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Novel chimeric antigen receptor and applications thereof

A technology of chimeric antigen receptors and antigens, applied in the field of biomedicine or biopharmaceuticals

Pending Publication Date: 2018-07-13
NANJING LEGEND BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the design of CAR using the basic structure of 4-1BB

Method used

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  • Novel chimeric antigen receptor and applications thereof
  • Novel chimeric antigen receptor and applications thereof
  • Novel chimeric antigen receptor and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of Chimeric Antigen Receptor

[0063] (1) Preparation of chimeric antigen receptor gene fragments

[0064] The present invention provides a novel chimeric antigen receptor comprising CD19 antigen binding domain. The antigen-binding domain antibody provided by the present invention consists of a heavy chain variable region (VH), a light chain variable region (VL) and a connecting peptide (G3S) of a single-chain antibody. 4 Linker composition. The sequences of the heavy chain variable region and the light chain variable region of the single-chain antibody were obtained from GenBank: Y14283.1 and GenBank: Y14284.1, respectively, and their codons and sequences were optimized to ensure that they are more suitable for expression in human cells .

[0065] The novel chimeric antigen receptor provided by the present invention can be designed and transformed according to the sequence of the following coding genes: 4-1BB signal peptide, V H -(G3S) 4 -V ...

Embodiment 2

[0093] Example 2 Construction of K562 cell line K562.CD19.Luc expressing CD19 antigen

[0094]K562 cells hardly express CD19. The gene of the present invention synthesizes the nucleotide sequence encoding human CD19 molecule (the protein sequence is NCBI No. NP_001171569.1), and Nanjing GenScript Biotechnology Co., Ltd. provides gene synthesis technical services. The synthesized CD19 nucleotide sequence was ligated to the pLVX-Puro (Clontech, Cat. No. #632164) lentiviral vector that had been digested with BamH1 and XbaI restriction enzyme sites overnight at 20°C under the action of T4 ligase. The ligation product was transformed into DH5α competent cells and spread on a bacterial plate, and multiple clone spots were picked for plasmid extraction (Qiagen EndofreeMegakit). After enzyme digestion identification and sequencing comparison, the successfully constructed vector was named pLVX-CD19-Puro.

[0095] The extracted pLVX-CD19-Puro expression plasmid, pCMV-ΔR-8.74 (J.Virol....

Embodiment 3

[0099] Example 3 Preparation of immune effector cells modified by chimeric antigen receptors and detection of killing activity in vitro

[0100] (1) Preparation of T lymphocytes

[0101] 50 mL of fresh blood from healthy individuals was collected, and peripheral blood mononuclear cells (PBMC) were separated by lymphocyte separation medium and density gradient centrifugation. The cells were labeled with magnetic beads using Pan T Cell Isolation Kit (purchased from Miltenyi Biotech), and T lymphocytes were isolated and purified. The purified T cells are then activated and proliferated using CD3 / CD28 magnetic beads.

[0102] (2) Lentiviral transduction of T lymphocytes

[0103] Activated T lymphocytes were collected and resuspended in RPMI1640 medium. Infect 1x10 with lentivirus 6 Activated T lymphocytes, add the cell suspension to a 6-well plate, set at 37°C, 5% CO 2 Incubate overnight in the incubator. On the second day, centrifuge again and replace with fresh medium, add...

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Abstract

The present invention discloses a novel chimeric antigen receptor and applications thereof, wherein the novel chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain and an intracellular signal domain, and comprises a 4-1BB signal peptide and / or a 4-1BB molecular transmembrane domain. According to the present invention, a variety of chimeric antigenreceptor nucleic acid sequences are separated and purified, the chimeric antigen receptor specifically for CD19 malignant tumor antigens and the CAR-T cells are provided, and the blood cell line malignant tumor killing test results show that the tumor-cell-targeting ability of immune cells is significantly enhanced, and the tumor cell killing activity is enhanced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to a novel chimeric antigen receptor and its application. Background technique [0002] According to the "2015 Global Cancer Statistics" data, in 2012, there were 14.1 million new cancer patients worldwide, and 8.2 million patients died. According to data from the China Cancer Registry Center, in 2015, there were 4.3 million new cancer cases in China, and more than 2.81 million cancer deaths, accounting for 28.82% of the annual death toll. first place. [0003] Traditional tumor treatment methods mainly include surgery, radiation therapy, chemotherapy, and stem cell transplantation, which has emerged in recent years. These treatments often treat the symptoms but not the root cause. With the development of science, great breakthroughs have been achieved in tumor immunotherapy in recent years, mainly including immune checkpoint inhibitors (such as anti-PD1 mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61K35/28A61K35/545A61P35/00A61P35/02
CPCA61K35/28A61K35/545C12N5/0638C07K14/7051C07K16/2815C07K16/2818C07K16/32C07K2319/02C07K2319/03A61K2239/31A61K2239/38A61K39/464412A61K39/4611A61K2239/48A61K39/4631A61P35/00A61P35/02C12N5/10C12N15/62C07K14/70596A61K35/17A61K45/06C07K16/30C12N5/0606C12N5/0663
Inventor 范晓虎庄秋传王平艳杨蕾郑修军郝佳瑛刘少波
Owner NANJING LEGEND BIOTECH CO LTD
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