A complex adjuvant for enhancing cellular immunity and its preparation method and application

Abstract

The application provides a kind of composite adjuvant for enhancing cellular immunity and its preparation method and application, belong to biological medicine technical field.The adjuvant includes polylysine hydroxymethyl cellulose polymyxin solution and PEG5000-PLA15000-GM-CSF nanoparticle solution;The concentration of the polylysine hydroxymethyl cellulose polymyxin solution is 0.25-4mg / mL, the concentration of the PEG5000-PLA15000-GM-CSF nanoparticle solution is 0.5-2mg / mL;The volume ratio of polylysine hydroxymethyl cellulose polymyxin solution and PEG5000-PLA15000-GM-CSF nanoparticle solution is (2.5-10):1.The composite adjuvant of the application can activate DC cells, induce stronger immune response, and has better antitumor effect when applied to tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a compound adjuvant that enhances cellular immunity, its preparation method, and its application. Background Technology

[0002] In modern immunology, cellular immunity is crucial for fighting diseases, especially tumors and chronic infectious diseases. Cellular immune responses depend on the activation, proliferation, and differentiation of T lymphocytes. This process involves antigen-presenting cells taking up and processing antigens, and then presenting antigen peptide-MHC complexes to T cells, thereby triggering a series of immune responses. In this process, adjuvants play an irreplaceable role as key substances for enhancing immunogenicity.

[0003] Traditional single adjuvants have significant limitations. Due to their singular mechanism of action, they are ill-suited to addressing complex immune regulatory needs. Taking tumor immunotherapy vaccines as an example, the tumor microenvironment not only requires the activation of antigen-presenting cells to promote antigen presentation and initiate an immune response, but also necessitates the regulation of the immune microenvironment to suppress the activity of immunosuppressive cells such as regulatory T cells (Tregs) and prevent tumor immune escape. However, single adjuvants can only exert their effects through a specific, single pathway. For instance, aluminum salt adjuvants primarily recruit immune cells through local inflammation but cannot effectively induce cytotoxic T lymphocyte (CTL) responses, making it difficult to finely regulate the immune system and meet the diverse immune regulatory needs in complex scenarios such as tumor immunotherapy.

[0004] Meanwhile, different antigens have specific requirements for adjuvants. Antigen types are diverse, including protein antigens, peptide antigens, and nucleic acid antigens, with significant differences in immunogenicity, requiring specific types of adjuvants to enhance their immunogenicity. For example, some protein antigens can only elicit an effective cellular immune response when the adjuvant can target specific immune cell receptors. If the adjuvant is not chosen appropriately, it may not only fail to effectively activate cellular immunity but may also inhibit the immune response. Universal adjuvants, lacking specific targeting capabilities for different antigens, are insufficient to meet the unique needs of various antigens.

[0005] To enhance immune efficacy, multiple adjuvants or adjuvants in combination with other immunomodulators are often used in practice. However, this strategy brings many complex problems. Different adjuvants may interact, altering their respective immunomodulatory mechanisms and affecting the final immune effect; optimizing the dosage when used in combination is also extremely challenging, requiring precise control of the dosage of each component. Excessive dosage may lead to immune overreaction and adverse reactions, while insufficient dosage may fail to achieve the expected immune enhancement effect; furthermore, combined use may increase the probability of potential adverse reactions, such as triggering excessive inflammatory responses or autoimmune reactions, which greatly hinders clinical application.

