Chimeric antigen receptor of targeted CD19 as well as method and use for jointly expressing IL-15

A single-chain antibody, fusion protein technology, applied in the direction of targeting specific cell fusion, polypeptides containing localization/targeting motifs, receptors/cell surface antigens/cell surface determinants, etc., can solve the secretion of cytokines less, less curative effect, poor durability and other problems

Active Publication Date: 2018-11-02
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the first-generation CAR can see some specific cytotoxicity, when it was summarized in clinical trials in 2006, it was found that the curative effect was not satisfactory.
The reason is that the first generation of CAR-T cells will soon be exhausted in the patient's body, and their persistence is so poor that the CAR-T cells have not yet had time to contact a large number of tumor cells before they die This kind of CAR-T cells can stimulate anti-tumor cytotoxic effect, but the secretion of cytokines is relatively small, but its survival period in the body is short and cannot stimulate long-lasting anti-tumor effect

Method used

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  • Chimeric antigen receptor of targeted CD19 as well as method and use for jointly expressing IL-15
  • Chimeric antigen receptor of targeted CD19 as well as method and use for jointly expressing IL-15
  • Chimeric antigen receptor of targeted CD19 as well as method and use for jointly expressing IL-15

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Determination of CD19scFv-CD8-41BB-CD3ζ-mbIL15 gene sequence

[0076] 1.1 Search the human CD8α hinge region and transmembrane region, human 41BB intracellular region, human CD3ζ intracellular region, IL15 and IL15Rα mature peptide gene sequence information from the NCBI website database. The anti-CD19 single-chain antibody clone number is FMC63. These sequences Codon optimization is performed on the website http: / / sg.idtdna.com / site to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.

[0077] See SEQENCE LISTING (SEQUENCE ID NO.1-2) for each amino acid and gene sequence information.

[0078] The above sequences were connected sequentially, and different enzyme cutting sites were introduced at the junctions of each sequence to form the complete CD19-BBz-mbIL15 gene sequence information.

[0079] 1.2 Sequencing of recombinant plasmids

[0080] The recombinant plasmid was sent to Shanghai Sangon B...

Embodiment 2

[0083] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0084] The nucleotide sequence of the CAR molecule prepared in Example 1 was double-digested by NotI (NEB) and EcoRI (NEB), connected by T4 ligase (NEB) and inserted into the NotI-EcoRI position of the retroviral RV (MSCV) vector point, transformed into competent E.coli (DH5α), after the sequencing was correct, 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.

[0085] The plasmid map constructed in this example is as follows figure 1 shown. figure 2 The peak diagram of partial sequencing results of the retroviral expression plasmid is shown.

Embodiment 3

[0086] Example 3: Retroviral Packaging

[0087] 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;

[0088] 2. On the second day, the confluence of 293T cells reached about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is 12.5ug for RV-CD19-BBz-mbIL15, 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;

[0089] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, s...

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Abstract

The invention discloses a chimeric antigen receptor CD19-CD8H&TM-41BB-CD3zeta-mbIL15 and use thereof. The chimeric antigen receptor is formed by series connection of a heavy chain and light chain variable region (CD19scFV) of a mouse anti-human CD19 monoclonal antibody (with a cloning number being FMC63), a human CD8alpha hinge region, a transmembrane region, a human 41BB intracellular region, a human CD3zeta intracellular region and an IL15+IL15Ralphla structure. The chimeric antigen receptor is used for modifying a human T-lymphocyte, and the modified T-cell (CAR-T cell) can be used for expressing CD19 positive acute / chronic lymphocytic leukemia and treating lymphoma. The prepared CD19 mbIL15 CAR-T cell has a strong killing function on specific tumor cells.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to CD19 chimeric antigen receptors and applications thereof. 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 the su...

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
CPCA61K35/17C12N5/0636C12N15/86C07K14/5443C07K14/7051C07K14/70517C07K14/7155C07K16/2803C07K16/2878C12N2510/00C12N2740/10043C12N2800/107C07K2319/03C07K2319/02C07K2317/622C07K2319/33
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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