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Target CD19 and CD22 chimeric antigen receptor and application thereof

A single-chain antibody and fusion protein technology, applied in the field of cell therapy, can solve the problems of variable remission and relapse of leukemia

Pending Publication Date: 2018-09-07
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical studies have found that CAR-T cells in the peripheral blood of patients decreased or disappeared, and leukemia relapse occurred immediately. To prevent these patients from relapse, researchers often injected CD19 CAR-T again, but the effect of maintaining leukemia remission was variable.

Method used

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  • Target CD19 and CD22 chimeric antigen receptor and application thereof
  • Target CD19 and CD22 chimeric antigen receptor and application thereof
  • Target CD19 and CD22 chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Determination of CD19 scFV-CD22 scFV-41BB-CD3ζ gene sequence

[0078] 1.1 The human CD8 hinge region, human CD8 transmembrane region, human 41BB intracellular region, and human CD3ζ intracellular region gene sequences were searched from the NCBI website database. The clone number of the anti-CD22 single-chain antibody is M971, and the clone number of the anti-CD19 single-chain antibody For the FMC63, these sequences are at the website http: / / sg.idtdna.com / site Codon optimization is carried out to ensure that it is more suitable for expression in human cells under the condition that the encoded amino acid sequence remains unchanged.

[0079] See SEQENCE LISTING (SEQUENCE ID NO.1-2) for each amino acid and gene sequence information.

[0080] The above sequences were connected in sequence, and different enzyme cutting sites were introduced at the junctions of each sequence to form complete CD19-CD22-BBz gene sequence information.

[0081] 1.2 Sequencing of re...

Embodiment 2

[0086] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0087] The nucleotide sequence of the CAR molecule prepared in Example 1 was double digested with NotI (NEB) and EcoRI (NEB), connected and inserted into the NotI-EcoRI site of the retroviral RV vector through T4 ligase (NEB), and transformed into After the competent E.coli (DH5α) was sequenced correctly, the plasmid was extracted and purified using the Qiagen company's plasmid purification kit, and the purified plasmid was transfected into 293T cells by the plasmid calcium phosphate method for retrovirus packaging experiments.

Embodiment 3

[0088] Example 3: Retroviral packaging

[0089] 1. On the first day, the 293T cells should be less than 20 passages and not overgrown. Plate with 0.6*10^6 cells / ml, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37 degrees;

[0090] 2. On the second day, the confluence of 293T cells reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is 12.5ug for RV-CD22-CD19-BBz, Gag- pol is 10ug, VSVg is 6.25ug, CaCl 2 250ul,H 2 O is 1ml and the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37 degrees for 4 hours, remove the medium, wash with PBS, and re-add the preheated fresh medium;

[0091] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, sto...

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Abstract

The invention relates to a double-target CD19 and CD22 chimeric antigen receptor and application thereof, in particular to a polynucleotide sequence which is selected from (1) a coding sequence comprising anti-CD22 and anti-CD19 single-chain antibodies which are connected sequentially, a coding sequence of a human CD8 hinge region, a coding sequence of human CD8 transmembrane region, a coding sequence of a human 41BB intracellular region, a coding sequence of a human CD3 zeta intracellular region and a polynucleotide sequence of a coding sequence of a fragment, comprising an extracellular domain III and a extracellular domain IV, of an optional EGFR; and (2) a complementary sequence of the (1) polynucleotide sequence. The invention further provides a relevant fusion protein, a carrier comprising the coding sequence and application of the fusion protein, the coding sequence and the carrier. The prepared CD19-CD22-BBzCAR-T cell has a strong killing function to the specific tumor cell, CD107a expression and IFN gamma secretion are high, and the killing efficiency to the target cell can reach about 80% under the situation that the effector-target ratio is 10:1.

Description

technical field [0001] The invention belongs to the field of cell therapy, and in particular relates to a chimeric antigen receptor dual-targeting CD19 and CD22 and its application. Background technique [0002] Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted manner by MHC after genetic modification, and continuously activate and expand T cells. In 2012, the annual meeting of the International Society for Cell Therapy pointed out that biological immune cell therapy has become the fourth means of tumor treatment besides surgery, radiotherapy, and chemotherapy, and will become a must-have means for future tumor treatment. CAR-T cell reinfusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C07K19/00A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02C12N7/00C12N15/86C07K14/7051C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2740/10043C12N2740/10021C12N5/0636A61K39/001112A61K39/001113C12N2510/00
Inventor 刘雅容金涛
Owner HRAIN BIOTECHNOLOGY CO LTD
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