Tumor antigen/MHC-I compound as well as preparation method and application thereof

A technology of MHC-I and tumor antigens, applied in the field of immunology, can solve the problems of inability to apply tumor neoantigens and limit wide application, and achieve the effect of enhanced killing function, wide application range, and simple steps

Pending Publication Date: 2022-07-08
青岛华赛伯曼医学细胞生物有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently used aAPC is an approach limited to identified and selected peptide-MHC complexes and cannot be applied in the case of unknown tumor neoantigens
Some researchers also combine known, genetically engineered peptide-MHC complexes on magnetic beads to enrich / stimulate T cells, but this process requires pre-identification of immunogenic tumor antigens and synthesis of corresponding patient-specific MHC in vitro -Class I molecular structure also limits the wide application of this method in clinical tumor immunotherapy

Method used

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  • Tumor antigen/MHC-I compound as well as preparation method and application thereof
  • Tumor antigen/MHC-I compound as well as preparation method and application thereof
  • Tumor antigen/MHC-I compound as well as preparation method and application thereof

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preparation example Construction

[0051] The present invention provides a method for preparing a tumor antigen / MHC-I complex, comprising:

[0052] (11) Lysing tumor cells to obtain a tumor cell lysate, the tumor cell lysate containing tumor antigen / MHC-I complexes;

[0053] (12) mixing the antibody-loaded carrier with the tumor cell lysate to obtain the tumor antigen / MHC-I complex; wherein the tumor antigen / MHC-I complex is loaded on the carrier, The antibody specifically binds to MHC-I in the tumor antigen / MHC-I complex.

[0054] Specifically, in step (11), tumor cells refer to tumor cells obtained from tumor tissue. The present invention does not limit the method for obtaining tumor cells from tumor tissue and the method for lysing tumor cells, and conventional methods in the art can be used.

[0055] The tumor antigen and MHC-I are already in a combined state in tumor cells, that is, the tumor antigen and MHC-I exist in the tumor cell lysate in the state of "tumor antigen / MHC-I complex (pMHC)".

[0056] ...

Embodiment 1

[0097] 1. Tumor tissue cell lysis

[0098] 1.1 Carry out the verification experiment with lung cancer tumor tissue samples, and cut the tumor tissue into 1mm by aseptic operation 3 Small and large tissue fragments were added to normal saline, and the tissue cells were collected through a 70 μm filter with a Pasteur pipette;

[0099] 1.2 The collected tissue cells were centrifuged at 400g, room temperature for 5min;

[0100] 1.3 After centrifugation, collect the cells and resuspend them in normal saline for cell counting, and resuspend the remaining cells in complete medium for use;

[0101] 1.4 Place cells in test tubes so that each tube contains 4*10 6 2 cells and lysed; washed with 1 mL of pre-cooled PBS (pH 7.4) before lysing, washed twice in total, centrifuged at 400g, 4°C for 5 min after each washing, and then collected the cells;

[0102] 1.5 Add 300 μl of conditioned cell lysis buffer (Lysis buffer: 2.5 mg / ml CHAPS, 25 mM Tris buffer and 150 mM NaCl) to the collected...

Embodiment 2

[0118] The effect of pMHC complexes on CD8 in TIL cells derived from the same tumor sample + The specific enrichment effect verification experiment of T cells:

[0119] 1. The TIL cells derived from the same lung cancer tumor tissue were counted by 1*10 6 per tube, centrifuged to remove the supernatant; washed once with 1 mL PBS (10 g / LHSA, pH 7.4); centrifuged at 400 g, 4°C for 5 min to remove the supernatant, and resuspended with 100 μl PBS (5 g / LHSA, pH 7.4).

[0120] 2. To remove interference background, add 0.5U avidin to each tube of resuspended cells, incubate at room temperature for 10 min; add 1 mL of PBS (5 g / L HSA, pH 7.4), centrifuge at 400 g for 5 min at 4°C to remove the supernatant; Cells were resuspended in 1 ml of PBS (5 g / L HSA, pH 7.4) and centrifuged twice.

[0121] 3. In order to enhance the binding of pMHC complex to TIL cells of the same source and reduce the interference background during flow staining and detection analysis, add dasatinib (50nM, 5g / L...

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Abstract

The invention relates to a tumor antigen / MHC-I compound as well as a preparation method and application thereof. On the basis of the design principle of a shotgun method, the compound of the tumor antigen expressed by the tumor cells and the MHC-I is grabbed from the tumor cell lysate at a time without difference, the immunogenic tumor antigen does not need to be identified in advance, the application range is wider, the steps are simpler and more convenient, and the cost is lower. The tumor antigen / MHC-I compound provided by the invention can specifically enrich CD8 < + > T cells in tumor infiltrating lymphocytes, and can induce and stimulate activation and proliferation of the tumor infiltrating lymphocytes and the CD8 < + > T cells in the tumor infiltrating lymphocytes, so that the problem of'precise targeting 'of the tumor antigen specific T cells is solved, the killing function of the tumor infiltrating lymphocytes on tumor cells is enhanced, and the tumor antigen / MHC-I compound can be used for preparing tumor cells. The compound can be used for treating tumor diseases and optimizing an ACT method.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a tumor antigen / MHC-I complex and a preparation method thereof and enrichment of CD8 in vitro + Use of T cells, stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source and / or preparing a medicament for treating tumors; also relates to an in vitro enriched CD8 + Methods of T cells and obtained CD8 + T cells, a method for stimulating the activation and proliferation of tumor-infiltrating lymphocytes from the same source in vitro, the obtained tumor-infiltrating lymphocytes, a corresponding pharmaceutical composition, and a method for treating tumors. Background technique [0002] The relationship between the immune system and tumors is intricate: on the one hand, the immune system can kill or eliminate tumor cells through a variety of immune effector mechanisms; Clear. In the early stage of tumors, the immune system attempts to eliminate tumo...

Claims

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Application Information

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IPC IPC(8): C07K14/74C07K14/47G01N33/543G01N33/569C12N5/0783C12N5/078A61K39/00A61P35/00
CPCC12N5/0636A61P35/00C12N2501/599C12N2501/2321C12N2501/2302A61K2039/5158G01N33/6848G01N33/6872A61K39/001111C12N2509/00
Inventor 李文静李斌高青
Owner 青岛华赛伯曼医学细胞生物有限公司
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