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A fully human chimeric antigen receptor targeting b7h3 co-expressing IL-21, inkt cells and its use

A chimeric antigen receptor, fully human technology, used in receptors/cell surface antigens/cell surface determinants, targeting specific cell fusion, genetically modified cells, etc. Eradication, adverse reactions and other problems, to achieve the effect of removing tumor cells

Active Publication Date: 2022-03-22
XUZHOU MEDICAL UNIV
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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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  • A fully human chimeric antigen receptor targeting b7h3 co-expressing IL-21, inkt cells and its use
  • A fully human chimeric antigen receptor targeting b7h3 co-expressing IL-21, inkt cells and its use
  • A fully human chimeric antigen receptor targeting b7h3 co-expressing IL-21, inkt cells and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1 Preparation of fully human B7H3.CAR / IL-21-iNKT targeting B7H3 and co-expressing IL-21

[0121] 1. Experimental method

[0122] (1) Preparation of iNKT

[0123] 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;

[0124] 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;

[0125] 3) Magnetic bead sorting of iNKT cells: Collect in...

Embodiment 2

[0140] Example 2 Detection of cytokine release ability and apoptosis of fully human B7H3.CAR / IL-21-iNKT targeting B7H3 co-expressing IL-21

[0141] 1. Experimental method

[0142] (1) Use ELISA kit to detect cytokine release ability

[0143] Collect 2×10 5 B7H3.CAR / IL-21-iNKT cells were mixed with 2×10 5Renal cancer cells 786-O and OSRC-2 were mixed and added to a 24-well plate for co-incubation, and duplicate wells were set up. After 24 hours, the culture supernatant was collected; the contents of IFN-γ and IL-2 were detected with ELISA kits.

[0144] (2) Detection of apoptosis of B7H3.CAR-iNKT cells by flow cytometry

[0145] The B7H3.CAR-iNKT and B7H3.CAR / IL-21-iNKT cells prepared in this example were collected, resuspended in T cell culture medium without cytokines (IL-2 / IL-7 / IL-15), and placed in in CO 2 incubator. Collect and wash the cells at 0 hour and 72 hours respectively, resuspend with 1×Annexin V Binding Buffer, add FITC-Annexin V and PI and incubate at room...

Embodiment 3

[0149] Example 3 Verification of the ability of B7H3.CAR / IL-21-iNKT cells to kill renal cancer cells in vitro

[0150] 1. Experimental method

[0151] First, add 50 μL DMEM complete medium to the E-Plate detection plate of the xCELLigence cell function analyzer, and measure the background impedance value; collect renal cancer cells 786-O and OSRC-2 in the logarithmic phase, and adjust the concentration of the cell suspension to 1 ×10 5 / mL, add 100 μL of cell suspension to the E-Plate detection plate, let it stand at room temperature for 30 minutes, and then place it on the detection platform; when the target cell proliferation is in the plateau stage for real-time dynamic observation, the experimental wells are divided according to the effect-to-target ratio of 5 / 1, 50 μL of effector cells iNKT, B7H3.CAR-iNKT and B7H3.CAR / IL-21-iNKT were added to 1 / 1 and 1 / 5, and tumor cells alone were set as the control group, and the cell-mediated cell killing effect curve was observed in ...

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PUM

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Abstract

The invention discloses a fully human chimeric antigen receptor targeting B7H3 and co-expressing IL-21, iNKT cells and applications thereof. The chimeric antigen receptor comprises a B7H3 antigen binding domain, a transmembrane domain, a cell Inner signaling domain and IL-21, it has been verified by experiments that the B7H3.CAR / IL-21-iNKT cells targeting B7H3 prepared by the present invention have strong cytokine release ability and tumor cell killing ability, and can effectively eliminate tumor cells , 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 specifically relates to a fully human chimeric antigen receptor targeting B7H3 and coexpressing IL-21, which comprises a B7H3 antigen binding domain, a transmembrane domain, a cell The internal signaling domain and IL-21, more specifically, relate to a fully human chimeric antigen receptor targeting B7H3 co-expressing IL-21, 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 er...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
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党元元
Owner XUZHOU MEDICAL UNIV
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