CD19 and CD22 double-target chimeric antigen receptor and application thereof

A technology of chimeric antigen receptors and antigens, applied in the field of biomedicine, can solve problems such as poor efficacy and tumor recurrence in patients

Active Publication Date: 2020-10-23
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, medical diagnosis shows that tumor cells in some hematological tumors do not express CD19 molecules, but express CD22 molecules. Only CAR-T cells targeting CD19 molecules are not effective, and some patients have tumor recurrence after a period of time Phenomenon

Method used

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  • CD19 and CD22 double-target chimeric antigen receptor and application thereof
  • CD19 and CD22 double-target chimeric antigen receptor and application thereof
  • CD19 and CD22 double-target chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Construction of CAR molecular vector

[0106] This example firstly synthesizes the coding gene of the anti-CD19 and CD22 dual target chimeric antigen receptor, and adds the restriction endonuclease Pme1 restriction site and its protective base and its protective base to the C-terminal and N-terminal of the coding gene. Restriction endonuclease Spe1 restriction site and its protective base;

[0107] The coding gene was double digested with restriction enzymes Pme1 and Spe1, and the digested product containing sticky ends was recovered by agarose gel electrophoresis, and ligated into the linearized pWPXLd-eGFP plasmid (containing the same double digestion with Pme1 and Spe1). In the sticky end), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent) to obtain a lentiviral vector containing CAR coding genes targeting CD19 and CD22 dual targets. The map is as follows figure 1 Shown.

[0108] In this example, the anti-CD19 scFv CA...

Embodiment 2

[0109] Example 2 Lentivirus packaging

[0110] In order to introduce CAR molecules into T cells, 293T cells are used to prepare recombinant lentivirus. When the 293T cells are spread on a 100mm culture dish to 80-90% of the bottom, the lentivirus is packaged:

[0111] 2h before virus packaging, change the medium to DMEM containing 1% fetal bovine serum, and add 6mL / 100mm petri dish;

[0112] Prepare the plasmid mixture as shown in Table 1. The pWPXLd-expression plasmid includes a lentiviral vector containing a CAR encoding gene targeting CD19 and CD22 dual targets, and a lentivirus containing a CAR encoding gene targeting CD19 single target Vectors, lentiviral vectors containing CAR encoding genes targeting CD22 single target, pWPXLd-eGFP plasmid is an empty vector without CAR encoding genes;

[0113] Table 1

[0114]

[0115] Add 36μg PEI to another 500μL opti-MEM medium, mix well, and let stand for 5min at room temperature;

[0116] Mix the pWPXLd-expression plasmid or pWPXLd-eGFP pla...

Embodiment 3

[0121] Example 3 T cell activation and lentiviral transfection

[0122] Use Ficoll density gradient centrifugation kit (GE) to separate peripheral blood mononuclear cells (PBMC) from whole blood, remove red blood cells, and then use MACS Pan-T magnetic beads to sort out T cells;

[0123] The sorted T cells are diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 Pieces / mL to be used;

[0124] Use CD2 / CD3 / CD28 T cell activation amplification kit (Miltenyi) to activate T cells, that is, the coated magnetic beads are mixed with T cells in a ratio of 1:2, and the final density of T cells is 5×10 6 Piece / mL / cm 2 , After mixing, place at 37℃, 5% CO 2 Incubate for 48h;

[0125] After T cells are activated for 48 hours, demagnetize the beads, centrifuge at 300g for 5 minutes, remove the supernatant, resuspend the T cells in fresh medium, add recombinant lentivirus expressing CAR or blank control eGFP lentivirus (MOI=10),...

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Abstract

The invention provides a CD19 and CD22 double-target chimeric antigen receptor and application thereof. The chimeric antigen receptor comprises an antigen binding structural domain, a transmembrane structural domain and a signal transduction structural domain; and the antigen binding structural domain comprises a light chain variable region of an anti-CD19 single-chain antibody, a heavy chain variable region of the anti-CD19 single-chain antibody, a light chain variable region of an anti-CD22 single-chain antibody and a heavy chain variable region of the anti-CD22 single-chain antibody. The anti-CD19 and anti-CD22 double-target chimeric antigen receptor disclosed by the invention has targeting activity on CD19 positive and/or CD22 positive cells; the T cells expressing the anti-CD19 and anti-CD22 double-target chimeric antigen receptor has a killing effect on tumor cells with low CD19 antigen expression quantity or no CD19 antigen expression quantity and tumor cells with low CD22 antigen expression quantity or no CD22 antigen expression quantity, so that the immune escape phenomenon is avoided, and the possibility of disease recurrence is reduced.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and relates to CD19 and CD22 dual-target chimeric antigen receptors and applications thereof. Background technique [0002] Chimeric antigen receptor (CAR) is composed of tumor-associated antigen binding domain, extracellular hinge domain, transmembrane domain and intracellular signal transduction domain. Generally, the single chain fragment variable (scFv) of the antibody contained in the CAR molecule has a specific binding effect to the tumor associated antigen (TAA), which interacts with the signal transduction molecule through the hinge and transmembrane region. Cytoplasmic domain coupling. [0003] In recent years, with the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. At present, CAR-T cell therapy has been widely used in the treatment of B-cell malignancies. CAR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N15/86C12N7/00A61K39/0011A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C12N2740/15021C12N2740/15043
Inventor 秦乐汤朝阳邓殷建魏志辉其他发明人请求不公开姓名
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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