Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell

一种免疫效应细胞、双靶向的技术，应用在血液/免疫系统细胞、用于靶向特定细胞融合、抗动物/人类的免疫球蛋白等方向，能够解决没有ASGPR1、没有CAR免疫效应细胞联合应用等问题

Active Publication Date: 2016-06-29
CARSGEN THERAPEUTICS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In the current prior art, there has never been a report on ASGPR1 as a target for immune cell therapy, let alone the combined application of GPC3, ASGPR1 and CAR immune effector cells

Method used

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  • Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell
  • Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell
  • Dual-targeting genetically modified immunologic effector cell aiming at GPC3 (Glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of dual-targeting genetically modified immunologic effector cell

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preparation example Construction

[0047] Conventional methods for preparing "chimeric antigen receptor immune effector cells" are known to those skilled in the art, including allowing them to express an extracellular antigen (such as a tumor-associated antigen) binding region; and intracellular costimulatory cell molecule intracellular structure Domains, such as one or more of CD28 (preferably including CD28a, CD28b), CD137, CD27, CD3ζ (preferably CD3ζ intracellular domain), CD8, CD19, CD134, CD20, FcRγ. Through their combination with corresponding ligands, the second signal of immune effector cells is activated, the proliferation ability of immune cells and the secretion function of cytokines are enhanced, and the survival time of activated immune cells is prolonged.

[0048] In the present invention, unless otherwise specified, the tumor refers to a tumor that is both positive for GPC3 and ASGPR1, for example, the tumor includes: liver cancer.

[0049] The present inventors unexpectedly found in the research...

Embodiment 1

[0066] Example 1, Detection of the expression levels of ASGPR1 and GPC3 in various liver cancer cell lines and the establishment of stably expressed liver cancer cell lines

[0067] 1. Detection of expression levels of ASGPR1 and GPC3 in various liver cancer cell lines

[0068] The inventors first detected the expression of ASGPR1 and GPC3 in various liver cancer cell lines.

[0069]Western-blot detection method: HepG2, Huh-7, Hep3B, PLC / PRF / 5, MHCC-97H, SK-HEP-1 and SMMC-7721 cells in good growth state were washed twice with D-PBS respectively , add T-REP tissue cell lysate, lyse on ice for 1h, centrifuge at 12000rpm for 10min, and collect the supernatant. The BCA method was used to quantify according to the instructions of the BCA assay kit, and the absorbance at a wavelength of 570 nm was detected by an ultraviolet spectrophotometer. Afterwards, the samples collected above were electrophoresed by 12% SDS-PAGE, and 20 μg of total protein were respectively loaded. After SD...

Embodiment 2

[0086] Embodiment 2, GCART cell (or claiming GZT cell), the establishment of ACCRT cell (or claiming 28BBT cell) and GZ+28BBT cell

[0087] 1. Construction of pWPT-eGFP-F2A-GPC3-CD3ζ

[0088] The amplification of the scFv (GPC3) sequence uses the single-chain bifunctional antibody nucleotide GPC3 / CD3 as a template. For the sequence of the template, refer to SEQ ID NO: 9 in Chinese patent application 201210480326.x. The primer pair used in the amplification is upstream primer 5'-gatgttgtgatgactcagtctc-3' (SEQ ID NO: 9) and downstream primer 5'-gcgctggcgtcgtggttgaggagacggtgaccag-3' (SEQ ID NO: 10) for the amplification of scFv (GPC3) (SEQ ID NO: 11, It encodes SEQIDNO: 12); the size of the target amplified band is 746bp. The PCR amplification conditions were pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 58°C, 40s; extension: 68°C, 40s; 30 cycles, and then a total extension of 68°C, 10min. The PCR amplified bands were confirmed by agarose gel electrophoresis...

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Abstract

The invention relates to a dual-targeting genetically modified immunologic effector cell aiming at GPC3 (glypican-3) and ASGPR1 (asialoglycoprotein receptor 1) and applications of the dual-targeting genetically modified immunologic effector cell. The invention discloses the gene-modified immunologic effector cell capable of simultaneously identifying GPC3 and ASGPR1 for the first time, and the gene-modified immunologic effector cell can be used for the treatment of the GPC3 and ASGPR1 double-positive tumor, such as liver cancer.

Description

technical field [0001] The present invention relates to the field of tumor immunotherapy, and more specifically, the present invention relates to a transgenic immune effector cell dual-targeting GPC3 and ASGPR1 and its application. Background technique [0002] The role of T lymphocytes in tumor immune response has been paid more and more attention. Adoptive immunotherapy based on T lymphocytes has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the curative effect in most tumors is still not satisfactory. In recent years, based on the discovery that the recognition of target cells by CTLs depends specifically on T lymphocyte receptors (TCell Receptor, TCR), the scFv of antibodies against tumor cell-associated antigens and CD3ζ or FcεRIγ of T lymphocyte receptors in cells The signal activation motif is fused into a chimeric antigen receptor (CAR), which is genetically modified on the su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/63A61K39/395A61P35/00
CPCA61K39/395C12N5/10C12N15/63C07K16/28C07K14/00A61K2039/505C07K2319/33A61P35/00C07K16/303C07K2317/622C07K2319/03C07K14/7051C12N5/0636A61K39/4644A61K39/4631A61K2239/31A61K39/4611A61K2239/38A61K39/464474A61K39/464402A61K35/17C07K14/70517C07K14/70521C07K14/70578C07K16/2851C07K2319/02C12N15/86C12N2740/15043
Inventor 王华茂蔡秀梅赵红霞宋波陈远梅朱寅玉
Owner CARSGEN THERAPEUTICS
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