Immunopotentiation adjuvant for tumor therapeutic vaccine

Abstract

The invention belongs to the field of biological medicine products, and relates to an immunological enhancement adjuvant for tumor therapeutic vaccines, which is formed by dissolving thymoquinone serving as an adjuvant active ingredient in a benign thymoquinone solvent in cooperation with necessary pharmaceutic adjuvants. The immunopotentiation adjuvant is suitable for being used as a polypeptide vaccine, a protein vaccine, a nucleic acid vaccine, a tumor cell vaccine, a dendritic cell vaccine and other tumor therapeutic vaccines, promotes IFN-gamma secretion by up-regulating immune-related gene expression in CD8 + T cells, enhances the killing function of the CD8 + T cells, inhibits tumor growth, and improves the tumor therapeutic effect. The immune effect of the tumor therapeutic vaccine is obviously enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical products, and relates to a vaccine adjuvant, in particular to a cell immune enhancement adjuvant for therapeutic vaccines. Background Art

[0002] Tumor therapeutic vaccines are biological agents that stimulate the body's own immune system to recognize and attack tumor cells, thereby achieving the goals of treating tumors, preventing tumor recurrence and metastasis, etc. They are mainly used for patients with existing tumors and belong to the active immunotherapy means for tumors.

[0003] Classified by vaccine components, tumor cell vaccines use tumor cells as antigens, including inactivated tumor cell vaccines that are inactivated by physical or chemical treatment but retain antigenicity, and genetically modified tumor cell vaccines that introduce genes enhancing immunogenicity through genetic engineering; tumor antigen vaccines are prepared by selecting tumor-associated or specific antigens, including protein polypeptide vaccines that induce immune responses with tumor antigen proteins or polypeptide fragments, and nucleic acid vaccines that introduce DNA or RNA encoding tumor antigens into the human body to stimulate immunity; dendritic cell vaccines are to transfuse dendritic cells (the most powerful antigen-presenting cells) cultured in vitro and loaded with tumor antigens back into patients to activate specific anti-tumor immunity.

[0004] However, regardless of the components, the mechanism of action of tumor therapeutic vaccines is to activate the body's own immune system to recognize and attack tumor cells. The tumor-associated or specific antigens contained in the vaccines are taken up and processed by antigen-presenting cells and presented to T cells in the form of antigen peptide-MHC complexes to activate CD8 + T cells differentiate into cytotoxic T lymphocytes to kill tumor cells, and CD4 + T cells assist in activation and secrete cytokines; memory T cells are induced to generate during the immune response and are rapidly activated when encountering the same antigen again to produce a persistent immune response to monitor tumor recurrence and metastasis; the activated immune cells secrete various cytokines such as TNF-α and IFN-γ to regulate the functions of immune cells, recruit more immune cells to the tumor site, break immune tolerance, and enhance the immune attack on tumor cells, etc.

[0005] The efficacy improvement of cancer therapeutic vaccines depends on the precise regulation of immune responses by adjuvants. However, there are core problems with existing adjuvants in cancer treatment: Traditional adjuvants such as aluminum salts and MF59 induce Th2-type humoral immunity through antigen depot effects or inflammatory responses (Brewer, J. M., et al. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. The Journal of Immunology , 1999, 163(12): 6448-6454.). Although they can adsorb antigens and release them slowly (such as aluminum adjuvants) or promote antigen uptake by APCs (such as oil-in-water emulsions like MF59 and AS03), they have limited ability to enhance the cytotoxic function of CD8 + T cells required for cancer therapeutic vaccines. Aluminum adjuvants mainly activate B cells to produce antibodies and are insufficient in inducing tumor-specific CTLs. Oil-in-water emulsions induce a mixed Th1 / Th2 response, but humoral immunity still dominates.

[0006] Although TLR agonists such as CpG can enhance Th1-type cellular immunity and CTL responses by activating the TLR pathway, there are safety controversies. For example, when combined with antigens in RSV vaccines, they can exacerbate pulmonary inflammation in some patients (Study on the immune safety of recombinant respiratory syncytial virus G protein vaccine [J]. Journal of Natural Science of Hunan Normal University , 2020, 43(4): 1-6.). In addition, traditional adjuvants cannot solve the problem that antigens are diffusely distributed after entering cells and are difficult to accurately locate to the endoplasmic reticulum to complete MHC class I molecule presentation (Halbroth BR., et al. Development of a molecular adjuvant to enhance antigen-specific CD8 + T cell responses. Sci Rep , 2018, Oct, 9; 8(1):15020.).

[0007] Novel adjuvants such as SABER have strong targeting properties. For example, endoplasmic reticulum-targeted delivery makes the CD8 + T cell response more than 5 times better than traditional adjuvants (Wang X., et al. STING agonist-based ER-targeting molecules boost antigen cross-presentation. Nature, 2025, Mar 26.); Manganese adjuvant activates multiple immune pathways (LvM., et al. Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy. Cell Res , 2020,Nov, 30(11): 966-979.), but it faces limitations such as high R&D costs, complex mechanisms of action, and long-term toxicity to be verified. There is an urgent need to develop new adjuvants that are both highly efficient and safe. Summary of the invention

[0008] The purpose of the present invention is to provide an immune enhancing adjuvant for tumor therapeutic vaccines, which can enhance the immune effect of therapeutic vaccines by regulating immune responses, inhibit tumor growth, and has good safety and stability, and is suitable for large-scale industrial production.

[0009] The immunoenhancing adjuvant for tumor therapeutic vaccine of the present invention is composed of thymoquinone (TQ) as an active ingredient, which is mixed with necessary pharmaceutical excipients and dissolved in a benign solvent of thymoquinone.

[0010] Wherein, further, the benign solvent of thymoquinone is preferably corn oil.

[0011] Furthermore, the necessary pharmaceutical excipients include, but are not limited to, suitable buffers, and stabilizers, etc., which may or may not be included, to ensure the stability and effectiveness of the adjuvant.

[0012] Furthermore, the buffer is preferably Tris-HCl buffer, and the stabilizer is preferably butylated hydroxytoluene.

[0013] Preferably, the content of thymoquinone in the immunoenhancing adjuvant of the present invention is 750-950 mM.

[0014] The present invention further provides a method for preparing the immunoenhancing adjuvant, which comprises dissolving the purified active ingredient thymoquinone in a benign solvent thereof, adding a buffer, adding or not adding a stabilizer, mixing evenly and adjusting the pH value of the solution to 6.8-7.2, and filtering and sterilizing to prepare the immunoenhancing adjuvant.

