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US-20260124253-A1 - METHOD FOR ENHANCING TRANSFERRIN RECEPTOR-BASED TRANSCYTOSIS ACROSS THE BLOOD BRAIN BARRIER

US20260124253A1US 20260124253 A1US20260124253 A1US 20260124253A1US-20260124253-A1

Abstract

Methods for enhancing transcytosis comprising modulating expression of Vesicle-associated membrane protein 3 (VAMP3) and syntaxin 4 (STX-4). Composition for increasing expression of VAMP3 and/or STX-4 in HBMECs and compositions comprising target molecules in a form suitable for transcytosis across the blood brain barrier.

Inventors

  • Lei Wang
  • Huamin Henry Li
  • Bin Liu
  • Lili Wan
  • Lu Feng
  • Shaobin Hou
  • J. Joanna Yu
  • Joseph Chiao

Assignees

  • NANKAI UNIVERSITY
  • MedBio Reference Laboratories, Inc.

Dates

Publication Date
20260507
Application Date
20241107

Claims (20)

  1. 1 . A method for modulating transcytosis of a target molecule in a cell comprising modulating Vesicle-associated membrane protein 3 (VAMP3) and/or Syntaxin 4 (STX-4) expression or modulating levels of VAMP3 or STX-4.
  2. 2 . The method of claim 1 , wherein the cell is a Human Brain Microvascular Endothelial Cell (HBMEC).
  3. 3 . The method of claim 1 , wherein VAMP3 expression or cellular levels of VAMP3 are increased in a HBMEC compared to otherwise similar HBMCs not treated to increase VAMP3 expression or VAMP3 level.
  4. 4 . The method of claim 1 , wherein VAMP3 expression or VAMP3 levels is increased by administering a drug, peptide or polynucleotide that increases VAMP3 expression.
  5. 5 . The method of claim 3 , wherein VAMP3 expression is increased by administering live, attenuated, or dead neonatal meningitis Escherichia coli (NMEC), Streptococcus pneumoniae , or group B Streptococcus , LPS, or components thereof which increases expression of VAMP3.
  6. 6 . The method of claim 1 , wherein level of VAMP 3 in HBMECs is increased by transiently or permanently transforming the HBMECs with a nucleic acid encoding VAMP3.
  7. 7 . The method of claim 1 , wherein VAMP3 level in HBMECs is increased by loading the HBMECs with exogenous VAMP3.
  8. 8 . The method of claim 1 , wherein VAMP3 level in HBMECs is increased by loading HBMEC vesicles comprising TfR with exogenous VAMP3.
  9. 9 . The method of claim 1 , wherein said transcytosis comprises movement of the target molecule or target molecule conjugate or complex that comprises an antibody or other protein molecule, or a conjugate thereof, from the blood compartment to a brain compartment.
  10. 10 . The method of claim 1 , wherein said transcytosis comprises movement of the target molecule or target molecule conjugate or complex that comprises a drug or pharmaceutical, or a conjugate thereof, from the blood or luminal compartment to a brain compartment.
  11. 11 . The method of claim 1 , wherein STX-4 expression or cellular levels of STX-4 is increased in a HBMEC compared to otherwise similar HBMCs not treated to increase STX-4 expression or STX-4 level.
  12. 12 . The method of claim 1 , wherein STX-4 expression or STX-4 level is increased by administering a drug, peptide or polynucleotide that increases STX-4 expression.
  13. 13 . The method of claim 1 , wherein STX-4 expression is increased by administering live, attenuated, or dead neonatal meningitis Escherichia coli (NMEC), Streptococcus pneumoniae , or group B Streptococcus , LPS, or components thereof which increases expression of STX-4.
  14. 14 . The method of claim 1 , wherein level of STX-4 in HBMECs is increased by transiently or permanently transforming the HBMECs with a nucleic acid encoding STX-4.
  15. 15 . The method of claim 1 , wherein STX-4 level in HBMECs is increased by loading the HBMECs with exogenous STX-4.
  16. 16 . The method of claim 1 , wherein STX-4 level in HBMECs is increased by loading HBMEC vesicles comprising TfR with exogenous STX-4.
  17. 17 . The method of claim 1 , wherein said transcytosis comprises movement of the target molecule or target molecule conjugate or complex that comprises an antibody or other protein molecule, or a conjugate or complex thereof, from the blood or luminal compartment to a brain compartment.
  18. 18 . The method of claim 1 , wherein said transcytosis comprises movement of the target molecule that is a drug or pharmaceutical, or a conjugate thereof, from the blood or luminal compartment to a brain compartment.
  19. 19 . The method of claim 1 , wherein both VAMP3 and STX-4 expression or levels are increased compared to an otherwise similar HBMCs not treated to increase VAMP3 expression or VAMP3 level and STX-4 expression or STX-4 level.
  20. 20 . A composition comprising an agent that increases the expression of VAMP3 and/or STX-when administered to HBMEC cells in combination with a target molecule or target molecule conjugate or complex that binds to TfR and initiates transcytosis through a blood brain barrier comprising said HBMECs.

