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Genetically modified dendritic cell vaccine

A technology of dendritic cells and vaccines, applied in the fields of biotechnology and medicine

Active Publication Date: 2019-07-02
上海尚泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using tumor cells or cell lysates, apoptotic tumor cells, tumor cell lysates, tumor proteins, recombinantly expressed tumor antigen dominant epitope peptides, tumor polypeptide mixtures, etc. to sensitize DC to make tumor vaccines, but subject to confirmed Tumor antigen restriction or autoimmune disease induced by mixed antigens including normal antigens of the body
Although the reinfusion of DC into the body can induce a specific immune response, the half-life of most MHC-restricted antigen peptides is only a few hours, and repeated reinfusion is required to induce a high level of persistent immune response

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A modified dendritic cell, the lentiviral vector is loaded with MG-7Ag antigen mimic epitope tandem sequence to infect the dendritic cell, so that the target antigen sequence is integrated into the dendritic cell genome.

[0033] 1. MG-7Ag antigen mimic epitope tandem sequence synthesis

[0034] The mimetope tandem sequence of the MG-7Ag antigen carrying the Asc I / Xba I double restriction site was synthesized and loaded with the PUC57 plasmid.

[0035] 2. Construction of MG-7Ag antigen mimic epitope tandem sequence lentiviral expression vector

[0036] The lentiviral pLVX-shRNA expression vector was purchased from Clontech Company, and the double plasmid was digested to obtain the target sequence and connection vector. The target sequence was recovered by the gel purification kit and treated with P, and the carrier sequence was recovered by the gel purification kit after the P treatment was removed, and the two were ligated to transform E.coli competent cells.

[0037...

Embodiment 2

[0072] A protocol for rapid in vitro culture of dendritic cells derived from peripheral blood monocytes.

[0073] 1. Peripheral blood-derived mononuclear cells induced DC culture in vitro

[0074] The sorted and enriched peripheral blood mononuclear cells were divided into 3-5×10 6 cells / mL cells were resuspended in AIM-V medium, spread in culture flasks at 37°C 5% CO 2 After incubation in the incubator for 1 h, the AIM-V differentiation induction medium containing 2% autologous plasma, 1000 U / mL rhIL-4, and 500 U / mL rhGM-CSF was replaced. After 48 hours of differentiation induction, lentivirus infection induced differentiated DCs. After differentiation induction for 72 hours, the differentiated DCs were collected by centrifugation at 300 g for 8 minutes, and replaced with fresh 12 mL of 2% autologous plasma containing 5 μg / mL poly(I:C), 1000 U / mL rhIL-4 , 500U / mL rhGM-CSF AIM-V medium, placed in a culture bottle, 37 ℃ 5% CO 2 Mature DCs were obtained after 24 h incubation ...

Embodiment 3

[0078] One active ingredient is DC-CTL immune function of dendritic cell vaccine modified by MG-7Ag mimetic epitope tandem sequence.

[0079] 1. In vitro tumor killing test of DC-CTL

[0080] 1.1 Use Ficoll to isolate autologous peripheral blood PBMCs, collect DCs on the fifth day of differentiation induction culture, and co-culture DCs and PBMCs in AIM-V medium containing 10% autologous plasma at a ratio of 1:10.

[0081] 1.2 Add 500U / mL rhIL-2 on the fourth day of culture, and continue to culture for 10 days. Supplement AIM-V medium during the period to maintain the cell density at 1-2×10 6 / mL by pipetting discrete large cell clumps.

[0082] 1.3 On the fourteenth day of DC-CTL co-culture, cells were collected and counted, and 50 μL of AIM-V medium resuspended in 96 wells was added to 5×10 4 , 2.5×10 4 , 5×10 3 For three gradient effector cells, add 50 μL AIM-V medium to the corresponding reaction wells to resuspend the target cells MKN45 and KATO-3 to 5×10 3 , set 6 ...

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Abstract

The invention relates to the fields of biotechnology and medicine, and provides a modified dendritic cell (DC); a dendritic cell is infected with MG-7Ag antigen mimic epitope tandem sequences loaded with a lentivirus vector, and a target antigen sequence is integrated into a dendritic cell genome. The invention provides a rapid culture scheme of the dendritic cell derived from a peripheral blood monocyte in vitro, and also provides a vaccine with an active ingredient of the modified dendritic cell. The vaccine is used for tumor prevention and active immunotherapy. The MG-7Ag antigen sequence-modified DC vaccine can obtain high-purity CTL cells and efficient target cell killing ability after DC-CTL co-culture, and the co-culture supernatant contains high-concentration IFN[gamma] secretion.After tumor attack, the tumorigenic volume of mice in a DC-CTL group is significantly smaller than that of mice in a control group, and the DC vaccine has great potential value in immunotherapy of MG-7Ag positive tumor.

Description

[0001] field of invention [0002] The invention relates to the fields of biotechnology and medicine, in particular, the invention relates to a genetically modified dendritic cell and a vaccine comprising the modified dendritic cell. [0003] Background of the invention [0004] Dendritic cells (DC) are the most functional professional antigen-presenting cells in the human body, which can activate CD8+ cytotoxic T cells (CTL) and CD4+ helper T cells (Th), and play an important role in anti-tumor and anti-virus important role in the immune response. Most studies have shown that tumors in vivo inhibit DC maturation rather than directly inhibit DC function. Studies have found that the number and function of dendritic cells in malignant tumor tissues are negatively correlated with the degree of infiltration of tumor cells into the surrounding tissues in the primary tumor tissue and metastases, and the clinical stage of the patient, while the infiltration dendritic cells in most so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61K39/00A61P35/00
CPCC12N5/0639A61K39/0011C12N2510/00C12N2501/2304C12N2501/22C12N2501/999
Inventor 李晨蔚梁九林
Owner 上海尚泰生物技术有限公司
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