Search

CN-121987788-A - Methods for enhancing transcytosis of transferrin-based receptors across the blood brain barrier

CN121987788ACN 121987788 ACN121987788 ACN 121987788ACN-121987788-A

Abstract

The present invention relates to methods of enhancing transcytosis comprising modulating the expression of vesicle-associated membrane protein 3 (VAMP 3) and synaptic fusion protein 4 (syntaxin, stx-4). Compositions for increasing VAMP3 and/or STX-4 expression in HBMEC, and compositions comprising a target molecule in a form suitable for transcytosis across the blood brain barrier.

Inventors

  • WANG LEI
  • H.H.Li
  • LIU BIN
  • L.WAN
  • FENG LU
  • S.Hou
  • J.J.Yu
  • J.Jiao

Assignees

  • 南开大学
  • 美德生物参考实验室股份有限公司

Dates

Publication Date
20260508
Application Date
20251107
Priority Date
20241107

Claims (20)

  1. 1. A method of modulating transcytosis of a target molecule in a cell comprising modulating expression of vesicle-associated membrane protein 3 (VAMP 3) and/or synaptobrevin 4 (Syntaxin 4, STX-4) 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 VAMP3 cell levels in HBMEC are increased as compared to other similar HBMC that have not been treated to increase VAMP3 expression or VAMP3 levels.
  4. 4. The method of claim 1, wherein VAMP3 expression or VAMP3 level is increased by administering an agent, 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 (ESCHERICHIA COLI) (NMEC), streptococcus pneumoniae (Streptococcus pneumoniae) or group B Streptococcus (Streptococcus), LPS or a component thereof that increases VAMP3 expression.
  6. 6. The method of claim 1, wherein the level of VAMP3 in the HBMEC is increased by transiently or permanently transforming the HBMEC with a nucleic acid encoding VAMP 3.
  7. 7. The method of claim 1, wherein the level of VAMP3 in the HBMEC is increased by loading exogenous VAMP3 to the HBMEC.
  8. 8. The method of claim 1, wherein the level of VAMP3 in HBMEC is increased by loading exogenous VAMP3 into HBMEC vesicles comprising TfR.
  9. 9. The method of claim 1, wherein the transcytosis comprises movement of a target molecule or a target molecule conjugate or complex comprising an antibody or other protein molecule or conjugate thereof from the blood chamber to the brain chamber.
  10. 10. The method of claim 1, wherein the transcytosis comprises movement of a target molecule or a target molecule conjugate or complex comprising a drug or agent or conjugate thereof from the blood chamber or chamber to the brain chamber.
  11. 11. The method of claim 1, wherein STX-4 expression or STX-4 cell levels in HBMEC are increased as compared to other similar HBMC that have not been treated to increase STX-4 expression or STX-4 levels.
  12. 12. The method of claim 1, wherein STX-4 expression or STX-4 levels are 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 (ESCHERICHIA COLI) (NMEC), streptococcus pneumoniae (Streptococcus pneumoniae) or group B Streptococcus (Streptococcus), LPS or a component thereof that increases STX-4 expression.
  14. 14. The method of claim 1, wherein the level of STX-4 in HBMEC is increased by transiently or permanently transforming HBMEC with a nucleic acid encoding STX-4.
  15. 15. The method of claim 1, wherein the level of STX-4 in the HBMEC is increased by loading exogenous STX-4 into the HBMEC.
  16. 16. The method of claim 1, wherein the level of STX-4 in HBMEC is increased by loading exogenous STX-4 into HBMEC vesicles comprising TfR.
  17. 17. The method of claim 1, wherein the transcytosis comprises movement of a target molecule or target molecule conjugate or complex comprising an antibody or other protein molecule or conjugate or complex thereof from the blood chamber or compartment to the brain chamber.
  18. 18. The method of claim 1, wherein the transcytosis comprises movement of a target molecule or conjugate thereof as a drug or agent from the blood chamber or cavity to the ventricle.
  19. 19. The method of claim 1, wherein the expression or level of both VAMP3 and STX-4 is increased as compared to an otherwise similar HBMC that has been untreated to increase VAMP3 expression or VAMP3 level and STX-4 expression or STX-4 level.
  20. 20. A composition comprising an agent that increases VAMP3 and/or STX-4 expression when administered to HBMEC cells, and a target molecule or target molecule conjugate or complex that binds to TfR and initiates transcytosis through a blood brain barrier comprising said HBMEC.