[0006] It is evident that current challenges include the limitations of single adjuvant mechanisms, the specificity of antigen-adjuvant matching, and the complexity of combined use. Therefore, developing a compound adjuvant that can effectively enhance cellular immunity and overcome these difficulties is of great practical significance for advancing the fields of vaccines and immunotherapy. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a compound adjuvant that enhances cellular immunity, can activate dendritic cells, induce a stronger immune response, and has a better anti-tumor effect when applied to tumor treatment.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] A compound adjuvant for enhancing cellular immunity, the adjuvant comprising a poly-L-lysine hydroxymethyl cellulose (poly I:C) solution and a PEG5000-PLA15000-GM-CSF nanoparticle solution; the concentration of the poly-L-lysine hydroxymethyl cellulose (poly I:C) solution is 0.25–4 mg / mL, and the concentration of the PEG5000-PLA15000-GM-CSF nanoparticle solution is 0.5–2 mg / mL; the volume ratio of the poly-L-lysine hydroxymethyl cellulose (poly I:C) solution to the PEG5000-PLA15000-GM-CSF nanoparticle solution is (2.5–10):1.

[0010] Preferably, the preparation method of the poly-L-lysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution includes: dissolving polyinosinic acid in a 0.9% NaCl solution by heating to obtain a polyinosinic acid solution; mixing equal volumes of the poly-L-lysine solution and the hydroxymethyl cellulose solution to obtain a poly-L-lysine hydroxymethyl cellulose complex; and adding the polyinosinic acid solution to the poly-L-lysine hydroxymethyl cellulose complex to obtain a poly-L-lysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution (PolyICLC).

[0011] Preferably, the concentration of the polyinosinic acid solution is 0.5–8 mg / mL, the concentration of the polylysine solution is 1–12 mg / mL, and the mass concentration of the hydroxymethyl cellulose solution is 2%.

[0012] Preferably, the preparation method of the PEG5000-PLA15000-GM-CSF nanoparticle solution includes: dissolving PEG5000-PLA15000 in an organic solvent to obtain a polymer solution; dissolving GM-CSF protein in an aqueous medium to obtain a protein solution; adding the protein solution dropwise to the polymer solution under stirring conditions, adding PVA solution, and continuing stirring to obtain a composite emulsion; adding the composite emulsion dropwise to the aqueous phase, stirring a second time, stirring overnight, centrifuging, and collecting the precipitate; dispersing the precipitate in an aqueous medium to obtain the PEG5000-PLA15000-GM-CSF nanoparticle solution.

[0013] Preferably, the organic solvent includes dichloromethane, acetone, and ethyl acetate; the concentration of the polymer solution is 5–40 mg / mL; the aqueous phase medium includes PBS buffer, 0.9% NaCl solution, and deionized water; the concentration of the protein solution is 0.5–2 mg / mL; and the working concentration of the PVA solution is 1%–5%.

[0014] Preferably, the stirring conditions are a stirring speed of 100-200 rpm and a second stirring speed of 300-500 rpm.

[0015] Another object of the present invention is to provide a method for preparing the composite adjuvant, comprising the following steps: preparing polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution and PEG5000-PLA15000-GM-CSF nanoparticle solution respectively, and mixing the polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution and the PEG5000-PLA15000-GM-CSF nanoparticle solution at a volume ratio of (2.5-10):1.

[0016] Another object of the present invention is to provide the application of the compound adjuvant or the preparation method thereof in the preparation of products with enhanced antitumor efficacy.

[0017] Another object of the present invention is to provide an anti-melanoma composition comprising the aforementioned adjuvant and polypeptide B16-M1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0018] Another object of the present invention is to provide a composition for resisting HPV16-overexpressing tumor cells, comprising the aforementioned adjuvant and HPV16-E7 polypeptide antigen, wherein the HPV16-E7 polypeptide antigen is composed of polypeptide antigens with amino acid sequences as shown in SEQ ID NO.2 to SEQ ID NO.5.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a compound adjuvant for enhancing cellular immunity. Through the synergistic effect of multiple active ingredients, it overcomes the shortcomings of single adjuvants, simultaneously activating antigen-presenting cells and regulating the immune microenvironment, effectively enhancing cellular immune responses and offering greater advantages in complex scenarios such as tumor treatment. The compound adjuvant of this invention is compatible with multiple antigen types, precisely targeting immune cell receptors, solving the antigen-adjuvant matching problem, and improving immunogenicity. Furthermore, the compound adjuvant of this invention features a rationally designed formulation and process, reducing mutual interference when adjuvants are used in combination, clarifying the optimal dosage ratio, reducing the risk of adverse reactions, and making it safer and more convenient to use. This invention also provides a method for preparing the compound adjuvant, which is simple, controllable, easy to industrialize, ensures stable product quality, reduces costs, improves efficiency, and has significant application value. Attached Figure Description

[0021] Figure 1 To enhance the anti-melanoma effect of the compound adjuvant, the top image shows mice treated in each group, and the bottom image shows the tumors of mice in each group.

