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Humanized CD19 antigen binding single chain antibody, chimeric antigen receptor thereof, immune cell and application

A single-chain antibody, humanized technology, applied in blood/immune system cells, receptors/cell surface antigens/cell surface determinants, anti-receptors/cell surface antigens/cell surface determinants immunoglobulins, etc. It can solve the problems affecting the long-term curative effect and recurrence of CAR-T

Active Publication Date: 2019-11-01
WUHAN SIAN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the antibody recognition sequences in the CD19-targeting CAR-T technology come from the mouse. Although the mouse antibody has been successfully applied to the clinic and has shown good results, due to individual differences, some patients have After transfusion, strong immunogenicity is produced, causing human anti-mouse antibody (HAMA) reaction and anti-antibody response (AAR), making CAR-T cells after transfusion easy to be recognized and eliminated by the autoimmune system, affecting CAR-T Long-term efficacy, there may be a risk of recurrence
According to reports, after receiving murine CAR-T reinfusion therapy, although the short-term (3 months) remission rate is very impressive, more than half of the patients relapsed within one year

Method used

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  • Humanized CD19 antigen binding single chain antibody, chimeric antigen receptor thereof, immune cell and application
  • Humanized CD19 antigen binding single chain antibody, chimeric antigen receptor thereof, immune cell and application
  • Humanized CD19 antigen binding single chain antibody, chimeric antigen receptor thereof, immune cell and application

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preparation example Construction

[0150] In the present invention, a method for preparing CAR-immune cells is also provided, including:

[0151] (A) providing an immune cell to be modified; and

[0152] (B) introducing a CAR expression cassette into the immune cell to be engineered, wherein the CAR expression cassette expresses the CAR construct of the present invention, thereby obtaining an engineered immune cell.

[0153]Preferably, the method comprises the following steps: synthesizing the coding sequence of a humanized anti-human CD19 antigen chimeric antigen receptor (CAR); transfecting the obtained CAR coding sequence into a virus preparation cell through a vector system, and packaging it into a The CAR virus, and then transduce the CAR sequence to the immune cells through the virus or directly transduce the CAR sequence to the immune cells through electroporation and other methods.

[0154] In the present invention, there is no special limitation on the transfection method, and the transfection method ...

Embodiment 1

[0169] Design of humanized scFv against human CD19 antigen

[0170] Using the mouse anti-human CD19 antigen FMC63 antibody as the parent antibody, on the basis of the amino acid sequence of its scFv chain, the framework region was humanized and modified to design a humanized antibody against human CD19 antigen.

[0171] Firstly, the core sequence of FMC63 was determined by molecular docking simulation, and the amino acid sequences of the CDR regions of the heavy chain and light chain variable regions of the scFv sequence were determined, that is, CDR1, CDR2, and CDR3 of the light chain VL and CDR1, CDR2, and CDR3 of the heavy chain VH ; Then, in the NCBI / lgBLAST software, the human sequence in the database is analyzed and compared with the scFv sequence of FMC63, and the framework region sequence corresponding to the human antibody variable region sequence with higher homology is selected as the human template . The framework sequence of FMC63 was modified and replaced with t...

Embodiment 2

[0211] The nucleotide sequence of the chimeric antigen receptor (CAR) artificially synthesized against human CD19 is shown in SEQ ID No: 21, and EcoRI and XbaI restriction sites are added at both ends:

[0212]

[0213]

[0214] Use restriction endonucleases EcoRI and XbaI to carry out double enzyme digestion reactions on the lentiviral vector and the synthesized DNA sequence respectively. After the reaction is completed, use T4 DNA ligase (purchased from TaKaRa) to ligate the digested lentiviral vector and DNA fragments In response, a lentiviral vector expressing the chimeric antigen receptor was obtained. The preparation method of the lentiviral vector plasmid expressing the chimeric antigen receptor (CAR) refers to the instructions of the QIAGEN endotoxin-free plasmid large-scale extraction kit.

[0215] The amino acid sequence of the chimeric antigen receptor (CAR) is shown in SEQ ID No:21.

[0216]

[0217] in,

[0218] The CD8 leader peptide is the 1st-21st po...

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Abstract

The invention provides a humanized CD19 antigen binding single chain antibody, a chimeric antigen receptor thereof, an immune cell and application. According to the humanized CD19 antigen binding single chain antibody, an FMC63 antibody of a mouse-anti-human CD19 antigen is taken as a basis, and a light chain skeleton region and a heavy chain skeleton region are humanized. The chimeric antigen receptor of the humanized anti-human CD19 antigen sequentially comprises a leading peptide, the humanized CD19 antigen binding single chain antibody, a hinge region, a transmembrane region and an intracellular region. The sequence of the chimeric antigen receptor of the humanized anti-human CD19 antigen is transduced to the immune cell, the prepared immune cell is applied to preparation of drugs fortreating a human CD19 positive neoplastic disease, the immune cell has the characteristics that lower immunogenicity is achieved, the immune cell can be survived longer in vivo, and the safer and moreefficient effects are achieved, and meanwhile, the immune cell has a higher killing effect on human CD19 positive tumor cells.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy, and in particular relates to a humanized CD19 antigen-binding single-chain antibody, a chimeric antigen receptor, immune cells and applications thereof. Background technique [0002] Immunotherapy is a new method for treating tumor diseases after surgery, radiotherapy and chemotherapy. Tumor cells can adopt various strategies for immune evasion, so that the human immune system cannot recognize tumor cells, and at the same time inhibit the function of the immune system, so that human immune cells cannot effectively kill tumor cells. [0003] Chimeric antigen receptors (CARs) are cell surface receptors that recognize specific proteins (antigens) that are made from parts of other receptors (chimeras). In the immune system, B cells and T cells have surface receptors, called B-cell receptors (BCRs) and T-cell receptors (TCRs), respectively, that recognize specific proteins associated with disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C07K16/2803C07K2317/24C07K2317/565C07K2317/567C07K2317/622C07K2317/92C07K2319/33C12N5/0636C12N5/0646C12N2510/00
Inventor 梅恒周棠怡陈雄波熊巍刘玉芝
Owner WUHAN SIAN MEDICAL TECH CO LTD
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