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A tumor precision T cell containing an efficient killing mechanism and its use

A cell and tumor technology that can be used in the fields of immunology and cell biology to solve problems such as decreased therapeutic efficacy

Active Publication Date: 2020-09-15
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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  • A tumor precision T cell containing an efficient killing mechanism and its use
  • A tumor precision T cell containing an efficient killing mechanism and its use
  • A tumor precision T cell containing an efficient killing mechanism and its use

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 GGGGGGGGG FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:1)。

[0090] Among them, the signal peptide (MALPVTALLLPLALLLHAARPS, SEQ ID NO:3) is underlined with a dotted line, and the CD20 epitope (NIYNCEPANPSEKNSPSTQYCYSI, SEQ ID NO:4) recognized by the CD20 commercial antibody MatThera is underlined with a wavy line, and the linker sequence with a single underline. Bold is HERIN (GTHSLPPRAAVVPLRMQPGPAHPVLSFLRPS...

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 (tumor infiltrating lymphocytes, TIL), a half-volume liquid change was performed every 1-2 days. Once the wells were overgrown with TILs and all adherent cells had been removed, the TILs in e...

Embodiment 3

[0104] Example 3: Genetic Modification of TIL Cells

[0105] Collect 1×10 7 For TIL cells (prepared in Example 2), transfect 6 μg of the pNB328-herinCAR plasmid (prepared in Example 1) into the nucleus through a Lonza 2b-Nucleofector instrument, place 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 Company) 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 tumor precision T cells containing efficient killing initiation mechanisms and their uses. Specifically, the present invention relates to a chimeric antigen receptor with medium affinity binding properties to membrane antigens broadly expressed on the surface of tumor cells and a new generation of tumor-precision T cells, also known as Baize T cells. T cell activation is quickly initiated under the action of chimeric antigen receptors with medium affinity binding properties. The chimeric antigen receptor activation signal will be superimposed with the TCR signal that naturally recognizes tumor antigens in tumor-specific T cells, activating tumor-specific T cells in Proliferate and grow in the tumor microenvironment, and accurately kill tumor cells through tumor antigen-specific TCR. The tumor-precision T cells of the present invention have good anti-tumor application prospects.

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 Patents(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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