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Dual-target chimeric antigen receptor and use thereof

A technology of single-chain antibody and fusion protein, which can be used to target specific cell fusion, antibody mimics/scaffolds, polypeptides containing positioning/targeting motifs, etc. Problems with high amplification

Pending Publication Date: 2019-03-05
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, anti-CD20 antibodies often have defects in the treatment of CD20-positive B-cell lymphoma due to their mild killing ability: they only have a therapeutic effect on some patients, and some CD20-positive patients are initially ineffective or quickly cause drug resistance; the cure rate Low; Tumor cells that evade antibody killing quickly cause relapse
However, this treatment has not been perfected, and T cells will go off target and attack other tissues, or expand too much, beyond the treatment needs

Method used

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  • Dual-target chimeric antigen receptor and use thereof
  • Dual-target chimeric antigen receptor and use thereof
  • Dual-target chimeric antigen receptor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Determination of CD20 scFV-CD19 scFV-41BB-CD3ζ-tEGFR gene sequence

[0084] 1.1 The human IgG4 hinge region, human CD28 transmembrane region, human 41BB intracellular region, human CD3ζ intracellular region and human truncated EGFR (tEGFR) gene sequence were searched from the NCBI website database. The anti-CD20 single chain antibody clone number is Leu -16, the anti-CD19 single chain antibody clone number is FMC63, these sequences are on 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.

[0085] See SEQENCE LISTING (SEQUENCE ID NO.1-2) for each amino acid and gene sequence information.

[0086] The above sequences were connected sequentially, and different enzyme cutting sites were introduced at the junctions of each sequence to form complete CD20-CD19-BBz-tEGFR gene sequence information.

[008...

Embodiment 2

[0092] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0093] 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 Qiagen’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

[0094] Example 3: Retroviral Packaging

[0095] 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;

[0096] 2. On the second day, the 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare the plasmid complex, the amount of various plasmids is 12.5ug for RV-CD20-CD19-BBz-tEGFR, 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;

[0097] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, ...

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Abstract

The present invention relates to a dual-target chimeric antigen receptor that targets CD19 and CD20 and use thereof. In particular, the present invention provides a polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence including coding sequences of anti-CD20 and anti-CD19 single-chain antibodies, a coding sequence of a human IgG4 hinge region, a coding sequence of a human CD28 transmembrane region, a coding sequence of a human 41BB intracellular region, a coding sequence of a human CD3[Zeta] intracellular region, and an optional coding sequence of an EGFRfragment including extracellular domains III and IV; and (2) a sequence complementary with the sequence (1). The invention also provides related fusion protein, vectors comprising the coding sequences, and use of the fusion protein, the coding sequences and the vectors. A CD20-CD19-BBz-tEGFR CAR-T cell prepared has strong killing function on specific tumor cells, high CD107a expression and IFN[gamma] secretion are achieved, and even when the multiplicity of infection is 5:1, the target cell killing efficiency reaches about 60%.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to a chimeric antigen receptor with dual targets of CD19 and CD20 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 si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N5/10A61P35/00A61P35/02
CPCA61K35/17C12N5/0636C12N15/86C07K14/7051C07K16/2803C07K16/2887C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C12N2730/00043C12N2740/00043C12N2510/00
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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