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B7H3-targeted fully-humanized chimeric antigen receptor, iNKT cell and application thereof

A chimeric antigen receptor, fully human technology, applied in the field of immunology and molecular biology, can solve the problems of cancer cell infiltration, curative effect decline, drug tolerance, etc., and achieve the effect of strong proliferation ability and elimination of tumor cells

Active Publication Date: 2021-10-15
江苏集萃崛创生物科技研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional tumor therapy, including surgery, chemotherapy and radiotherapy, has limitations: surgery often cannot eradicate cancer cells due to infiltration into adjacent or metastatic tissues; chemotherapy and radiotherapy are limited by toxicity and damage to other normal tissues in the body
The popular targeted therapy in recent years can design corresponding therapeutic drugs for the identified carcinogenic sites at the cell molecular level. When the drugs enter the body, they will specifically select the carcinogenic sites to bind and act, causing tumor cells to specifically die. However, molecular targeted drugs can only have an effect on tumors with specific gene mutations. If the target tumor gene is mutated, drug resistance will occur, resulting in a decline in curative effect and even serious adverse reactions.

Method used

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  • B7H3-targeted fully-humanized chimeric antigen receptor, iNKT cell and application thereof
  • B7H3-targeted fully-humanized chimeric antigen receptor, iNKT cell and application thereof
  • B7H3-targeted fully-humanized chimeric antigen receptor, iNKT cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Preparation of fully human B7H3-CAR-iNKT

[0122] 1. Experimental method

[0123] (1) Preparation of iNKT

[0124] 1) Separation of PBMCs: collect peripheral blood from the donor, dilute the whole blood with an equal volume of normal saline, add the lymphocyte separation solution and the diluted blood to the centrifuge tube at a ratio of 1:2, centrifuge at 2000rpm / min for 20 minutes, and collect the buffy coat cells , washed twice with normal saline, and centrifuged at 1500rpm / min for 8 minutes to obtain PBMCs from peripheral blood mononuclear cells;

[0125] 2) Induce iNKT cells: resuspend PBMCs with lymphocyte medium, adjust the concentration to 2×10 6 / mL, add α-Galcer, IL-2, IL-21, IL-4 and GM-CSF, seed the cells in a 24-well plate, and place at 37°C, 5% CO 2 Incubator, observe the cell state every day, and change the medium in half every other day;

[0126] 3) Magnetic bead sorting of iNKT cells: Collect induced cells on the 10th day, resuspend with 5...

Embodiment 2

[0141] Example 2 Verification of the ability of fully human B7H3-CAR-iNKT cells to kill renal cancer cells in vitro

[0142] 1. Experimental method

[0143] (1) CFSE test to detect proliferation ability

[0144] CFSE staining: Collect B7H3.CAR-iNKT cells, wash the cells with 0.1% FBS / PBS and resuspend, add CFSE working solution for staining to a final concentration of 1.5 μM, incubate at room temperature for 10 minutes, add FBS and incubate at 37°C for 10 minutes to terminate After staining, wash the cells twice with 2% FBS / PBS, and finally resuspend the T cell medium for use;

[0145]Kidney cancer cells 786-O and OSRC-2 were plated overnight, and the above stained effector cells were added according to the effect-to-target ratio of 1:2, and the effector cell group alone was used as a control. After 5 days, the cells were collected, washed, and CFSE fluorescence was detected by flow cytometry Signal, to analyze the proliferation ability of B7H3.CAR-iNKT cells.

[0146] (2) ...

Embodiment 3

[0152] Example 3 Verification of fully human B7H3-CAR-iNKT cells clearing renal cancer xenografts in vivo

[0153] 1. Experimental method

[0154] Purchase 6-week-old male NCG mice and inject 2 × 10 via subcutaneous injection 6 OSRC-2-Ffluc-GFP was used to construct the subcutaneous xenograft tumor model of renal cancer in mice. After tumor formation on the 12th day, the mice were randomly divided into Ctrl group, iNKT group, and B7H3.CAR-iNKT group, with 5 mice in each group, 3 groups in total; On days 0 and 8, respectively, iNKT and B7H3.CAR-iNKT cells were infused into the tail vein for treatment, 5×10 6 / mouse; the Ctrl group was only infused with PBS; the therapeutic effect was observed by measuring the tumor volume twice a week, and the survival of CAR-iNKT in vivo was detected by blood collection from the submandibular vein, and the survival period of the mice was recorded.

[0155] 2. Experimental results

[0156] See the experimental results Figure 7 A-D, Figur...

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PUM

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Abstract

The invention discloses a B7H3-targeting fully human chimeric antigen receptor, an iNKT cell and application thereof, and the chimeric antigen receptor comprises a B7H3 antigen binding structural domain, a transmembrane structural domain, an intracellular signal structural domain and IL-15, experiments prove that the CAR-iNKT cell targeting B7H3 prepared by the invention has strong proliferation ability, cytokine release ability and tumor cell killing ability, can effectively remove tumor cells, and has important application prospects in the field of tumor cell immunotherapy.

Description

technical field [0001] The invention belongs to the technical field of immunology and molecular biology, and in particular relates to a chimeric antigen receptor targeting B7H3, which comprises a B7H3 antigen binding domain, a transmembrane domain, an intracellular signaling domain and IL-15 , more specifically, relate to a fully human chimeric antigen receptor targeting B7H3, iNKT cells and uses thereof. Background technique [0002] Tumor cell immunotherapy is the fourth largest tumor treatment technology after surgery, radiotherapy and chemotherapy. It is a tumor-specific tumor-specific therapy that uses biotechnology and biological agents to separate and activate in vitro and reinfuse the patient's own or allogeneic immune system. Or a new treatment method that non-specifically kills cells. Traditional tumor therapies, including surgery, chemotherapy and radiotherapy, all have limitations: surgery often fails to eradicate cancer cells due to their infiltration into adja...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2827C07K14/7051C12N15/86A61P35/00C07K2317/622C07K2319/03C07K2319/02C07K2319/33C12N2740/10043C12N2510/00C07K14/5443C12N5/0636A61K39/001111C12N2800/107A61K2039/5156A61K2039/5158
Inventor 李慧忠郑骏年王刚刘宜林
Owner 江苏集萃崛创生物科技研究所有限公司
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