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Specific antibody with CD20 as target, CAR-NK cell and preparation and application of CAR-NK cell

A specificity and antibody technology, applied in the field of biomedicine, can solve the problems of high production cost, inability to reinfuse the allogeneic body, unfavorable large-scale application, etc., and achieve the effect of stable traits

Inactive Publication Date: 2018-07-17
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the T cells used to prepare CAR-T can only come from the patient's own body and cannot be reinfused, which leads to high production costs and is not conducive to its large-scale application.

Method used

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  • Specific antibody with CD20 as target, CAR-NK cell and preparation and application of CAR-NK cell
  • Specific antibody with CD20 as target, CAR-NK cell and preparation and application of CAR-NK cell
  • Specific antibody with CD20 as target, CAR-NK cell and preparation and application of CAR-NK cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of lentiviral expression vector

[0060]Gene synthesis ScFV (Anti CD20)-CD8-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 11, and the gene sequence is shown in SEQ ID NO: 12). Through enzyme digestion, it was transformed and connected to the PRRSLIN vector, and the upstream of the gene was the EP-1α promoter. Transform the Stbl3 E. coli strain with the vector, screen with ampicillin, obtain positive clones, extract the plasmids, identify the clones by restriction enzyme digestion, and obtain the PRRLSIN-SCFV (Anti CD20)-CD8-4-1BB-CD3ζ lentiviral transfection vector (such as figure 1 shown).

Embodiment 2

[0061] The preparation of embodiment 2 lentiviruses

[0062] (1) 24 hours before transfection, use about 8×10 per dish 6 293T cells were seeded into 15cm culture dishes. Make sure that the cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

[0063] (2) Prepare solution A and solution B

[0064] Solution A: 6.25ml 2×HEPES buffer (the amount packed in 5 large dishes, the effect is the best).

[0065] Solution B: Add the mixture of the following plasmids: 112.5 μg PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ (target plasma); 39.5 μg pMD2.G (VSV-G envelope); 73 μg pCMVR8.74 (gag , pol, tat, rev); 625 μl of 2M calcium ion solution. Total volume of solution B: 6.25ml.

[0066] Mix solution B thoroughly, and while vortexing solution A gently, add solution B drop by drop and let stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, add dropwise to the culture dish containing 293T cells, gently shake the culture dish back a...

Embodiment 3

[0067] Example 3 Preparation of CD20 CAR NK-92 cells

[0068] Adjust the NK-92 cell density to 2-3 x 10 5 / ml, add the virus vector (prepared in Example 2) according to the ratio of volume ratio (V / V) virus vector: cell culture medium=1:5-10, and add polybrene 8 μg / ml at the same time. After 4 h, add an equal amount of fresh complete medium to adjust the cell density to 1×10 5 / ml to continue the culture. The next day, all the cells were centrifuged, fresh medium was added, and the culture was continued. Replenish fluid every 1-2 days to maintain cell density at 2-3×10 5 / ml. After 72 hours, CAR antibody staining was performed, and CD20 CAR NK-92 positive cells were sorted by flow cytometry and expanded for culture. Observe the color change of the medium, cell density, and cell shape every day and make corresponding records.

[0069] The positive rate of CAR NK-92 cells was detected by flow cytometry, and the results of flow cytometry were as follows: Figure 2a and F...

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Abstract

The invention provides an anti-CD20 antibody with CD20 as a target or an antigen binding fragment of the antibody. The antibody comprises a VH1 chain and / or a VL1 chain and / or a VH2 chain and / or a VL2chain, and the VH1 chain and / or the VL1 chain and / or the VH2 chain and / or the VL2 chain are directly connected or connected through linker peptide. An Anti CD20 CAR-NK cell established from the anti-CD20 antibody with CD20 as the target or the antigen binding fragment of the antibody is obtained by culturing and amplifying a monoclonal cell strain, is also stable in property, and can be used forlarge-scale production and preparation. The Anti CD20 CAR-NK cell can specifically kill or wound lymphoma cells by taking CD20 molecules as target antigens, can be used as a therapeutic drug for treating lymphoma diseases and is used for treating CD20 molecule high-expression tumors.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a specific antibody targeting CD20, a CAR-NK cell targeting CD20, and preparation and application thereof. Background technique [0002] Chimeric antigen receptor (CAR) modified immune cells use genetic engineering to modify immune cells to express exogenous anti-tumor genes. CAR genes mainly include an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize specific molecules on the tumor surface and the latter is used to initiate immune cell responses after recognizing tumor surface molecules and exert cytotoxic effects, which are mainly used for T-cells are carriers. [0003] CAR-T, the full name is Chimeric AntigenReceptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. Chimeric antigen receptor T cells (CAR-T cells) combine the antigen-binding portion of an antibody that can recognize a certain tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70578C07K16/2887C07K2317/56C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0646C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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