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Preparation method and application of antigen reactive T cell based on RNA vaccine

A reactive and cell-based technology, applied in the direction of genetically modified cells, blood/immune system cells, biochemical equipment and methods, etc., can solve the problems of long polypeptide vaccine preparation cycle, low T cell expansion efficiency, low degree of cell activation, etc. problems, to achieve the effect of short preparation cycle, high preparation success rate and good repeatability

Inactive Publication Date: 2021-09-07
浙江格源致臻生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 1. The production cycle of polypeptide vaccine is long and the cost is high
[0010] 2. DC transfection efficiency is low, safety is not high, applicability is not strong
[0011] 3. After traditional co-incubation, the expansion efficiency of T cells is low, and the degree of cell activation is not high

Method used

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  • Preparation method and application of antigen reactive T cell based on RNA vaccine
  • Preparation method and application of antigen reactive T cell based on RNA vaccine
  • Preparation method and application of antigen reactive T cell based on RNA vaccine

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preparation example Construction

[0043] A method for preparing antigen-reactive T cells based on RNA vaccine, comprising the following steps:

[0044] Construct the RNA vaccine in vitro expression vector, and obtain the RNA vaccine after plasmid linearization and in vitro transcription; wherein, the construction of the RNA vaccine includes direct synthesis and construction of the expression vector for in vitro transcription. This application uses the construction of the expression vector for in vitro transcription, which has a short production cycle. Low cost, large-scale production and other advantages; mammalian expression vector can be pcDNA TM 6. pcDNA TM 5. pCEP4, pcDNA3.1, etc., this application is preferably pcDNA3.1, which has the advantages of being applicable to high-level and constitutive expression in various mammalian cell lines.

[0045] T cells and RNA vaccine-modified DC cells were obtained based on the collected PBMCs; among them, the common methods for RNA vaccines to enter DC cells include...

Embodiment 1

[0048] Construct RNA vaccine in vitro expression vector, and obtain RNA vaccine after plasmid linearization and in vitro transcription:

[0049] (1) At room temperature, take a 200ul PCR tube, add 15ul of double-distilled water, 2ul of 10X buffer, 1ug ​​of plasmid (2ul), 1ul of XhoI enzyme in turn, shake and mix, and incubate at 37°C for 5min. Plasmid linearization;

[0050] (2) The linearized plasmid was purified and recovered using a PCR purification kit (qiagen, 28104);

[0051] (3) At room temperature, add the linearized pcDNA3.1+ plasmid, T7 transcription premix, T7 RNA polymerase, add double distilled water to the system of 20ul, 37°C, warm bath for 1h, and perform in vitro transcription of the plasmid ;

[0052] (4) The in vitro transcribed RNA vaccine was purified and recovered using an RNA purification kit (qiagen, 74134).

[0053] The obtained RNA vaccine was tested for integrity.

[0054] The present invention has been tested for integrity: as figure 1 The frag...

Embodiment 2

[0056] Obtain T cells and RNA vaccine-modified DC cells based on harvested PBMC:

[0057] (1) On day 0, 80-100 ml of peripheral blood mononuclear cells were collected by an apheresis machine. PBMCs were separated by Ficoll-Histopaque, adhered to the wall for 2 hours, and non-adherent cells (T cells) were collected and frozen for future use. Adherent cells (DC cells) are added to the DC culture medium for the preparation of RNA vaccines;

[0058] (2) On the second day, absorb the DC culture supernatant, centrifuge for 5 minutes, remove half the volume of medium, add a corresponding volume of fresh medium and supplement cytokines to maintain a certain solubility of cytokines (rhGM-CSF and IL-4) ;

[0059] (3) On day 4, DC cells were induced to mature. Add TNF-α, IL-1b, IL-6 to induce DC cell maturation overnight;

[0060] (4) On the 5th day, mature DC cells were collected, and RNA vaccine (DC cells modified by RNA vaccine) was electroporated.

[0061] Take 10ml of whole blo...

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Abstract

The invention discloses a preparation method and application of an antigen reactive T cell based on an RNA vaccine. The preparation method comprises the following steps of constructing an in-vitro expression vector, and carrying out plasmid linearization, in-vitro transcription and purification to obtain the RNA vaccine; obtaining a T cell and a DC cell modified by the RNA vaccine based on a collected PBMC; and carrying out multiplication culture on the DC cell modified by the RNA vaccine and the T cell to obtain the antigen reactive T cell based on the RNA vaccine. According to the preparation method and application of the antigen reactive T cell based on the RNA vaccine, pcDNA3.1 + plasmids are used for constructing the mammal in-vitro expression vector, and compared with traditional polypeptide synthesis, the preparation period is short, the cost is low, and the preparation success rate is high; an electrotransfection instrument is used for electrotransfection, and compared with a traditional transfection mode, the repeatability is good, reagent cost is avoided, and the electrotransfection rate is high; the RNA vaccine is used for electrotransfecting the DC cell, compared with other biological treatment drugs, in-vitro production and purification are easy, and the production process is high in universality and high in safety; and the DC cell and the T cell which are subjected to electrotransfection are co-incubated and amplified, and the obtained activated vaccine is high in proportion and strong in immunogenicity.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to a preparation method and application of an RNA vaccine-based antigen-reactive T cell. Background technique [0002] Malignant tumor is one of the diseases with the highest incidence rate in the world, and it ranks in the top three along with cardiovascular disease and cerebrovascular disease. In our country, these three diseases are also ranked in the top three. At present, the clinical effect of comprehensive treatment based on surgery, radiotherapy, and chemotherapy is not ideal. Although the wide application of molecular targeted therapy in recent years has significantly improved the prognosis of some malignant tumors, the number of people who benefit from targeted therapy is small and the final average facing the problem of drug resistance. Most patients with non-malignant tumors do not have these oncogene mutations, and the treatment methods are still based on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N15/85C12N5/10A61K35/17A61P35/00
CPCC12N5/0636C12N5/0639C12N15/85A61K35/17A61P35/00C12N2502/1121C12N2510/00C12N2501/515C12N2501/51C12N2501/2302C12N2501/2307
Inventor 苏小平李锐李伟迎张齐
Owner 浙江格源致臻生物医药科技有限公司
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