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Method for preparing HPV (human papillomavirus) antigen specific CTL (cytotoxic T lymphocyte)

A technology of cytotoxicity and lymphocytes, which is applied in the field of biotechnology development and application research, can solve the problems of long preparation cycle, low cell expansion efficiency, and limited materials, and achieve the effects of low cost, prolonged survival time, and repeatability

Active Publication Date: 2018-07-20
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Commonly used preparation methods for CTL include tumor infiltrating lymphocyte (TIL) in vitro expansion method, TCR gene modification method, and in vitro antigen sensitization method. Disadvantages, which limit its large-scale application

Method used

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  • Method for preparing HPV (human papillomavirus) antigen specific CTL (cytotoxic T lymphocyte)
  • Method for preparing HPV (human papillomavirus) antigen specific CTL (cytotoxic T lymphocyte)
  • Method for preparing HPV (human papillomavirus) antigen specific CTL (cytotoxic T lymphocyte)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of HLA-A2402-restricted anti-HPV antigen-specific CTL

[0044] (1) HLA-A2402 + Typing test: collect 2mL of blood from the subject (EDTA anticoagulant), and send it out for testing of HLA typing (Beijing Boao Jingdian Biotechnology Co., Ltd.).

[0045] (2) Synthesis of antigenic peptide: HPV16E6 antigenic peptide, the site is 49-57, and the sequence is 9 peptides of VYDFAFRDL (SEQ IDNO: 1, hereinafter referred to as HPV16E6 49-57 Peptide), chemically synthesized (Shanghai Gill Biochemical Co., Ltd.), fully dissolved in sterile double distilled water, the peptide concentration was 2mg / ml, and stored in -80°C in aliquots.

[0046] (3) Peripheral blood collection and peripheral blood mononuclear cell (PBMC) isolation: 50 mL of peripheral venous blood was collected in a vacuum blood collection tube anticoagulated with heparin, and PBMC were obtained after Ficoll density gradient centrifugation:

[0047] a. Transfer the collected blood sample to a 50m...

Embodiment 2

[0056] Example 2: Detection of CD8 after cell expansion by flow cytometry + / IFNγ + cell ratio

[0057] (1) Get the cell sample obtained on the 14th day of cultivation in Example 1, 1×10 6 Cells / tube, wash 2 times with PBS.

[0058] (2) Add antigenic peptide (HPV16E6 49-57 Peptide, Shanghai Golgi Biochemical Co., Ltd.) and protein transport inhibitor Golgi-stop (BD, product number 554715) stimulated and activated cells, and incubated in a cell culture incubator for 3h.

[0059] (3) Flow detection antibody was added for flow detection: human CD8-APC antibody (BD, Cat. No. 555369), incubated at room temperature in the dark for 30 min, washed twice with PBS.

[0060] (4) Add fixed permeabilization solution (BD, Cat. No. 554715), incubate at room temperature in the dark for 30 min, and wash twice with PBS.

[0061] (5) Add flow detection antibody for flow detection of intracellular factors: human IFNγ-PE antibody (BD, Cat. No. 559327), incubate at room temperature in the dark...

Embodiment 3

[0064] Embodiment 3: Detection of cell killing activity by lactate dehydrogenase (LDH) method

[0065] Take the Caski cell line in the logarithmic growth phase (purchased from the China Institute for Food and Drug Control) as the target cells, and adjust the cell density to 3 × 10 4 cells / mL, 100 μL per well was spread in a 96-well culture plate. Take the CTL cells obtained in Example 1 of the present invention on the 14th day and adjust the density to 3×10 4 pcs / mL, 3×10 5 pcs / mL, 6×10 5 pcs / mL, 1.2×10 6 1 / mL, added to a 96-well culture plate, 100 μL per well, so that the effect-to-target ratios were 1:1, 10:1, 20:1, and 40:1, respectively, and three replicate wells were set up for each group.

[0066] (1) Analysis board settings

[0067] a. Spontaneous LDH release by effector cells: different effector-target ratios, take the corresponding number of effector cells and add them to the reaction plate;

[0068] b. Experimental well: cells with different effect target ratio...

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Abstract

The invention discloses a method for preparing HPV (human papillomavirus) antigen specific CTL (cytotoxic T lymphocyte) and particularly discloses a preparation method of HLA-A2402 restrictive anti-HPV antigen specific CTL. According to the method, a peripheral blood mononuclear cell is collected through single blood collection or venous blood collection, the antigen presentation function of B cells is enhanced with CpG ODN 2395, the B cells supporting HLA-A2402 restrictive HPV antigen peptide are used for stimulating the peripheral blood mononuclear cell, rhIL-2, rhIL-7, rhIL-15 and rhIL-21 are jointly used to promote growth of the T cell. The target CTL prepared with the method has the characteristics that preparation is simple, the preparation cycle is short, the cost is low, the multiplication capacity and the killing activity are high, the cell viability is high and the like, and can be used for immunotherapy of HPV infection related diseases including cervical cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology development and application research, and specifically relates to a preparation method of human leukocyte antigen-A2402-restricted anti-HPV antigen-specific cytotoxic T lymphocytes. Background technique [0002] Cervical cancer is a malignant tumor that seriously threatens women's life and health, and is the third most common malignant tumor in women worldwide after breast cancer and colorectal cancer. In developing countries, it is the second most common malignant tumor after breast cancer and the most common malignant tumor of female reproductive system. According to the World Health Organization (WHO), there are 500,000 new cases of cervical cancer and 250,000 deaths from it each year. Developing countries are the hardest-hit areas of this disease, and 200,000 people die from it every year, accounting for 80% of the global death toll. A large number of studies have shown that the occurrence of ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0638C12N2501/2302C12N2501/2307C12N2501/2321C12N2501/2315
Inventor 陈立敏毕薇薇
Owner JILIN TUO HUA BIOTECH
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