Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method and application of immune cell exosomes carrying chimeric antigen receptors

An immune cell and exosome technology, applied in the field of biomedicine, can solve the problems of no tumor lethality, low immune cell activity, and no specific targeting.

Active Publication Date: 2020-11-06
PHARCHOICE THERAPEUTICS INC
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently significant problems as follows: such as using autologous immune cells, 1) It takes 10-14 days for the patient to complete the cell reinfusion after blood collection, which may miss the best time for patient treatment during this period
2) Patients often undergo multiple treatments such as radiotherapy and chemotherapy, etc., their physical condition is poor, and the activity of immune cells themselves is low, so the effectiveness of reinfusion cells cannot be guaranteed
3) It may not be appropriate to collect blood from patients with advanced malignant diseases
4) Large-scale application of immune cell reinfusion and massive proliferation of immune cells may cause inflammatory storm and become a dangerous complication of clinical treatment
5) The use of donor-derived CAR immune cells is likely to cause immune rejection
However, in subsequent studies, the exosomes secreted by CAR-T cells have complex components, no specific targeting, and no real tumor killing effect.
The use of exosomes directly isolated from CAR-T cells to treat tumors did not observe a significant tumor-suppressive effect in vivo experiments (attached Figure 4-5 )
And so far, there has not been any report that exosomes derived from CAR immune cells are effective in treating diseases
Exosomes produced by CAR-T cells face great challenges as a new method for cell-free treatment of diseases

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of immune cell exosomes carrying chimeric antigen receptors
  • Preparation method and application of immune cell exosomes carrying chimeric antigen receptors
  • Preparation method and application of immune cell exosomes carrying chimeric antigen receptors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Preparation of CAR exosomes derived from CAR-T cells

[0066]1) Whole gene synthesis including the CAR sequence of the anti-EGFR single-chain antibody (commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.), where the single-chain antibody sequence is derived from the anti-EGFR antibody cetuximab (Li et al., 2005, Structural basis for inhibition of the epidermal growth factor receptor by cetuximab, Cancer Cell, 7:301-311), the specific structure of CAR includes: anti-EGFR single-chain antibody scFv-CD8α hinge region and transmembrane region-4-1BB co-activation domain and CD3ζ signaling molecule cytoplasm inner segment. The specific sequence is roughly consistent with that reported in the literature Johnson L A, etal. Science translational medicine, 2015, 7 (275) (except scFv). In order to facilitate detection, a Myc tag was inserted between the scFv and the hinge region. The position of the tag was the same as that in the literature Chu J, et al.CS1-speci...

Embodiment 2

[0078] Example 2: CAR exosomes inhibit the viability of EGFR-positive MDA-MB-231 and HCC827 cells

[0079] Take MDA-MB-231 and HCC827 cells (ATCC) in good growth state, and adjust the cell concentration to 5×10 3 / ml, seeded in 96-well cell culture plate, 200μl / well, at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, EGF with a final concentration of 5 nmol and exosomes with different concentration gradients were added to the culture medium, and cetuximab antibody drug was used as a control (Cetuximab was purchased from Merck). After 4 days, the cell viability was measured by CellTiter -Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI) detection. Experimental results such as image 3 shown. The experimental results showed that CAR exosomes could significantly inhibit the viability of MDA-MB-231 and HCC827 cells (P Figure 4 ).

Embodiment 3

[0080] Example 3: Experiment of CAR exosomes inhibiting tumor growth in vivo

[0081] In order to detect the anti-tumor activity of CAR exosomes in vivo, HCC827 and MDA-231 cells were firstly inoculated subcutaneously on the right flank of BALB / c nude mice (Experimental Animal Center, Chinese Academy of Sciences), and 3500 mg / kg of CAR was injected into the tail vein after tumor formation. Exosomes and antibody drug cetuximab (10 mg / kg) were injected once a week, and the mice were sacrificed until the tumor was too large. The length and width of the tumor were measured every day, and the tumor volume was calculated.

[0082] tumor growth curve Figure 5 shown. The results showed that the tumor growth rate of the activated CAR exosome treatment group was significantly smaller than that of the cetuximab treatment group (after 40 days, P<0.01, Bonferroni test).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
diameteraaaaaaaaaa
Login to View More

Abstract

The present invention relates to the field of biomedicine, in particular to a preparation method and application thereof for obtaining immune cell exosomes carrying CAR through separation. Specifically, after the activation of CAR immune cells with specific antigens, the exosomes produced are further analyzed, separated, purified, and enriched, and finally immune cell exosomes carrying CAR are obtained. The exosomes prepared by the present invention can be used for the treatment of various diseases, such as cancer, severe infectious diseases, etc., and the exosomes can overcome adverse reactions such as immune and inflammatory storms of CAR cell therapy, and enhance the tissue penetration ability of CAR, It also has the advantages of convenient storage and transportation, and provides a new strategy for the treatment of related diseases.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular, to a method for preparing exosomes containing chimeric antigen receptor (Chimeric Antigen Receptor, CAR) through separation and its application in treating diseases. Background technique [0002] Surgical treatment, radiotherapy and chemotherapy, supplemented by new targeted treatment schemes are the basic strategies for the treatment of malignant tumors in recent years, and important progress has been made in clinical practice. However, the recurrence, metastasis and therapeutic resistance of malignant tumors are still difficult problems for clinical and scientific researchers. In recent years, the method of genetically modifying various immune cells for the treatment of diseases has been proposed, such as through the expression of Chimeric Antigen Receptor (CAR) on T cells, so that the genetically modified T cells target tumor cells Expressed antigens for the treatme...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00A61P31/00A61P37/02
CPCA61K35/17C12N5/0636C12N5/0646C07K14/7051C12N2510/00C12N2501/2302A61K39/001124A61K39/001104A61K39/001106A61K2039/5158A61K2039/5156A61K2039/804A61K2039/812A61K31/704A61P35/00A61K2300/00C07K14/70517C07K14/70521C07K14/70578
Inventor 胡适傅文燕
Owner PHARCHOICE THERAPEUTICS INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com