Ev-mediated delivery of binding protein-small molecule conjugates

A technology that combines proteins and small molecules, applied in the field of protein-drug conjugates, can solve the problems of EV loading with less small molecules and practical therapeutic applications

Inactive Publication Date: 2019-04-26
EVOX THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as is often the case in the field, there is very little information on how to load exosomes, and little, if any, are available for the loading and practical therapeutic application of small molecule-bearing EVs.

Method used

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  • Ev-mediated delivery of binding protein-small molecule conjugates
  • Ev-mediated delivery of binding protein-small molecule conjugates
  • Ev-mediated delivery of binding protein-small molecule conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Apomorphine-loaded EVs for Parkinson's disease

[0064] MSCs endogenously expressing dopamine receptor D2 and MSCs genetically engineered to express a fusion protein comprising EV protein CD63 and dopamine receptor D2 were cultured in MSCGM growth medium. To endogenously load MSC-EVs with apomorphine (APO), a drug used to treat Parkinson's disease, MSC medium was replaced with Opti-EM medium and incubated with 2 μM APO for 12 hours. Thereafter, conditioned medium was collected and APO-EVs were isolated by tangential flow filtration and size exclusion chromatography. To exogenously load MSC-EVs with APO, EVs from untreated MSCs were isolated as above, then incubated with 2 μM APO for 2 h, and re-isolated to remove APO molecules not loaded into EVs. APO loading to EVs is facilitated through the interaction of its dopamine receptor D2 on the EV surface, which results in a "precharge" of the dopamine receptors capable of signaling.

[0065] The activity of APO-...

Embodiment 2

[0066] Example 2: Loading of Imatinib into EVs for Cancer Therapy

[0067] MSCs endogenously expressing ABL1 receptor tyrosine kinase and MSCs genetically engineered to express a fusion protein of the EV protein syntenin and ABL1 receptor tyrosine kinase were cultured in MSCGM growth medium. To endogenously load MSC-EVs with imatinib (IMA), MSC medium was replaced with Opti-MEM medium and incubated with 2 mg IMA. Thereafter, conditioned medium was collected and IMA-EVs were isolated by tangential flow filtration and size exclusion chromatography. To exogenously load MSC-EVs with IMA, EVs from untreated MSCs were isolated as described above, then incubated with 2 mg IMA for 2 h, and re-isolated to remove IMA molecules not loaded into EVs

[0068] IMA is a drug used in the treatment of chronic myelogenous leukemia (CML) because it binds to ABL1 and subsequently stabilizes the Bcr-Abl receptor tyrosine kinase complex, making IMA a potent inhibitor of the kinase. Loading of IMA ...

Embodiment 3

[0070] Example 3: Imatinib Loading to EVs for Prevention of Intravenous LPS-Induced Sepsis

[0071] Cell culture, EV loading and EV isolation were performed as in Example 2, but the activity of EV loaded IMA was tested in an intravenous LPS-induced acute sepsis model. LPS treatment is known to induce high levels of reactive oxygen species associated with septic shock and ARDS. Increased IMA function of catalase activity was associated with reduced DNA damage and decreased production of the pro-inflammatory cytokines TNF-α and IL-6.

[0072] Mice were sensitized to LPS by retro-orbital injection of 5 mg / kg LPS. IMA and EV-IMA treatments were initiated the day before daily administration with an equivalent amount of 200 mg / kg / day of IMA, and mouse survival was monitored.

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Abstract

The present invention relates to extracellular vesicles (EVs) comprising a binding protein which may be used for delivery of protein-drug conjugates comprising the binding protein and a small moleculeagent, typically a small molecule drug. The present invention also relates to methods for producing such EVs as well as pharmaceutical compositions and medical uses of such EVs.

Description

technical field [0001] The present invention relates to extracellular vesicles (EVs) comprising binding proteins for the delivery of protein-drug conjugates comprising binding proteins and small molecule agents, typically small molecule drugs. Background technique [0002] Extracellular vesicles (EVs) regulate cell-to-cell communication in normal physiology and pathology by presenting their contents (mainly RNA, proteins, and lipids) to recipient cells in target tissues. The modification of EVs to incorporate various types of pharmacological agents has been investigated in many contexts, for example WO2013 / 084000 discloses the use of exosomes for the intracellular delivery of biotherapeutics, or WO2010 / 119256 describes the use of exosomes Delivery of exogenous genetic material. [0003] The utility of EVs as drug delivery vehicles is impeccable in the case of, for example, nucleic acid-based drugs such as siRNAs, large protein-based drugs targeting intracellular components,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/50
CPCA61K38/22A61K31/436A61K31/473A61K31/506C07K14/705C07K14/70571C07K14/70596C12N15/00C07K2319/00A61K47/6901A61P25/16A61P35/00A61P3/06A61P37/00A61K47/64A61K9/1277A61K31/438A61K31/485
Inventor O.维克兰德
Owner EVOX THERAPEUTICS LTD
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