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Preparation method of tumor-enhanced tumor-infiltrating lymphocytes

A lymphocyte and tumor infiltration technology, which is applied in the field of tumor infiltrating lymphocyte preparation, can solve the problems of short survival time, immune cells do not have tumor killing function, and TILs of different quality, so as to accelerate clearance, improve tumor killing activity, and improve The effect of lethality

Active Publication Date: 2021-12-07
THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] However, clinical trials have also found many shortcomings of TILs: ①The first is the strong heterogeneity. Although TILs contain tumor-specific killing T cells, most of the immune cells do not have tumor-killing functions, and the tumors contained in TILs in different patients The number of specific killer T cells is different, and the preparation is non-selective expansion, which eventually leads to different quality of TILs prepared from different patients, which also leads to great differences in subsequent clinical effects; Inhibition of the PD1 signaling pathway in the microenvironment prevents the tumor killing effect to the greatest extent; ③ TILs prepared by traditional techniques have poor activity and short survival time in vivo. Due to the above reasons, the clinical efficacy of traditional TILs is different, and breakthrough treatment needs to be upgraded through technological upgrading bottleneck

Method used

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  • Preparation method of tumor-enhanced tumor-infiltrating lymphocytes
  • Preparation method of tumor-enhanced tumor-infiltrating lymphocytes
  • Preparation method of tumor-enhanced tumor-infiltrating lymphocytes

Examples

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Effect test

Embodiment 1

[0074] The patient, male, 66 years old, the prostate tumor tissue was fully washed with normal saline, and the normal tissue and necrotic area around the prostate cancer specimen were removed. Put the prostate cancer specimen in a sterile petri dish, add a small amount of normal saline, and use sterilized surgical scissors to cut out a size of about 1-8mm from different areas around the specimen 3 The number of small tissue blocks is generally not less than 8. A small piece of prostate cancer tissue was taken and placed in a well of a 24-well plate, each well containing 2ml of X-VIVO15 medium, IL-7 (10ng / mL), IL-15 (20ng / mL), IL- 21 (20ng / mL), 1% double antibody, 5% human serum albumin, β-mercaptoethanol (55uM) and hepes (10mM), and other wells were operated in the same way. Place the 24-well plate at 37°C, 5% CO 2 cultured in a cell culture incubator. The proliferation of lymphocytes around the tissue blocks in each well was observed microscopically every other day. Regar...

Embodiment 2

[0076] The patient, male, 58 years old, the enhanced TILs culture method was the same as the above method (harvested cells 78×10 8 indivual).

Embodiment 3

[0078] The patient, male, 74 years old, the enhanced TILs culture method was the same as the above method (harvested cells 46×10 8 indivual).

[0079] like figure 2 As shown, General Biosystems (Anhui) Co., Ltd. was commissioned to construct the PBR and IL-15 super complex, according to the extracellular region of PD1 (SEQ ID NO: 1), the intracellular region of 41BB (SEQ ID NO: 2), △ Truncation (SEQ ID NO: 3) of IL2RB (YRHQ), P2A linking peptide (SEQ ID NO: 21), signal peptide (SEQ ID NO: 7), "suicide gene" R structure (SEQ ID NO: 8), Linker1 (SEQ ID NO: 9), Interleukin-15 mutant IL-15-M-N72D (SEQ ID NO: 10), Linker2 (SEQ ID NO: 11), Sushi structure of interleukin-15 receptor alpha The sequence of the domain (SEQ ID NO: 12), CD-4 transmembrane region (SEQ ID NO: 13) was assembled. After optimization, the gene is routinely synthesized and cloned into the vector plasmid vector.

[0080] 所述的PD1 DNA序列ATCCCAATGCAGCAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAG...

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Abstract

The invention discloses a preparation method of tumor-enhanced tumor-infiltrating lymphocytes. The preparation method comprises the following steps: (1) extracting tumor-infiltrating lymphocytes TILs from tumor tissues; (2) sorting and enriching PD1 positive T cells in the TILs; (3) integrating a PBR transformed fusion protein and an IL-15 super compound into the PD1 positive T cell at one time; and (4) carrying out multiplication culture to obtain the tumor enhanced TILs. The preparation method has the advantages that (1) by sorting the PD1 positive T cells, the proportion of tumor-specific T cells is increased; (2) the designed PBR fusion protein can relieve the inhibition from a PD1 pathway in a tumor microenvironment and improve the killing ability of the TILs to tumor cells; (3) the IL-15 super compound not only can improve the tumor killing activity of T cells, but also can stimulate other immune cells in the body, such as endogenous NK, T cells and the like, and accelerates the removal of the tumor cells in the body; and (4) the added 'suicide gene' R structure solves the problem of subsequent clinical safety after T cell gene modification.

Description

technical field [0001] The invention relates to a method for preparing tumor-enhanced tumor-infiltrating lymphocytes, and belongs to the technical field of preparing tumor-infiltrating lymphocytes. Background technique [0002] Tumor is currently one of the most threatening diseases to human beings. Conventional treatment such as surgery and chemotherapy can only delay the disease if it is detected in the early stage, and there is no better treatment method in the advanced stage. In recent years, immunotherapy technology has developed rapidly, whether CAR-T or PD1 / PD-L1 immune card control antibody drugs have been launched one after another, bringing hope for tumor treatment. Tumor-targeted immune cell research is in full swing, and some progress has been made, including DC vaccines and CAR-T currently on the market globally. However, most immune cell technologies currently target a single tumor target or a few targets. More aggressive tumors have limited efficacy. [0003...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N5/10C12N15/62
CPCC12N15/87C12N5/0636C07K14/70521C07K14/70578C07K14/5443C07K14/7155C07K14/70514C07K2319/02C07K2319/03C07K2319/00C12N2510/00
Inventor 徐振宇伍耀何银梅
Owner THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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