[0015] Furthermore, the filtration sterilization is preferably performed using a 0.22 μm sterile filter.

[0016] Furthermore, the present invention also provides the use of the immune enhancing adjuvant as an adjuvant for tumor therapeutic vaccines.

[0017] Furthermore, the present invention also provides the use of the immune enhancing adjuvant in the preparation of a tumor therapeutic vaccine.

[0018] More specifically, the present invention provides the use of the immune enhancing adjuvant in the preparation of a therapeutic cervical cancer vaccine.

[0019] Furthermore, the present invention also provides a tumor therapeutic vaccine composition, comprising the immune enhancing adjuvant of the present invention and an immunologically effective amount of a tumor therapeutic vaccine.

[0020] Furthermore, in the tumor therapeutic vaccine composition of the present invention, the mass ratio of the immune enhancing adjuvant to the active ingredient of the tumor therapeutic vaccine is (10 - 50):1.

[0021] More preferably, the mass ratio of the immune enhancing adjuvant to the active ingredient of the tumor therapeutic vaccine is (20 - 25):1.

[0022] The immune enhancing adjuvant of the present invention is applicable to various tumor therapeutic vaccines, including but not limited to at least one of polypeptide vaccines, protein vaccines, recombinant viral vector vaccines, recombinant bacterial vector vaccines, DNA vaccines, RNA vaccines, tumor cell vaccines or dendritic cell vaccines, etc.

[0023] Furthermore, the tumor therapeutic vaccine is preferably a therapeutic cervical cancer vaccine.

[0024] The present invention uses TQ as an immune enhancing adjuvant for tumor therapeutic vaccines and has multiple breakthrough points, which are specifically reflected in the following aspects:

[0025] Enhancing immune effect: The combined application of TQ and a tumor therapeutic vaccine can significantly enhance the anti-tumor efficacy. Animal experiments show that in the combined treatment group, the tumor volume and weight are significantly reduced, and the spleen weight and spleen index are decreased, indicating a reduced tumor burden. At the same time, in the tumor tissues of mice in the combined treatment group, the infiltration of CD4 + and CD8 + T cells increases, the number of Treg cells decreases, and the proportions of CD4 + IFN-γ + and CD8 + IFN-γ + T cells in the spleen are significantly increased, and the Treg cell population is decreased, effectively promoting the anti-tumor immune response.

[0026] Regulating the functions of immune cells: In terms of mechanism, TQ can up-regulate the expression of immune-related genes such as interferon-stimulated gene 15 (ISG15) in CD8 + T cells. RNA-Seq analysis shows that after treatment with TQ, CD8 +The expression level of ISG15 in T cells was significantly increased; qRT-PCR and WB analysis confirmed that TQ upregulated the expression of ISG15 at the mRNA and protein levels, promoting CD8 + T cells to secrete IFN-γ, enhancing CD8 + T cell killing function, thereby improving the efficacy of therapeutic vaccines.

[0027] Good safety: As a natural compound, compared with traditional aluminum adjuvants, etc., TQ has better safety. Animal experiments showed that it had no adverse effects on the overall health of mice at effective doses, had a low risk of causing adverse reactions with long-term or high-dose use, had no risk of cytokine storm, which was of great significance for cancer patients who needed long-term treatment.

[0028] Stability and applicability: The adjuvant of the present invention has good stability under suitable conditions, with a stability of more than 18 months at 2 - 8°C, facilitating storage and transportation; and the preparation method is simple, suitable for large-scale industrial production, and can meet the clinical demand for novel adjuvants for therapeutic vaccines that enhance cellular immune responses. Brief Description of the Drawings

[0029] Figure 1 shows the combined application of TQ and tumor therapeutic vaccine to enhance anti-tumor efficacy.

[0030] Figure 2 shows the distribution and proportion of immune cells in tumor tissues of TQ combined with tumor therapeutic vaccine.

[0031] Figure 3 shows the proportion of immune cells in the spleen of TQ combined with tumor therapeutic vaccine.

[0032] Figure 4 shows the effect of TQ on the cytotoxic function of CD8 + T cells.

[0033] Figure 5 shows the changes in the contents of IFN-γ and TNF-α in the co-culture supernatant of CD8 + T cells and tumor cells treated with TQ. Embodiments

[0034] The following further describes in detail the specific embodiments of the present invention in conjunction with the drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can well understand and utilize the present invention, rather than limiting the protection scope of the present invention.

[0035] In the embodiments of the present invention, the production processes, experimental methods or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0036] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0037] In the following embodiments, the main active ingredient of the immunopotentiating adjuvant is thymoquinone (TQ), which can be used in combination with other pharmaceutical excipients. TQ is a natural active ingredient extracted from Nigella sativa seeds, with a purity of over 98%, and has pleiotropic immunomodulatory properties.

[0038] In the following embodiments, in practical applications, the content of TQ in the immunopotentiating adjuvant can be optimized according to specific requirements. For example, in animal experiments, a dose of 80 mg / kg was administered to mice by gavage, which can significantly enhance the anti-tumor effect of the vaccine.

[0039] In the following embodiments, other pharmaceutical excipients include food-grade corn oil as a solvent; 20 mM Tris-HCl buffer; butylated hydroxytoluene stabilizer; and 1 M HCl solution and 1 M NaOH solution as pH regulators.

[0040] In a sterile and dry environment, purified TQ was slowly added to corn oil and stirred until completely dissolved into a homogeneous and transparent solution. Then, Tris-HCl buffer was slowly added and stirred. BHT stabilizer was added and stirred evenly. Under the monitoring of a high-precision pH meter, the pH was adjusted to 6.8 - 7.2 with 1 M HCl solution or 1 M NaOH solution. Finally, in a sterile operating table, the solution was filtered through a 0.22 μm sterile filter by a positive pressure filtration device to obtain a clear and sterile TQ immunopotentiating adjuvant.

[0041] The polypeptide working solution used in the following examples was prepared specifically according to the following method: Take the HPV16 E7 (E743-77, sequence GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR) polypeptide powder, perform a transient centrifugation to gather the powder at the bottom of the tube, weigh 2.5 mg of the polypeptide powder, add 50 μL of DMSO and dissolve it fully, then perform another transient centrifugation, add 950 μL of autoclaved PBS buffer, and repeatedly pipette to mix the solution evenly to obtain 1000 μL of the polypeptide working solution, which is stored in a -80 °C refrigerator. When in use, 20 μL of the working solution containing 50 μg of the polypeptide is taken for each mouse.

[0042] The CpG ODN working solution used in the following examples was prepared specifically according to the following method: Take the CpG ODN1826 (sequence 5′-TCCATGACGTTCCTGACGTT-3′) powder, first perform a transient centrifugation to precipitate the powder to the bottom of the tube before use, according to the ratio of adding 412.5 μL of nuclease-free water for every 5 OD of powder, after pipetting and mixing evenly and dissolving fully, store it in a -20 °C refrigerator. When in use, 50 μL of the working solution containing 20 μg of CpG ODN 1826 is taken for each mouse.

[0043] In the animal experiments of the embodiments of the present invention, the administration dose of TQ is 80 mg / kg, and the equivalent dose for humans is 6.486 mg / kg. According to the currently published clinical trial data and references (Ali M., et al. Phase Isafety and clinical activity study of thymoquinone in patients with advancedrefractory malignant disease. Shiraz E-MedJ ., 10(3): 107-11.), it is reported that the oral dose of TQ in humans is usually 1-10 mg / kg / day. Another report (Badary, O.A., et al. Acute and subchronictoxicity of thymoquinone in mice. Drug Dev. Res ., 1998, 44: 56-61.) shows that its oral median lethal dose is 2.4 g / kg. The use concentration of TQ in the present invention is within the above range and far lower than the oral median lethal dose.

[0044] The immune enhancing adjuvant of the present invention is applicable to various tumor therapeutic vaccines, including polypeptide vaccines, protein vaccines, recombinant viral vector vaccines, recombinant bacterial vector vaccines, DNA vaccines, RNA vaccines, tumor cell vaccines, dendritic cell vaccines, etc.

[0045] The scope of protection of the immune-enhancing adjuvant of the present invention covering a variety of tumor therapeutic vaccines is scientifically reasonable: The core mechanism of tumor therapeutic vaccines is to activate the immune system to recognize and attack tumors. Whether its components are tumor cells, antigen polypeptides / proteins, nucleic acids or antigen-loaded dendritic cells, they all rely on common pathways such as antigen presentation, T cell activation, immune memory formation, and microenvironment regulation. The immune-enhancing adjuvant of the present invention targets the common pathway of the immune system through dual immune regulatory functions of promoting the expression of CD8 + T cell function genes and inhibiting immunosuppressive cell subsets. The effects such as upregulation of the ISG15 gene, inhibition of Treg cells, and induction of Th1-type cytokines are common mechanisms across tumor types. Although the present invention takes the cervical cancer vaccine as an example to verify its effect, its action targets are generally present in the anti-tumor immune response dependent on CD8 + T cells, which perfectly fits the treatment requirements of inducing CTL responses and regulating the microenvironment by various vaccines such as polypeptides, proteins, recombinant vectors, nucleic acids, tumor cells, and dendritic cells. Therefore, the limitation of the scope of application of the immune-enhancing adjuvant described in the present invention conforms to the common immune activation mechanism based on tumor therapeutic vaccines.

[0046] The immune-enhancing adjuvant of the present invention is stably preserved under the condition of 2-8 °C, which is beneficial to conventional cold chain transportation. Examples

[0047] Example 1

[0048] 1. Raw material preparation

[0049] TQ: TQ crystals with a purity of 98% or above were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and re-inspected by gas chromatography to ensure the stability of quality and activity.

[0050] Corn oil: Cold-pressed and unrefined food-grade corn oil was selected. Such corn oil is rich in unsaturated fatty acids, has stable properties, good solubility for TQ, and high safety, which can reduce the risk of adverse reactions.

[0051] Buffer: Weigh an appropriate amount of tris(hydroxymethyl)aminomethane (Tris) and dissolve it in deionized water, and precisely adjust the pH value to 7.4 with hydrochloric acid to prepare a buffer solution with a concentration of 20 mM. The Tris-HCl buffer has a wide buffering range, can provide a stable pH environment for TQ, and has good compatibility with the corn oil system.

[0052] Stabilizer: Butylated hydroxytoluene (BHT) with a purity of more than 99% was selected as the stabilizer. BHT is a fat-soluble antioxidant that can prevent TQ from being oxidized in the solution and maintain its chemical stability.

[0053] pH regulator: 1M hydrochloric acid (HCl) solution and 1M sodium hydroxide (NaOH) solution, used to precisely adjust the pH value of the adjuvant solution.

[0054] Sterile filter: Select a 0.22 μm filter meeting pharmaceutical grade standards for sterilization to ensure the sterility quality of the final adjuvant product.

[0055] 2. Preparation of adjuvant

[0056] In a sterile and dry environment, weigh 324.3 g of purified TQ and slowly add it to 1.5 L of corn oil. Use a magnetic stirrer to stir at a speed of 250 - 350 revolutions per minute for about 30 - 60 minutes until TQ is completely dissolved, forming a homogeneous and transparent TQ-corn oil solution.

[0057] Slowly add the prepared 20 mM Tris-HCl buffer solution to the TQ-corn oil solution at a ratio of 30 - 40% of the total solution volume, while starting stirring and increasing the stirring speed to 400 - 500 revolutions per minute to fully mix the two. Then add the BHT stabilizer at a ratio of 0.05 - 0.1% of the total solution volume and continue stirring for 45 - 60 minutes to ensure that the stabilizer is evenly dispersed in the solution system and enhance the chemical stability of TQ.

[0058] Use a high-precision pH meter to monitor the pH value of the solution in real time, and slowly drip 1M HCl or NaOH solution to finely adjust the pH value of the solution to 6.8 - 7.2. This pH range can not only ensure the chemical activity of TQ but also conform to the physiological environment of most immune cells, which helps to exert the subsequent immune enhancement effect.

[0059] In a sterile operating table, filter the solution with adjusted pH value through a 0.22 μm sterile filter. Use a positive pressure filtration device to slowly press the solution into the filter to ensure that the entire filtration process is carried out under sterile conditions, and finally obtain a clear and sterile TQ immune enhancement adjuvant.

[0060] 3. Quality inspection

[0061] Appearance inspection: Observe the adjuvant solution under natural light, which should be clear, transparent, without precipitation, turbidity or foreign matters. The normal TQ adjuvant should be a slightly light yellow transparent liquid.

[0062] Re-measurement of pH: Re-detect the pH value of the filtered adjuvant using a calibrated pH meter to ensure that its pH value is within the target range of 6.8 - 7.2, with a deviation not exceeding ±0.1.

[0063] Sterility test: The membrane filtration method was adopted. An appropriate amount of adjuvant was filtered through a 0.22 μm sterile filter membrane, and then the filter membrane was inoculated into fluid thioglycollate medium and tryptic soy broth, and cultured at 30 - 35 °C and 20 - 25 °C for 14 days respectively. Observe whether there is microbial growth in the medium to confirm that the adjuvant meets the sterility requirements.

[0064] Assay for content: The content of TQ in the adjuvant was determined by high performance liquid chromatography (HPLC) method to ensure compliance with the formulated target concentration, with the deviation controlled within ±5%, so as to ensure the effectiveness and batch - to - batch consistency of the adjuvant.

[0065] The amount of the above - prepared immune - enhancing adjuvant is the dose for 1000 person - times of use.

[0066] Example 2: Study on the anti - tumor immune effect of TQ - enhanced tumor therapeutic vaccine

[0067] 1. Establishment of mouse tumor model

[0068] Select C57BL / 6 female mice at 6 - 8 weeks old. After one - week adaptive feeding in the experimental environment, shave the hair on their backs.

[0069] Collect TC - 1 cells (mouse lung epithelial cells) in the logarithmic growth phase, wash them 3 times with PBS buffer, resuspend the cells, and adjust the cell density to 5×10 6 / mL to obtain the TC - 1 cell suspension.

[0070] On the 0th day of the experiment, use a 1 mL sterile syringe to aspirate 100 μL of the above - mentioned TC - 1 cell suspension, and slowly inject it into the subcutaneous tissue on the right back of the mouse to construct a subcutaneous transplanted tumor model.

[0071] 2. Grouping of experimental animals

[0072] Randomly divide the modeled mice into four groups, with 6 mice in each group:

[0073] 1) PBS control group (PBS): The mice in this group do not receive special treatment and are only used as a control to observe the tumor growth under normal circumstances;

[0074] 2) Adjuvant alone group (TQ): The mice in this group only receive TQ treatment to observe the effect of using the adjuvant alone on tumor growth;

[0075] 3) Vaccine alone group (Vax): The mice in this group only receive the tumor therapeutic vaccine treatment to study the inhibitory effect of using the vaccine alone on tumors;

[0076] 4) Vaccine and adjuvant co - immunization group (Vax + TQ): The mice in this group receive both the tumor therapeutic vaccine and TQ adjuvant treatment, aiming to explore the effect of their combination on tumors.

[0077] 3. Vaccination and Treatment

[0078] The body weight of the mice was approximately 20 g. Calculated according to the dosing concentration of 80 mg / kg, each mouse needed to be given 1.6 mg of TQ.

[0079] Accurately weigh 1.6 mg of TQ into a sterile EP tube, add 100 μL of corn oil, and stir to fully dissolve TQ to make a uniform solution.

[0080] Using a gavage needle, administer TQ by oral gavage to the mice in the TQ group and the Vax+TQ group. During the 25-day experimental period, gavage 100 μL of the TQ corn oil solution containing 1.6 mg of TQ three times a week. Gavage can allow the drug to accurately enter the stomach of the mice, ensure absorption, and ensure the accurate consistency of the drug dose in the experiment.

[0081] Take 20 μL of the prepared polypeptide working solution into a centrifuge tube, add 50 μL of the prepared CpG ODN working solution, and then add 30 μL of high-pressure sterilized PBS buffer. Mix well by pipetting to prepare a therapeutic vaccine containing 20 μg of CpG ODN 1826 and 50 μg of HPV16 E7 polypeptide.

[0082] On the 7th, 14th, and 21st days of the experiment, perform subcutaneous injections on the mice in the Vax group and the Vax+TQ group, with each injection dose being 100 μL.

[0083] During the 25-day experimental period, use a vernier caliper to measure the major axis and minor axis of the tumors of the mice three times a week, and calculate the tumor volume (Volume = 0.5 × major axis × minor axis 2 )). At the same time, carefully observe and record in detail the time when the tumors appear and the growth rate of each mouse.

[0084] At the end point of the experiment, on the 25th day, carefully separate along the edge of the tumor to ensure complete resection of the tumor tissue, avoid residue or mixing with the surrounding normal tissue, and use sterile filter paper to absorb the surface blood and body fluid, and weigh to obtain the final weight of the mouse tumor.

[0085] 4. Result Analysis

[0086] Figure 1 Intuitively presents the anti-tumor effect of the combined application of TQ and the tumor therapeutic vaccine. Among them, the tumor volume and weight of the mice in the group immunized with the vaccine and adjuvant together (Vax+TQ) were significantly lower than those in the PBS control group, the single adjuvant group, and the single vaccine group, directly proving that TQ used as an adjuvant in combination with the vaccine can enhance the anti-tumor efficacy.

[0087] Figure 1In (a), over time, the tumor volume in the PBS group continuously increased, with the average value increasing from nearly 0 initially to 1082.04 mm at 25 days 3 and the growth rate gradually accelerating; the tumor volume in the TQ group treated only with adjuvant also increased with time, but the growth amplitude was slightly slower than that of the PBS group. The average tumor volume at 25 days was 733.586 mm 3 , which was smaller than that of the PBS group; the growth trend of the tumor volume in the Vax group treated only with the tumor therapeutic vaccine was relatively gentle, and the tumor volume at 25 days was significantly smaller than that of the PBS group and the TQ group, with an average value of 223.248 mm 3 ; while the tumor volume in the Vax+TQ group with the combined use of the tumor therapeutic vaccine and adjuvant increased extremely slowly throughout the time period, almost maintaining at a low level, significantly lower than the other three groups. The average tumor volume at 25 days was 5.609 mm 3 .

[0088] Figure 1 In (b), the tumor weight in the PBS group was relatively high, with an average value of 1715.7 mg, and the data points were relatively scattered, indicating a large difference in tumor weight among individuals; the tumor weight in the TQ group was lower than that in the PBS group, with an average value of 1094.12 mg. Although the data points were relatively more concentrated, there was still a certain degree of dispersion, indicating that TQ had a certain inhibitory effect on tumor growth, but individual differences still existed; the tumor weight in the Vax group was further reduced, with an average value of 434.75 mg, and the data points were relatively more concentrated, indicating that the tumor therapeutic vaccine had a good inhibitory effect on tumor growth; the tumor weight in the Vax+TQ group was the lowest, with an average value of 30.15 mg, a reduction of more than 93% compared with the single vaccine group, and the data points were almost concentrated in a very low value area, indicating that the combined use of the tumor therapeutic vaccine and TQ could significantly inhibit tumor growth and the individual differences were small.

[0089] Analysis of the experimental data found that the single use of the vaccine already had a certain inhibitory effect on the mouse tumor model, and when TQ was combined with the vaccine for immunization, the induced tumor inhibitory effect in mice was better than that of the single use of the vaccine, indicating that the TQ adjuvant of the present invention could significantly enhance the anti-tumor immune effect of the tumor therapeutic vaccine.

[0090] Example 3: Study on the distribution and proportion of immune cells in tumor tissues with TQ combined with tumor therapeutic vaccine

[0091] At the end point of the experiment in Example 2, that is, at 25 days, the tumor tissues of each group of mice were taken out, fixed with 4% paraformaldehyde, embedded in paraffin, continuously sectioned into slices with a thickness of 4 μm, fished with adhesive glass slides, and dried in an environment at 65°C.

[0092] After dewaxing and hydrating the dried sections, immerse them in sodium citrate antigen retrieval solution and perform high-pressure retrieval for 15 min. Wash the sections with PBS buffer, place them in a humid box, add an endogenous peroxidase blocker, ensure that the blocker completely covers the tissue, incubate at room temperature for 10 min, then wash with PBS buffer again, add 5% bovine serum albumin to cover the tissue, and block at room temperature for 30 min.

[0093] Add anti-CD4, anti-CD8, and anti-Foxp3 primary antibodies to the blocked sections, incubate overnight at 4 °C, and wash with PBS to remove unbound primary antibodies. Add a biotinylated secondary antibody, incubate at room temperature for 30 min, and wash with PBS to remove unbound secondary antibody. Add freshly prepared DAB chromogenic solution, observe the chromogenic situation under a microscope, and immediately rinse the sections with tap water to terminate the chromogenic reaction when appropriate chromogenic intensity is observed.

[0094] Put the chromogenic sections into hematoxylin for counterstaining and differentiation, dehydrate, mount, air-dry, and then perform scanning analysis.

[0095] Quantitatively analyze the immunohistochemical results through two scoring methods: staining intensity score and positive cell percentage score.

[0096] 1) Staining intensity score: Score according to the color depth of positive staining on the sections. No positive staining is recorded as 0 points; light yellow is recorded as 1 point; brownish yellow is recorded as 2 points; brownish brown is recorded as 3 points.

[0097] 2) Positive cell percentage score: Score according to the proportion of positive cells in the sections. A positive cell ratio of 0% is recorded as 0 points; 1 - 25% is recorded as 1 point; 26 - 50% is recorded as 2 points; 51 - 75% is recorded as 3 points; 76 - 100% is recorded as 4 points.

[0098] The results showed that the numbers of CD4 + and CD8 + T cells in the Vax+TQ group were significantly higher than those in other treatment groups, while the number of Foxp3 + Treg cells was significantly reduced. This indicates that the combination therapy promoted the anti-tumor immune response by increasing the number of tumor-infiltrating CD8 + T cells (with anti-tumor effects) and inhibiting Treg cells (inhibiting the immune response), revealing the mechanism by which TQ enhances the vaccine effect at the level of local tumor immune cells.

[0099] Figure 2 In the PBS group, the immunohistochemical score of CD4 was relatively low, indicating that the number of CD4 + T cells in the tumor tissue of mice in this group was small; the score of the TQ group was higher than that of the PBS group, indicating that when using TQ alone, the number of CD4 +The number of T cells increased; the score of the Vax group further increased, indicating that the vaccine alone could increase the number of CD4 + T cells; while the score of the Vax+TQ group was the highest and significantly higher than that of other groups, suggesting that when the vaccine was combined with TQ, the number of CD4 + T cells in the tumor tissue increased significantly.

[0100] Meanwhile, the immunohistochemical score of CD8 in the PBS group was very low, indicating that the number of CD8 + T cells in the tumor tissue was extremely low; the score of the TQ group was higher than that of the PBS group, suggesting that treatment with TQ alone increased the number of CD8 + T cells to a certain extent; the score of the Vax group was higher than that of the TQ group, showing that treatment with the vaccine alone was effective in increasing the number of CD8 + T cells; the score of the Vax+TQ group was the highest and significantly higher than that of other groups, with obvious statistical differences, indicating that the combined treatment significantly increased the number of CD8 + T cells in the tumor tissue.

[0101] Furthermore, the immunohistochemical score of Foxp3 in the PBS group was relatively high, indicating that the number of Foxp3 + Treg cells (regulatory T cells, which can inhibit the immune response) in the tumor tissue of mice in this group was relatively large; the score of the TQ group was lower than that of the PBS group, suggesting that treatment with TQ alone could reduce the number of Foxp3 + Treg cells; the score of the Vax group was further reduced, showing that treatment with the vaccine alone could reduce the number of Foxp3 + Treg cells; the score of the Vax+TQ group was the lowest and significantly lower than that of other groups, with statistical differences, indicating that the combined treatment could significantly reduce the number of Foxp3 + Treg cells in the tumor tissue.

[0102] Example 4: Study on the Enhancement of Specific Cellular Immune Response of Tumor Therapeutic Vaccine by TQ

[0103] 1. Preparation of mouse spleen cell suspension

[0104] At the end of the experiment in Example 2, on the 25th day, the spleens of mice in each group were dissected and removed, ground and broken on a 70μm sieve to release cells, and the sieve was rinsed with PBS buffer. The rinsing solution containing cells was collected into a 15mL centrifuge tube.

[0105] Place the centrifuge tube containing cells in a centrifuge and centrifuge at 300 g for 5 min at 4 °C. Discard the supernatant, add 2 mL of red blood cell lysis buffer, let it stand at 4 °C for 10 min, and add 8 mL of PBS buffer to terminate the lysis reaction. Centrifuge again at 300 g for 5 min at 4 °C, discard the supernatant, and leave the lymphocytes to be detected at the bottom of the centrifuge tube. Add 1 mL of PBS buffer to the centrifuge tube to resuspend the cells and adjust the cell concentration to 1×10 7 / mL to prepare a mouse spleen cell suspension for flow cytometry detection.

[0106] 2. Detect CD4 + IFN-γ + and CD8 + IFN-γ + T cell ratio

[0107] Take 100 μL of the mouse spleen cell suspension and place it in a 24-well plate. Add 900 μL of 1640 culture medium containing 10% fetal bovine serum and 10 μM of HPV 16 E7 polypeptide, and place it in an incubator at 37 °C for 4 h.

[0108] After incubation, transfer the cells to a 2 mL centrifuge tube, wash the cells with 2% FBS-PBS (PBS buffer containing 2% fetal bovine serum) to remove possible impurities or excess culture medium components on the cell surface, and perform live / dead staining and FcR Blocking (blocking Fc receptors) operations to distinguish live and dead cells and prevent non-specific binding. Then add CD45, CD4, and CD8 antibodies to the cells for incubation to specifically bind the antibodies to the corresponding cell surface markers. After incubation, use IC Fixation Buffer (cell fixation buffer) and 1×Permeabilization Buffer (membrane permeabilization buffer) for membrane permeabilization treatment to expose the internal structure of the cells, add IFN-γ antibody for incubation to bind to the IFN-γ produced inside the cells. Finally, fix the cells with 4% paraformaldehyde to maintain the cell morphology and structure stable.

[0109] Filter the fixed cell suspension through a 300-mesh filter to remove possible cell clumps or impurities, and then perform flow cytometry detection on the machine to obtain CD4 + IFN-γ + and CD8 + IFN-γ + T cell ratio.

[0110] 3. Detect CD4 + CD25 + Foxp3 +Treg cell ratio

[0111] Take 100 μL of mouse spleen cell suspension and add it to a 2 mL centrifuge tube. Add 1 mL of 2% FBS-PBS buffer to each tube, vortex to mix evenly, centrifuge at 4°C and 300 g for 5 min, and remove the supernatant. Add 0.1 μL of live / dead dye to each tube and incubate in the dark at 4°C for 30 min. After incubation, add 1 mL of 2% FBS-PBS buffer to each tube again, vortex to resuspend the cells, and discard the supernatant after centrifugation. Add 0.5 μL of FcR Blocking reagent to each tube and continue to incubate in the dark at 4°C for 15 min.

[0112] Add CD45, CD4, and CD25 antibodies to each tube and incubate in the dark at 4°C for 30 min. Add 1 mL of 2% FBS-PBS buffer to each tube, vortex and centrifuge, and discard the supernatant. Add 1 mL of 1×Fixation / Permeabilization to each tube, vortex to mix evenly, and incubate in the dark at 4°C for 40 min. Add 1 mL of 1×Permeabilization Buffer to each tube, vortex and centrifuge, and discard the supernatant.

[0113] Add Foxp3 antibody to the treated cells, vortex, and incubate in the dark at 4°C for 30 min. Add 2 mL of 1×Buffer, vortex and centrifuge, and discard the supernatant. Finally, add 200 μL of 4% paraformaldehyde to each tube for fixation, and vortex to evenly distribute the cells in the fixative.

[0114] Filter the cell suspension through a 300-mesh filter into a flow tube, load it onto the machine, and detect and analyze it by flow cytometry to obtain the proportion of Treg cells in the mouse spleen.

[0115] 4. Result analysis

[0116] Figure 3 It shows that the percentage of CD4 + IFN-γ + cells in the PBS group is very low, with an average value of 0.07%, almost approaching 0, indicating that the number of such cells in the mouse spleen treated only with PBS is extremely small; the cell percentage in the TQ group is slightly higher than that in the PBS group, with an average value of 0.14%, but the value is still low, suggesting that the effect of using TQ alone in increasing the number of such cells is limited; the cell percentage in the Vax group is higher than that in the TQ group, with an average value of 0.32%, indicating that using the vaccine alone can increase CD4 + IFN-γ +The number of T cells; however, the percentage of cells in the Vax+TQ group was significantly higher than that in the PBS control group, with a mean of 0.95%, which was approximately twice that of the Vax group treated with the vaccine alone, indicating that the combined use of the vaccine and TQ significantly increased the proportion of CD4 + IFN-γ + T cells in the spleen.

[0117] Similarly, the percentage of CD8 + IFN-γ + cells in the PBS group was extremely low, with a mean of 0.07% and almost undetectable; the percentage of cells in the TQ group was slightly higher than that in the PBS group, with a mean of 0.11%, indicating that the treatment with TQ alone slightly increased the cell number; the percentage of cells in the Vax group was higher than that in the TQ group, with a mean of 0.43%, showing that the treatment with the vaccine alone had a certain effect on increasing the number of CD8 + IFN-γ + T cells; the percentage of cells in the Vax+TQ group was the highest, with a mean of 1.26%, which was approximately twice that of the Vax group treated with the vaccine alone and was significantly higher than that of other groups, showing obvious statistical differences, indicating that the combined treatment significantly increased the proportion of CD8 + IFN-γ + T cells in the spleen.

[0118] The percentage of CD4 + CD25 + Foxp3 + cells in the PBS group was relatively high, with a mean of 12.93%, indicating that there were more Treg cells in the spleen of mice treated with PBS alone; the percentage of cells in the TQ group was lower than that in the PBS group, with a mean of 10.88%, indicating that the use of TQ alone could reduce the number of Treg cells; the percentage of cells in the Vax group was further reduced, with a mean of 9.36%, showing that the use of the vaccine alone could also reduce the number of Treg cells; the percentage of cells in the Vax+TQ group was the lowest, with a mean of 8.80% and was significantly lower than that in the PBS control group, indicating that the combined treatment could significantly reduce the proportion of Treg cells in the spleen.

[0119] The results showed that the adjuvant alone basically did not induce mice to produce specific IFN-γ, the vaccine alone could induce mice to produce specific IFN-γ, and the proportions of CD4 + IFN-γ + and CD8 + IFN-γ + T cells in the Vax+TQ group were significantly increased, while the proportion of Treg cells was significantly reduced, indicating that the combination therapy could activate CD4 + IFN-γ + and CD8 + IFN-γ +Equivalent effector T cells reduce immunosuppression by decreasing Treg cells. The adjuvant of the present invention can effectively enhance the specific cellular immune level of tumor therapeutic vaccines.

[0120] Example 5: TQ enhances CD8 + Study on the in vitro killing effect of T cells

[0121] Using EasySep TM Mouse CD8 + T cell isolation kit to isolate CD8 + T cells from the spleens of tumor-bearing mice. Take 100 μL of the isolated CD8 + T cells, add 1 μL of FITC Anti-Mo CD8 antibody, incubate at 4 °C in the dark for 30 min, add 1 mL of 2% FBS-PBS, vortex and centrifuge, discard the supernatant, resuspend with PBS, and detect the sorting efficiency of CD8 + T cells by flow cytometry. After verifying that the purity is ≥90%, adjust the concentration of CD8 + T cells to 4×10 5 cells / mL for standby.

[0122] Meanwhile, collect TC-1 cells and adjust the concentration to 2×10 4 / mL.

[0123] The experiment was set up with 4 groups in total. Among them, the TC-1 group added 100 μL of the suspension containing 2000 TC-1 tumor cells per well as a blank control to observe the natural growth state of tumor cells; the TC-1+TQ group added 100 μL of TC-1 cell suspension (2000 cells / well) and 20 μM TQ working solution per well to verify the direct effect of TQ on tumor cells; the TC-1+CD8 + T group added 100 μL of TC-1 cell suspension and 100 μL of the suspension containing 40000 CD8 + T cells per well to detect the killing ability of CD8 + T cells alone on tumor cells; the TC-1+CD8 + T+TQ group added 100 μL of TC-1 cell suspension, 100 μL of CD8 + T cell suspension and 20 μM TQ working solution per well to verify the synergistic killing effect of TQ combined with CD8 + T cells.

[0124] Place the 96-well plate in a 37 °C, 5% CO2 cell culture incubator. After co-culturing for 48 h, according to the requirements of the cell toxicity kit instructions, measure the OD values (optical density values) of different samples by an enzyme-linked immunosorbent assay (ELISA) reader, and substitute them into the formula "Cytotoxicity rate (%) = (OD E+T -OD E -ODT ) / (OD Emax - OD E )× 100%”, where OD E+T represents the OD value when CD8 + T cells co - exist with TC - 1 cells, OD E is the OD value of CD8 + T cells alone, OD T is the OD value of TC - 1 cells alone, OD Emax is the OD value after all TC - 1 cells are lysed, thereby calculating the cytotoxicity rate, which intuitively reflects the killing ability of CD8 + T cells against TC - 1 cells.

[0125] Figure 4 The following shows the cytotoxicity rate data of the above 4 different treatment groups (single tumor cells TC - 1, TC - 1+TQ group with TQ co - cultured with tumor cells, TC - 1+CD8 + T group with CD8 + T cells co - cultured with tumor cells, TC - 1+CD8 + T+TQ group with CD8 + T cells combined with TQ co - cultured with tumor cells). The results show that the cytotoxicity percentage of the TC - 1 group is extremely low, with an average value of 2.16%, almost approaching 0, indicating that there is almost no cytotoxicity when only TC - 1 cells are present without adding TQ and CD8 + T cells; the cytotoxicity percentage of the TC - 1+TQ group has increased, with an average value of 23.63%, but the value is relatively not very high, indicating that when using TQ alone to treat TC - 1 cells, the cytotoxicity increases to a certain extent, but the increase amplitude is limited; the cytotoxicity percentage of the TC - 1+CD8 + T group is significantly higher than that of the TC - 1+TQ group, with an average value of 52.93%, indicating that after adding CD8 + T cells, the cytotoxicity is greatly enhanced; the cytotoxicity percentage of the TC - 1+CD8 + T+TQ group is the highest, with an average value of 79.66%, increasing by about 50% compared with the TC - 1+CD8 + T group, and the difference is highly statistically significant, indicating that when TQ and CD8 + T cells act together on TC - 1 cells, the killing ability against TC - 1 cells is significantly improved, providing evidence at the cellular function level for TQ as an immune - enhancing adjuvant to enhance the immune effect of vaccines.

[0126] The present invention aims to study the effects and mechanisms of TQ as an immune - enhancing adjuvant, and CD8 +The enhancement of T cell killing function is an important manifestation of immune enhancement. The above results intuitively show the effect of TQ on CD8 + The positive effect of T cells supplements the functional evidence of TQ in immune regulation, further illustrates that TQ has potential application value in tumor immunotherapy, and provides strong support for the development of TQ as an immune-enhancing adjuvant for tumor therapeutic vaccines.

[0127] Example 6: TQ enhances CD8 + Study on the secretion of pro-inflammatory cytokines IFN-γ and TNF-α by T cells

[0128] Take the CD8 prepared in Example 5 + After co-culture of T cells and TC-1 tumor cells, mouse IFN-γ and TNF-α ELISA kits were used to detect CD8 + IFN-γ and TNF-α levels in the supernatant of co-culture of T cells and TC-1 tumor cells.

[0129] The mouse IFN-γ and TNF-α ELISA kits contain ELISA plates coated with antibodies against IFN-γ and TNF-α, standards, standard diluents, sample diluents, enzyme-labeled antibodies, colorimetric solution A, colorimetric solution B, stop solution and other reagents and materials. After taking the kit out of the refrigerated environment, equilibrate it at room temperature for 15-30 minutes before use.

[0130] 1. Co-cultivation process

[0131] Put the prepared CD8 + T cells and TC-1 tumor cells were inoculated in a sterile 96-well plate at an appropriate ratio (effector-target ratio 20:1) and conditions. The inoculation number was 2000 TC-1 cells / well, CD8 + 40,000 T cells / well were co-cultured in 1640 cell culture medium containing multiple nutrients that can maintain normal cell growth and metabolism. The culture environment was maintained at 37°C in a cell culture incubator containing 5% CO2 to allow the cells to fully interact with each other and promote CD8 + T cells are stimulated by TC-1 tumor cells to secrete proinflammatory cytokines IFN-γ and TNF-α.

[0132] 2. Collection of supernatant

[0133] After the co-culture, carefully collect the culture supernatant from each well into a clean centrifuge tube using a pipette, centrifuge at 300 g for 5 min at 4 °C, and pipette the supernatant into a new centrifuge tube.

[0134] 3. Enzyme-linked immunosorbent assay (ELISA)

[0135] 1) Gradiently dilute the standard product according to the kit instructions to prepare standard product solutions of different concentrations: Take 5 autoclaved 1.5 mL centrifuge tubes, labeled as 1 - 5 respectively. Add 150 μL of standard product diluent to each tube. Add 150 μL of the standard product to tube 1. After vortex mixing and instantaneous centrifugation, take 150 μL and add it to tube 2. After vortex mixing and instantaneous centrifugation, take 150 μL and add it to tube 2, and so on. Dilute the 5 sample concentrations to 800 pg / mL, 400 pg / mL, 200 pg / mL, 100 pg / mL, and 50 pg / mL. Add the standard product solutions of different concentrations to the designated wells of the ELISA plate. Set 2 - 3 replicates for each concentration to improve accuracy, and reserve 2 blank wells to subtract the OD value of the chromogenic solution.

