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GPC3-targeted CAR and CAR-NK cell using same

A NK cell, targeted technology, applied in the field of CAR-NK cells, can solve the problems of lack of research on NK cell CAR, and achieve the effect of efficient killing, large release and high toxicity

Active Publication Date: 2021-01-12
SHANTOU PROCAPZOOM BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, there is still a lack of research on the structural modification of NK cell CARs.

Method used

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  • GPC3-targeted CAR and CAR-NK cell using same
  • GPC3-targeted CAR and CAR-NK cell using same
  • GPC3-targeted CAR and CAR-NK cell using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Sequence selection of CAR

[0034]The sequence structure of a conventional chimeric antigen receptor for GPC3 (abbreviated as conventional CAR-GPC3) is shown in Figure 1. However, the structure of this chimeric antigen receptor mainly follows the structure of CAR-T, and when it is applied in NK cells, the transduction rate is not high and the lethality is not strong. After continuous exploration and testing, the present invention screened out a chimeric antigen receptor (called CAR1-GPC3) composed of the amino acid sequence shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2.

[0035] 2. Construct sequence vector

[0036] The nucleotide sequence of CAR1-GPC3 was amplified by PCR technology, and then the CAR1-GPC3 sequence was cloned into a vector containing T2A sequence, EF1a promoter sequence, GFP sequence, Puro sequence and IRES sequence (pWPXLd-2A-EGFP plasmid ), a sequence vector (called pWPXLd-CAR1-GPC3-2A-EGFP vector) with T2A sequence...

Embodiment 2

[0044] Example 2: Detection of the killing effect of CAR1-GPC3-NK 92 cells constructed in Example 1 and conventional CAR-GPC3-NK 92 cells on liver cancer cells

[0045] (1) Primary tumor cell culture: tumor tissue samples were obtained during surgery, placed in DMEM+10% FBS preservation solution, placed in an ice box and taken to the cell chamber. Tissues such as blood clots and fat were removed in D-PBS solution, and the complete tissue was rinsed in a centrifuge tube filled with cleaning solution for 3-4 times, then transferred to a petri dish for cleaning, and washed twice.

[0046] After cleaning, put the tissue in a clean Petri dish, add a small amount of medium, and use scissors and tweezers to cut the tissue until there are no obvious large pieces of tissue. Add freshly prepared digestive solution, put it in the incubator for digestion for 30min, and observe the digestion situation during this period. After digestion is complete, transfer it to a centrifuge tube to col...

Embodiment 3

[0058] Example 3: Detection of the killing effect of the CAR1-GPC3-NK 92 cells constructed in Example 1 on different cancer cells

[0059] The specific operation is the same as that in Example 2, except that the killing target is replaced by other cancer cells.

[0060] image 3 Comparison chart of killing of different tumor cells by CAR1-GPC3-NK 92 cells constructed for the present invention. It can be seen from the comparison chart that the CAR1-GPC3-NK 92 cells of the present invention have a better killing effect in hepatocellular carcinoma than other tumors.

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PUM

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Abstract

The invention provides a GPC3-targeted chimeric antigen receptor. The GPC3-targeted chimeric antigen receptor comprises a single-chain antibody for specifically recognizing GPC3, a transmembrane region structural domain and an intracellular region structural domain which are sequentially connected in series; and the chimeric antigen receptor has a full-length amino acid sequence as shown in SEQ IDNO: 1. The GPC3-targeted chimeric antigen receptor provided by the invention can significantly improve the lethality of NK cells to liver cancer cells, and has high transduction rate.

Description

technical field [0001] The invention belongs to the field of cellular immunotherapy, and in particular relates to a CAR targeting GPC3 and a CAR-NK cell using the same. Background technique [0002] Hepatocellular carcinoma (HCC) is a primary epithelial neoplasm of the liver, often associated with underlying viral, metabolic, autoimmune, or alcoholic chronic liver disease. Hepatocellular carcinoma is one of the major forms of primary liver cancer, accounting for 85-90% of all primary liver cancers. [0003] Treatment for HCC depends on the stage of the disease, usually very early, early, intermediate, advanced, and terminal (terminal), with appropriate treatment for each stage. Patients with early-stage HCC (stage A, asymptomatic early-stage tumors) can choose among possible treatment options (tumor resection, transplantation, or ablation). Patients with intermediate-stage HCC (stage B, asymptomatic multinodular HCC) are generally treated with chemoembolization (TACE). Pa...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/303C07K14/7051C12N15/86C12N5/0646A61K39/001174A61P35/00C07K2319/03C07K2319/33C07K2317/622C12N2740/15043C12N2800/107C12N2510/00A61K2039/5158
Inventor 沈振波
Owner SHANTOU PROCAPZOOM BIOSCIENCES CO LTD
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