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Cell subset for immunotherapy of primary hepatocellular carcinoma and preparation method thereof

A technology for hepatocellular carcinoma and cell subgroups, which is applied in the field of immune cells, cell subgroups for immunotherapy of primary hepatocellular carcinoma and its preparation, can solve the problems of poor tumor effect and achieve strong tumor killing effect, good effect

Active Publication Date: 2019-11-22
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a cell for immunotherapy and a preparation method thereof. The cell for immunotherapy and its preparation method should solve the problem of using immunotherapy in the prior art. Technical problems of ineffective treatment of tumor diseases

Method used

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  • Cell subset for immunotherapy of primary hepatocellular carcinoma and preparation method thereof
  • Cell subset for immunotherapy of primary hepatocellular carcinoma and preparation method thereof
  • Cell subset for immunotherapy of primary hepatocellular carcinoma and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] CD8 + T cells are key cells in the anti-tumor immune response, capable of secreting cytokines such as interferon gamma (IFN-γ), attacking tumor cells with major histocompatibility complex class I (MHCI) on their surface. CD8 infiltrating the tumor microenvironment + T cells are susceptible to inhibitory factors, which eventually lead to functional exhaustion and immune escape of tumors. In 2016, Speiser et al proposed that exhausted T cells in tumors are heterogeneous. Finding T cells with functional plasticity will be very helpful for immunotherapy.

[0020] CD3 isolated from tumors of patients with primary hepatocellular carcinoma (HCC) + CD45RO + Single-cell sequencing of cells found CD8 in tumor patients + T cells are mostly exhausted.

[0021] Experimental operation procedure: the tumor tissue and paracancerous tissue of the HCC patient were chopped and ground, filtered through a 70 μM filter, and treated with erythrocyte lysate to obtain a single cell suspen...

Embodiment 2

[0029] 1) Process the surgically resected HCC tumor tissue into a single cell suspension. Cut the tissue block into a slurry with sterile surgical scissors, add 20ml of collagenase type IV with a concentration of 1mg / ml, and place it on a constant temperature shaker at 37°C for 45 minutes for digestion. Then take it out, use a 40um filter to filter the digested product into a 50ml centrifuge tube, and place the filtrate in a centrifuge for 10 minutes at 1100rpm. Then remove the supernatant, add 2ml erythrocyte lysate (containing 150mM NH 4 Cl, 10mM KHCO 3 ,

[0030] 100uMEDTA), lyse at room temperature for 5 minutes, add 10% FBS RPMI1640 medium to 10ml, and centrifuge at 1100rpm for 10 minutes. Discard the supernatant, add 10ml of normal saline, centrifuge at 1100rpm for 10 minutes, and discard the supernatant.

[0031] 2) Perform flow cytometric antibody labeling.

[0032] The labeling methods of several markers to be tested are as follows:

[0033] a) Add the surface f...

Embodiment 3

[0041] Example 3 CD137 + CD3 + CD8 + CD45RO + Preparation method of T lymphocyte subsets

[0042] The cell population obtained by flow cytometry sorting in Example 2 was cultured in vitro to amplify tumor-specific TILs for immunotherapy of HCC. Amplification methods are described below.

[0043] Primary culture (enzymatic tumor digestion solution and tumor fragments (1-8mm) produced by mechanical grinding 3 )):

[0044] 1. After placing the tumor in enzyme digestion solution (RPMI 1640, 2mM glutamine, 10mg / mL gentamicin, 30U / mLDNase, 1.0mg / mL collagenase), immediately mechanically dissociate for about 1 minute;

[0045] 2. Incubate the solution at 37°C in 5% carbon dioxide by volume for 30 minutes, then mechanically separate again for 1 minute;

[0046] 3. After incubating again at 37°C in 5% carbon dioxide by volume for 30 minutes, mechanically disturb the tumor for a third time for 1 minute;

[0047] 4. If there is still a large piece of tissue after the third mechan...

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Abstract

The invention provides a cell subset for immunotherapy of primary hepatocellular carcinoma, namely a CD137 + CD3 + CD8 + CD45RO + T cell subset. The invention also provides a preparation method of theabove cell subset. The method comprises the following steps: firstly labeling a T cell subset in tumor tissues with a specific marker, and then carrying out flow cell sorting on the CD137 + CD3 + CD8+ CD45RO + T cell subset, and carrying out in-vitro expansion culture to obtain the cell subset for immunotherapy of primary hepatocellular carcinoma. The cell subset of the invention has stronger tumor-killing effect, anti-apoptosis capacity and multiplication capacity, and can survive in vivo for a long time. The invention aims to provide effective candidate cell populations for immunotherapy of liver cancer, especially T cell immunotherapy of tumor infiltration, and provide new ideas for treatment of other tumors.

Description

technical field [0001] The invention belongs to the field of biology, and relates to an immune cell, in particular to a cell subgroup for immunotherapy of primary hepatocellular carcinoma and a preparation method thereof. Background technique [0002] Immunotherapy refers to the treatment of disease by activating the immune system. In tumor treatment, CAR-T therapy is a widely used immunotherapy. Traditional CAR-T therapy transforms PBMC cells through chimeric antigen receptors to improve the ability of T cells to recognize target cells. After a large number of CAR-T cells are expanded in vitro, they are reinfused into the patient. This method has achieved remarkable curative effect in the treatment of both solid tumors and hematological tumors, but the therapeutic effect is greatly affected by individual differences, and it only has a significant effect in some patient populations, and the overall treatment response rate is not high. There are two main reasons for this: i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K39/00A61P35/00
CPCC12N5/0636A61K39/001111A61P35/00C12N2501/515C12N2501/52C12N2501/505C12N2501/599
Inventor 武多娇刘芳铭王向东刘卫仁史颖弘
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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