WO2022073254A1

WO2022073254A1 - Method for preparing nanosized tumor vaccine

Info

Publication number: WO2022073254A1
Application number: PCT/CN2020/122251
Authority: WO
Inventors: 乐龙; 尹鸿萍; 刘永军; 杨美家; 徐红星
Original assignee: 江苏艾洛特医药研究院有限公司
Priority date: 2020-10-06
Filing date: 2020-10-20
Publication date: 2022-04-14
Also published as: CN112691188A

Abstract

A method for preparing a tumor vaccine by nanosizing Flagellin and a cytokine and/or a cytokine inducer and combining same with a tumor antigen in vitro. The tumor vaccine prepared by the method has the following features: after nanosizing, Flagellin and the cytokine and/or the cytokine inducer have the characteristics of targeting and sustained release; Flagellin and the cytokine and/or the cytokine inducer are respectively used as a second signal and a third signal for T cell activation, and can activate non-specific immunity more strongly; Flagellin can enhance the antigen presentation capability of APC, and enhance specific immunity; and the tumor vaccine is widely suitable for preventing recurrence and preventing metastasis and treatment of various types of tumors, and has a good application prospect.

Description

A kind of preparation method of nano-sized tumor vaccine

technical field

The application belongs to the field of biomedicine, and in particular relates to a preparation method and application of a nano-sized tumor vaccine.

Background technique

Since the pioneering work of William B. Coley, the father of cancer immunotherapy in 1893, supporting tumors expressing specific antigens that make them naturally immunogenic when sufficient immune stimulation is provided, activation of the body's own immune system to suppress tumors Occurrence and development, and the elimination of tumor cells in the body has become a research hotspot in tumor therapy. Immune checkpoint blockade (CPB) drugs and adoptive cell transfer (ACT) therapy occupy a very important position in the treatment of tumors, but CPB drugs have a narrow scope of application and are representative of ACT The existing CAR-T therapy also has disadvantages such as high cost, high target selectivity, and complicated in vitro culture operations, which greatly limit its application in tumor treatment. A tumor vaccine is a vaccine that enhances specific T cell responses through active immunization. The active components mainly include tumor antigens, immune adjuvants, carriers, and dosage forms. Tumor antigens can generally be divided into tumor-associated antigens and tumor-specific antigens.

Immune adjuvants can activate immune cells nonspecifically. It is mainly divided into five types: the first activation signal adjuvant; the second activation signal/costimulatory signal adjuvant; the third activation signal adjuvant; and the tumor immunosuppressive microenvironment adjuvant. Flagellin belongs to the second activation signal/costimulatory signal adjuvant, which can bind to the TLR5 receptor on the cell membrane to promote the dimerization of TLR5, thereby activating MyD88, and MyD88 recruits downstream activators

(IRAK1/IRAK4) phosphorylates IKK, activates IKK, catalyzes the phosphorylation of IκB, and then dissociates from NF-κB after phosphorylation of IκB. Start gene transcription and release cytokines such as TNF-ɑ, IL-6, IL-12.

There are a large number of TLR5 receptors on the surface of most antigen-presenting cells (APCs), and Flagellin can activate and promote the proliferation and maturation of these APCs, increase their antigen uptake ability, enhance their ability to migrate to the draining lymph nodes, and complete the TLR5 receptors. Helper cells recruit, activate, promote differentiation, and ultimately initiate adaptive immunity. So that the body's immune system exerts a more powerful function to kill and remove tumor cells. With people's understanding and development of nanomaterials, nanotechnology has been widely used in the field of medical research.

Nano-drugs with targeting and sustained release are designed based on the differences in the physiological environment of tissues or lesions. Scholars have found that some nanomaterials can exert the proton sponge effect under specific pH conditions, destroy the cell membrane, and promote the cytoplasmic transport of substances. This material has great potential for antigen cytoplasmic transport and transport to lymph nodes. In addition, the use of nanomaterials to encapsulate proteins, peptides, etc., which are easily degraded by the body, such as antigens or adjuvants, prolong the half-life of the metabolism of such substances in the body or play a slow-release effect. Therefore, nanosized tumor vaccines are very feasible and advantageous.

SUMMARY OF THE INVENTION

1. The purpose of this application is to provide a nano-sized tumor vaccine and its application.

2. The preparation method of the nano-sized tumor vaccine provided by this application specifically includes the following steps:

The flagellin Flagellin and/or cytokines and/or cytokine inducers obtained by expressing and purifying by molecular biological means are encapsulated with nanomaterials;

The nanoformulation is combined with tumor antigen in vitro to obtain tumor vaccine.

3. Construct the expression vector of the flagellin Flagellin of Salmonella typhimurium by conventional means of molecular biology, and introduce the successfully constructed expression vector into the expression host by chemical and/or physical methods, which can be a prokaryotic expression host or a eukaryotic expression vector expression host.

4. After fermentation to obtain Flagellin Flagellin, according to the type of protein tag and the physicochemical properties of the protein itself, select appropriate separation and purification conditions to obtain electrophoresis pure Flagellin Flagellin, and use the existing in vitro activity detection method to detect Flagellin Flagellin. active.

5. Construct, express and purify various cytokines and/or cytokine inducers that activate T cells and/or bind to antigen-transporting cells by the same method as in steps 1 to 4 above.

6. Tumor antigens can be tumor cells inactivated by repeated freezing and thawing, X-ray irradiation, treatment with radioactive chemicals such as cisplatin, etc.; they can also be peptides synthesized by conventional molecular biology methods. tumor antigens.

7. Using nanomaterials that are positively charged and/or specifically bound to tumor antigens, explore suitable conditions to combine flagellin Flagellin; flagellin Flagellin and one or several cytokines; flagellin Flagellin and one or more Cytokine inducers; the flagellin Flagellin and one or more cytokines and cytokine inducers are encapsulated with suitable nanomaterials.

