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SiRNA of humanized interleukin 6, recombination expression carrier CAR-T and construction method and application of recombination expression carrier CAR-T

An interleukin and expression vector technology, applied in the field of tumor immunotherapy, can solve the problems of high cost, unbearable for ordinary families, and easy infection of patients

Active Publication Date: 2017-05-10
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But tocilizumab also has some obvious disadvantages, the first is the high cost
The price of a 10kg dose of tocilizumab is about 2,000 RMB, and adult patients generally use 5 at a time, which is unaffordable for ordinary families
Secondly, after the use of tocilizumab, due to the blocking of IL-6 receptors, patients are susceptible to infection in the later stage

Method used

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  • SiRNA of humanized interleukin 6, recombination expression carrier CAR-T and construction method and application of recombination expression carrier CAR-T
  • SiRNA of humanized interleukin 6, recombination expression carrier CAR-T and construction method and application of recombination expression carrier CAR-T
  • SiRNA of humanized interleukin 6, recombination expression carrier CAR-T and construction method and application of recombination expression carrier CAR-T

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Construction of recombinant lentiviral vector

[0083] 1. Materials

[0084] 1. Lentiviral backbone plasmid pLenti-3G ​​silencer, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, homologous recombination enzyme, Oligo Annealing Buffer are provided by Shiao (Shanghai) Bio Provided by Medical Technology Co., Ltd.;

[0085] 2. Primers: Design the primers required for amplifying DNA fragments and target sites according to the primer design principles, specifically:

[0086] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.25)

[0087] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.26)

[0088] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.27)

[0089] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.28)

[0090] VL-F: 5'-CACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATC-3' (SEQ ID NO.29)

[0091] VL-R: 5'-TGTGATCTCCAGCTTGGTCC-3' (SEQ ID NO.30)

[0092] OLC-VH-F...

Embodiment 2

[0196] Example 2 Concentration and detection of recombinant lentiviral vector

[0197] 1. Purification of recombinant lentiviral vectors by ion exchange chromatography (such as Figure 8 shown);

[0198] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;

[0199] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

[0200] (3) Place two ion exchange columns in series, and pass through the columns sequentially with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl25mM Tris-HCl (pH 6-8);

[0201] (4) The solution obtained in step 2 is loaded on the ion exchange column with a speed of 1-10ml / min by a peristaltic pump;

[0202] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;

[0203] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;

[02...

Embodiment 3

[0255] Example 3 Functional detection of recombinant lentiviral vectors lvCAR19-1761-lvCAR19-1769.

[0256] 1. Cell-level expression detection of CAR gene:

[0257] (1) After the recombinant lentiviral vector lvCAR19-1761~lvCAR19-1769 and the control virus MOCK infected PBMC cells, the collected cells were detected by RT-PCR for CAR mRNA transcription level to verify the expression of CAR gene. If the CAR mRNA transcription level increased, then It shows that the transcription level expression of CAR gene is successful;

[0258] (2) After the recombinant lentiviral vector lvCAR19-1761~lvCAR19-1769 and the control virus MOCK infected PBMC cells, the cells were collected to detect the expression level of CAR protein by western blot to verify the expression of CAR gene. If the expression level of CAR protein increased, then It shows that the translation level expression of CAR gene is successful;

[0259] (3) Cells were infected with lvCAR19-1761~lvCAR19-1769 at MOI=15 and the ...

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Abstract

The invention discloses an siRNA of humanized interleukin 6, a recombination expression carrier CAR-T and a construction method and application of the recombination expression carrier CAR-T. An IL-6 knock-down siRNA expression cassette and an siRNA expression product not only can be applied to eliminating or alleviating of CRS symptoms in treatment of B lineage acute lymphoblastic leukemia (ALL) through CAR19-T, but also can be applied to alleviating of CRS symptoms caused in treatment of all types of tumors like B lymphoma, pancreatic cancer, brain glioma and myeloma through CAR-T, and is even applied to alleviating of CRS caused by other types of treatment.

Description

technical field [0001] The invention belongs to the technical field of tumor immunotherapy, and specifically relates to a human interleukin 6 (IL-6) siRNA, a recombinant expression CAR-T vector (especially a kind of siRNA for alleviating CRS by knocking down IL-6) CAR-T transgene vector) and its construction method and application. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies, including cytokine the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/7088C12N15/867C12N15/66C12N5/10A61K35/17A61P35/00
CPCC12N5/0636C12N15/1136C12N15/66C12N15/86C12N2310/14C12N2740/15043C12N2510/00A61K39/464412A61K39/4611A61K39/4631A61P35/02A61P35/00C07K14/70517C07K16/2896C07K2319/03C07K2317/622A61K39/4635A61K2121/00A61K2300/00C12N15/85
Inventor 祁伟康立清俞磊夏荣华
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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