Nasal spray WT1 polypeptide preparation as well as preparation method, use method and application thereof

Abstract

The invention belongs to the field, and discloses a nasal spray WT1 polypeptide preparation, a preparation method, a use method and application, the preparation contains a polypeptide fragment of a specific WT1 antigen, through nasal spray administration, the vaccine can rapidly stimulate local and whole body immune response, and therefore the effect of preventing various tumors is achieved. The vaccine preparation disclosed by the invention has the advantages of local administration, quick effect, strong targeting property, easiness in storage and transportation and the like, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of vaccine technology, and specifically relates to a nasal spray WT1 polypeptide preparation and a preparation method, a use method, and an application. Background Art

[0002] With the deepening of oncology research, scientists have gradually realized the importance of enhancing the body's specific immunity to prevent tumor occurrence. WT1 (Wilms tumor gene 1) is a gene that is abnormally highly expressed in many cancers, and the protein it encodes (WT1 protein or WT1 antigen) has become an important target for tumor immunotherapy. WT1 antigen was not only first discovered in nephroblastoma, but is also widely expressed in many solid tumors and blood cancers including leukemia, lung cancer, breast cancer, etc., showing its potential as a broad-spectrum tumor antigen.

[0003] At present, immunotherapy strategies targeting WT1 antigen mainly include WT1 antigen peptide vaccine, WT1 antigen-loaded dendritic cells and WT1-specific T cell therapy. These therapies have shown significant anti-tumor effects in preclinical and clinical trials, but their application is mostly limited to the treatment of confirmed cancer patients rather than preventive vaccination. In addition, peptide drugs have attracted much attention in the biopharmaceutical field due to their high activity, low toxicity and easy synthesis. Summary of the invention

[0004] In order to overcome the above-mentioned technical problems, the present invention provides a nasal spray WT1 polypeptide preparation and a preparation method, a use method, and an application. The nasal spray WT1 polypeptide preparation is designed based on extensive safety research on the WT1 antigen and the low toxicity characteristics of polypeptide drugs. It has high safety under normal conditions of use, can significantly enhance the body's specific immunity to the WT1 antigen, and enhance the immune system's ability to recognize and eliminate tumor cells expressing the WT1 antigen, thereby reducing the risk of various tumors.

[0005] The present invention adopts the following technical solutions:

[0006] The method for preparing a nasal spray WT1 polypeptide preparation comprises the following steps:

[0007] S1. WT1 polypeptide sequence design: Based on the results of antigenic epitope analysis of WT1 protein, the polypeptide sequence with the strongest immunogenicity was selected for synthesis;

[0008] S2. Peptide synthesis and purification: WT1 peptide was synthesized by solid phase peptide synthesis (SPPS) and purified by high performance liquid chromatography (HPLC) to ensure the purity and activity of the peptide;

[0009] S3. Preparation: The purified WT1 polypeptide is mixed with appropriate excipients, stabilizers and immune adjuvants to prepare a liquid preparation suitable for nasal spray.

[0010] The volume ratio of the WT1 polypeptide, excipient, stabilizer and immune adjuvant is 0.01-0.05: 0.50-0.90: 0.001-0.05: 0.001-0.05.

[0011] The excipient is a buffer solution.

[0012] The stabilizer is a polymer or an amino acid and its derivatives.

[0013] The immune adjuvant is a cytokine adjuvant.

[0014] Nasal spray WT1 polypeptide preparation, the preparation contains polypeptide fragments of WT1 antigen.

[0015] The present invention also discloses the application of the nasal spray WT1 polypeptide preparation in preventing tumors.

[0016] The present invention also discloses a method for using the nasal spray WT1 polypeptide preparation, which comprises spraying the nasal spray WT1 polypeptide preparation into the nasal cavity.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Regular nasal injection of WT1 peptide preparations can significantly improve the body's specific immunity to WT1 antigens, enhance the immune system's ability to identify and eliminate tumor cells expressing WT1 antigens, and thus reduce the risk of various tumors. The specific expected effects include but are not limited to: increasing the number and activity of specific T cells and prolonging the tumor latency period in healthy people;

[0019] 1. Local administration: administration through nasal spray avoids systemic side effects and improves patient compliance;

[0020] 2. Rapid onset of effect: The nasal mucosa absorbs quickly and can quickly stimulate the immune response;

[0021] 3. Strong targeting: Peptide vaccines can specifically activate immune responses against tumor cells expressing WT1 antigens, reducing the impact on normal cells;

[0022] 4. Easy to store and transport: Nasal spray vaccines are stable at room temperature and easy to store and transport. DETAILED DESCRIPTION

[0023] The embodiments of the present invention are described in detail below. Unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments are conventional methods in the art unless otherwise specified. The embodiments are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0024] The WT1 gene is located on human chromosome 11p13 and can regulate cell proliferation, growth, differentiation, and apoptosis. Mutations in this gene can lead to dysfunction and induce tumors. WT1 is not expressed in normal tissues, so tumor vaccines targeting WT1 will not produce off-target toxicity while specifically activating T cell immune responses, and are highly safe.

[0025] The method for preparing a nasal spray WT1 polypeptide preparation comprises the following steps:

[0026] S1. WT1 polypeptide sequence design: According to the results of antigenic epitope analysis of WT1 protein, the polypeptide sequence with the strongest immunogenicity is selected for synthesis. The present invention uses a combination of multiple optimized WT1 polypeptide sequences to improve the strength and breadth of the immune response;

[0027] S2. Peptide synthesis and purification: WT1 peptide was synthesized by solid phase peptide synthesis (SPPS) and purified by high performance liquid chromatography (HPLC) to ensure the purity and activity of the peptide;

[0028] S3. Preparation: The purified WT1 polypeptide is mixed with suitable excipients, stabilizers and immune adjuvants, and the volume ratio of WT1 polypeptide, excipients, stabilizers and immune adjuvants is 0.01-0.05: 0.50-0.90: 0.001-0.05: 0.001-0.05 to prepare a liquid preparation suitable for nasal spray.

