Molecular brake for rapidly stopping killing effect of CAR-T (T cell engineered with chimeric antigen receptors) and application of molecular brake

A technique for cellular and intracellular signaling, used in the fields of immunology and cell biology

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

[0009] However, the cytokine "cascade" effect caused by off-target CAR-T cell

Method used

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  • Molecular brake for rapidly stopping killing effect of CAR-T (T cell engineered with chimeric antigen receptors) and application of molecular brake
  • Molecular brake for rapidly stopping killing effect of CAR-T (T cell engineered with chimeric antigen receptors) and application of molecular brake
  • Molecular brake for rapidly stopping killing effect of CAR-T (T cell engineered with chimeric antigen receptors) and application of molecular brake

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Design and expression of CD19-specific CAR containing CD20 molecular brake Vector construction

[0083] According to the different insertion positions of the CD20 molecular brake, 10 types of CD19-specific CARs were designed and constructed (see figure 1), were spliced ​​into the entire fused amino acid sequence and the coding DNA expression frame, and were named CAR1920-1, CAR1920-2, CAR1920-3, CAR1920-4, CAR1920-5, CAR1920-6, CAR1920-7, CAR1920-8, CAR1920- 9. CAR1920-10. in:

[0084] The amino acid residue sequence of CAR1920-1 is: (546aa)

[0085] MALPVTALLLPLALLLHAARPS GG GGGGGGG DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS...

Embodiment 2

[0147] Example 2: Genetic modification of T cells

[0148] Prepare 1×10 7 Peripheral blood mononuclear cells (PBMC) obtained by fresh isolation, respectively, 6 μg of pNB328-CAR1920-1, pNB328-CAR1920-2, pNB328-CAR1920-3 prepared in Example 1, The pNB328-CAR1920-4 plasmid was transfected into the nucleus, and 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. After 3 passages, the proportion of CAR-positive cells was detected by flow cytometry (goat anti-mouse Fab2' antibody, purchased from Jackson, USA).

Embodiment 3

[0150] Example 3: In vitro brake function test of CAR19-T cells containing CD20 molecular brake Measurement

[0151] 1. Construction of HEK293 cells (target cells) stably expressing CD19 gene

[0152] 1) According to the DNA coding sequence of the extracellular region and transmembrane region of CD19, Shanghai Jereh Biotechnology Co., Ltd. was commissioned to synthesize the whole gene and insert it into the pNB328 vector. The constructed vector was named pNB328-CD19.

[0153] The coding sequence of CD19 extracellular region and transmembrane region is as follows: (960bp)

[0154] ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCT...

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Abstract

The invention belongs to the fields of immunology and cytobiology and relates to a molecular brake for rapidly stopping the killing effect of CAR-T (T cell engineered with chimeric antigen receptors) and an application of the molecular brake. Specifically, the molecular brake serving as an element is inserted into the specific position in the CAR and can rapidly stop the killing effect of the CAR-T on a target cell, thereby effectively improving the safety of CAR-T treatment. The invention further relates to immunoreactive cells expressing the CAR and an application of the immunoreactive cells in preparation of drugs for treating malignant tumors or virus infectious diseases.

Description

technical field [0001] The invention belongs to the field of immunology and cell biology, and relates to a molecular brake for quickly stopping the killing effect of CAR-T cells and its application. Specifically, the molecular brake is inserted as a component into a specific position inside chimeric antigen receptors (chimeric antigen receptors, CAR), which can quickly stop the killing effect of chimeric antigen receptor-modified T cells on target cells. The present invention also relates to the use of the CAR-T cells expressing the molecular brake for the medicine of malignant tumors and viral infectious diseases. Background technique [0002] Chimeric antigen receptor engineering T cells (chimeric antigen receptor engineering T cells, referred to as CAR-T cells) have achieved great success in the treatment of refractory B cell malignancies, with a complete remission rate of 90%; they are also used in the treatment of other solid tumors It shows a good application prospect...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K35/17A61P35/00A61P31/12G01N33/68
Inventor 钱其军金华君李林芳叶真龙胥阶英吕赛群吴红平吴孟超
Owner SHANGHAI CELL THERAPY RES INST
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