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Allogeneic antigen-presenting cell targeting KRAS mutation, construction method and preparation method of intestinal cancer specific CTL cell

A construction method and technology of allogeneic antigens, which can be applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., which can solve the problems of low proportion and number of specific cells, and it is difficult for patients with KRAS mutation to benefit. , to achieve the effect of a strong immune response

Active Publication Date: 2019-09-06
焦顺昌 +1
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  • Abstract
  • Description
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Problems solved by technology

[0002] In colorectal cancer patients, KRAS mutation is one of the reasons for resistance to targeted drugs; therefore, patients with KRAS mutation are difficult to benefit from targeted drugs and have a short survival period. Therefore, it is urgent to develop immune cells targeting KRAS mutations treatment approach
At the same time, the patient-derived dendritic cell DC has a small number and poor ability. If autologous DC is used as the antigen of KARS, the prepared cytotoxic T lymphocyte CTL will have a small proportion of specific cells and weak killing ability.
If it is an allogeneic DC, the HLA does not match and cannot be used

Method used

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  • Allogeneic antigen-presenting cell targeting KRAS mutation, construction method and preparation method of intestinal cancer specific CTL cell
  • Allogeneic antigen-presenting cell targeting KRAS mutation, construction method and preparation method of intestinal cancer specific CTL cell
  • Allogeneic antigen-presenting cell targeting KRAS mutation, construction method and preparation method of intestinal cancer specific CTL cell

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preparation example Construction

[0032] The present invention also provides a method for preparing colon cancer-specific CTL cells, comprising the following steps: using the allogeneic antigen-presenting cells targeting KRAS mutations to co-culture T cells in peripheral blood mononuclear cells for 20-20~ On 22d, colon cancer-specific CTL cells were obtained. In the present invention, the peripheral blood mononuclear cells are derived from the intestinal cancer patient described in step 1) or the mononuclear cell provider described in step 3). The method for collecting the peripheral blood mononuclear cells is not particularly limited in the present invention, and a conventional method for collecting peripheral blood mononuclear cells in the art can be used (Ficoll-Pague, GE: 17544202). In the specific implementation of the present invention, there is no special limitation on the method for isolating T cells from the peripheral blood mononuclear cells, and conventional methods in the field can be used (Milteny...

Embodiment 1

[0036] Construction of a heterogeneous antigen-presenting cell targeting KRAS mutation

[0037] According to the patient's HLA typing results and KRAS mutation sequence, use the online prediction software NetMHCpan 4.0Server to analyze the affinity of all HLA typing and KRAS combinations, and sort them;

[0038] HLA typing includes: HLA-A0206, HLA-A2601, HLA-B4001, HLA-B1301, HLA-C0304, HLA-C0702

[0039] KRAS mutations include:

[0040] KRAS-12A: TEYKLVVVGAAGVGKSALTIQ (SEQ ID NO: 3)

[0041] KRAS-12C: TEYKLVVVGACGVGKSALTIQ (SEQ ID NO: 4)

[0042] KRAS-12D: TEYKLVVVGADGVGKSALTIQ (SEQ ID NO: 5)

[0043] KRAS-12S: TEYKLVVVGASGVGKSALTIQ (SEQ ID NO: 6)

[0044] KRAS-12V: TEYKLVVVGAVGVGKSALTIQ (SEQ ID NO: 7)

[0045] KRAS-13D: EYKLVVVGAGDVGKSALTIQL (SEQ ID NO: 8)

[0046] KRAS-61H: CLLDILDTAGHEEYSAMRDQY (SEQ ID NO: 9)

[0047] The combination of HLA typing and KRAS mutation antigen includes: HLA-A0206+KRAS-G12D, HLA-A0206+KRAS-G13D, HLA-A0206+KRAS-G12V, HLA-A0206+KRAS-G12C, ...

Embodiment 2

[0071] Colorectal cancer-specific CTL cells

[0072] 1. Isolation of T cells:

[0073] 1) Collect the patient's own PBMC by centrifugation, resuspend in PBS, pass the cells through a 30 μm cell sieve, and count;

[0074] 2) centrifuge at 300g for 10min, completely remove the supernatant;

[0075] 3) Resuspend the cells with separation buffer (PBS, pH 7.2, 0.5% BSA2mM EDTA), every 10 7 Cells were resuspended in 80 μL of separation buffer;

[0076] 4) Every 10 7 cells, add CD3 magnetic beads (Germany Miltenyi Biotechnology Co., Ltd.);

[0077] 5) After mixing, incubate for 15min (2-8°C)

[0078] 6) Every 10 7Add 1-2mL separation buffer to resuspend the cells, centrifuge at 300g for 10min, and completely remove the supernatant;

[0079] 7) will be 10 8 Resuspend cells within 500 μL of separation buffer;

[0080] 8) Rinse the separation column with separation buffer; MS: 500 μL, LS: 3 mL

[0081] 9) transfer the cells to the separation column;

[0082] 10) Collect unbou...

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Abstract

The invention provides an allogeneic antigen-presenting cell targeting KRAS mutation, a construction method and a preparation method of intestinal cancer specific CTL cell, which belong to the technical field of tumor targeting, the method for constructing the allogeneic antigen-presenting cell comprises the following steps: 1) analyzing the affinity of a combination of HLA typing and KRAS mutation sequence of a certain intestinal cancer patient, and selecting a combination of synthetic fusion genes with the highest affinity; 2) ligating the synthetic fusion gene to an expression vector to obtain a recombinant vector; and 3) transferring the recombinant vector to a healthy-derived monocyte, and performing induced culture to obtain the allogeneic antigen-presenting cell targeting KRAS mutation. The allogeneic antigen-presenting cell targeting KRAS mutation solve the problem that the allogeneic DC does not match the HLA typing of the patient, and can stimulate a stronger immune response.The colon cancer-specific CTL cell has the good killing effect on target cells.

Description

technical field [0001] The invention belongs to the technical field of tumor targeting therapy, and in particular relates to a KRAS mutation-targeted allogeneic antigen-presenting cell, a construction method, and a preparation method of colon cancer-specific CTL cells. Background technique [0002] In colorectal cancer patients, KRAS mutation is one of the reasons for resistance to targeted drugs; therefore, patients with KRAS mutation are difficult to benefit from targeted drugs and have a short survival period. Therefore, it is urgent to develop immune cells targeting KRAS mutations treatment approach. At the same time, the patient-derived dendritic cell DC is small in number and poor in ability. If autologous DC is used as the antigen of KARS, the proportion of specific cells in the prepared cytotoxic T lymphocyte CTL is small and the killing ability is weak. If it is an allogeneic DC, the HLA does not match and cannot be used. [0003] Therefore, how to prepare DCs tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783
CPCC12N5/0638C12N5/0645C12N2502/1114C12N2510/00
Inventor 焦顺昌张嵘周子珊
Owner 焦顺昌
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