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Tumor precision T cell containing efficient killing starting mechanism and application of tumor precision T cell

A cell and tumor technology, applied in the field of immunology and cell biology, can solve the problem of declining therapeutic efficacy

Active Publication Date: 2016-02-17
SHANGHAI CELL THERAPY RES INST +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CAR + The "cascade" effect caused by off-target T cells is very fast, and these suicide systems may not be able to function in time
Another approach is to reduce CAR + The number of T cells reinfused, this program will reduce the efficacy of treatment

Method used

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  • Tumor precision T cell containing efficient killing starting mechanism and application of tumor precision T cell
  • Tumor precision T cell containing efficient killing starting mechanism and application of tumor precision T cell
  • Tumor precision T cell containing efficient killing starting mechanism and application of tumor precision T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Synthesis of CAR expression cassette and construction of expression vector

[0087] According to the composition structure of herinCAR (for the model diagram, see figure 1 ), spliced ​​into the whole fusion amino acid sequence and coding DNA expression frame:

[0088] The amino acid residue sequence of herinCAR is:

[0089] GGGGGGGGG GGG GGGGGG FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQIDNO:1)。

[0090] Among them, the signal peptide (MALPVTALLLPLALLLHAARPS, SEQIDNO:3) is underlined with a dotted line, and the CD20 epitope (NIYNCEPANPSEKNSPSTQYCYSI, SEQIDNO:4) recognized by the CD20 commercial antibody MabThera is underlined with a wavy line, and the linker sequence is underlined. Bold is HERIN (GTHSLPPRAAVVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLPLA...

Embodiment 2

[0101] Example 2: Isolation and culture of cholangiocarcinoma tissue-derived TIL

[0102] Collect freshly resected cholangiocarcinoma specimens and process them immediately under sterile conditions. The specific method is as follows: remove the normal tissue and necrotic area around the cholangiocarcinoma specimen, and remove the 1-2mm in size from different areas of the specimen 3 Place a small tissue block in each well of a 24-well plate. Add 2 mL of complete medium (GT-T551 medium containing 10% FBS) and 3000 IU / mL IL-2 to each well. Place the 24-well plate at 37 °C, 5% CO 2 cultured in an incubator. On the 5th to 6th day after the initiation of culture, a half-volume medium change was performed for all wells. Afterwards, according to the growth of tumor infiltrating lymphocytes (tumorinfiltratinglymphocyte, TIL), a half volume of medium was changed every 1-2 days. Once the wells were overgrown with TILs and all adherent cells had been removed, the TILs in each overg...

Embodiment 3

[0104] Example 3: Genetic Modification of TIL Cells

[0105] Collect 1×10 7 TIL cells (prepared in Example 2), transfected 6 μg of pNB328-herinCAR plasmid (prepared in Example 1) into the nucleus by Lonza2b-Nucleofector instrument, placed at 37 ° C, 5% CO 2 Culture in an incubator; transfer to a 6-well plate containing 30ng / mL anti-CD3 antibody and 3000IU / mL IL-2 (purchased from Novoprotein) after 6 hours, and place at 37°C, 5% CO 2 Incubator culture. After the cells were confluent, they were subcultured at a ratio of 1:5. The TIL cells containing herinCAR, referred to as Bz-T cells, were obtained and used in the following Examples 4-7.

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Abstract

The invention belongs to the fields of immunology and cell biology, and relates to a tumor precision T cell containing an efficient killing starting mechanism and an application of the tumor precision T cell, in particular to a CAR (chimeric antigen receptor) having moderate-affinity binding characteristic with a broad-spectrum expression membrane antigen on the tumor cell surface as well as a new-generation tumor precision T cell, namely, Baize T. T cell activation is started rapidly under the action of the CAR having the moderate-affinity binding characteristic, an activation signal of the CAR is superposed with a TCR (T cell receptor) signal with tumor antigen natural recognition capacity in a CTL (tumor-specific cytotoxic T lymphocyte), the CTL is activated to proliferate and grow in a tumor microenvironment, and a tumor cell is killed preciously by the tumor-antigen-specific TCR. The tumor precision T cell has broad anti-tumor application prospect.

Description

technical field [0001] The invention belongs to the field of immunology and cell biology, and relates to tumor precision T cells containing an efficient killing initiation mechanism and uses thereof. Specifically, it relates to a T cell therapy technology, in particular, this technology targets tumor-specific T cells, and transfers elements that can quickly initiate and enhance the killing function of T cells through transgenic modification methods, such as recognizing the broad-spectrum expression of tumor cells, Moderate affinity chimeric antigen receptor gene. The present invention also relates to the use of the obtained T cells for treating malignant tumors. Background technique [0002] In recent years, tumor immunotherapy has made major breakthroughs. Using chimeric antigen receptors (chimeric antigen receptors, CAR) modified T cells (CAR-T for short) to treat relapsed and refractory B-cell leukemia, the effective rate reached 90%; using PD-1 antibody monotherapy to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K35/17A61K39/395A61P35/00
CPCA61K39/395C07K16/28C12N5/10A61K39/4631A61K2239/38A61K2239/31A61K39/4644A61K2239/53A61K39/4611
Inventor 钱其军金华君李林芳叶真龙章浩王颖吴红平吴孟超
Owner SHANGHAI CELL THERAPY RES INST
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