[0022] Figure 2 The compound adjuvant enhances the effect against tumor cells with high HPV16 expression. Detailed Implementation

[0023] This invention provides a composite adjuvant for enhancing cellular immunity. The adjuvant comprises a poly(L-lysine-hydroxymethylcellulose) polyinosinic-polycytidylic acid (PolyICLC) solution and a PEG5000-PLA15000-GM-CSF nanoparticle solution. The concentration of the PolyICLC solution is 0.25–4 mg / mL, and the concentration of the PEG5000-PLA15000-GM-CSF nanoparticle solution is 0.5–2 mg / mL. The volume ratio of the PolyICLC solution to the PEG5000-PLA15000-GM-CSF nanoparticle solution is (2.5–10):1. This composite adjuvant, using PolyICLC as the core raw material and compounded with PEG5000-PLA15000-GM-CSF nanoparticles, induces a stronger immune response and effectively improves anti-tumor efficacy.

[0024] In this invention, the concentration of the PolyICLC solution is preferably 1–3 mg / mL, more preferably 2 mg / mL; the concentration of the PEG5000-PLA15000-GM-CSF nanoparticle solution is preferably 0.8–1.5 mg / mL, more preferably 1 mg / mL; the volume ratio of the PolyICLC solution to the PEG5000-PLA15000-GM-CSF nanoparticle solution is preferably 5:1. Preferably, the PolyICLC solution and the PEG5000-PLA15000-GM-CSF nanoparticle solution are stored separately and mixed only before use.

[0025] In this invention, the preferred method for preparing the poly-L-lysine hydroxymethyl cellulose (PolyICLC) solution includes: dissolving polyinosinic acid (polyIC) in a 0.9% NaCl solution by heating to obtain a polyinosinic acid solution; mixing equal volumes of poly-L-lysine solution and hydroxymethyl cellulose solution to obtain a poly-L-lysine hydroxymethyl cellulose complex; and adding an equal volume of polyinosinic acid solution to the poly-L-lysine hydroxymethyl cellulose complex to obtain a poly-L-lysine hydroxymethyl cellulose (PolyICLC) solution.

[0026] In the preparation method of the PolyICLC solution of the present invention, the concentration of the polyIC solution is preferably 0.5-8 mg / mL, and in some examples, it can be 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL or 8 mg / mL; the heating dissolution is preferably carried out by heating to 60°C, preferably for 1 hour; the concentration of the Poly-L-Lysine solution is preferably 1-12 mg / mL, and in some examples, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL or 12 mg / mL. The mass concentration of the hydroxymethyl cellulose solution is preferably 2%. In the present invention, the PolyICLC solution is preferably stored at 4°C for later use.

[0027] In this invention, the preferred method for preparing the PEG5000-PLA15000-GM-CSF nanoparticle solution includes: dissolving PEG5000-PLA15000 in an organic solvent to obtain a polymer solution; dissolving GM-CSF protein in an aqueous medium to obtain a protein solution; adding the protein solution dropwise to the polymer solution under stirring conditions, adding PVA solution, and continuing stirring to obtain a composite emulsion; adding the composite emulsion dropwise to the aqueous phase, stirring a second time, stirring overnight, centrifuging, and collecting the precipitate; dispersing the precipitate in an aqueous medium to obtain the PEG5000-PLA15000-GM-CSF nanoparticle solution.

