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Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier as well as establishment method and application thereof

A technology of recombinant lentivirus and transgenic vector, which is applied in the field of medical biology to achieve the effect of narrow expression range and low expression level

Active Publication Date: 2016-09-28
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

It is worth noting that the above differences are only the conclusions obtained from in vitro experiments, and there is no report comparing the second-generation and third-generation CARs in vivo.

Method used

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  • Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier as well as establishment method and application thereof
  • Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier as well as establishment method and application thereof
  • Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier as well as establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction of recombinant lentiviral vector

[0069] 1. Materials

[0070] 1. The lentiviral backbone plasmid pLenti-3G ​​basic, the lentiviral packaging plasmids pPac-GP, pPac-R and the membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzyme were supplied by Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0071] 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:

[0072] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.26)

[0073] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0074] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.28)

[0075] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0076] VH-F: 5'-CACGCCGCCAGGCCGCAAGTCCAACTGGTCCAAAGT-3' (SEQ ID NO.30)

[0077] VH-R: 5'-AGAGCTAA...

Embodiment 2

[0160] Example 2 Concentration and detection of recombinant lentiviral vector

[0161] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0162] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0163] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0164] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray 70% ethanol on the surface to sterilize them, put them on a clean table and irradiate them with ultraviolet light for 30 minutes to sterilize them. It can also be sterilized by high temperature and moist heat;

[0165] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0166] (5) Cover the metal cover, balance the centrifuge tube together with the metal cover, and adjust with 1XPBS to make the weight deviation within 0.02g;

[0167] (6) Place the balanced centrifuge tubes symmetrically in the ul...

Embodiment 3

[0244] Example 3 Functional detection of recombinant lentiviral vectors lvCARmeso-CLA, lvCARmeso-CLB, and lvCARmeso-OLC.

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

[0246] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCARmeso-CLA, lvCARmeso-CLB, and lvCARmeso-OLC, collect the cells and detect CAR mRNA transcription levels by RT-PCR to verify the expression of CAR genes. If the CAR mRNA transcription levels increase, then It shows that the transcription level expression of CAR gene is successful;

[0247] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCARmeso-CLA, lvCARmeso-CLB, and lvCARmeso-OLC, collect the cells and detect the expression level of CAR protein by western blot to verify the expression of CAR gene. If the expression level of CAR protein increases, then It shows that the translation level expression of CAR gene is successful;

[0248] (3) Infect the cells with lvCARmeso-CLA, lvCARmeso-CLB, lvCARmeso-OL...

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Abstract

The invention discloses a Mesothelin-targeted replication-defective recombinant lentivirus CAR-T transgenic carrier which comprises a pronucleus replicon pUC Ori sequence for plasmid replication, an amicillin resistance gene AmpR containing sequence for mass amplification of target strain, virus replicon SV40Ori sequence for enhancing replication in eukaryocyte, a lentivirus packaging cis-element for lentivirus packaging, a ZsGreen1 green fluorescent protein for green fluorescence expression of eukaryocyte, an IRES ribosome combination sequence for joint transcriptional expression of protein, a human EF1(alpha) promoter for the eukaryotic transcription of chimeric antigen receptor gene, a chimeric antigen receptor for forming a second-generation CAR or third-generation CAR integrating identification, transfer and start, and an eWPRE element for improving the transgenic expression efficiency. Moreover, the invention also discloses an establishment method and application of the carrier. In the invention, the secretion of cell factors and the in-vitro killing effect of CAR-T cells can be remarkably enhanced, and the effect of clinical treatment of malignant pleural mesothelioma and pancreatic cancer is outstanding.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a vector, in particular to a CAR-T transgene vector of a replication-deficient recombinant lentivirus targeting Mesothelin. 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). This biological process is complex and is still under investigation. In the 1990s, several scientific research groups have discovered tumor antigens, and T lymphocytes can recognize these tumor antigens in a major histocompatibility complex (MHC)-dependent manner. [0003] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Non-specific immunotherapy ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66A61K35/17A61P35/00
CPCA61K35/17C12N15/66C12N15/86C12N2740/15043C12N2800/107C12N2810/40C12N2820/10
Inventor 祁伟俞磊欧黄思
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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