, subcutaneously, or by inhalation; and/or the EV is delivered to the brain or the central nervous system of the subject.

Description

RELATED APPLICATION This application claims the benefit of priority to U.S. Provisional Application No. 63/015,936, filed on Apr. 27, 2020, which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 20, 2021, is named A106525_1050WO_SL.txt and is 621,333 bytes in size. BACKGROUND Macromolecules can cross the blood-brain barrier (BBB) by exploiting a small number of known pathways (see, e.g., Abbott, N. J., et al., Astrocyte-endothelial interactions at the blood-brain barrier. Nature Reviews, Neurosicence, 7(1):41-53, 2006)). It is largely accepted that extracellular vesicles (EVs) cross the blood-brain barrier (BBB) via receptor mediated transcytosis (RMT) (Morad, G., et al., Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier via Transcytosis. ACS Nano, 2019), as other known pathways are not feasible given inherent size and charge limitations. However, only a handful of RMT pathways and/or receptors have been identified, including the insulin receptor, transferrin receptor, and low density lipoprotein receptor, and none has been shown to be involved in EV RMT. Co-opting these pathways for drug delivery is generally regarded as high risk, given the key roles these proteins play in the brain. Accordingly, there is a need for improved methods of delivering EVs across the BBB. Little is known regarding the molecular underpinnings of EV RMT. Understanding the molecules and pathways involved in EV RMT can be beneficial for, e.g., increased drug delivery to the CNS, lowered effective dose, decreased manufacturing demands, and decreased off-target effects. SUMMARY OF THE INVENTION In various aspects, the invention provides novel targets and binding agents that facilitate passage of associated molecules across the blood brain barrier (BBB). For example, to improve the delivery of extracellular vesicles across the blood brain barrier of a subject (e.g., to the brain or central nervous system of the subject), provided herein are extracellular vesicles (EVs) comprising one or more binding agents that specifically bind to one or more proteins (e.g., receptors) expressed by brain endothelial cells, allowing enhanced uptake across the BBB. In one aspect, provided herein is an extracellular vesicle composition that comprises a binding agent (e.g., ligand, antibody, aptamer) that binds to a receptor expressed by brain endothelial cells. In another aspect, provided herein is a method of delivering EVs (e.g., EVs comprising a therapeutic agent) across the blood brain barrier of a subject, comprising administering to the subject an EV of the present disclosure. In some embodiments, the extracellular vesicle is derived from a neural cell. In one aspect, provided herein is an extracellular vesicle (EV) comprising an exogenous binding agent that specifically binds to a protein expressed by a brain endothelial cell. In some embodiments, the exogenous binding agent enhances transport of the EV across the blood brain barrier. In certain embodiments, the exogenous binding agent specifically binds to a target protein set forth in Table 1. In some embodiments, the exogenous binding agent comprises all or a portion of a binding agent set forth in Table 1. For example, the exogenous binding agent can comprise a binding agent set forth in Table 1, or a fragment or portion thereof that retains the ability to bind to a target protein described herein. In some embodiments, the exogenous binding agent specifically binds to a target protein set forth in Table 2. In some embodiments, the EV comprises a plurality of exogenous binding agents, each of which specifically binds to a target protein set forth in Table 1. For example, the EV can comprise two, three, four, five, six, seven, eight, nine, or ten exogenous binding agents. In certain embodiments, the exogenous binding agent specifically binds to a target protein set forth in Table 2. In some embodiments, the EV comprises a plurality of exogenous binding agents, each of which specifically binds to a target protein set forth in Table 2. For example, the EV can comprise two, three, four, five, six, seven, eight, nine, or ten exogenous binding agents. In some embodiments, the exogenous binding agent specifically binds to an endothelial cell protein selected from ARHGEF18, ASB12, BAD, CD74, CD164, DCAF12L1, ECHS1, GORASP2, GPHA2, GRID2IP, HLA-DOA, HOXD4, IFNLR1, GPR37L1, HTR6, KCNT2, LIPJ, MCHR2, MESDC2, MTHFS, OCM, OR4X2, SCLT1, SERAC1, SFTD2, SHOC2, SPRYD3, STAG1, TMED10, TRIM67, TTLL7, VLDLR or ZP2, a B3GAT1-modified protein, a ST8SIA3-modified protein, or a combination thereof. In some embodiments, the exogenous binding agent specifically binds to one or more endothelial cell proteins selected from ARHGEF18, ASB12, BAD, CD74, CD164, DCAF12L1, ECHS1, GORASP2