[0136] 2) Sample addition: Add 40 μL of sample diluent to each well of the sample wells. Add the collected co - culture supernatant to the corresponding wells of the ELISA plate at 10 μL / well, and set replicates in the same way. Control the sample addition time within 15 min to avoid the reaction time difference between the front and back wells.

[0137] 3) Incubation and washing: After adding the standard product and the sample, seal the ELISA plate with a sealing film and gently shake the ELISA plate. Place the ELISA plate in an incubator at 37 °C for 30 min to allow the antigen - coated plate to fully bind to IFN - γ or TNF - α in the sample. Measure 20 mL of concentrated washing solution and 580 mL of deionized water and pour them into a beaker, stir to make the washing buffer. After incubation, wash the ELISA plate 5 times with the washing buffer, and thoroughly shake dry each time to remove unbound substances and reduce background interference.

[0138] 4) Enzyme conjugate addition and re - incubation: After washing, add the enzyme - labeled antibody to each well of the ELISA plate and gently oscillate to mix evenly. Place the ELISA plate in an incubator at 37 °C for 30 min to allow the enzyme - labeled antibody to react with the antibody - cytokine complex that has bound to the well wall. After incubation, wash the ELISA plate 5 times again with the washing buffer to remove the unbound enzyme conjugate.

[0139] 5) Color development and termination of reaction: Add chromogenic solution A and chromogenic solution B to each well of the ELISA plate respectively, and gently oscillate to allow the chromogenic solution to react with the enzyme conjugate for color development. Develop color for 10 min at 37 °C in the dark, observe the color change of the ELISA plate. The color depth is proportional to the cytokine content in the sample. When the color development is appropriate, add the termination solution to each well to terminate the reaction.

[0140] 4. Result detection

[0141] 1) OD value detection: Within 15 min after adding the termination solution, use an ELISA reader to detect the absorbance (OD value) of each well of the ELISA plate at 450 nm, and the ELISA reader automatically reads and records it.

[0142] 2) Standard curve drawing and sample content calculation: According to the standard product concentration and the corresponding OD value, use professional data analysis software to draw the standard curve. Substitute the OD value of the sample into the standard curve equation to calculate the content of IFN-γ and TNF-α in the sample. If the content of the substance to be measured in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard product), it needs to be diluted by a certain multiple (n times) with the sample diluent before measurement, and the total dilution multiple (×n×5) is multiplied during calculation.

[0143] Figure 5 Shows the differences in the concentrations of cytokines IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α) in different treatment groups.

[0144] TC-1+CD8 + The IFN-γ concentration in group T had a certain value, indicating that when there was no TQ treatment, CD8 + T cells co-cultured with TC-1 tumor cells had a certain secretion of IFN-γ in the supernatant; TC-1+CD8 + The IFN-γ concentration in group TC-1+CD8 + T+TQ was higher than that in group TC-1+CD8 + T cells, and there was statistical significance between the two groups, indicating that after adding TQ treatment, the secretion of IFN-γ in the supernatant of co-cultured CD8

[0145] TC-1+CD8 + The TNF-α concentration in group TC-1+CD8 + T was at a certain level, reflecting the basal secretion of TNF-α in the co-culture system without adding TQ; TC-1+CD8 + The TNF-α concentration in group TC-1+CD8 + T+TQ was significantly higher than that in group TC-1+CD8

[0146] ELISA results showed that compared with the control group, the levels of IFN-γ and TNF-α in the supernatant of co-cultured CD8 + T cells in the TQ treatment group were significantly increased, indicating that TQ could promote the secretion of pro-inflammatory cytokines. In tumor immunotherapy, pro-inflammatory cytokines IFN-γ and TNF-α play important roles in activating the immune system and enhancing the functions of immune cells. The above experimental results of the present invention show that TQ may activate the body's immune cells and enhance the immune response by promoting the secretion of these cytokines, thereby playing the role of an immune-enhancing adjuvant, revealing the mechanism of TQ enhancing the immune effect from the perspective of promoting cytokine secretion, and further supporting the role of TQ as an immune-enhancing adjuvant.

[0147] The above embodiments of the present invention do not describe all the details in detail, nor do they limit the present invention to the above-described embodiments. All changes, modifications, substitutions, and variations made to these embodiments by those of ordinary skill in the art without departing from the principles and spirit of the present invention shall be included within the protection scope of the present invention.

Claims

1. An immunoenhancing adjuvant for tumor therapeutic vaccine, wherein the immunoenhancing adjuvant has thymoquinone as an active ingredient, which is mixed with necessary pharmaceutical excipients and dissolved in a benign solvent of thymoquinone.

2. The immunoenhancing adjuvant according to claim 1, characterized in that The good solvent of thymoquinone is corn oil.

3. The immunoenhancing adjuvant according to claim 1, characterized in that The pharmaceutical excipients include a buffer and may or may not include a stabilizer. The buffer is a Tris-HCl buffer and the stabilizer is butylated hydroxytoluene.

4. The method for preparing the immunoenhancing adjuvant according to claim 1 is to dissolve the active ingredient thymoquinone in a benign solvent thereof, add a buffer, add or not add a stabilizer, mix well and adjust the pH value of the solution to 6.8-7.2, and filter and sterilize.

5. Use of the immune enhancing adjuvant according to claim 1 in the preparation of tumor therapeutic vaccines.

6. Use of the immune enhancing adjuvant according to claim 1 in the preparation of a therapeutic vaccine for cervical cancer.

7. A tumor therapeutic vaccine composition, comprising the immune enhancing adjuvant according to claim 1 and an immunologically effective amount of a tumor therapeutic vaccine.

8. The tumor therapeutic vaccine composition according to claim 7, characterized in that The tumor therapeutic vaccine includes at least one of a polypeptide vaccine, a protein vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a DNA vaccine, an RNA vaccine, a tumor cell vaccine or a dendritic cell vaccine.

9. The tumor therapeutic vaccine composition according to claim 7, characterized in that The tumor therapeutic vaccine is a therapeutic cervical cancer vaccine.

10. The tumor therapeutic vaccine composition according to claim 7, characterized in that The mass ratio of the active ingredients of the immune enhancing adjuvant to the tumor therapeutic vaccine is (10-50):1.

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