[0028] In the preparation method of the PEG5000-PLA15000-GM-CSF nanoparticle solution of the present invention, the organic solvent preferably includes dichloromethane, acetone, and ethyl acetate; the aqueous phase medium preferably includes PBS buffer, 0.9% NaCl solution, and deionized water; the concentration of the polymer solution is preferably 5-40 mg / mL, more preferably 10-30 mg / mL, and even more preferably 20 mg / mL; the concentration of the protein solution is preferably 0.5-2 mg / mL, and in some examples... The concentrations are 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, or 2 mg / mL; the preferred stirring conditions are a stirring speed of 100–200 rpm; under stirring conditions, the GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor) protein solution is preferably added dropwise to the polymer solution at a dropping rate of 1–2 mL / min; the working concentration of the PVA solution is preferably 1%–5%, and in some examples, it can be 1%, 2%, 3%, 4%, or 5%. The present invention stabilizes the emulsion by adding the emulsifier PVA solution. The emulsion is added to an aqueous medium, and a second stirring is performed to allow the organic solvent to gradually evaporate, the polymer to gradually form nanoparticles, and the GM-CSF protein to be encapsulated within them. The second stirring speed is preferably 300–500 rpm, more preferably 400 rpm. The dropping rate of the emulsion is preferably 1–2 mL / min; after the emulsion has completely decreased, stirring is continued overnight at 4°C, and the stirring speed is preferably 300–500 rpm, more preferably 400 rpm. After stirring, centrifugation is performed. The preferred centrifugation speed is 10,000–15,000 rpm, more preferably 12,000 rpm, and the preferred centrifugation time is 15–30 min, more preferably 20 min. The precipitate is collected after centrifugation to obtain PEG5000-PLA15000-GM-CSF nanoparticles. Preferably, the PEG5000-PLA15000-GM-CSF nanoparticles are washed 2–3 times with an aqueous medium to remove micro-reaction substances and impurities. The washed PEG5000-PLA15000-GM-CSF nanoparticles are dispersed in an aqueous medium to obtain a PEG5000-PLA15000-GM-CSF nanoparticle solution.

[0029] The present invention also provides a method for preparing the composite adjuvant, comprising the following steps: preparing a PolyICLC solution and a PEG5000-PLA15000-GM-CSF nanoparticle solution respectively, and mixing the PolyICLC solution and the PEG5000-PLA15000-GM-CSF nanoparticle solution at a volume ratio of (2.5-10):1.

[0030] The present invention also provides the application of the aforementioned compound adjuvant or the aforementioned preparation method in the preparation of products with enhanced antitumor efficacy.

[0031] The present invention also provides an anti-melanoma composition comprising the aforementioned adjuvant and polypeptide B16-M1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0032] The present invention also provides a composition for resisting HPV16-overexpressing tumor cells, comprising the aforementioned adjuvant and HPV16-E7 polypeptide antigen, wherein the HPV16-E7 polypeptide antigen is composed of polypeptide antigens with amino acid sequences as shown in SEQ ID NO.2 to SEQ ID NO.5.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] A compound adjuvant for enhancing cellular immunity contains a PolyICLC solution and a PEG5000-PLA15000-GM-CSF nanoparticle solution, wherein the volume ratio of the PolyICLC solution to the PEG5000-PLA15000-GM-CSF nanoparticle solution is 5:1.

[0036] The method for preparing PolyICLC solution is as follows:

[0037] PolyIC was dissolved in a 0.9% NaCl solution by heating at 60°C for 1 hour to obtain a polyIC solution (4 mg / mL).

[0038] A poly-L-Lysine solution (6 mg / mL) and a 2% hydroxymethyl cellulose solution were mixed in equal volumes to obtain a poly-L-Lysine hydroxymethyl cellulose complex.

[0039] Add an equal volume of polyIC solution to the polylysine hydroxymethyl cellulose complex to obtain a polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution (2 mg / mL).

