Preparation and application of CD19-targeting humanized chimeric antigen receptor

A chimeric antigen receptor and humanization technology, applied in the biological field, can solve the problems of antibody affinity and specificity reduction

Pending Publication Date: 2020-10-02
天津英科赛奥科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, after antibody humanization, due to the change of amino acid sequence, the peptide chain size, charge, hydrophobicity, spatial conformation, etc. are usually changed, and the formation of hydrogen bonds is also different from that of the mouse source, thus affecting the complementarity determining region of the antibody ( CDR) conformation, therefore, the affinity and specificity of the humanized antibody are usually reduced by more than 10 times compared with the mouse antibody (Vahideh Ahmadzadeh et al., Monoclonal antibodies in immunodiagnosis and immunotherapy, volume 33, number 2, 2014)

Method used

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  • Preparation and application of CD19-targeting humanized chimeric antigen receptor
  • Preparation and application of CD19-targeting humanized chimeric antigen receptor
  • Preparation and application of CD19-targeting humanized chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1: Preparation of humanized chimeric antigen receptor lentiviral vector targeting CD19 antigen

[0135] (1) Targeting CD19 humanized chimeric antigen receptor gene consists of CD19 single-chain antibody scFv, CD8α hinge region, CD8α transmembrane region, CD28 intracellular co-stimulatory signal domain, CD3ζ intracellular primary signal domain, the full length is 1428bp ;

[0136] (2) Synthesize the CD19 humanized chimeric antigen receptor gene fragment (hereinafter referred to as "fragment") by artificial gene, add BamH1 enzyme cleavage site to the 5' end of the fragment, and add Sal1 enzyme to the 3' end of the fragment Cutting site: The empty lentiviral expression plasmid pLenti6 / V5-p53_R273H was double-digested with BamH1 and Sal1, and the restriction system was as follows:

[0137] pLenti6 / V5-p53_R273H 4 μg,

[0138] BamH1 1μl,

[0139] Sal1 1μl,

[0140] 10×buffer 5μl,

[0141] wxya 2 O supplemented to 50 μl;

[0142] (3) Perform agarose gel electr...

Embodiment 2

[0145] Example 2: Packaging of pLv6-EF1α-HuCD19 lentivirus

[0146] (1) Culture Lenti-293T (hereinafter referred to as 293T) to a fusion degree of 70% to 80% for transfection;

[0147] (2), pMD2.G, pΔR 8.91 (pMD2.G and pΔR 8.91 are biological wind products, used to provide the protein particles required by the virus coat) and pLv6-EF1α-HuCD19 prepared in Example 1 according to the mass ratio of each plasmid pMD2.G: pΔR8.91: pLv6-EF1α-HuCD19 prepared in Example 1 = 1:3:4, mixed together to obtain 40 μg of co-transfection plasmid;

[0148] (3) Take two 15ml centrifuge tubes and mark tube 1 and tube 2 respectively. Add the co-transfection plasmid of the above 1 to tube 1 and supplement serum-free DMEM to 1.5ml. Add 1mg / mL PEI aqueous solution (PEI is sigma Product No. GF70215825) 120μl and add serum-free DMEM to 1.5ml, mix thoroughly and vortex tube 1, and at the same time add PEI in tube 2 to tube 1 drop by drop; get plasmid-PEI mixture, mix plasmid-PEI Let the solution stand ...

Embodiment 3

[0150] Example 3: Isolation, infection and expansion of T cells

[0151] (1) Put 100ml of healthy adult peripheral blood containing sodium citrate anticoagulant into a 250ml centrifuge cup, centrifuge at 1500rpm, 20°C for 15min, and collect the upper plasma;

[0152] (2) Add an equal volume of normal saline to the lower blood cell layer and mix thoroughly; add 20ml of Ficoll-Hypaque lymphocyte separation medium (density 1.077, Tianjin Haoyang) into a 50ml centrifuge tube, and then put an equal volume of blood cell suspension along the tube wall Slowly add to the upper layer of the lymphocyte separation medium, be careful not to break the separation liquid level; then centrifuge the centrifuge tube at 2000rpm for 20min, and adjust the speed of the centrifuge to 0;

[0153] (3) Discard the supernatant layer of the cell suspension after centrifugation, and carefully absorb the middle buffy coat layer, which is the peripheral blood mononuclear cell layer. Trypan blue staining is...

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Abstract

The invention relates to preparation and application of a CD19-targeting humanized chimeric antigen receptor, and concretely relates to construction of a human CD19-antigen-targeting humanized lentiviral vector expression plasmid. The human CD19-antigen-targeting humanized lentiviral vector expression plasmid is applied to the preparation of chimeric antigen receptor T cells; and then, the effectsare verified through the chimeric antigen receptor T cells in in-vitro proliferation tests, cell phenotype tests and cell killing tests. A CAR sequence of the invention consists of a leader peptide sequence, a human CD19-antigen-targeting single-chain scFv sequence, a hinge region, a transmembrane region structure domain and an intracellular signal conduction structure domain sequence. The chimeric antigen receptor is built into a lentivirus system expression vector, and immune cells are infected; the human CD19-antigen-targeting chimeric antigen receptor can be expressed on the surface of the immune cells; the effects of killing tumor cells can be achieved by recognizing the CD19 antigen expressed on tumor tissues; the goal of tumor treatment is achieved; and a novel measure is providedfor cancer treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and discloses the construction of a lentiviral vector expression plasmid with a humanized chimeric antigen receptor targeting human CD19 antigen, and its application in the preparation of chimeric antigen receptor T cells , and then verify the effect of the chimeric antigen receptor T cells in vitro proliferation test, cell phenotype test, and cell killing test. The humanized chimeric antigen receptor (CAR) sequence targeting human CD19 antigen consists of a leader peptide sequence, a scFv sequence targeting human CD19 antigen (including heavy chain variable region sequence and light chain variable region sequence), hinge region , transmembrane region and intracellular signaling domain (co-stimulatory signaling domain and primary intracellular signaling domain). The chimeric antigen receptor is constructed as a lentiviral system expression vector and infected with immune cells, which can express the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10
CPCC07K16/2803C07K14/7051C12N15/86C12N5/0636C07K2319/02C07K2319/03C12N2740/15043C12N2510/02
Inventor 魏卿许晓椿赵梦莲刘庆喜牛翰婕
Owner 天津英科赛奥科技有限公司
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