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Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor

A technology of chimeric antigen receptor and chondroitin sulfate, which is applied in the direction of antibody medical components, resistance to vector-borne diseases, and medical preparations containing active ingredients. , good targeted effect

Active Publication Date: 2016-07-13
BLUE ELEGANT 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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  • Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor
  • Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor
  • Chimeric antigen receptor resistant to placenta chondroitin sulfate and application of chimeric antigen receptor

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 SEQUENCELISTING (SEQID 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. Appropriate restriction sites are 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 of 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 the MaxPurePlasmidMaxiKit of Guangzhou Meiji Biotechnology Co., Ltd. to extract plasmids. The specific steps for extracting plasmids are:

[0055] (1) Add 20...

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Abstract

The invention relates to the field of cellular immunotherapy of tumors, in particular to a chimeric antigen receptor resistant to placenta chondroitin sulfate (p1-CS) and application of the chimeric antigen receptor.The chimeric antigen receptor is mainly formed by serially connecting an antigen recognition area resistant to pl-CS, a hinge area, a transmembrane area and an intracellular area, the antigen recognition area resistant to p1-CS is one of plasmodium protein VAR2CSA, part of peptide segment of the plasmodium protein VAR2CSA or p1-CS antibody, and the peptide segment of the plasmodium protein VAR2CSA is a peptide segment with number of amino acid in the plasmodium protein VAR2CSA greater than 500.T cells expressing the chimeric antigen receptor can kill tumor cells to maximum extent, are quite low in degree of damage to normal tissue, can break tumor immune to inhibit microenvironment so as to have better treatment effect on solid tumors and can almost treat tumors of various types.

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): C07K19/00A61K39/00A61P35/00
CPCY02A50/30
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 BLUE ELEGANT BIOTECH CO LTD
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