[0040] The preparation method of PEG5000-PLA15000-GM-CSF nanoparticle solution is as follows:

[0041] The PEG5000-PLA15000 complex was dissolved in acetone to prepare a polymer solution with a concentration of 20 mg / mL.

[0042] The GM-CSF protein was dissolved in PBS buffer to prepare a protein solution with a concentration of 1 mg / mL.

[0043] Under stirring conditions (150 rpm), the protein solution was slowly added dropwise (at a rate of 1 mL / min) to the polymer solution to form a water-in-oil (W / O) emulsion; then, an aqueous PVA emulsion (working concentration of 3%) was added, and stirring was continued (150 rpm) for 2 hours to stabilize the emulsion.

[0044] Under stirring conditions (400 rpm), the emulsion was added dropwise (1 mL / min) to PBS buffer. After the addition was completed, stirring was continued at 4°C (400 rpm) overnight. After stirring, the mixture was centrifuged at 12000 rpm for 20 min, and the nanoparticle precipitate was collected. The nanoparticle precipitate was washed twice with PBS buffer.

[0045] The washed nanoparticles were dispersed in PBS buffer to obtain a PEG5000-PLA15000-GM-CSF nanoparticle solution (1 mg / mL).

[0046] Example 2

[0047] A compound adjuvant that enhances cellular immunity differs from Example 1 in that:

[0048] The method for preparing PolyICLC solution is as follows:

[0049] PolyIC was dissolved in a 0.9% NaCl solution by heating at 60°C for 1 hour to obtain a polyIC solution (2 mg / mL).

[0050] A poly-L-Lysine solution (3 mg / mL) and a 2% hydroxymethyl cellulose solution were mixed in equal volumes to obtain a poly-L-Lysine hydroxymethyl cellulose complex.

[0051] Add an equal volume of polyIC solution to the polylysine hydroxymethyl cellulose complex to obtain a polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution (1 mg / mL).

[0052] The preparation method of PEG5000-PLA15000-GM-CSF nanoparticle solution is as follows:

[0053] PEG5000 and PLA15000 were dissolved in dichloromethane to prepare a polymer solution with a concentration of 10 mg / mL.

[0054] The GM-CSF protein was dissolved in a 0.9% NaCl solution to prepare a protein solution with a concentration of 1 mg / mL.

[0055] Under stirring conditions (100 rpm), the protein solution was slowly added dropwise (at a rate of 2 mL / min) to the polymer solution to form a water-in-oil (W / O) emulsion; then, an aqueous PVA emulsion (working concentration of 2%) was added, and stirring was continued (100 rpm) for 2 hours to stabilize the emulsion.

[0056] Under stirring conditions (300 rpm), the emulsion was added dropwise (2 mL / min) to a 0.9% NaCl solution. After the addition was completed, stirring was continued overnight at 4°C (300 rpm). After stirring, the mixture was centrifuged at 10,000 rpm for 30 min to collect the nanoparticle precipitate. The nanoparticle precipitate was washed three times with a 0.9% NaCl solution.

[0057] The washed nanoparticles were dispersed in a 0.9% NaCl solution to obtain a PEG5000-PLA15000-GM-CSF nanoparticle solution (1 mg / mL).

[0058] Example 3

[0059] A compound adjuvant that enhances cellular immunity differs from Example 1 in that:

[0060] The method for preparing PolyICLC solution is as follows:

[0061] PolyIC was dissolved in a 0.9% NaCl solution by heating at 60°C for 1 hour to obtain a polyIC solution (6 mg / mL).

[0062] A poly-L-Lysine solution (9 mg / mL) and a 2% hydroxymethyl cellulose solution were mixed in equal volumes to obtain a poly-L-Lysine hydroxymethyl cellulose complex.

[0063] Add an equal volume of polyIC solution to the polylysine hydroxymethyl cellulose complex to obtain a polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution (3 mg / mL).

[0064] The preparation method of PEG5000-PLA15000-GM-CSF nanoparticle solution is as follows:

[0065] Dissolve PEG5000-PLA15000 in ethyl acetate to prepare a polymer solution with a concentration of 30 mg / mL;

[0066] Dissolve GM-CSF protein in deionized water to prepare a protein solution with a concentration of 2 mg / mL;

[0067] Under stirring conditions (200 rpm), the protein solution was slowly added dropwise (at a rate of 2 mL / min) to the polymer solution to form a water-in-oil (W / O) emulsion; then, an aqueous PVA emulsion (working concentration of 5%) was added, and stirring was continued (200 rpm) for 2 hours to stabilize the emulsion.

[0068] Under stirring conditions (500 rpm), the emulsion was added dropwise (2 mL / min) to deionized water. After the addition was completed, stirring was continued at 4°C (500 rpm) overnight. After stirring, the mixture was centrifuged at 12000 rpm for 20 min, and the nanoparticle precipitate was collected. The nanoparticle precipitate was washed three times with deionized water.

[0069] The washed nanoparticles were dispersed in deionized water to obtain a PEG5000-PLA15000-GM-CSF nanoparticle solution (0.5 mg / mL).

[0070] Example 4

[0071] A compound adjuvant that enhances cellular immunity differs from Example 1 in that:

[0072] The method for preparing PolyICLC solution is as follows:

[0073] PolyIC was dissolved in a 0.9% NaCl solution by heating at 60°C for 1 hour to obtain a polyIC solution (1 mg / mL).

[0074] An equal volume of poly-L-Lysine solution (10 mg / mL) and a 2% hydroxymethyl cellulose solution were mixed to obtain a poly-L-Lysine hydroxymethyl cellulose complex.

[0075] Add an equal volume of polyIC solution to the polylysine hydroxymethyl cellulose complex to obtain a polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution (0.5 mg / mL).

[0076] The preparation method of PEG5000-PLA15000-GM-CSF nanoparticle solution is as follows:

[0077] Dissolve PEG5000-PLA15000 in acetone to prepare a polymer solution with a concentration of 40 mg / mL;

[0078] The GM-CSF protein was dissolved in PBS buffer to prepare a protein solution with a concentration of 2 mg / mL.

[0079] Under stirring conditions (150 rpm), the protein solution was slowly added dropwise (at a rate of 1 mL / min) to the polymer solution to form a water-in-oil (W / O) emulsion; then, an aqueous PVA emulsion (working concentration of 5%) was added, and stirring was continued (150 rpm) for 2 hours to stabilize the emulsion.

[0080] Under stirring conditions (400 rpm), the emulsion was added dropwise (1 mL / min) to PBS buffer. After the addition was completed, stirring was continued at 4°C (400 rpm) overnight. After stirring, the mixture was centrifuged at 12000 rpm for 20 min to collect the nanoparticle precipitate. The nanoparticle precipitate was washed twice with PBS buffer.

[0081] The washed nanoparticles were dispersed in PBS buffer to obtain a PEG5000-PLA15000-GM-CSF nanoparticle solution (2 mg / mL).

[0082] Example 5

[0083] Compound adjuvants enhance the anti-melanoma effect.

[0084] Polypeptide B16-M1 (SEQ ID NO.1): IGSYYTVFDRDNNRVSFANAVVL.

[0085] C57BL / 6 mice were randomly divided into 4 groups, with 2 mice in each group. The treatments for each group were as follows:

[0086] Treatment group 1: 200 μL of peptide B16-M1 (1 mg / mL) and 20 μL of PEG5000-PLA15000-GM-CSF nanoparticle solution (prepared in Example 1, 1 mg / mL) were mixed, and then 180 μL of physiological saline was added, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0087] Treatment group 2: 200 μL of peptide B16-M1 (1 mg / mL) and 100 μL of PolyICLC solution (prepared in Example 1, 2 mg / mL) were mixed, and then 100 μL of physiological saline was added to make a total volume of 400 μL. 200 μL was injected into each mouse.

