FLT3 chimeric antigen receptor and application thereof

A chimeric antigen receptor and carrier technology, applied in the field of biomedicine, can solve the problems of poor expected therapeutic effect and prone to off-target effects.

Active Publication Date: 2018-07-06
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

[0008] One aspect of the present invention is aimed at the problem that CAR-T in the prior art is expected to have poor curative effect on acute myeloid leukemia and is prone to off-target effects, and provides a FLT3 chimeric antigen receptor and its application

Method used

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  • FLT3 chimeric antigen receptor and application thereof
  • FLT3 chimeric antigen receptor and application thereof
  • FLT3 chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Cloning of antigen recognition region FLT3 ligand (FLT3L) in chimeric antigen receptor

[0082] 1. Extract the total RNA of the patient's bone marrow mononuclear cells: at 5×10 6 Add 1ml of RNA iso 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.

[0083] 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 minutes, 70°C, 10...

Embodiment 2

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

[0089] 1. Digest the plasmid containing the CD8α-4-1BB-CD3ζ fragment with Nhe 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 FLT3L fragment obtained in Example 1 with the destination vector, and construct the FLT3L-CD8α-4-1BB-CD3ζCAR destination vector. NheI and NotI were used for enzyme digestion 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 FLT3L-CD8α-4-1BB-CD3ζ Lentiviral Modified T Cells

[0092] 1. Use EndoFree Plasmid Maxi Plasmid Extraction Kit (QIAGEN Company) to extract FLT3L-CD8α-4-1BB-CD3ζ expression plasmid and packaging plasmid psPAX2, PMD.2G. The three plasmids were transfected with Turbofect Transfection Reagent (Thermo Company) at a ratio of 4:3:1 (see the manual of Turbofect Transfection Reagent for specific methods). The virus supernatant was collected 24 hours and 48 hours after transfection, centrifuged at 4°C, 3000rpm for 10 minutes, filtered through a 0.45μm filter, concentrated 10 times by ultracentrifugation at 50000g, 4°C for 3 hours, and then transferred to - Store at 80°C.

[0093] 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 cells (specific steps follow RosetteSep T cell enrichment Cocktai...

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Abstract

The invention discloses an encoding chimeric antigen receptor nucleic acid molecule. The chimeric antigen receptor comprises an extracellular region, a transmembrane domain and an intracellular signaltransduction domain, wherein the encoded extracellular region comprises an FLT3 binding structural domain, and the FLT3 binding structural domain is an FLT3 ligand or an amino acid sequence with the90 to 99% identity as the FLT3 ligand. A flow cytometry, a degranulation analysis experiment and cytokines secreted by ELISA detection T cells prove that T cells modified by the chimeric antigen receptor have a strong killing effect of leukemia cells for expressing FLT3, especially have a specific killing effect on AML cells carrying FLT3 mutant and can effectively prevent an off-target effect. The chimeric antigen receptor FLT3L-CD8 alpha-4-1BB-CD3 zeta disclosed by the invention can be applied to treating FLT3+leukemia, especially leukemia carrying the FLT3 mutant.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to FLT3 chimeric antigen receptor and its application. Background technique [0002] Acute myeloid leukemia (AML) is a malignant clonal disease of the hematopoietic system. The long-term survival rate of patients under 60 years old is less than 40%, and the cure rate of patients over 60 years old is less than 15%. Fms-like tyrosine kinase 3 (FLT3) is an early hematopoietic growth factor receptor gene discovered in recent years, and its expression is low in normal hematopoietic stem / progenitor cells (Haemopoietic Stem / progenitor Cells, HSPCs). Current studies have shown that more than 90% of AML patients express FLT3, and about 30% of AML patients have FLT3 mutations. FLT3 mutations known to be associated with the pathogenesis of AML are mainly internal tandem duplications (ITD) mutations and tyrosine kinase domain (TKD) mutations, among which FLT3-ITD mutations can be found in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00A61K35/17A61P35/02
CPCA61K35/17C07K14/52C07K14/7051C07K14/70517C07K14/70578C07K2319/74C07K2319/33C07K2319/02C07K2319/00C07K2319/03
Inventor 王建祥王敏王颖徐颖茜饶青
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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