Chimeric antigen receptor targeting CD22 and application of chimeric antigen receptor

A chimeric antigen receptor, targeting technology, applied in the field of biomedicine, can solve the problems of recurrence or ineffectiveness, poor killing effect, etc.

Active Publication Date: 2018-10-30
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

[0007] One aspect of the present invention is aimed at the chimeric antigen receptor targeting CD22 in the prior art, which has poor killing effect on CD22-expressing B-cell lymphoma cells and B-cell lymphocytic leukemia cells, and at the same time during CD19 CAR-T treatment The

Method used

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  • Chimeric antigen receptor targeting CD22 and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting CD22 and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting CD22 and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Cloning of CD22 scFv in antigen recognition region in chimeric antigen receptor

[0075] 1. Extract the total RNA of the mouse anti-human CD22 monoclonal antibody hybridoma cell line: at 5×10 6 Add 1ml of RNAiso Plus (Takara) to the cells, and mix well by pipetting. Add 200 μl of chloroform, invert up and down, and vortex to mix. Centrifuge at 12000 rpm for 5 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.

[0076] 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): 2μl; First strand buffer (5×): 4μl; CD20-RNA: 2μg; DEPC water: make up to 20μl. Reaction conditions: 37°C, 60 min...

Embodiment 2

[0091] Example 2: Construction of Chimeric Antigen Receptor Vector

[0092] 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.

[0093] 2. Ligate the CD22 scFv fragment obtained in Example 1 with the destination vector, and identify the constructed CD22 scFv-CD8α-4-1BB-CD3ζ CAR destination vector with XbaI and NotI. 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

[0094] Example 3: Preparation of chimeric antigen receptor CD22 scFv-CD8α-4-1BB-CD3ζ lentivirus modified T cells

[0095] 1. The CD22scFv-CD8α-4-1BB-CD3ζ expression plasmid and packaging plasmid psPAX2 and pMD.2G were extracted using the EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company). The three plasmids were transfected with PEI transfection reagent (polyscience company) at a ratio of 4:3:1 (see the instructions of PEI transfection reagent for specific methods). Replace the fresh culture medium 12 hours after transfection, collect the virus supernatant 24 hours and 48 hours later, centrifuge at 4°C, 3000rpm for 15 minutes, filter through a 0.45μm filter, and use 50000g, 4°C, 1.5 hours after ultracentrifugation Concentrate 10 times, then transfer to -80°C for storage.

[0096]2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment Cocktail (Stemcell) and Ficoll-Paque PLUS (GE Healthcare) to extract T ce...

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Abstract

The invention provides a nucleic acid molecule for encoding a chimeric antigen receptor targeting CD22. The chimeric antigen receptor comprises an extracellular region, a transmembrane region and a intracellular signal transduction region, the extracellular region encoded by the nucleic acid molecule comprises a CD22 binding domain, and the CD22 binding domain is amino acid sequence as shown in SEQ ID No. 3. By using flow cytometry, a degranulation analysis experiment and ELISA to detect cell factors secreted by a T cell, a fact that the CAR-T cell has a high lethal effect on B cell lymphoma cells and acute B cell lymphoma cell leukemia cells expressing CD22 and hardly has a lethal effect on cells which do not express CD22, and an off-target effect is prevented effectively is proved. The chimeric antigen receptor CD22scFv-CD8alpha-4-1BB-CD3zeta can be used for treating CD22<+>B cell hematologic neoplasms and is applicable to combined treatment with a CD19 CAR-T cell.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor targeting CD22 and its application. Background technique [0002] Chimeric antigen receptor (CAR)-modified T cells, as an immunotherapeutic strategy, have received extensive attention and application in tumor treatment. The structure of CAR generally consists of four parts: an extracellular targeting linker region (usually a single-chain antibody with antigen recognition function), a hinge region, a transmembrane region, and an intracellular signal transduction region. Currently, according to the number of co-stimulatory molecules added to the intracellular signal transduction region, CARs are divided into one generation (no co-stimulatory molecule), second generation (with one co-stimulatory molecule) and third-generation (with two co-stimulatory molecules). Currently the most widely used is the second-generation CAR. [0003] Despite great adva...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2803C07K2319/02C07K2319/03
Inventor 王建祥王敏李赛赛徐颖茜饶青廖小龙
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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