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In-situ vaccine and preparation method thereof

A vaccine preparation and in-situ technology, which is applied in the fields of pharmaceutical formulations, cancer antigen components, vertebrate antigen components, etc., can solve the problem of unsatisfactory vaccination system, low specific immune response of vaccination response, reduction of vaccine immunogenicity and effective In order to achieve the effect of green preparation method, improve the utilization rate of antigen, improve safety and curative effect

Pending Publication Date: 2021-12-10
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons include: (1) Vaccination was performed on patients with "cold" tumors, resulting in low response to vaccination or ineffective specific immune responses
(2) Use of a suboptimal vaccination system that reduces the immunogenicity and effectiveness of the vaccine

Method used

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  • In-situ vaccine and preparation method thereof
  • In-situ vaccine and preparation method thereof
  • In-situ vaccine and preparation method thereof

Examples

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Effect test

preparation example Construction

[0030] The present invention provides a method for preparing an in situ vaccine, comprising the following steps:

[0031] S1. Cultivation: resuscitate and culture the tumor cells in the culture medium to reach the logarithmic growth phase, collect the cells with the digestive fluid and wash to obtain the tumor cell fluid;

[0032] S2, inactivation: inactivating the tumor cell liquid collected in step S1, incubating the inactivated tumor cell liquid, centrifuging the incubated tumor cell liquid to take the supernatant to obtain the inactivated antigen stock solution;

[0033]S3. Preparation of nanomaterial @Mal solution: use nanomaterial and PEG-Mal ligand in a mass ratio of 1:10-1:15 to form a mixed solution, add 10-15 times the volume of the mixed solution of cyclohexane to mix Precipitate the liquid, remove the supernatant by centrifugation to obtain the precipitate, which is dried, dissolved in ultrapure water, centrifuged and passed through a membrane, and then ultrafilter...

Embodiment 1

[0044] Prepare an in situ vaccine as follows:

[0045] (a) The breast cancer tumor cells (4T1) were recovered and cultured to reach their logarithmic growth phase. After the 4T1 cells were observed under the microscope and covered the bottom of the culture dish, the unattached tumor cells were washed with PBS buffer, and then used with a mass fraction of The adherent tumor cells were brought into suspension by digestion with 0.2% trypsin solution, collected by centrifugation, and washed 4 times with PBS buffer to remove the complete medium components in the culture dish, so that the tumor cells were suspended in PBS buffer The tumor cell fluid was formed, and then counted, and the adjusted cell volume was about 4M;

[0046] (b) irradiating the collected cells with X-rays, then incubating in a cell incubator for 0.5 h, then centrifuging to remove cells and cell debris, collecting the supernatant and centrifuging again to remove residual cell debris, for later use;

[0047] (c)...

Embodiment 2

[0051] Prepare an in situ vaccine as follows:

[0052] (a) with embodiment one;

[0053] (b) with embodiment one;

[0054] (c) First, draw 2 mL of oil-phase Fe with a concentration of 6.6 mg / mL 3 O 4 into a 15mL centrifuge tube, the oil phase Fe 3 O 4 Contain cyclohexane, add 15 mL of acetone solution to the centrifuge tube to complete precipitation, then centrifuge at 5000g for 5 min to remove the supernatant, and then add 2 mL of tetrahydrofuran solution until the precipitation is completely dissolved. Then weigh 158 mg of PEG-Mal ligand, at this time the oil phase Fe 3 O 4 The mass ratio of PEG-Mal ligand is =1:12, and tetrahydrofuran solution is added dropwise to it and shaken to dissolve it completely. Fe dissolved in tetrahydrofuran 3 O 4 Added to PEG-Mal also dissolved in tetrahydrofuran, and sonicated for 1 h. Cool to room temperature, then add 10 times the volume of cyclohexane, remove the supernatant by centrifugation, add an appropriate amount of ultrapure...

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Abstract

The invention relates to an in-situ vaccine and a preparation method thereof. The preparation method comprises the following steps: culturing and inactivating a tumor cell antigen stock solution; preparing a nano-material (at) Mal solution; adding an adjuvant to synthesize nano-material (at) Mal-adjuvant particles; and mixing and incubating the tumor cell antigen stock solution and the nano-material (at) Mal-adjuvant particles in a volume ratio of (5: 1)-(5: 3) to obtain a nano-material (at) Mal-adjuvant (at) antigen. The preparation method of the in-situ vaccine is environment-friendly, simple and easy to operate, and is suitable for various tumors and has universality. The maleimide on the surface of the nano-material (at) Mal can form a stable thioether bond with sulfydryl on protein so as to capture tumor-associated antigens, so that the utilization rate of the antigens is improved, and co-delivery of the adjuvant and the antigens can be realized. The nano material (at) Mal can be used as an adjuvant to activate antigen presenting cells and enhance the phagocytosis, so that the antigen capabilty is represented, T cells are further activated, and the safety and curative effect of cancer immunotherapy are improved.

Description

technical field [0001] The invention relates to an in situ vaccine and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] In recent years, with the development of immune checkpoint inhibitors and CAR-T, immunotherapy has shown great promise in the field of tumor treatment. The rapid development of new strategies for tumor immunotherapy has become the most attractive field of tumor therapy. [0003] In order to achieve the most ideal anti-tumor immune effect, "passive" immunotherapy such as adoptive cell therapy, genetically engineered T cells, etc. are usually used to directly attack tumor cells, or "active" immunotherapy such as cytokines, tumor vaccines, immune checkpoints are used. Inhibitors etc. regulate and activate the immune system. Tumor vaccines, as an active tumor immunotherapy strategy, have made great progress with the identification of more and more tumor neoantigens. Common types of tumor vaccines include pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/39A61P35/00B82Y5/00B82Y30/00B82Y40/00
CPCA61K39/0011A61K39/39A61P35/00B82Y5/00B82Y30/00B82Y40/00A61K2039/55505A61K2039/55561Y02A50/30
Inventor 汪勇马洁高明远王杨云张乐帅
Owner SUZHOU UNIV
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