Description

REFERENCE TO A SEQUENCE LISTING In accordance with 37 CFR § 1.831-1.835 and 37 CFR § 1.77(b)(5), this specification includes a Sequence Listing as part of the application. The Sequence Listing is provided as an XML file named “549922US_110724_ST26.xml” in compliance with WIPO Standard ST.26. This file is 12,754 bytes in size and was generated on Nov. 7, 2024. The information recorded in the Sequence Listing XML file is identical to the sequence information described in the application as filed. The entire contents of the Sequence Listing are hereby incorporated by reference. BACKGROUND OF THE INVENTION Field of the Invention. The invention pertains broadly to the field of medicine and especially to the fields of drug or biologic delivery, cell biology, neuroscience, and pharmacology. Description of Related Art. The blood-brain barrier (BBB) is a highly selective semipermeable border that separates the circulating blood from the brain and extracellular fluid in the central nervous system. Bacterial meningitis occurs when pathogenic bacteria penetrate this barrier and invade the brain, leading to inflammation of the meninges and potentially severe neurological complications. The BBB is composed of human brain microvascular endothelial cells (HBMECs) and is an essential gatekeeper for the central nervous system (CNS). It uniquely separates brain's internal milieu from the circulating blood. Kim K S (2008) Mechanisms of microbial traversal of the blood-brain barrier. Nat Rev Microbiol 6(8):625-634. With the features of numerous intercellular tight-junctions and low rates of transcytosis, the BBB is characterized by a very low permeability for biomolecules, microorganisms, and toxins in order to protect and regulate the metabolism of the brain and maintain the neural microenvironment. Sweeney M D, Zhao Z, Montagne A, Nelson A R, & Zlokovic B V (2019) Blood-Brain Barrier: From Physiology to Disease and Back. Physiol Rev 99(1):21-78; Langen U H, Ayloo S, & Gu C (2019) Development and Cell Biology of the Blood-Brain Barrier. Annu Rev Cell Dev Biol 35:591-613. To guarantee the proper functioning of the brain, several low-rate transcytosis pathways across the BBB are employed under tight regulation to ensure an adequate supply of ions, nutrients, and essential signaling molecules required by nervous tissue. Zhao Z & Zlokovic B V (2020) Therapeutic TVs for Crossing Barriers in the Brain. Cell 182(2):267-269. Among these pathways, transcytosis that is mediated by the transferrin receptor (TfR) provides an important route for delivering iron to the brain, which is essential for multiple neurological functions. Zuchero Y J, et al. (2016) Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies. Neuron 89(1):70-82; Preston J E, Joan Abbott N, & Begley D J (2014) Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol 71:147-163. The impermeable nature of the BBB poses a significant challenge to the uptake of therapeutic agents into the brain. Andreone B J, et al. (2017) Blood-Brain Barrier Permeability Is Regulated by Lipid Transport-Dependent Suppression of Caveolae-Mediated Transcytosis. Neuron 94(3):581-594 e585. The TfR transcytosis-mediated delivery system has been extensively utilized for the transport of drugs across the BBB and is considered one of the most promising brain delivery approaches (Zhao, 2020 #4969). Terstappen G C, Meyer A H, Bell R D, & Zhang W (2021) Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov 20(5):362-383; Fishman J B, Rubin J B, Handrahan J V, Connor J R, & Fine R E (1987) Receptor-mediated transcytosis of transferrin across the blood-brain barrier. J Neurosci Res 18(2):299-304. Several TfR-targeting antibodies and antibody-drug conjugates have demonstrated encouraging outcomes for brain delivery in clinical trials. Terstappen G C, Meyer A H, Bell R D, & Zhang W (2021) Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov 20(5):362-383; Yu Y J, et al. (2014) Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci Transl Med. 2014 Nov. 5; 6(261):261ra154; Rawal S U, Patel B M, & Patel M M (2022) New Drug Delivery Systems Developed for Brain Targeting. Drugs 82(7):749-792. However, efficiency remains low despite significant efforts that have been made to improve the TfR transcytosis-mediated delivery system through methods such as antibody engineering. Zhou Q H, et al. (2011), Receptor-mediated Abeta amyloid antibody targeting to Alzheimer's disease mouse brain. Mol Pharm 8(1):280-285; Yu Y J, et al. (2014); Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates; Sci Transl Med. 2014 Nov. 5; 6(261):261ra154; Ullman J A-O, et al. (2020). Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice Sci Transl Med. 2020 May 27; 12(545):eaay1163. There remain