[0029] S4. Filling of long nozzle spray bottle.

[0030] WT1 peptide: As the main drug ingredient, its proportion is relatively high and may account for 1%-5% of the total volume in the preparation, depending on the activity, stability and required dose of the peptide.

[0031] Excipients: Excipients are used to increase the volume of a formulation, improve its physical properties, or increase patient compliance. Common excipients are buffers. Excipients account for the majority of the total volume of a formulation, 50%-90%.

[0032] Stabilizer: Stabilizers are polymers or amino acids and their derivatives, which are used to improve the stability of the preparation and prevent the degradation of the polypeptide during storage and use. The proportion of stabilizers is usually low, accounting for 0.1%-5% of the total volume in the preparation.

[0033] -Amino acids and their derivatives: such as glycine, L-arginine, etc., which can neutralize the acidic or alkaline substances that may be produced by the polypeptide during storage, thereby maintaining the stability of the polypeptide.

[0034] -Polymers: such as polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), etc., which can reduce the contact of polypeptides with adverse factors in the environment and improve the stability of polypeptides by binding to polypeptides.

[0035] Immune adjuvant: Immune adjuvants are cytokine adjuvants, such as interleukin-2 (IL-2), interferon-γ (IFN-γ), etc. They can enhance the immune effect of antigens by regulating the function of the immune system. Its proportion is also relatively low, accounting for 0.1%-5% of the total volume in the preparation, depending on the type of adjuvant and the desired immune effect.

[0036] The prepared nasal spray WT1 polypeptide preparation contains polypeptide fragments of WT1 antigen and can effectively prevent tumors:

[0037] Regular nasal inoculation of WT1 peptide preparations can significantly improve the body's specific immunity to WT1 antigens, enhance the immune system's ability to identify and eliminate tumor cells expressing WT1 antigens, and thus reduce the risk of various tumors. Specific expected effects include but are not limited to: increasing the number and activity of specific T cells, prolonging the tumor latency period in healthy people, and even completely preventing the occurrence of tumors in some cases. These expected effects need to be further verified through large-scale clinical trials.

[0038] Mechanism of action

[0039] After being absorbed by the nasal mucosa, the nasal spray WT1 peptide preparation enters the blood circulation system, is taken up by dendritic cells (DCs) and presented to the lymph nodes. In this process, the WT1 peptide binds to the HLA molecules on the surface of DCs, activates specific T cells (including CD4+ helper T cells and CD8+ cytotoxic T cells), and triggers a strong immune response. The activated T cells recognize and eliminate tumor cells expressing WT1 antigens in the body, thereby achieving the purpose of preventing tumor occurrence.

[0040] The present invention also discloses a method for using the nasal spray WT1 polypeptide preparation:

[0041] Preparation: Wash your hands, blow your nose and clean your nasal cavity before use;

[0042] Nasal spray: Open the protective cap of the nasal spray device and gently shake the spray. Tilt your head forward slightly and press gently on the side of your nose with one finger to close one nostril. Insert the tip of the nasal spray into the open nostril and breathe in slowly through the open nostril while squeezing the nasal spray to spray one dose. Repeat the above steps for the other nostril;

[0043] Follow-up treatment: After use, dry the nozzle with a tissue and cover the nasal spray. Try not to blow your nose within a few minutes after using the spray.

[0044] The first-generation tumor nano-vaccine product nasal spray based on WT1 developed by the team adds nano-carriers to the traditional tumor vaccine to improve the immunogenicity of the vaccine. Preclinical research data show that it has good immune activation and tumor killing effects, and almost no toxic side effects. Therefore, the nasal spray based on WT1 can be used for the treatment of patients with advanced solid tumors, and can also be used for prevention of high-risk cancer populations, such as those with a family history and those who have been immunocompromised for a long time.

[0045] Although the embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the above embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a nasal spray WT1 polypeptide preparation, characterized in that: Includes the following: S1. WT1 polypeptide sequence design: Based on the results of antigenic epitope analysis of WT1 protein, the polypeptide sequence with the strongest immunogenicity was selected for synthesis; S2. Peptide synthesis and purification: WT1 peptide was synthesized by solid phase peptide synthesis (SPPS) and purified by high performance liquid chromatography (HPLC) to ensure the purity and activity of the peptide; S3. Preparation: The purified WT1 polypeptide is mixed with appropriate excipients, stabilizers and immune adjuvants to prepare a liquid preparation suitable for nasal spray.

2. The method for preparing a nasal spray WT1 polypeptide preparation according to claim 1, characterized in that: The volume ratio of WT1 polypeptide, excipient, stabilizer and immune adjuvant is 0.01-0.05:0.50-0.90:0.001-0.05:0.001-0.

05.

3. The method for preparing a nasal spray WT1 polypeptide preparation according to claim 1, characterized in that: The excipient is a buffer solution.

4. The method for preparing a nasal spray WT1 polypeptide preparation according to claim 1, characterized in that: The stabilizer is a polymer or an amino acid and its derivatives.

5. The method for preparing a nasal spray WT1 polypeptide preparation according to claim 1, characterized in that: The immune adjuvant is a cytokine adjuvant.

6. Nasal spray WT1 polypeptide preparation, which contains polypeptide fragments of WT1 antigen.

7. Application of nasal spray WT1 polypeptide preparation in tumor prevention.

8. Method of use of the nasal spray WT1 polypeptide preparation: spray the nasal spray WT1 polypeptide preparation into the nasal cavity.