Anti AFP (alpha-fetoprotein) CAR-T (chimeric antigen receptor T) cell, preparation method thereof and application of cell

A technology of cells and lymphocytes, applied in the field of gene-modified cells and tumor treatment, which can solve the problems of high cost of immune rejection, shortage of donors, surgical injuries, etc., and achieve the effect of high-efficiency tumor-killing activity and simple preparation method

Active Publication Date: 2017-10-17
刘未斌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although liver transplantation has become an important method for the treatment of primary liver cancer, problems such as donor shortage, surgical injury, immune rejection and high cost constitute the main obstacles to the development of liver transplantation technology

Method used

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  • Anti AFP (alpha-fetoprotein) CAR-T (chimeric antigen receptor T) cell, preparation method thereof and application of cell
  • Anti AFP (alpha-fetoprotein) CAR-T (chimeric antigen receptor T) cell, preparation method thereof and application of cell
  • Anti AFP (alpha-fetoprotein) CAR-T (chimeric antigen receptor T) cell, preparation method thereof and application of cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Preparation of Anti AFP CAR-T cells of the present invention

[0046] 1. Construction of lentiviral expression vector

[0047] Through conventional genetic engineering methods, the SCFV-IgD-CD28-OX40-CD3ζ fusion protein gene sequence was synthesized, and the SCFV-IgD-CD28-OX40-CD3ζ fusion protein gene sequence was cloned into a lentiviral expression vector to obtain SCFV-IgD-CD28 –OX40–CD3ζ lentiviral expression vector pGreen puro-CAR. Wherein, each sequence of the SCFV-IgD-CD28-OX40-CD3ζ fusion protein is as described above.

[0048] 2. Lentiviral packaging

[0049] 1) Culture 293T cells in 1640 medium with a mass fraction of 10 wt% fetal bovine serum (fetal bovine serum, FBS for short);

[0050] 2) 293T cells were treated with 3x 10 5 / cm 2 Transfer the density to a culture dish with a diameter of 15cm and cultivate it for 20h to ensure that the cell confluence is 80-90% during transfection;

[0051] Replace the medium with serum-free 1640 medium and set aside; ...

Embodiment 2

[0065] The in vitro anti-tumor effect of the Anti AFP CAR-T cells prepared in Example 1 was evaluated, including the following steps:

[0066] With the liver cancer cell lines HepG-2 and Hep3B stably expressing AFP as target cells, T cells infected with the lentiviral vector pGreen puro-CAR (that is, the Anti AFP CAR-T cells prepared in Example 1) and uninfected T cells were respectively used. Make effector cells from cells, and target cells at a density of 1x 10 5 cells / ml inoculated in 96-well plate, 100 μl per well, added effector cells to target cells according to 5:1, 10:1, 20:1 effect-to-target ratio, placed in 5% CO 2 , cultured in a 37°C incubator for 4 hours, using WST-1 to detect cell viability, and calculate the killing efficiency.

[0067] figure 2 It is a schematic diagram of the killing effect of Anti AFP CAR-T cells and uninfected T cells on the target cell HepG-2 (liver cancer) according to the embodiment of the present invention; image 3 It is a schematic...

Embodiment 3

[0070] The anti-tumor effect of the Anti AFP CAR-T cells prepared in Example 1 was evaluated in vivo, including the following steps:

[0071] Take 15 6-week-old female nude mice and inject 5x 10 subcutaneously in the right armpit 6 HepG-2 (liver cancer) cells, when the tumor grows to 60mm 3 Size, tumor model was randomly divided into 3 groups: control group, Anti AFP CAR-T cell group, and T cell group; control group was injected with saline 200ul / time, twice a week; Anti AFP CAR-T cell group was injected with tail vein Anti AFP CAR-T cells 1×10 7 T cells / time, 2 times a week; T cells in the T cell group were injected into the tail vein 1×10 7 Each time, twice a week; the survival status of the mice within 100 days was counted, and the survival rate curve was made. Figure 4 It is a schematic diagram of the survival period of the mice treated with liver cancer (HepG-2) xenografted tumor model mice by the Anti AFP CAR-T cells of the embodiment of the present invention, the co...

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Abstract

The invention provides an anti AFP (alpha-fetoprotein) CAR-T (chimeric antigen receptor T) cell, a preparation method thereof and an application of the cell. According to the anti AFP CAR-T cell, SCFV-IgD-CD28-OX40-CD3 zeta fusion protein is expressed in a T cell, a SCFV (single chain Fv domain) is combined with AFP, and the amino acid sequence of the SCFV is a first sequence. The T cell is modified and transformed to obtain the anti AFP CAR-T cell, liver cancer can be specifically recognized and killed, and the anti AFP CAR-T cell is applicable to prevention and treatment of corresponding tumor diseases.

Description

technical field [0001] The invention relates to an Anti AFP CAR-T cell and its preparation method and application, belonging to the technical field of gene modified cells and tumor treatment. Background technique [0002] Primary liver cancer (hepatocellular carcinoma, referred to as: HCC) ranks fifth in the incidence of malignant tumors, and the annual death toll is about 1 million worldwide. Currently, most patients with primary liver cancer lack effective treatments, and the overall survival rate is only 3%-5%. Although liver transplantation has become an important method for the treatment of primary liver cancer, problems such as donor shortage, surgical injury, immune rejection and high cost constitute the main obstacles to the development of liver transplantation technology. Therefore, it is necessary to explore a new and effective treatment approach. With the rapid development of molecular biology, genetic engineering technology and immunology, biological therapy ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70521C07K2319/02C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 刘未斌
Owner 刘未斌
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