[0088] Treatment group 3: 200 μL of peptide B16-M1 (1 mg / mL), 100 μL of PolyICLC solution (prepared in Example 1, 2 mg / mL) and 20 μL of PEG5000-PLA15000-GM-CSF nanoparticle solution (prepared in Example 1, 1 mg / mL) were mixed, and then 80 μL of physiological saline was added, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0089] Control group: 200 μL of peptide B16-M1 (1 mg / mL) was mixed with 200 μL of physiological saline, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0090] Mice in each group were inoculated with the corresponding reagents, denoted as D0. Five days later (D5), they were inoculated with B16F10 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), with each mouse receiving 1×10⁶ cells. 5 Cells. On day 7 (D7) and day 14 (D14), mice were inoculated with the corresponding formulations for each group. Mice were sacrificed on day 25, and the weight was calculated using the formula V = 0.52 × L × W. 2 Tumor size was measured, where L is length and W is width. The euthanized mice and the tumors within them are shown in the image. Figure 1 As shown in Table 1, the tumor volume is as follows.

[0091] Table 1 Tumor Volume

[0092] Treatment group 1 Treatment group 2 Treatment group 3 control group <![CDATA[Tumor volume (mm 3 )]]> 130 33.28 0.52 624

[0093] As shown in Table 1, the antigen peptide combined with the compound adjuvant of this invention can better activate T cells, reduce tumor volume, and has a better anti-tumor effect.

[0094] Example 6

[0095] The compound adjuvant enhances the effect against tumor cells with high HPV16 expression.

[0096] HPV16-E7 polypeptide antigen is obtained by mixing equal masses of polypeptide antigens with the following sequences:

[0097] HPV16-E7-1 (SEQ ID NO.2): MHGDTPTLHEYMLDLQPETTDLYCYEQL NDSSEEE;

[0098] HPV16-E7-2 (SEQ ID NO.3): LYCYEQLNDSSEEEDEIDGPAGQAEPDRA HYNIVT;

[0099] HPV16-E7-3 (SEQ ID NO.4): GQAEPDRAHYNIVTFCCKCDSTLRLCVQ STHVDIR;

[0100] HPV16-E7-4 (SEQ ID NO. 5): TLRLCVQSTHVDIRTLEDLLMGTLGIVCP ICSQKP.

[0101] C57BL / 6 mice were randomly divided into 4 groups, with 2 mice in each group. The treatments for each group were as follows:

[0102] Treatment group 1: 200 μL of HPV16-E7 polypeptide antigen (1 mg / mL) and 20 μL of PEG5000-PLA15000-GM-CSF nanoparticle solution (prepared in Example 2, 1 mg / mL) were mixed, and then 180 μL of physiological saline was added, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0103] Treatment group 2: 200 μL of HPV16-E7 polypeptide antigen (1 mg / mL) and 100 μL of PolyICLC solution (prepared in Example 2, 1 mg / mL) were mixed, and then 100 μL of physiological saline was added to make a total volume of 400 μL. 200 μL was injected into each mouse.

[0104] Treatment group 3: 200 μL of HPV16-E7 polypeptide antigen (1 mg / mL), 100 μL of PolyICLC solution (prepared in Example 2, 1 mg / mL) and 20 μL of PEG5000-PLA15000-GM-CSF nanoparticle solution (prepared in Example 2, 1 mg / mL) were mixed, and then 80 μL of physiological saline was added, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0105] Control group: 200 μL of HPV16-E7 polypeptide antigen (1 mg / mL) was mixed with 200 μL of physiological saline, with a total volume of 400 μL. 200 μL was injected into each mouse.

[0106] Mice in each group were inoculated with the corresponding reagents, denoted as D0. Five days later (D5), they were inoculated with TC-1 cells (purchased from Xiamen Yimo Biotechnology Co., Ltd., catalog number IM-M085), with each mouse receiving 9 × 10⁶ cells. 5 Cells. On day 7 (D7) and day 14 (D14), mice were inoculated with the corresponding formulations for each group. Mice were sacrificed on day 25, and the weight was calculated using the formula V = 0.52 × L × W. 2 The size of the tumor was measured, where L is the length and W is the width. Tumors in mice are shown below. Figure 2 As shown in Table 2, the tumor volume is as follows.

