Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof

A chimeric antigen receptor, intracellular domain technology, applied in the field of genetic engineering, can solve the problem of no report showing the promoting effect of CD27 molecule

Active Publication Date: 2016-09-21
CHONGQING PRECISION BIOTECH CO LTD
View PDF3 Cites 22 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no combination of CD27 molecule and other co-stimulatory molecules for the construction of chimeric antigen receptors, and there is no report showing that CD27 molecule can promote the formation of Tscm

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
  • Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof
  • Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof
  • Chimeric antigen receptor containing CD27 intracellular domain, lentiviral vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of CAR virus containing four stimulating signals of CD28-CD137-CD27-CD3

[0041] In order to prove that the CAR-T cells containing the four stimulating signals of CD28-CD137-CD27-CD3 are compared with the previously reported three stimulating signals of CD28-CD137-CD3, the two stimulating signals of CD28-CD3 and the single CD3 stimulation Signaling CAR-T cells are more advantageous, so it is necessary to construct viral vectors containing different combinations of stimulating signals. In this example, the single-chain antibody targeting carcinoembryonic antigen (CEA) is used as a unified extracellular recognition antigen structure, and the following four chimeric antigen receptors need to be constructed respectively ( figure 1 ):

[0042] LP-CEA(scfv)-CD8a-TM-CD3(CEA1);

[0043] LP-CEA(scfv)-CD8a-TM-CD28-CD3(CEA2);

[0044] LP-CEA(scfv)-CD8a-TM-CD28-CD137-CD3(CEA3);

[0045] LP-CEA(scfv)-CD8a-TM-CD28-CD137-CD27-CD3(CEA4).

[0046] 1) Synthes...

Embodiment 2

[0066] Embodiment 2, virus infection T cell

[0067] 1) Isolation and activation of T cells and virus infection

[0068] (1) Isolation of human peripheral blood mononuclear cells

[0069] Collect about 60ml of peripheral blood with anticoagulant-added blood collection tubes, divide into 30ml of 50ml centrifuge tubes, add 7.5ml of hydroxyethyl starch to dilute; naturally settle at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and The collected upper plasma was centrifuged at 1400rpm / min for 15min; then the pellet was resuspended with normal saline, added to the lymphocyte separation medium at a volume ratio of 1:1, gradient centrifuged at 400g / min, and the speed of the centrifuge was set to 1 , centrifuged for 20min; after centrifugation, the centrifuge tube was divided from top to bottom: the first layer: plasma layer; the second layer: annular milky white lymphocyte layer; the third layer: transparent separation liquid layer; the fourth layer...

Embodiment 3

[0074] Example 3. Effect of Virus Infection of CAR-T Cells on Cell Proliferation

[0075] After each group of viruses infected T cells, T cells were cultured for 10 days with RPMI 1640 complete medium containing 10% fetal bovine serum and 500 IU / ml recombinant human IL-2, and counted every 2-3 days . Then observe the growth of T lymphocytes, the results are as follows: image 3shown. The results showed that after the cells were infected with CAR-expressing viruses, they could still form a typical proliferating clonal cluster. By counting the cells and drawing the cell proliferation curve, it can be seen that the cells infected with Lv-CEA(scfv)-CD8a-TM-CD28-CD137-CD27 -CD3(CEA4) virus T cell proliferation compared with Lv-CEA(scfv)-CD8a-TM-CD3(CEA1) virus, Lv-CEA(scfv)-CD8a-TM-CD28-CD3(CEA2) virus, Lv- CEA(scfv)-CD8a-TM-CD28-CD137-CD3(CEA3) virus and T cells not infected with virus are better.

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
wavelengthaaaaaaaaaa
Login to view more

Abstract

The invention discloses a chimeric antigen receptor containing a CD27 intracellular domain, a lentiviral vector and application thereof. The immune receptor tyrosine activation sequence motif of the chimeric antigen receptor containing CD27 intracellular domain contains CD27molecule intracellular signal domain and T cell costimulatory signal molecules. The CD27molecule intracellular signal domain is connected with costimulatory signals related to T cell activation, so that the in-vitro proliferation and apoptosis effect of T cells can be obviously enhanced, and the ratio of young sample Tscm cells (CD45RA+CD62L+) with in vivo therapeutic action and central memory cells (CD45RO+CD62L+) in CAR-T cells is increased; expression of IL-10 factors capable of restraining immunization is reduced, an obvious effect of improving the CAR-T (T cell chimeric antigen receptor) cellular therapy effect is achieved, and a new idea and a new selection are provided for the field of CAR-T cellular therapy.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a chimeric antigen receptor comprising the CD27 intracellular domain, and also relates to a lentiviral vector and application of the chimeric antigen receptor comprising the CD27 intracellular domain. Background technique [0002] Adoptive cell therapy (ACT) is a kind of biotherapy technology, which expands autologous immune cells (mainly T cells) in vitro, and then reintroduces them to tumor patients to achieve therapeutic purposes. It is the fourth treatment after surgery, radiotherapy and chemotherapy, and it is widely used in clinical treatment. Adoptive cell therapy is widely used: lymphokine activated killer cells (lymphokine activated killer cells, LAK) combined with IL-2 have achieved certain curative effect in advanced malignant tumors; tumor infiltrating lymphocytes (tumor infiltrating lymphocytes, TIL) clinical trials In the treatment of metastatic melan...

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 Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61K2039/5156A61K2039/5158C07K14/70503C07K14/70521C07K14/70578C07K16/3007C07K2317/622C07K2319/02C07K2319/33C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 钱程杨智沈俊杰单娟娟
Owner CHONGQING PRECISION BIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap