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A chimeric antigen receptor, chimeric antigen receptor umbilical cord blood nucleated cells and application thereof

A chimeric antigen receptor and nucleated cell technology, applied in the field of cord blood nucleated cells and their preparation, can solve the problem that the killing effect of solid tumors cannot meet the expected requirements, and achieve the effect of strong targeting and killing effect.

Active Publication Date: 2021-07-06
GZ CEDICINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent CN105384826A also discloses a chimeric antigen receptor and its related nucleic acid and expression vector, umbilical cord blood nucleated cells, and injection, which can be used to prevent and treat a variety of EGFRvIII-positive tumors such as glioma, breast cancer, ovarian cancer, lung cancer, etc., but its killing effect on solid tumors in vivo still cannot meet the expected requirements

Method used

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  • A chimeric antigen receptor, chimeric antigen receptor umbilical cord blood nucleated cells and application thereof
  • A chimeric antigen receptor, chimeric antigen receptor umbilical cord blood nucleated cells and application thereof
  • A chimeric antigen receptor, chimeric antigen receptor umbilical cord blood nucleated cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design and Construction of Chimeric Antigen Receptor and Its Carrier Plasmid

[0044] 1. CAR design

[0045] The chimeric antigen receptor (CAR) of the present invention comprises an antigen recognition domain, a transmembrane domain and an intracellular signaling domain of the human antibody EGFRvIII, and its amino acid sequence is shown in SEQ ID NO: 1, through patent CN105384826A The CAR mutation was obtained and achieved unexpected effects.

[0046] Chimeric antigen receptors are artificially constructed fusion proteins or polypeptides that exploit the antigen-binding properties of monoclonal antibodies to alter the specificity and reactivity of T cells to selected targets in a non-MHC-restricted manner, thereby bypassing the barriers of tumor escape. mechanism.

[0047] The CAR of the present invention has antigen specificity for epidermal growth factor receptor variant III (EGFRvIII). EGFRvIII, a variant of the epidermal growth factor receptor (EGFR),...

Embodiment 2

[0062] Example 2 Lentivirus transfection of umbilical cord blood nucleated cells

[0063] Isolation of nucleated cells from healthy human cord blood by gradient density centrifugation. Take 1×10 6 The cell / mL density was added to the T lymphocyte medium GT-T551 (Takara) containing 300IU / mL IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.) for culture, and 5% of the same cord blood plasma was added, and 80ng / mL CD3mAb (Shanghai Weike Biotechnology Co., Ltd.) activated T cells and stimulated culture for 48 hours, then infected umbilical cord blood nucleated cells with the recombinant lentivirus of Example 1 at MOI=5, and the cells after infection were changed the next day, every 2-3 days passaging.

[0064] The cells are harvested after about 10 days of culture, and the cells can be expanded by 30-80 times, and the cells can still be expanded after reactivation. Under the fluorescent microscope, it can be observed that the isotype control cells highly express green fluorescent...

experiment example 1

[0067] Experimental example 1 in vitro tumor cell killing

[0068] The chimeric antigen receptor cord blood nucleated cells prepared in Example 1 and Example 2 of the present invention, the chimeric antigen receptor cord blood nucleated cells prepared in Comparative Example 1, and the unprepared cord blood nucleated cells ( Negative control group) in vitro tumor cell killing effect was compared. The specific steps are as follows: 1×10 5 Effector cells (chimeric antigen receptor umbilical blood nucleated cells prepared in the present invention, chimeric antigen receptor umbilical blood nucleated cells prepared in Comparative Example 1, EGFP control cells, unprepared umbilical cord blood nucleated cells) and Target cells (U251 human glioblastoma cells) at 37°C, 5% CO 2 The co-cultivation was carried out under the conditions, and the cells were collected 15-18 hours after the culture, and the cell killing was detected. The result is as Figure 5 As shown, it is shown that the...

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Abstract

The present invention provides a chimeric antigen receptor targeting EGFRvIII with high affinity. The chimeric antigen receptor is mutated from a commonly used chimeric antigen receptor targeting EGFRvIII. Its amino acid sequence is as shown in SEQ ID NO:1 shown. The present invention also provides a cord blood nucleated cell containing the chimeric antigen receptor, which can efficiently target the EGFRvIII antigen through the chimeric antigen receptor, has a stronger ability to kill EGFRvIII-positive tumor cells, and can be used to prepare prophylactic and Drugs for the treatment of a variety of EGFRvIII-positive tumors.

Description

technical field [0001] The invention relates to the field of tumor cell therapy, in particular to a chimeric antigen receptor targeting EGFRvIII, umbilical cord blood nucleated cells expressing the chimeric antigen receptor, and a preparation method and application thereof. Background technique [0002] From the perspective of tumorigenesis, tumor cells are transformed from normal cells. This process from "self" to "non-self" is often monitored by the body's immune system and eliminated by an effective immune response. In recent years, due to the rapid development and cross-infiltration of theories and technologies in oncology, immunology, cytology, molecular biology and other related disciplines, with the in-depth understanding of the body's anti-tumor immune response, as well as the understanding of tumor immune escape mechanisms and tumor microbiology. With an in-depth understanding of the environment, new strategies and new ideas for tumor immunotherapy have been further...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N5/078A61K39/00A61P35/00
CPCA61K2039/5158A61P35/00A61K39/001104C07K14/7051C07K16/2863C07K2319/00C12N5/0634
Inventor 钟慧霖邹清雁刘海霞邹庆剑杜少茵梁海燕吴志君叶俊杰
Owner GZ CEDICINE BIOTECH CO LTD
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