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

Bispecific chimeric antigen receptor for treating hematologic tumor complicated with HIV infection, gene thereof, and construction method and application of gene

A chimeric antigen receptor and bispecific technology, applied in the field of medicine and biology, can solve the problems of large side effects, high mortality of patients, and easy recurrence, and achieve the effect of accelerated clearance

Active Publication Date: 2020-05-26
WUHAN UNIV OF SCI & TECH
View PDF2 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main treatment for lymphoma is chemotherapy, which has large side effects and is prone to relapse, and there is no available therapy
However, patients with HIV combined with malignant tumors are generally in serious condition, their immune system is severely damaged, their disease progresses rapidly, and their mortality rate is high. For this, how to choose an effective treatment plan is imminent

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
  • Bispecific chimeric antigen receptor for treating hematologic tumor complicated with HIV infection, gene thereof, and construction method and application of gene
  • Bispecific chimeric antigen receptor for treating hematologic tumor complicated with HIV infection, gene thereof, and construction method and application of gene
  • Bispecific chimeric antigen receptor for treating hematologic tumor complicated with HIV infection, gene thereof, and construction method and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of PTK881-EF1α-C5 and PTK881-EF1α-C6 plasmids

[0043] 1. The sequences SP1-N6 (nucleotide sequence SEQ ID NO: 1), SP2-FMC63-28Z (nucleotide sequence SEQ ID NO: 2) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the synthesized sequences were cloned into pUC57 on the carrier.

[0044] 2. Using the human cDNA library as a template, design primers to amplify the fragments CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, CD8 transmembrane region, CD3ζ intracellular signaling domain, and Primer complementation method to obtain Strep II, connecting peptide 3×G 4 S, P2A;

[0045] 3. Using Overlap PCR technology to combine SP1-N6, Strep II, and connecting peptide 3×G 4 S, SP2-FMC63-28Z and CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, and CD3ζ intracellular signaling domain were sequentially amplified and connected to obtain the code of chimeric antigen recepto...

Embodiment 2

[0052] Example 2: Preparation and sequencing of plasmids

[0053] 1. Preparation of plasmid

[0054] Inoculate the DH5α strains containing plasmids PTK881-EF1α-C5-1 and PTK881-EF1α-C6-1 into 250 mL of LB culture medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 220 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0055] Take out Buffers P1 from the EndoFree plasma mega kit (Qiagen), add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0056] Add 120mL Buffers P2 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to 50rpm, mix thoroughly and place at room temperature for 5min.

[0057] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of...

Embodiment 3

[0070] Example 3, Preparation of Lenti3-C5, Lenti3-C6 Lentiviral Vectors and Live Droplet Detection

[0071] 1. Preparation of lentiviral vector

[0072] 130.0~140.0×10 6 Number of 293T cells (Takara), a total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)), at 37 °C with 5% CO 2 Incubate for 24 hours in the incubator. Mix 320 μg of plasmids (PTK881-EF1α-C5: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6 and PTK881-EF1α-C6: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5 : 6) The DMEM basal medium was added to two 960 μg PEI tubes, vortexed, and equilibrated at room temperature for 10 minutes. Mix the above-mentioned two tubes of 35mL PEI and plasmid mixture with 525mL DMEM complete medium respectively, and replace them into the above-mentioned multi-layer cell culture flask. Place the multilayer cell culture flask at 37 °C with 5% CO 2 After 3 days in the incubator, the cell culture supernatant was collected.

[0073] After t...

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

No PUM Login to View More

Abstract

The invention discloses a construction method and an application of a recombinant gene of a bispecific chimeric antigen receptor for treating HIV infection complicated with blood tumor. The chimeric antigen receptor is formed by sequentially connecting a signal peptide, an HIV gp120 antigen specific single-chain antibody and an anti-CD19 single-chain antibody and then sequentially connecting witha CD28 transmembrane region, a CD28 intracellular structural domain (ICD), a 4-1BB costimulatory structural domain and a CD3zeta intracellular signal transduction structural domain in series, or the chimeric antigen receptor comprises: first CAR composed of the signal peptide, the chimeric antigen receptor, the HIV gp120 antigen specific single-chain antibody, the CD28 transmembrane region, the CD28-ICD, the anti-CD19 single-chain antibody, the CD8 transmembrane region, the CD28-ICD, the 4-1BB costimulatory structural domain and the CD3zeta intracellular signal transduction domain; and secondCAR composed of and the signal peptide, the anti-CD19 single-chain antibody, the CD8 transmembrane region in parallel, connecting the CD28-ICD, the 4-1BB costimulatory structural domain and the CD3zeta intracellular signal transduction domain, wherein the first CAR and the second CAR are sequentially connected in parallel.

Description

technical field [0001] The invention relates to the field of medicine and biology, and specifically refers to a bispecific chimeric antigen receptor for treating blood tumors complicated with HIV infection, its coding gene, its construction method and its application. Background technique [0002] After the human body is infected with HIV, CD4+ T lymphocytes are attacked by the virus, resulting in reduced immunity of the body, prone to various viral infections or malignant tumor diseases, and lymphoma is one of them. The risk of AIDS-related lymphoma is 165 times that of the general population. Malignant lymphoma is currently one of the leading causes of death in AIDS patients. [0003] According to the opinions of oncologists, patients with HIV complicated with malignant tumors were treated with a medical treatment plan, and the treatment plan was anti-tumor combined with ART treatment; According to the results of drug susceptibility test, etc., give patients immune suppor...

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
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K39/42A61P35/00A61P31/18
CPCA61K39/42A61K2039/505A61K2039/5156A61P31/18A61P35/00A61K39/001112A61K2039/804C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/1063C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 顾潮江张同存
Owner WUHAN UNIV OF SCI & TECH
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