Description

Methods for enhancing transcytosis of transferrin-based receptors across the blood brain barrier [ Reference sequence Listing ] According to 37 CFR ≡ ≡ 1.831-1.835 and 37 CFR ≡1.77 (b) (5), this specification includes sequence listings as part of the application. The sequence listing is provided in an XML file conforming to WIPO standard st.26, entitled "54992US_110724_st26. XML". The file size was 12,754 bytes, generated at 2024, 11, 7. The information recorded in the sequence table XML file is the same as the sequence information described in the application file. The entire contents of the sequence listing are incorporated herein by reference. Technical Field The present invention relates generally to the field of medicine, in particular to the fields of drug or biological delivery, cell biology, neuroscience and pharmacology. Background The Blood Brain Barrier (BBB) is a highly selective semi-permeable boundary that separates circulating blood from brain and extracellular fluids in the central nervous system. Bacterial meningitis occurs when pathogenic bacteria penetrate the barrier and invade the brain, leading to meningitis and potentially serious neurological complications. The BBB is composed of Human Brain Microvascular Endothelial Cells (HBMECs) and is an important gatekeeper of the Central Nervous System (CNS). It uniquely isolates the internal environment of the brain from circulating blood （Kim KS（2008）Mechanisms of microbial traversal of the blood-brain barrier. Nat Rev Microbiol 6（8）：625-634）. The BBB has many characteristics of tight intercellular junctions and low transcytosis rate, and is characterized by very low permeability to biomolecules, microorganisms and toxins, thereby protecting and regulating brain metabolism and maintaining the neural microenvironment （Sweeney MD,Zhao Z,Montagne A,Nelson AR,& Zlokovic BV（2019）Blood-Brain Barrier：From Physiology to Disease and Back. Physiol Rev 99（1）：21-78;Langen,UH,Ayloo,S,＆ Gu C（2019） Development and Cell Biology of the Blood-Brain Barrier. Annu Rev Cell Dev Biol 35：591-613）. To ensure proper functioning of the brain, several low rate transcytosis pathways across the BBB are employed under stringent regulation to ensure adequate supply of ions, nutrients and essential signal molecules required by the nervous tissue （Zhao Z ＆ Zlokovic BV（2020）Therapeutic TVs for Crossing Barriers in the Brain. Cell 182（2）：267-269）. Among these pathways, transferrin receptor (TfR) -mediated transcytosis provides an important pathway for iron delivery to the brain, which is critical for a variety of neurological functions (Zuchero YJ et al ,（2016） Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies. Neuron 89（1）：70-82;Preston JE,Joan Abbott N,&Begley DJ（2014）Transcytosis of macromolecules at the blood-brain barrier. Adv Pharmacol 71：147-163）. The impermeable nature of the BBB poses a significant challenge for therapeutic agents to enter the brain (Andreone BJ et al ,（2017）Blood-Brain Barrier Permeability Is Regulated by Lipid Transport-Dependent Suppression of Caveolae-Mediated Transcytosis. Neuron 94（3）：581-594 e585）. TfR transcytosis mediated delivery systems have been widely used for drug transport across the BBB and are considered one of the most promising brain delivery methods （Zhao,2020,#4969）（Terstappen GC,Meyer AH,Bell RD,＆ Zhang W（2021）Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov 20（5）：362-383;Fishman JB,Rubin JB,Handrahan JV,Connor JR,＆ Fine RE（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 shown encouraging brain delivery results （Terstappen GC,Meyer AH,Bell RD,&Zhang W（2021）Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov 20（5）：362-383;Yu YJ et al ,（2014）Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci Transl Med. 2014, 11, 5 in clinical trials ;6（261）：261ra154;Rawal SU,Patel BM,&Patel MM（2022）New Drug Delivery Systems Developed for Brain Targeting. Drugs 82（7）：749-792）. However, despite significant efforts to improve TfR transcytosis-mediated delivery systems by methods such as antibody engineering, the efficiency is still low (Zhou QH et al ,（2011）,Receptor-mediated Abeta amyloid antibody targeting to Alzheimer's disease mouse brain. Mol Pharm 8（1）：280-285;Yu YJ, ,（2014）;Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates;Sci Transl Med. 2014, 11, 5; 6 (261): 261ra154;Ullman JA-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, 5, 27; 12 (545): eaay, 1163). There remains a great need for a more efficient method of TfR transcytosis across the BBB. Advantages of increasing efficiency of TfR transcytosis include, but are not l