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Preparation method of immune cell exosome carrying chimeric antigen receptor and application thereof

A technology of immune cells and exosomes, applied in the field of biomedicine, can solve the problems of non-specific targeting, dangerous clinical treatment, and no tumor lethality.

Active Publication Date: 2018-07-24
PHARCHOICE THERAPEUTICS INC
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  • 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

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  • Preparation method of immune cell exosome carrying chimeric antigen receptor and application thereof
  • Preparation method of immune cell exosome carrying chimeric antigen receptor and application thereof
  • Preparation method of immune cell exosome carrying chimeric antigen receptor and application thereof

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).

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Abstract

The invention relates to the field of biological medicine, in particular to a preparation method for obtaining immune cell exosome carrying a CAR through separation. Specifically CAR immune cell is activated through specific antigen, produced exosome is further analyzed, separated, purified and enriched, and finally the immune cell exosome carrying the CAR is obtained. The exosome can be applied to the treatment of various diseases, such as cancer and severe infectious diseases, overcomes adverse reactions, such as immune inflammation storm, during CAR cell therapy, enhances the CAR tissue infiltration capacity, further has the advantages of high convenience in storage and transportation, and provides a novel strategy for the treatment of relevant 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

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/17A61P35/00A61P31/00A61P37/02
CPCC12N5/0636C12N5/0646C07K14/7051C12N2510/00A61K2239/38A61K2239/31A61K2239/13A61K2239/55A61K39/4611A61K39/464406A61K2239/17A61K39/464404A61K39/4631A61K39/4613A61K39/464424A61K2239/46C12N2501/2302A61K2039/804A61K2039/812A61K31/704A61P35/00A61K2300/00A61K35/17C07K14/70517C07K14/70521C07K14/70578
Inventor 胡适傅文燕
Owner PHARCHOICE THERAPEUTICS INC
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