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Molecule, cell expressing same, and preparation method and use thereof

A cell and receptor molecule technology, applied in chimeric immunosuppressive checkpoint receptor molecules, in the field of pharmaceutical applications, can solve problems such as defects, increase cell killing effect, and cells lose body functions, achieve low off-target rate, strong immunity Activating effect, promoting anti-tumor immune effect

Active Publication Date: 2018-01-23
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In addition, CAR T co-expressing diabody PD-1 and CD19 / Mesothelin / PSCA has also been reported in the prior art, although this expands the range of targets (expressing PD-1 or CD19 / Mesothelin / PSCA or both at the same time) tumor cells), but ignored the side effects caused by blindly increasing cell killing: cytokine storm, off-target phenomenon (normal cells also express two antigens or one of them, causing CAR T to kill normal cells, causing the body's functional defects in the absence of normal cells )

Method used

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  • Molecule, cell expressing same, and preparation method and use thereof
  • Molecule, cell expressing same, and preparation method and use thereof
  • Molecule, cell expressing same, and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, preparation of S-CTL cells

[0072] Plasmid construction

[0073] 1) Through gene synthesis, "immunosuppressive checkpoint molecule (signal peptide + extracellular segment + transmembrane region) - immunostimulatory signal 1 intracellular segment - immune stimulatory signal 2 (usually TLR1 or TLR2) intracellular segment - CD3ζ ” DNA sequence, the beginning and end of the DNA sequence contain PmeI and Spe1 restriction enzyme sites respectively. Synthetic sequence maps such as figure 1 shown.

[0074]For the gene sequence synthesized in the examples of the present invention, please refer to SEQ NO.1 (in order to prepare S-CTL targeting PD-1, that is, SP-CTL, the synthetic DNA sequence has a structure of PD-1 signal peptide + extracellular Segment+transmembrane region-CD28-TLR1 / 2-CD3ζ) and SEQNO.2 (for the preparation of S-CTL targeting CTLA-4, i.e. SC-CTL, and the synthetic DNA sequence, the structure is CTLA-4 signal peptide+ Extracellular segment+tr...

Embodiment 2

[0095] Example 2. In vitro killing effect of SP-CTL cells on human leukemia cell line JURKAT

[0096] 1) Transduce and express Luciferase in the leukemia cell line JURKAT to construct the JURKAT-GL reporter cell line;

[0097] 2) SC-CTL GFP positive cells and JURKAT-GL cells were mixed and co-cultured at a ratio of 1:2 (the control group added GFP T cells, the blank group did not add T cells, and each group set up three replicate wells) ;

[0098] 3) After 18 hours, gently use a pipette to remove half the volume of the medium supernatant, add an equal volume of luciferase substrate (concentration: 300 μg / ml), and mix well;

[0099] 4) The RLU (relative light unit) was measured by a multi-functional microplate reader, and the measurement time was set to 1 second.

[0100] 5) Calculation formula of killing ratio: 100%×(blank well reading-experimental well reading) / blank well reading, the in vitro killing ratio of SP-CTL cells to human leukemia cell line JURKAT is as follows ...

Embodiment 3

[0101] Example 3. In vitro killing effect of SP-CTL cells on non-small cell lung cancer H460

[0102] 1) transduction and expression of luciferase (Luciferace) in non-small cell lung cancer H460, and construction of H460-GL reporter cell line;

[0103] 2) SP-CTL GFP-positive cells and H460-GL cells were mixed and co-cultured at a ratio of 1:2 (the control group was added with GFP T cells, the blank group was not added with T cells, and three replicate wells were set for each group) ;

[0104] 3) After 18 hours, gently use a pipette to remove half the volume of the medium supernatant, add an equal volume of luciferase substrate (concentration: 300 μg / ml), and mix well;

[0105] 4) The RLU (relative light unit) was measured by a multi-functional microplate reader, and the measurement time was set to 1 second.

[0106] 5) Calculation formula of killing ratio: 100%×(blank well reading-experimental well reading) / blank well reading (such as Figure four ), the ratio of SP-CTL cel...

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Abstract

Provided are a chimeric immune inhibitory checkpoint receptor molecule, a nucleic acid encoding the same, a construct, expression vector and transformed cell containing the nucleic acid, and a pharmaceutical use thereof. The chimeric immune inhibitory checkpoint receptor molecule comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain and the optional transmembrane domain are corresponding domains of an immune inhibitory checkpoint receptor molecule or are genetically engineered on the basis of the corresponding domains. The intracellular domain is one or more immune activation signal domains. The transformed cell has a strong tumor-killing effect and can abrogate a tumor-immunosuppressive microenvironment, thereby strengthening an immune response in the tumor microenvironment. The transformed cell also has a self-regulation function and can alleviate side effects such as an overactive, non-specific immune response and a cytokinestorm.

Description

technical field [0001] The present invention relates to the technical field of tumor cell immunotherapy, in particular, to a chimeric immunosuppressive checkpoint receptor molecule, a nucleic acid encoding it, a construct containing the nucleic acid, an expression vector, and a transformed cell, as well as their Pharmaceutical use. Background technique [0002] Current status of CAR-T cell tumor therapy [0003] In October 2015, the National Cancer Center released the cancer prevalence data of Chinese residents for the first time in the famous cancer professional journal "Cancer Letters". The results show that the number of cancer cases diagnosed and still alive in my country within 5 years is about 7.49 million (including 3.68 million male patients and 3.81 million female patients). The overall 5-year cancer prevalence rate is 556 / 100,000. This reflects the urgent needs of cancer patients for research progress in new treatments. [0004] Immunotherapy has become a new ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/70503C07K14/7051C07K14/70596C12N5/0636C12N15/86C07K2319/02C07K2319/03C12N2510/00C12N2740/15043C12N2800/107A61K38/17C07K19/00A61P35/00
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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