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Anti-placenta-like chondroitin sulfate chimeric antigen receptor and applications thereof

A technology of chimeric antigen receptor and chondroitin sulfate, which is applied in the field of tumor cell immunotherapy, can solve the problem of inhomogeneity, and achieve the effect of small damage, good curative effect and good targeting

Inactive Publication Date: 2017-07-25
GUANGZHOU CAS LAMVAC BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the main chain structure of the polysaccharide is not complex, it shows a high degree of heterogeneity in terms of the degree of sulfation, the distribution of sulfate groups and the two different isomeric uronic acids within the chain

Method used

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  • Anti-placenta-like chondroitin sulfate chimeric antigen receptor and applications thereof
  • Anti-placenta-like chondroitin sulfate chimeric antigen receptor and applications thereof
  • Anti-placenta-like chondroitin sulfate chimeric antigen receptor and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Determination of the CAR gene sequence targeting pl-CS

[0046] Plasmodium VAR2CSA gene, human CD8α signal peptide gene, human CD8α hinge region gene, human CD28 transmembrane region and intracellular region gene, human 4-1BB intracellular region gene and human CD3ζ intracellular region gene were searched from GenBank database and literature gene sequence. It is codon-optimized to ensure that it is more suitable for expression in human cells under the condition that the encoded amino acid sequence remains unchanged.

[0047] For the sequence information of each gene, see SEQUENCE LISTING (SEQ ID NO.1-22 in the sequence listing).

Embodiment 2

[0048] Example 2: Construction of lentiviral expression vector pLHlentiCAR004

[0049] The above gene sequences were sequentially linked according to the human CD8α signal peptide gene, VAR2CSA, human CD8α hinge region gene, human CD28 transmembrane region and intracellular region gene, human 4-1BB intracellular region gene and human CD3ζ intracellular region gene sequence, A chimeric antigen receptor is formed, and the nucleic acid sequence of the antigen receptor is shown in SEQ ID NO.2. A suitable restriction site is introduced, artificial whole gene synthesis is performed, and the pLHlentiCAR004 plasmid is obtained by cloning into a lentiviral expression vector. For a schematic diagram of the plasmid see figure 1 .

[0050] The recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing, and the sequencing result was compared with the fitted sequence to confirm that the sequence was completely correct.

Embodiment 3

[0051] Example 3: Large-scale extraction of lentiviral expression vectors and packaging vectors

[0052] The pLHlentiCAR004 plasmid and the stbl3 Escherichia coli of the pLHlentiCAR004 plasmid and the psPAX2 and pMD2.G packaging plasmids were mass-cultured in LB culture medium, and a large amount of plasmids were extracted by alkaline lysis method for transfection. The specific process is as follows:

[0053] 1) Inoculate stbl3 Escherichia coli strains with pLHlentiCAR004, psPAX2, and pMD2.G into culture flasks containing 500mL LB / antibiotic culture solution, and culture on a shaker at 37°C for 12-16 hours;

[0054] 2) Centrifuge at 3000*g for 10 minutes to collect the bacteria, discard the culture medium, turn it upside down on absorbent paper and pat lightly to absorb the residual liquid. The collected bacteria used MaxPure Plasmid MaxiKit of Guangzhou Meiji Biotechnology Co., Ltd. to extract plasmids. The specific steps for extracting plasmids are as follows:

[0055] (1) ...

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Abstract

The present invention relates to the field of cellular immunotherapy for tumors, particularly to an anti-placenta-like chondroitin sulfate (pl-CS) chimeric antigen receptor and applications thereof, wherein the chimeric antigen receptor is mainly formed by linking an anti-pl-CS antigen recognition region, a hinge region, a transmembrane domain and an intracellular region in series, the anti-pl-CS antigen recognition region is plasmodium protein VAR2CSA, the partial peptide fragment of the plasmodium protein VAR2CSA, or any one of pl-CS antibodies, and the partial peptide fragment of the plasmodium protein VAR2CSA is the peptide fragment having an amino acid number of greater than 500 in the plasmodium protein VAR2CSA. According to the present invention, the T cells expressing the chimeric antigen receptor can kill the tumor cells to the greatest extent, causes little damage to normal tissues, and can break through tumor immunosuppressive microenvironments, such that the god treatment effects can be provided for solid tumors, and almost various types of tumors can be treated.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a chimeric antigen receptor against placenta-like chondroitin sulfate and its application, specifically a chimeric antigen receptor T( Construction method of CAR-T) cell technology and its application in anti-tumor therapy. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) technology is a new type of cell therapy currently undergoing large-scale clinical trials. It has remarkable efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment methods. The basic technology is to transform T cells to recognize tumor antigens in an HLA-independent manner through genetic engineering, so that CAR-T cells have a stronger ability to recognize and kill tumor cells than natural T cells. The core component of CAR-T is CAR, which generally includes a tumor-associated antigen bindi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/445C07K14/7051C07K14/70517C07K14/70521C07K14/70596C07K2319/03C12N5/0636Y02A50/30
Inventor 姚永超戚何妹邓萃兰郭文中黄烁洲刘立宝贺倩倩秦莉陈小平
Owner GUANGZHOU CAS LAMVAC BIOTECH CO LTD
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