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Chimeric antigen receptor targeting CD5 and application thereof

A chimeric antigen receptor, CD5 technology, applied in the field of biomedicine, can solve the problem of poor killing effect of lymphoma cells and lymphocytic leukemia cells

Active Publication Date: 2019-01-25
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The CD5-targeted chimeric antigen receptors currently on the market have poor killing effects on CD5-expressing lymphoma cells and lymphocytic leukemia cells, and cannot better meet the requirements of clinical treatment. Therefore, it is urgent to find a new CD5-targeted chimeric antigen receptor. Chimeric antigen receptors to solve the above problems

Method used

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  • Chimeric antigen receptor targeting CD5 and application thereof
  • Chimeric antigen receptor targeting CD5 and application thereof
  • Chimeric antigen receptor targeting CD5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Cloning of CD5 scFv in antigen recognition region in chimeric antigen receptor

[0072] 1. Extract the total RNA of the mouse anti-human CD5 monoclonal antibody hybridoma cell line (HI211): at 5×10 6 Add 1ml of RNAiso Plus (Takara) to the cell mass, and mix by pipetting. Add 200 μl of chloroform, invert up and down, shake and mix. After standing at room temperature for 5 minutes, centrifuge at 12000 rpm for 15 minutes at 4°C. Pipette the supernatant into a 1.5ml EP tube, add the same volume of isopropanol, and mix by gently inverting up and down. Centrifuge at 12000 rpm for 15 minutes at 4°C. Pre-cool 75% ethanol to precipitate RNA at 4°C, and dissolve total RNA in 50 μl DEPC water.

[0073] 2. Synthesize the first strand of cDNA by reverse transcription: Prepare the PCR reaction system (20 μl) as follows: Oligo d(T)15Primers: 2 μl; M-MLV (200u / μl): 1 μl; dNTP (each 2.5mM): 1 μl; DTT ( 0.1M): 2 μl; Firststrand buffer (5×): 4 μl; CD5-RNA: 2 μg; DEPC water...

Embodiment 2

[0088] Example 2: Construction of Chimeric Antigen Receptor Vector

[0089] 1. Digest the plasmid containing the CD8α-4-1BB-CD3ζ fragment with BamH I and EcoR I endonucleases to obtain the CD8α-4-1BB-CD3ζ fragment, the amino acid sequence of which is shown in SEQ ID NO.5. The plasmid containing the CD8α-4-1BB-CD3ζ fragment can be prepared by any suitable method in the prior art.

[0090] 2. Ligate the CD5 scFv fragment obtained in Example 1 with the destination vector, and carry out double enzyme digestion with the constructed CD5 scFv-CD8α-4-1BB-CD3ζ CAR destination vector with BamH I, EcoR I, Xba I, and Not I, respectively Identification. The result is as figure 2 As shown, the enzyme digestion results showed that the positive clone contained the target band and was correctly identified by sequencing. The schematic diagram of the carrier is as image 3 shown.

Embodiment 3

[0091] Example 3: Preparation of Chimeric Antigen Receptor CD5 scFv-CD8α-4-1BB-CD3ζ Lentivirus Modified NK-92 Cells

[0092] 1. The CD5 scFv-CD8α-4-1BB-CD3ζ expression plasmid and packaging plasmid psPAX2, pMD.2G were extracted using the EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company). The three kinds of plasmids were transfected with PEI transfection reagent (polyscience company) at a ratio of 4:3:1 (see the manual of PEI transfection reagent for specific methods). Replace the fresh culture medium 12 hours after transfection, collect the virus supernatant 48 hours later, centrifuge at 4°C, 3000rpm for 20 minutes, filter through a 0.45μm filter, and concentrate 10 times after ultracentrifugation at 50000g, 4°C, 2.5 hours, Store at -80°C.

[0093] 2. The cultivation of NK-92 cells: use 12.5% ​​fetal bovine serum (Gibco company), 12.5% ​​horse serum (Gibco company), 0.2mM inositol, 0.1mM 2-mercaptoethanol, 0.02mM folic acid and 200U / ml human IL-2 MEM-α (Gibco com...

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Abstract

The present invention provides a nucleic acid molecule encoding a chimeric antigen receptor targeting CD5, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular signal transduction domain, and the extracellular domain encoded by the chimeric antigen receptor comprises a CD5 binding domain which is an amino acid sequence as shown in SEQ ID NO:3. The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular signal transduction domain, and the extracellular domain encoded by the chimeric antigenreceptor comprises a CD5 binding domain which is an amino acid sequence shown in SEQ ID NO:3. The cytokines secreted by NK cells were detected by flow cytometry, degranulation assay and ELISA. The results showed that the CD5 CAR NK cells had strong cytotoxic effect on hematological tumor cells expressing CD5, but had weak cytotoxic effect on cells not expressing CD5. The cytokines secreted by CD5CAR NK cells could effectively prevent the missed target effect. A chimeric antigen receptor CD5 scFv-CD8[alpha]-4-1BB-CD 3 Xi can be used in the treatment of lymphocytic hematologic tumors with positive CD 5.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor targeting CD5 and its application. Background technique [0002] In recent years, chimeric antigen receptor (chimeric antigen receptor, CAR) immunotherapy has become a powerful new adoptive immunotherapy technology, which has obvious therapeutic effects on many solid tumors, blood system tumors, especially B cell lymphoma . CAR therapy utilizes improved patient T cells that can break through the restriction of MHC to directly recognize tumor antigens and target and eliminate malignant tumors. CAR-T cells have been widely used in hematological tumors, especially the CAR-T cell immunotherapy with CD19 as the target antigen has achieved a breakthrough. However, the problem of self-targeted killing caused by the co-expression of target antigens between CAR-T cells and malignant T cells limits the application of CAR-T in T-cell hematological tumors. ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N5/10A61K35/17A61P35/02
CPCA61K35/17A61P35/02C07K14/7051C07K16/2896C07K2319/02C07K2319/03C12N5/0636C12N15/86
Inventor 王建祥王敏刘倩徐颖茜饶青廖小龙
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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