Culturing method of adenovirus gene-modifying tumor specificity cytotoxic T lymphocyte

A tumor-specific and cytotoxic technology, applied in the field of tumor-specific cytotoxic T lymphocyte culture, can solve the problems of patient influence, low tumor cell targeting, low antigen specificity, etc. Therapeutic effect, the effect of reducing the burden

Inactive Publication Date: 2016-05-25
山东省齐鲁细胞治疗工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Looking at the existing technology, it is not difficult to find that there are many deficiencies in the existing technology: the immune cells used in the existing immune cell therapy mainly include CIK, NK, TIL, and DC-CIK, which are a type of immune cells, but their antigens Low specificity and low targeting of tumor cells; iAPA-DC / CTL technology has certain antigen specificity, but CTL simply proliferates in vitro and does not have tumor antigen specificity; moreover, it is limited by the blood component apheresis machine , not every hospital is able to collect mononuclear cells, resulting in limitations; tumor patients have weak autoimmunity, and extracting a large number of immune cells at one time may have a worse impact on patients

Method used

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  • Culturing method of adenovirus gene-modifying tumor specificity cytotoxic T lymphocyte
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  • Culturing method of adenovirus gene-modifying tumor specificity cytotoxic T lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Obtaining mononuclear cells from cord blood or autologous peripheral blood

[0063] ①Wipe the blood collection bag with dust-free paper soaked in alcohol and put it into the biological safety cabinet. Use a 50ml syringe to draw the blood in the blood bag and divide it into 50ml centrifuge tubes. Each tube has 30ml of blood. Centrifuge for 10 minutes;

[0064] ② After centrifugation, put the upper layer plasma into a new 50ml centrifuge tube. Suction until 0.5cm away from the interface, if the remaining blood cells are more than 15ml, take the upper 15ml of blood cells and place them in a new centrifuge tube, discard the remaining red blood cells at the bottom;

[0065] ③Dilute the white blood cells with normal saline at a ratio of 1:1, take a centrifuge tube containing 15ml of ficoll and tilt it, and slowly add the mixed blood cells to the surface of the ficoll to make it a clear interface. The ratio of blood sample to ficoll is not the same. More than 2:1,...

Embodiment 2

[0069] Example 2: A method for obtaining immature DCs from umbilical cord blood mononuclear cells induced by cytokines

[0070] ①Set the cell density to 10 7 / ml of umbilical cord blood mononuclear cells according to 10 6 / cm 2 The number of cells added to the culture flask, 37 ° C, 7.5% CO 2 Cultivate in a concentration incubator;

[0071] ② After standing for 2 hours, gently shake the culture bottle to shake up the non-adhered cells, absorb the upper layer of non-adhered cells and transfer them to a new culture bottle; add X-VIVO15 containing GMCSF1000U / ml and IL-41000U / ml Medium, at 37°C, 7.5% CO 2 Cultivate in a concentration incubator;

[0072] ③The next day, shake the culture bottle to shake up the non-adherent cells, discard the culture medium, and refill the X-VIVO15 medium containing GMCSF1000U / ml and IL-41000U / ml to continue the culture;

[0073] ④Change the medium every two and a half days, add X-VIVO15 medium containing GMCSF1000U / ml and IL-41000U / ml, complet...

Embodiment 3

[0074] Example 3: A method for inducing maturation of immature DCs by adenovirus infection

[0075] ①Collect the immature DC cultured to the 5th day, take a sample and count, calculate the total amount of DC, centrifuge at 1500rpm for 10min;

[0076] ② Discard the supernatant after centrifugation, add X-VIVO15 medium containing 1000U / mlGMCSF, 1000U / mlIL-4 and 100ng / ml TNF-α to resuspend the cells, and adjust the cell density to 5×10 6 / ml; According to the MOI is 10 4 Add the virus dosage of vp / cell to the cell suspension, mix thoroughly, spread in culture flasks, and store at 37°C, 7.5% CO 2 Cultivate in a concentration incubator;

[0077] ③ After culturing for 48 hours, collect the suspended cells, scrape up the unsuspended cells with a cell scraper, and mix the two types of cells into mature DC cells. Sampling and counting, flow cytometry detection of cell phenotype;

[0078] ④Determine the requirement of DC according to the amount of T cells, resuspend the remaining ma...

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Abstract

The invention relates to a lymphocyte culture method, and in particular discloses a culturing method of adenovirus gene-modifying tumor specificity cytotoxic T lymphocyte. The method comprises: taking 150-200ml of bleeding of the umbilicus or autologous peripheral blood for single karyocyte separation, separately culturing DC cells by adhering to a wall for 2h, and performing T lymphocyte preculture on the non-adhered cells in an IL-2-contained culture medium; collecting mature DC after the DC cells are mature through induction of iAPA factor adenovirus infection, stimulating the autologous primary T lymphocyte to differentiate into cytotoxic T lymphocyte according to an effect target ratio of DC to T being 1 to 10, and observing and recording multiplication capacity of CTL and detecting the cytotoxicity of the CTL. The limitation of a blood component single sampling machine is solved, collected blood volume is less, burden of a patient is reduced, and iAPA-DC can be observed in vitro to stimulate and differentiate the primary T lymphocyte into tumor specificity cytotoxic T lymphocyte.

Description

(1) Technical field [0001] The invention relates to a method for culturing lymphocytes, in particular to a method for cultivating tumor-specific cytotoxic T lymphocytes modified by adenovirus genes. (2) Background technology [0002] Immune cell therapy is a new treatment for cancer. In 2008, Baylor College of Medicine in the United States filed a patent application for DC vaccine combination: the technology of silencing immune negative regulatory genes (inhibition of Antigen Presentation Attenuators, iAPA), commonly known as "cancer fear" technology, is to use small interference Ribonucleic acid (siRNA) specifically blocks the key negative regulator SOCS1 gene in DC cells, silences its gene expression, induces the body to break through its own immune tolerance to tumors, enhances antigen-specific immune responses, and improves the efficacy of tumor immunotherapy. Clinical efficacy. Compared with traditional DC-CIK cells, iAPA technology can prevent DC cells from entering a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/22C12N2501/2304C12N2501/25C12N2502/1114C12N2502/1121
Inventor 张剑慧王芝辉武建明谭毅张慧慧
Owner 山东省齐鲁细胞治疗工程技术有限公司
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