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Chimeric antigen receptor, immune cell modified by chimeric antigen receptor and application of immune cell to treatment of advanced pancreatic cancer

A chimeric antigen receptor and immune cell technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, and antibody medical components, etc., can solve the problems of tumor cell immune escape, increase high affinity, overcome the The effect of clearing too fast and improving the curative effect

Active Publication Date: 2021-02-05
山东仁济生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Current research shows that chimeric antigen receptor modified immune cell therapy has shown significant curative effect in the clinical treatment of blood tumors, but it encounters many difficulties in the treatment of advanced pancreatic cancer, one of the important reasons is transforming growth factor -β (transforming growth factor-β, TGF-β), TGF-β is an immunosuppressive factor, which can be secreted by advanced pancreatic cancer cells. Once the level of TGF-β increases, it can block immature T cells to Th1 Cell differentiation, promotes its conversion to Treg subsets, and inhibits the antigen presentation function of dendritic cells, leading to immune escape of tumor cells

Method used

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  • Chimeric antigen receptor, immune cell modified by chimeric antigen receptor and application of immune cell to treatment of advanced pancreatic cancer
  • Chimeric antigen receptor, immune cell modified by chimeric antigen receptor and application of immune cell to treatment of advanced pancreatic cancer
  • Chimeric antigen receptor, immune cell modified by chimeric antigen receptor and application of immune cell to treatment of advanced pancreatic cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of scFv (TGF-β) and verification of affinity

[0033] 1. Preparation of pLent-scFv plasmid

[0034] Select the VH-VL part of the antibody and fuse it with CD8Leader to obtain the fusion gene piece CD8Leader-VH-VL, the gene fragment is the nucleotide sequence shown in SEQ ID NO.2;

[0035] Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize the entire expression cassette of the pre-designed fusion gene, and inserted it into the NotI-AsiSI site of the pLent-C-GFP vector (Invitrogen) to construct the pLent-scFv (TGF-β) vector (See image 3 ), transformed into E.coli (DH5α) for expression, after correct sequencing, use Omega’s Endo-Free Plasmid Maxi Kits kit to extract and purify the plasmid to obtain plasmid pLent-scFv, and use Tecan Infinite 200PRO microplate reader to measure the extracted plasmid The concentration (>2mg / ml) and purity (A260 / A280 is 1.8-2.0), the scFv (TGF-β) in this process is designed based on the variable region ...

Embodiment 2

[0043] Example 2 Construction of Lentiviral Plasmid Expressing Chimeric Antigen Receptor Protein

[0044] 1. Insert the fusion gene sheet CD8Leader-scFv(TGF-β)-T2A-CD8Leader-scFv(GPC-1)-CD8-CD28-CD3ζ into the lentiviral expression vector pLent-EF1a-FH-CMV-RFP-P2A-Puro vector ;

[0045] CD8Leader-scFv(TGF-β)-T2A-CD8Leader-scFv(GPC-1)-CD8-CD28-CD3ζ CAR module representation image 3 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence);

[0046] The sequences of the CAR modules of CD8Leader-scFv(TGF-β)-T2A-CD8Leader-scFv(GPC-1)-CD8-CD28-CD3ζ are as follows:

[0047] (1) Artificial sequence of self-cleaving polypeptide T2A nucleic acid (SEQ ID NO.3);

[0048] (2) Nucleic acid artificial sequence (SEQ ID NO.2) of scFv (TGF-β);

[0049] (3) artificial sequence of the leader CD8Leader nucleic acid (SEQ ID NO.4);

[0050] (4) scFv (GPC-1) nucleic acid artificial sequence (SEQ ID NO.5);

[0051] (5) CD8 Hinge region nucleic acid artificial sequence (SEQ ID NO.6);

...

Embodiment 3

[0059] Example 3 Preparation and detection of CAR (TGF-β-GPC-1)-T cells

[0060] 1. Lymphocytes were separated by Ficoll density gradient centrifugation, and CAR(TGF-β-GPC-1)-T cells were prepared

[0061] After signing the informed consent form from healthy adult volunteers, draw 30ml of peripheral blood with an EDTA anticoagulant tube, mix it with normal saline 1:1; then take an equal volume of lymphocyte separation medium, and gently add the diluted peripheral blood sample to the separation medium On the surface, to prevent the peripheral blood sample from breaking through the interface of the separation liquid, centrifuge at 2500rpm at room temperature for 20 minutes; after the centrifugation, the liquid surface is divided into 4 layers, which are diluted plasma layer, mononuclear cell layer, separation liquid layer and red blood cell layer from top to bottom. ;Collect the mononuclear cell layer carefully, and transfer it to a new centrifuge tube, resuspend and wash twice ...

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Abstract

The invention provides a chimeric antigen receptor, an immune cell modified by the chimeric antigen receptor and application of the immune cell to treatment of advanced pancreatic cancer. The chimericantigen receptor is formed by sequentially connecting a hinge region of CD8leader, scFv(TGF-beta), T2A, CD8leader, scFv(GPC-1) and CD8, a CD28 transmembrane-stimulus structural domain and a CD3-zetastimulus signal transduction region in series; a preparation method of the immune cell modified by the chimeric antigen receptor includes immune cell separation, and infection with lentiviral plasmids; Two antibodies are carried in the chimeric antigen receptor; and by applying the immune cell provided by the invention to treatment of the advanced pancreatic cancer, the in-vitro killing efficiencyof positive pancreatic cancer cells reaches 90% or above, and in the aspect of in vivo killing efficiency, it can be seen by nude mouse experiments that the tumor is rapidly shrunk, and the nude mouse tumor completely disappears after 21 days.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor and its modified immune cells and the application of the immune cells in the treatment of advanced pancreatic cancer. Background technique [0002] Pancreatic cancer is a malignant tumor of the digestive system with a very high mortality rate. Due to the lack of specific symptoms and effective biomarkers in the early stage of the disease, more than 80% of the patients are diagnosed at an advanced stage and cannot be given surgical treatment. Known as "the king of cancer" and "the stubborn fortress of the medical field in the 21st century". [0003] In recent years, with the rapid development of cell biology and tumor immunology, the important role of tumor immunity in the occurrence, development and treatment of tumors has been gradually confirmed, and accordingly the chimeric antigen receptor (CAR) has been developed. ) to modify immune cells and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K35/17A61P35/00
CPCA61K35/17A61K39/0011A61P35/00A61K39/001134C07K14/7051C07K16/22C07K16/303C07K2317/622C07K2317/92C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 席广民张才波王丽萍
Owner 山东仁济生物科技有限公司
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