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Chimeric antigen receptor comprising Fcgamma RI and application thereof

A chimeric antigen receptor and chimeric receptor technology, applied in the field of biomedicine, can solve problems such as low affinity

Active Publication Date: 2019-09-06
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FcγRI has high affinity for human IgG1, IgG3 and mouse IgG2a, but low affinity for human IgG4 and IgG2

Method used

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  • Chimeric antigen receptor comprising Fcgamma RI and application thereof
  • Chimeric antigen receptor comprising Fcgamma RI and application thereof
  • Chimeric antigen receptor comprising Fcgamma RI and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Construction of pCAR-Fcγ receptor I lentiviral overexpression vector

[0116] NCBI obtained the signal peptide region and extracellular region gene sequence and corresponding amino acid sequence of Fcγ receptor Ⅰ, and obtained the gene sequence and corresponding amino acid sequence of transmembrane region CD28, intracellular signal region 4-1BB and CD3ζ by the same method to form Fcγ receptor Ⅰ chimeric gene, figure 1 It is a schematic diagram of the chimeric gene structure of Fcγ receptor Ⅰ. BstBI and XbaI restriction sites were added to both ends of the gene, the total length was 1692bp, and it was sent to Shanghai Jierui Bioengineering Co., Ltd. for synthesis, and the vector PGH-Z1528G for cloning the target gene was obtained. The lentiviral overexpression vector pCAR was purchased from Aikangde Biomedical Technology (Suzhou) Co., Ltd. and Hanbio, and the specific map refers to figure 2 .

[0117] The PGH-Z1528G and pCAR plasmid vectors were respectivel...

Embodiment 2

[0120] Example 2 Preparation of pCAR-Fcγ receptor I lentivirus

[0121] The lentivirus preparation, purification and concentration of the chimeric gene Fcγ receptor I adopt the following methods:

[0122] 1. Cryopreservation of HEK293T cells

[0123] When the cells are frozen, the cell density is preferably 60% to 80%. At this time, the cells are in the logarithmic growth phase, the proliferation is vigorous, and the cells are in good condition.

[0124] 1) Take out the cells, add trypsin to digest, stop the digestion, transfer the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min;

[0125] 2) Prepare the cell cryopreservation solution according to the ratio of DMSO to FBS of 1:9 (V / V), and the prepared cell cryopreservation solution needs to be pre-cooled in a 4°C refrigerator;

[0126] 3) After centrifugation, discard the supernatant of the culture medium, add pre-cooled cryopreservation solution, resuspend the cell pellet with a pipette gun, dispense...

Embodiment 3

[0133] Example 3 Preliminary verification of overexpression of Fcγ receptor Ⅰ in Jurkat cells

[0134] Jurkat cells are human peripheral blood leukemia cells, which are widely used in the research of blood tumors and immunology. Because their physiological characteristics are the closest to those of T lymphocytes, this disclosure uses Jurkat cells to initially verify the overexpression of Fcγ receptor Ⅰ. On the one hand, it can be judged whether the packaged virus is correct and infectious; on the other hand, it can be judged whether the Fcγ receptor I protein can be correctly transcribed and translated.

[0135] Recovery of Jurkat cells

[0136] For the specific steps of recovery of Jurkat cells, refer to the recovery of HEK293T cells above, but it should be noted that the medium used for Jurkat cells is RPMI 1640, and 10% FBS can be added before use.

[0137] Passaging of Jurkat cells

[0138] Jurkat cells are suspension cells. When the cell density is 60%-80% under a micr...

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Abstract

The present disclosure relates to the field of biomedicine, and in particular to a chimeric antigen receptor comprising an Fcgamma receptor I and uses thereof. The chimeric antigen receptor comprisesA) a Fc gamma receptor I extracellular domain, B) a hinge region, C) a transmembrane domain, and D) an intracellular signaling domain. The chimeric antigen receptor can improve the therapeutic effectof antibody drugs through the ADCC effect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric antigen receptor containing FcγRI and its application. Background technique [0002] Tumors, especially malignant tumors, are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve. [0003] A large number of studies have shown that tumors can escape the surveillance of the immune system through multiple mechanisms during t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70535C07K2319/02C07K2319/03C12N15/86C12N2740/15043
Inventor 汪洋
Owner REGENECORE BIOTECH CO LTD
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