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Human T lymphocytes carrying CD20/CD19 bispecific chimeric antigen receptor as well as preparation method and application of human T lymphocytes

A chimeric antigen receptor and bispecific technology, which is applied in the field of human T lymphocytes and preparation, can solve the problems of limited clinical application, high price, and low efficiency of lentivirus infection, so as to enhance the killing effect and improve transduction Efficiency, good therapeutic effect

Active Publication Date: 2018-08-10
北京双赢科创生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0004] At present, the lentiviral vector commonly used in the preparation of CAR-T cells is to integrate CAR into the T cell genome, so that it can express CAR molecules stably for a long time. CD8+ T lymphocytes play a role in CAR-T cells, so through Specific cell activation and culture methods to increase the proportion of CD3+CD8+T lymphocytes in T lymphocytes can help improve the performance of CAR-T cells. In the process of preparing CART, T lymphocyte activation is a key link. The existing The activation of T lymphocytes mostly uses commercial magnetic beads coated or coupled with anti-human CD3 and anti-human CD28 monoclonal antibodies, such as Invitrogen’s Dynabeads, Miltenyi’s Transact beads, etc., which are convenient to use but expensive. Among them, Dynabeads is also protected by patents and can only be used in the production of CAR-T produced by Novartis. The proportion of CD3+CD8+T lymphocytes in T lymphocytes activated by the magnetic bead method is often not high
[0005] The vector with lentivirus as the backbone is mainly improved from human immunodeficiency virus HIV, which can infect non-dividing cells and dividing cells in vivo, and has a very broad application prospect in the field of gene therapy. Currently, lentivirus is used as a gene transduction vector Clinical experiments have proved its safety as a gene therapy carrier to a certain extent. Although lentivirus can infect non-dividing and dividing cells, the lentivirus infection efficiency of primary T cells has been relatively low, with an average of less than 20%. To a certain extent, the wide clinical application of lentivirus in chimeric antigen receptor immune cell therapy technology is limited. In addition, the method of adding polybrene and centrifugation to transduce lymphocytes with lentivirus is time-consuming and labor-intensive. The concentration of polybrene is cytotoxic, and long-term centrifugation will cause greater damage to the cells, and the transduction efficiency of lentiviruses containing large foreign fragments has not been significantly improved

Method used

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  • Human T lymphocytes carrying CD20/CD19 bispecific chimeric antigen receptor as well as preparation method and application of human T lymphocytes
  • Human T lymphocytes carrying CD20/CD19 bispecific chimeric antigen receptor as well as preparation method and application of human T lymphocytes
  • Human T lymphocytes carrying CD20/CD19 bispecific chimeric antigen receptor as well as preparation method and application of human T lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of lentiviral expression vector plvx-EF1a-CD20CD19CAR

[0052] The amino acid sequences of each part of the second-generation bispecific chimeric antigen receptor are connected to form the amino acid sequence of the chimeric antigen receptor, as shown in SEQ ID No.2, and the nucleic acid sequence of the chimeric antigen receptor is optimized , the nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID No.3, after introducing restriction sites SpeI and BamHI, artificial whole gene synthesis is performed, and cloned into a lentiviral vector to obtain plvx-EF1a-CD20CD19CAR recombinant lentivirus The expression vector plasmid, the recombinant plasmid was sent to Sangon Bioengineering Company for sequencing, and the sequencing results were compared with the fitted sequence and confirmed to be completely correct.

Embodiment 2

[0053] Example 2: Peripheral Blood Mononuclear Cells (PBMC) Isolation and Culture

[0054] Before separating PBMCs, in a biosafety cabinet in a GMP laboratory, coat a 6-well plate with anti-human CD3 monoclonal antibody and human fibronectin fragments, and take anti-human CD3 monoclonal antibody (OKT-3, Acrobiosystem ) 30ul and 500ug / ml human fibronectin fragment (Novoprotein) 600ul, diluted to 6ml with PBS, mixed well and added to each well, 1ml per well, sealed the plate with a sealing film, and placed at 4 degrees Refrigerate overnight.

[0055] In the biosafety cabinet of the GMP laboratory, separate PBMCs from 60ml of peripheral blood of healthy donors with human lymphocyte separation medium Ficollpaque plus (GE), resuspend with lymphocyte medium, place them in T150 culture flasks, and incubate at 37 degrees for 2h , retain unattached cells, count the cells and resuspend them in serum-free lymphocyte medium, take half of the cells and divide them into each well of a six-...

Embodiment 3

[0059] Embodiment 3: CART cell preparation

[0060] Take CD3+Fibronectin (fibronectin fragment) activated lymphocytes for 48h 2×10 6 For each, add 40ul of virus solution and 0.5ul of DEAE-dextran, incubate at 37°C for 10min, then add AIM-V medium (ie, serum-free medium) (containing 10% autologous plasma, 300U / ml hIL-2 and 10ng / ml hIL-15) 400ul, incubate at 37°C for 3.5h, then replace with fresh medium AIM-V (containing 10% autologous plasma, 300U / ml hIL-2 and 10ng / ml hIL-15), adjust the cell density to 1× 10 6 cells / ml, and then replenish or replace the medium every 2-3 days to keep the cell density at (1-3)×10 6 pieces / ml.

[0061] On the 8th day of culture, the positive rate of CAR+ and the positive rate of CAR in CD4+ and CD8+ T lymphocytes were detected by flow cytometry. The results are summarized in Table 2. For the results of flow cytometry, please refer to image 3 ,pass image 3 It can be seen that CAR+ (%) is 91.9; CD4+CAR+ (%) is 48.4%, and CD8+CAR+ (%) is 42.5...

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Abstract

The invention discloses human T lymphocytes carrying a CD20 / CD19 bispecific chimeric antigen receptor as well as a preparation method and application of the human T lymphocytes. The human T lymphocytes are activated by means of an antibody, and the CD20 / CD19 bispecific chimeric antigen receptor is obtained by means of tandem of an anti-human CD20 antibody and an anti-human CD19 antibody. The preparation method provided by the invention can significantly increase the proportion of CD3+CD8+T cells in the T lymphocytes, obviously increases the virus transduction efficiency, can improve the coverage rate of CART cells on tumors, and prevents tumor recurrence after CD19 or CD20 single-target-point CART treatment.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to human T lymphocytes carrying CD20 / CD19 bispecific chimeric antigen receptors, a preparation method and application thereof. Background technique [0002] Targeted immune cell tumor therapy technology (mainly T lymphocyte (CART)) genetically modified by chimeric antigen receptor (CAR) is a new type of tumor cell immunotherapy technology that has been verified by a large number of preclinical studies and some clinical trials in recent years. , is also a current research hotspot; this technology is to genetically modify immune cells with chimeric antigen receptors containing scfv targeting tumor surface antigens, so that they have targeting and killing activity, among which T lymphocytes are most commonly used, Among them, CD8+ T lymphocytes mainly play a killing role. The technology of targeted immune cell tumor therapy with chimeric antigen receptor genetic modification provid...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K14/70521C07K14/70578C07K16/2803C07K16/2887C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2501/51C12N2501/515C12N2510/00C12N2533/52C12N2740/15043C12N2800/107
Inventor 赵欣黄欣欣高志慧朱永波盖丽云李刚毅
Owner 北京双赢科创生物科技有限公司
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