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Chimeric antigen receptor targeting CD5 and its application

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, and achieve the effect of preventing off-target effects and strong killing effect

Active Publication Date: 2021-08-03
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 its application
  • Chimeric antigen receptor targeting CD5 and its application
  • Chimeric antigen receptor targeting CD5 and its application

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 region, a transmembrane region and an intracellular signal transduction region, the extracellular region encoded by it The region comprises a CD5 binding domain, and the CD5 binding domain is the amino acid sequence shown in SEQ ID NO.3. The cytokines secreted by NK cells were detected by flow cytometry, degranulation analysis experiments, and ELISA, which proved that the CD5 CAR NK cells had a strong killing effect on blood tumor cells expressing CD5, but had a strong killing effect on cells that did not express CD5. Weak, effectively preventing off-target effects. The chimeric antigen receptor CD5 scFv-CD8α-4-1BB-CD3ζ of the present invention can be used for the treatment of CD5-positive lymphocytic blood tumors.

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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Patent Type & Authority Patents(China)
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晓龙
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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