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BCMA-targeting chimeric antigen receptor, method for double modification of BCMA-targeting chimeric antigen receptor, and uses of BCMA-targeting chimeric antigen receptor

A single-chain antibody and heavy chain technology, applied in genetically modified cells, targeting specific cell fusion, polypeptides containing positioning/targeting motifs, etc. T cell attack and other issues

Inactive Publication Date: 2018-08-14
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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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  • BCMA-targeting chimeric antigen receptor, method for double modification of BCMA-targeting chimeric antigen receptor, and uses of BCMA-targeting chimeric antigen receptor
  • BCMA-targeting chimeric antigen receptor, method for double modification of BCMA-targeting chimeric antigen receptor, and uses of BCMA-targeting chimeric antigen receptor
  • BCMA-targeting chimeric antigen receptor, method for double modification of BCMA-targeting chimeric antigen receptor, and uses of BCMA-targeting chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Determination of BCMA CAR-tEGFR-aPD1 gene sequence

[0084] 1.1 The anti-BCMA single-chain antibody clone number J22.9, human CD8α hinge region, human CD8α transmembrane region, human 41BB intracellular region and human CD3ζ intracellular region, and human EGFR intracellular region were found from the NCBI website database. Outer region genes, anti-PD1 antibody heavy chain and light chain variable region gene sequence information, these sequences are codon optimized on the website http: / / sg.idtdna.com / site to ensure that the encoded amino acid sequence remains unchanged More suitable for human cell expression. The whole gene of the above sequence was synthesized, and different enzyme cutting sites were introduced at the beginning and end to form the complete BCMA-41BB-tEGFR-aPD1 gene sequence information.

[0085] 1.2 Sequencing of recombinant plasmids

[0086] The recombinant plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and t...

Embodiment 2

[0089] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0090] The nucleotide sequence of the CAR molecule prepared in Example 1 and Example 2 was double digested with NotI (NEB) and EcoRI (NEB), connected with T4 ligase (NEB) and inserted into the NotI-EcoRI position of the retroviral RV 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.

[0091] 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

[0092] Example 3: Retroviral packaging

[0093] 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;

[0094] 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 the backbone plasmid (MSCV), and 12.5ug for Gag-pol 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;

[0095] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with ...

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Abstract

The present invention relates to a chimeric antigen receptor targeting BCMA (J22.9)-tEGFR-aPD1, and uses thereof, and specifically provides a polynucleotide sequence, which is selected from (1) a polynucleotide sequence containing the coding sequence of an anti-BCMA single-chain antibody, the coding sequence of a human CD8[alpha] hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of human 41BB intracellular region and human tEGFR, and the coding sequence of an anti-human PD1 single chain antibody, wherein the coding sequences are sequentially linked; and(2) the complementary sequence of the polynucleotide sequence (1). The invention further provides a related fusion protein, a vector comprising the coding sequence, and uses of the fusion protein, the coding sequence and the vector.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor targeting BCMA(J22.9)-BBz-tEGFR-aPD1 and its application. Background technique [0002] Multiple myeloma is a malignant plasma cell disease, manifested as malignant clonal hyperplasia of bone marrow plasma cells, secreting monoclonal immunoglobulin or its fragments (M protein), resulting in bone, kidney and other related target organs or tissue damage, common clinical Manifested as bone pain, anemia, renal insufficiency, infection, etc. [N Engl J Med.2011.364(11):p.1046-60.]. Currently, multiple myeloma is the second most malignant tumor of the hematological system, accounting for 10% of hematological malignancies, and it mostly occurs in men. CA Cancer JClin.2014.64(1):p.9-29.]. [0003] B-cell maturation antigen (BCMA), also known as CD269, consists of 184 amino acid residues, its intracellular region contains 80 amino acid residues, and t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/71C07K16/2818C07K16/2878C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/00043
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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