Closed IL6R CAR-T transgenic vector for relieving CRS as well as construction method and application of CAR-T transgenic vector

A transgenic carrier and carrier technology, applied in the field of medical biology, can solve the problems of unaffordable and high costs for ordinary families, and achieve the effects of avoiding low delivery efficiency in the body, saving expensive expenses, and saving costs

Active Publication Date: 2018-06-12
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost of using tocilizumab is very high. The price of a 10kg dose of tocilizumab is abo

Method used

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  • Closed IL6R CAR-T transgenic vector for relieving CRS as well as construction method and application of CAR-T transgenic vector
  • Closed IL6R CAR-T transgenic vector for relieving CRS as well as construction method and application of CAR-T transgenic vector
  • Closed IL6R CAR-T transgenic vector for relieving CRS as well as construction method and application of CAR-T transgenic vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention. Example 1 Construction of recombinant lentiviral vector

[0066] 1. Materials

[0067] 1. The lentiviral backbone plasmid pLenti-3G ​​Basic2, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzyme were provided by Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0068] 2. Primers: According to the principles of primer design, the primers required for amplifying DNA fragments and target sites are designed. The primers are synthesized by Shanghai Biological Company, specifically:

[0069] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTC...

Embodiment 2

[0184] Example 2 Concentration and detection of recombinant lentiviral vector

[0185] 1. Purification of recombinant lentiviral vectors by ion exchange chromatography (such as Figure 7 shown):

[0186] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;

[0187] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

[0188] (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);

[0189] (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;

[0190] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;

[0191] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;

[01...

Embodiment 3

[0244] Example 3 Functional detection of recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, and lvCAR19-scFv0.

[0245] 1. Cell-level expression detection of CAR gene:

[0246] (1) After the recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 and the control virus Mock infected PBMC cells, the collected cells were detected by RT-PCR for the mRNA transcription levels of CAR gene and scFv gene, and verified The expression of CAR gene and scFv gene, if the transcription level of CAR gene and scFv gene mRNA increases, it means that the transcription level of CAR gene and scFv gene is successfully expressed;

[0247] (2) After the recombinant lentiviral vectors lvCAR19-IL6RscFv1, lvCAR19-IL6RscFv2, lvCAR19-IL6RscFv3, lvCAR19-scFv0 and the control virus Mock infected PBMC cells, the collected cells were detected by western blot to detect the expression level of CAR protein to verify the expression...

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Abstract

The invention discloses a closed IL6R CAR-T transgenic vector for relieving CRS. The vector consists of an amicillin resistance gene AmpR sequence (SEQ ID NO.1), a prokaryotic replicon pUC Ori sequence (SEQ ID NO.2), a virus replicon SV40 Ori sequence (SEQ ID NO.3), an eWPRE-enhanced marmot hepatitis B virus post-transcriptional regulatory element (SEQ ID NO.11), a human EF1[alpha] promoter (SEQ ID NO.12), a lentivirus packaging cis element applied to lentivirus packaging, a human IL6R humanized single-chain antibody IL6RscFv1 (SEQ ID NO.21) or IL6RscFv2 (SEQ ID NO.22) or IL6RscFv3 (SEQ ID NO.23), an IRES ribosome bonding sequence (SEQ ID NO.25), an IL6 signal peptide (SEQ ID NO.26), a humanized antibody Fc segment (SEQ ID NO.27) and a chimeric antigen receptor for constituting a second-generation or third-generation CAR integrating recognition, transferring and promoting. In addition, the invention also discloses a construction method of the vector and an application of the vector inpreparing medicines for relieving CRS.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a carrier, in particular to a CAR-T transgene carrier for alleviating CRS by blocking IL6R. In addition, the present invention also relates to the construction method and application of the carrier. 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 therapy, monoclonal antibody th...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A61K48/00A61K39/00A61K35/17A61P35/00
CPCA61K35/17A61K39/0005A61K48/005C12N15/66C12N15/86C12N2740/15043C12N2800/107A61K38/1774A61K2035/124A61K2039/5156A61P29/00A61P35/00A61K39/001112C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2803C07K16/2866C07K2317/24C07K2317/622C07K2317/76C07K2319/03C07K2319/30C07K2319/33C12N2740/16043A61K48/00C07K14/70596C07K2317/624C12N2740/15041A61K39/00C07K14/5412C07K2317/21C07K2317/52C07K2317/73C07K2319/02C12N15/113C12N15/867C12N2760/00043C12N2760/00051
Inventor 祁伟俞磊康立清余宙
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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