[0107] Table 2 Tumor Volume

[0108] Treatment group 1 Treatment group 2 Treatment group 3 control group <![CDATA[Tumor volume (mm 3 )]]> 780 676 332.8 1318.2

[0109] As shown in Table 1, the antigen peptide combined with the compound adjuvant of this invention can better activate T cells, reduce tumor volume, and has a better effect on anti-HPV16 highly expressed tumors.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a compound adjuvant in the preparation of products that enhance the antitumor efficacy of vaccines, characterized in that, The tumor is melanoma; The composite adjuvant is a polylysine hydroxymethyl cellulose polyinosinic-polycytidylic acid solution and a PEG5000-PLA15000-GM-CSF nanoparticle solution. The poly-lysine hydroxymethyl cellulose polyinosinic-polycytidylic acid (poly-ICLC) is the same as Poly-ICLC; the concentration of the Poly-ICLC solution is 0.25~4 mg / mL, and the concentration of the PEG5000-PLA15000-GM-CSF nanoparticle solution is 0.5~2 mg / mL; the volume ratio of the Poly-ICLC solution to the PEG5000-PLA15000-GM-CSF nanoparticle solution is (2.5~10):1; The preparation method of the PEG5000-PLA15000-GM-CSF nanoparticle solution includes: dissolving PEG5000-PLA15000 in an organic solvent to obtain a polymer solution; dissolving GM-CSF protein in an aqueous medium to obtain a protein solution; adding the protein solution dropwise to the polymer solution under stirring conditions, adding PVA solution, and continuing stirring to obtain a composite emulsion; adding the composite emulsion dropwise to the aqueous phase, stirring a second time, stirring overnight, centrifuging, and collecting the precipitate; dispersing the precipitate in an aqueous medium to obtain the PEG5000-PLA15000-GM-CSF nanoparticle solution.

2. The application according to claim 1, characterized in that, The preparation method of the Poly-ICLC solution includes: dissolving polyinosinic acid in a 0.9% NaCl solution by heating to obtain a polyinosinic acid solution; mixing equal volumes of polylysine solution and hydroxymethyl cellulose solution to obtain a polylysine-hydroxymethyl cellulose complex; and adding the polyinosinic acid solution to the polylysine-hydroxymethyl cellulose complex to obtain the Poly-ICLC solution.

3. The application according to claim 2, characterized in that, The concentration of the polyinosinic acid solution is 0.5~8 mg / mL, the concentration of the polylysine solution is 1~12 mg / mL, and the mass concentration of the hydroxymethyl cellulose solution is 2%.

4. The application according to claim 1, characterized in that, The organic solvents include dichloromethane, acetone, and ethyl acetate; the concentration of the polymer solution is 5-40 mg / mL; the aqueous medium includes PBS buffer, 0.9% NaCl solution, and deionized water; the concentration of the protein solution is 0.5-2 mg / mL; and the working concentration of the PVA solution is 1%-5%.

5. The application according to claim 1, characterized in that, The stirring conditions are a stirring speed of 100-200 rpm and a second stirring speed of 300-500 rpm.

6. The application according to claim 1, characterized in that, The preparation method of the composite adjuvant includes the following steps: preparing Poly-ICLC solution and PEG5000-PLA15000-GM-CSF nanoparticle solution respectively, and mixing Poly-ICLC solution and PEG5000-PLA15000-GM-CSF nanoparticle solution at a volume ratio of (2.5~10):

1.

7. An anti-melanoma composition, characterized in that, It includes the compound adjuvant of claim 1 and peptide B16-M1, wherein the amino acid sequence of peptide B16-M1 is shown in SEQ ID NO.1.

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