8. Using the intermolecular interaction force between the selected nanomaterials and various tumor antigens through physical adsorption or chemical binding, the adjuvant and tumor antigens are combined to produce different tumor vaccine compositions.

9. The tumor vaccine provided by this application effectively combines the adjuvant and tumor antigen, and since the half-life in vivo is prolonged after the nano-adjuvant is nano-sized, the nano-formulation can also play a targeting role, thereby more activating the body's immune system, clearing the tumor.

Description of drawings

Figure 1 is a schematic diagram of the construction of a nanosized tumor vaccine.

Figure 2 is the Western blotting of the expression of Flagellin, wherein Line1: Marker; Line2: the expression of Flagellin on the first day of fermentation; Line3: the expression of Flagellin on the second day of fermentation; Line4: the expression of Flagellin on the third day of fermentation; Line5: the expression of Flagellin on the fourth day of fermentation Day Flagellin expression level; Line6: Flagellin expression level on the 5th day of fermentation.

Figure 3 is the SDS-PAGE chart of the purification result of Flagellin, in which Line1: Marker; Line2: fermentation stock solution; Line3: loading flow-through solution; Line4: 50mM imidazole stepwise elution to collect the first tube eluate; Line5: 50mM imidazole The eluate from the second tube was collected by staged elution; Line6: the eluate of the third tube was collected by the staged elution of 50mM imidazole; Line7: the eluate of the fourth tube was collected by the staged elution of 50mM imidazole; Elution and collect the 5th tube eluate; Line9: 50mM imidazole stepwise elution and collect the 6th tube eluate.

Figure 4 is a graph showing the detection results of Flagellin activity after purification.

Detailed ways

Unless otherwise specified in this application, the nouns, terms and phrases used in this application have their ordinary meanings in the art. In addition, unless otherwise specified in this application, the reagents, instruments, etc. used in this application are conventional reagents or instruments in the field, and they are freely available in the market.

Example

Example 1

Expression of Flagellin in eukaryotic cells

The constructed 293-F-Flagellin-expressing cell line was taken out of the liquid nitrogen tank and then recovered, and counted after recovery for 48 hours. The density reached 1×10 ⁶ –3×10 ⁶ cells/mL and then passaged, and the number of cells after passage was 0.2×10 ⁶ – 0.5×10 ⁶ cells/mL, cultured in a shaker at 37°C, 5% CO ₂ , and 125 rpm. Take 500 μL of cell suspension on the 1st, 2nd, 3rd, 4th, and 5th days after passage, 1000-1500rpm, 4 Centrifuge at °C for 5 min, take the supernatant, centrifuge the supernatant at 8000 rpm for 10 min at 4 °C to take the supernatant, and detect the expression of Flagellin in the supernatant by Western blotting. The results are shown in Figure 1.

Example 2

Purification of Flagellin

On the 5th day of fermentation, the protein was collected. The specific operation was as follows: the fermentation broth was centrifuged at 400G for 5 min at 4°C and the supernatant was taken. The supernatant was centrifuged at 8000 rpm for 10 min at 4°C, and then passed through 0.8 μm, 0.45 μm and 0.22 μm aqueous filter membranes, and 10kDa TFF supernatant. It was concentrated by filtration and purified by Ni column. Elution Buffer (20mM PB+50mM imidazole) was eluted stepwise. The purification results are shown in Figure 2.

Example 3

Flagellin activity assay in vitro

Flagellin can stimulate THP-1 cells to release TNF-α, so the content of TNF-α can be detected by ELISA to reflect the activity of Flagellin. Specific operation: Spread the well-grown THP-1 cells into a 24-well plate, add different concentrations of Flagellin and control proteins when the confluence reaches 80-90%, and incubate at 37°C, 5% CO ₂ in a static incubator After culturing for 48 h, the supernatant was collected to detect the expression of TNF-α in the supernatant by ELISA kit. The results are shown in Figure 3.

The results showed that the Flagellin we expressed and purified was active.

Claims

A preparation method of a nano-sized tumor vaccine, specifically comprising the following steps:

(1) The flagellin Flagellin and/or cytokines and/or cytokine inducers obtained by expressing and purifying by molecular biological means are wrapped with nanomaterials;

(2) Combining the nano-formulation with tumor antigen in vitro to obtain a tumor vaccine.
The preparation method of claim 1, wherein the flagellin Flagellin is derived from Salmonella typhimurium.
The preparation method according to any one of claims 1 or 2, wherein the flagellin Flagellin can be obtained by biomolecular means.
The preparation method according to claim 1, wherein the cytokines and/or cytokine inducers used include various types of cytokines and/or cytokine inducers that activate T cells to bind to antigen-transporting cells.
The preparation method according to any one of claims 1 or 4, wherein the cytokines and/or cytokine inducers used are other cytokines such as IL2, IL15, INF-γ, TNF-α, GM-CSF and/or or cytokine inducers.
The preparation method according to any one of claims 1 to 4, wherein the flagellin Flagellin can be nanosized alone, or can be nanosized together with one or more cytokines and/or cytokine inducers.
The preparation method according to any one of claims 1 or 5, characterized in that: the nanosized material is a substance that is positively charged and/or specifically combined with tumor antigens.
The preparation method of claim 1, wherein the tumor antigens include inactivated tumor cells, tumor cell lysates and tumor antigens designed and produced by means of molecular biology.
The tumor vaccine according to any one of claims 1 or 7, wherein tumor cells can be inactivated by various physical and chemical methods.
The tumor vaccine obtained by the preparation method according to any one of claims 1 to 7.
The application of the tumor vaccine according to claim 10 in the prevention and/or treatment of tumor drugs.