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Cytotoxic T lymphocytes with CA9 tumor antigen specificity

A cytotoxic and lymphocyte technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problem of poor targeting of renal cancer cells, reduce blood collection and inhibit tumors Growth, pain relief effects

Inactive Publication Date: 2020-08-21
山东省齐鲁细胞治疗工程技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem that current immune cell therapy has poor targeting of kidney cancer cells, the present invention provides a CA9 tumor antigen-specific tumor antigen modified by dendritic cells modified by an adenovirus vector carrying an immune-suppressing negative regulatory gene and a carbonic anhydrase IX gene. Culture method of cytotoxic T lymphocytes

Method used

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  • Cytotoxic T lymphocytes with CA9 tumor antigen specificity
  • Cytotoxic T lymphocytes with CA9 tumor antigen specificity
  • Cytotoxic T lymphocytes with CA9 tumor antigen specificity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Culture of CA9-DC-CTL cells

[0026] The culture process of CA9-DC-CTL cells is as follows: figure 1 As shown, it includes the following steps: prepare recombinant adenovirus Ad-siSOCS1-CA9-IRES-F and control adenovirus Ad-GFP respectively; isolate mononuclear cells from whole blood, and obtain DC cells after 7 days of induction; DC cells are treated with adenovirus Cultured for 24 hours after infection, and mixed with T cells to obtain CA9-specific cytotoxic T lymphocytes.

[0027] 1. Preparation of AD5-siSOCS1-CA9 Recombinant Adenovirus

[0028] The siSOCS1-CA9 recombinant adenovirus target gene sequence design is as follows: U6 promoter-SOCS1 shRNA-CMV promoter-CA9 gene-IRES sequence-Flagellin flagellin gene sequence. Then the outsourcing company packaged the above-mentioned target gene and AD5 adenovirus backbone into adenovirus to obtain Ad-siSOCS1-CA9-IRES-F recombinant adenovirus carrying the target gene. At the same time, an irrelevant siRNA control...

Embodiment 2

[0035] Example 2 Killing activity of different CTL cells on target cell CA9-293FT

[0036] The pHR-CA9 expression vector was constructed, lentivirus was packaged, and HEK293 cells were infected to obtain CA9-expressing HEK293-FT cells as target cells of CA9-specific DC-CTL cells. The target cell CA9-293FT was inoculated into the 16-well plate provided with the RTCA instrument, and after 6-8 hours of culture, it completely adhered to the wall.

[0037] According to the set effect-to-target ratio of 1:3 and 1:1, add 100 μL of CA9-DC-CTL cells and GFP-DC-CTL cells (control) into the corresponding wells, and RTCA will dynamically detect the effect of the two cells on the tumor in real time. cell killing process. Figure 4 It is a real-time record of the killing of CA9-293FT on the 1st to 3rd day after the mixed culture of DC and T. The results showed that the killing effect of CA9-DC-CTL cells on target cells was significantly higher than that of control cells when the effect-to...

Embodiment 3

[0038] Example 3 Different treatment of T cells to obtain the killing activity of CTL cells on the target cell CA9-293FT

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Abstract

The invention provides a preparation method of renal cell carcinoma tumor antigen CA9 specific cytotoxic T lymphocytes, which comprises the following steps: infecting mature DC cells with a recombinant vector containing siRNA targeting SOCS1 and carbonic anhydrase 9 to obtain iSOCS1-CA9-DC cells; and then carrying out mixed culture on the iSOCS1-CA9-DC cells and T cells to obtain CA9-DC-CTL cells,namely the cytotoxic T lymphocytes with antigen specificity. According to the invention, replication-defective adenovirus mediated RNAi is adopted to down-regulate SOCS1 gene expression, and CA9 andS-Flagellin are combined to stimulate DC cell maturation so as to induce specific CTL to resist renal cell carcinoma. The targeting property of CTL to kidney cancer cells is effectively enhanced, damage to autologous cells is avoided, growth of tumors can be effectively inhibited, and the lifetime of a patient is prolonged.

Description

technical field [0001] The invention belongs to the field of immune cell therapy, and specifically relates to a CA9 tumor antigen-specific cytotoxic T lymphocyte activated by dendritic cells modified by an adenovirus vector carrying an immune-suppressing negative regulatory gene and a carbonic anhydrase IX gene, and its training method. Background technique [0002] Immune cell therapy is a new treatment for cancer. Lymphocytes isolated from peripheral blood of patients have a good ability to respond to a variety of foreign antigens. Therefore, culturing DC cells in vitro, inducing high-efficiency cytotoxic T lymphocytes (CTL), and returning them to patients has become a good treatment method. At present, tumor cell lysate is often used to load DC, but it has shortcomings, such as many types of non-tumor-associated antigens, which are easy to induce autoimmune diseases, and multiple antigens bring disadvantages such as low immunogenicity. Another stimulus inducer uses tumo...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783C12N15/113C12N15/861
CPCC07K14/00C12N5/0638C12N5/0639C12N9/88C12N15/113C12N15/86C12N2310/14C12N2310/531C12N2502/1114C12N2502/1121C12N2510/00C12N2710/10043C12Y402/01001
Inventor 张剑慧孔群芳马贺然谭毅
Owner 山东省齐鲁细胞治疗工程技术有限公司
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