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Autologous dendritic cell activated tumor-infiltrating T-lymphocyte preparation method and application of T-lymphocyte

A technology of dendritic cells and lymphocytes, which is applied to the preparation and application of autologous dendritic cells to activate tumor-infiltrating T lymphocytes. It can solve the problems of limited activation growth, low killing function of TIL cells, and long culture time. question

Inactive Publication Date: 2014-11-19
YANTAI BOYU BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The number and killing function of TIL cells are directly related to the curative effect. The problems in the treatment are limited access to a large number of TIL cells and too long culture time
Therefore, the traditionally used low-dose interleukin-2 activation growth factor is limited, and the long culture time causes the low killing function of TIL cells

Method used

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  • Autologous dendritic cell activated tumor-infiltrating T-lymphocyte preparation method and application of T-lymphocyte
  • Autologous dendritic cell activated tumor-infiltrating T-lymphocyte preparation method and application of T-lymphocyte
  • Autologous dendritic cell activated tumor-infiltrating T-lymphocyte preparation method and application of T-lymphocyte

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preparation example Construction

[0035] The invention relates to a preparation method for the proliferation of autologous tumor-infiltrating T lymphocytes, which is characterized in that the autologous tumor antigen-loaded dendritic cells and interleukin 2 are used to amplify and activate tumor-infiltrating T lymphocytes to make tumor-infiltrating T lymphocytes Lymphocytes proliferate in large numbers.

[0036]The highly expressed CD80, CD83, CD86 and CD137L polypeptides on the autologous tumor antigen-loaded dendritic cell membrane of the present invention, and the processed tumor antigen epitope, will be able to activate a large number of T lymphocytes and have high activity. Antitumor cytotoxic response. Dendritic cells are collected by strong acid, strong alkali and / or irradiation, and high-speed centrifugation, and treated as TIL-activated and expanded trophoblast cells.

[0037] The tumor-infiltrating T lymphocytes in the method of the present invention are derived from fresh autologous tumor tissues o...

Embodiment 1

[0064] Example 1 Preparation of autologous tumor-infiltrating T lymphocytes

[0065] Fresh autologous tumor tissues were collected from each of breast cancer, lung cancer, melanoma and liver cancer patients (who signed informed consent), and cut into 1mm pieces with ophthalmic scissors 3 The small pieces of the resulting shredded tissue were digested with 0.25% (mass volume ratio) trypsin or 100 activity units / mL type I collagenase solution at a weight volume ratio of 1 g: 0.5 mL for 30 minutes, and then added to the digestion solution Calf serum with a volume of 1 / 5 of the solution was terminated, the digestion solution was passed through a 10 μM cell sieve, and washed twice with 5 mL of 1×PBS (pH=7.4), the filtered solution was centrifuged at 1000 rpm to collect the cell pellet, and the AIM-V medium (US Life Company) was resuspended to 16 mL to prepare a single cell suspension.

[0066] Use a 5mL pipette to take 5mL of lymphocyte separation solution and add it to a 15mL cen...

Embodiment 2

[0069] Example 2 Preparation of autologous tumor antigen-loaded dendritic cells

[0070] The tumor cells obtained in Example 1 were diluted with AIM-V medium to obtain a cell concentration of 2×10 6 / mL cell suspension, divided into cryopreservation tubes, sealed tightly, quickly frozen with liquid nitrogen, slowly thawed at room temperature, repeated freezing and thawing 3-5 times, centrifuged at 5000rpm for 10 minutes, took the supernatant, added to 1.5mL centrifuged Autologous tumor antigens were prepared in tubes and stored at -80°C for loading dendritic cells.

[0071] Mononuclear cells were obtained by centrifuging 100 mL of peripheral blood from tumor patients through Ficoll-Hypaque density gradient. Peripheral blood mononuclear cells were resuspended in AIM-V medium, and the adherent cells were induced by adding complete medium, containing 1000IU / mL GM-CSF, 500IU / mL IL-4, placed at 37°C, 5% CO 2 Harvest immature dendritic cells after five days in the incubator. Afte...

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Abstract

The invention provides an autologous dendritic cell activated tumor-infiltrating T-lymphocyte preparation method. The method has the following characteristics that: a tumor-infiltrating T-lymphocyte from a cancer patient is activated and amplified in a large quantity by using a dendritic cell loaded by an autologous tumor antigen as a trophoblast cell and interleukin-2, and has a highly active anti-tumor cell toxic response. The invention further provides an autologous tumor-infiltrating T-lymphocyte kit prepared according to the method, and the kit has an efficient anti-tumor function.

Description

technical field [0001] The invention relates to a preparation method for autologous dendritic cells to activate tumor-infiltrating T lymphocytes, which includes the following features: the infiltrating T lymphocytes isolated from fresh autologous tumor tissues after surgery of cancer patients are loaded with autologous tumor antigens The dendritic cells are activated as trophoblast cells and massively expanded under the action of interleukin 2, and have a highly active anti-tumor cytotoxic response; The kit can be used for immunotherapy of various cancers including solid tumors and blood tumors. Background technique [0002] Tumor-infiltrating lymphocytes (Tumor-infiltrating lymphocytes, TIL) cultured and expanded in vitro plus interleukin-2 (IL-2), its anti-tumor effect is 50-100 times stronger than LAK cells, and only a small Only a small amount of IL-2 can play a role, and because of its specificity to its own tumor target cells, it has become a hot spot in tumor biology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K39/00A61P35/00
Inventor 张增明方志明
Owner YANTAI BOYU BIOTECH CO LTD
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