H1-pIL-24 / pCAIX nano vaccine, anti-tumor medicine and application

Abstract

The invention relates to the technical field of vaccine preparation, and particularly discloses an H1-pIL-24 / pCAIX nano vaccine, an anti-tumor medicine and application, and the H1-pIL-24 / pCAIX nano vaccine is prepared through the following steps: constructing an expression plasmid containing an mIL-24 sequence and an expression plasmid containing a CAIX sequence; the expression plasmid containing the mIL-24 sequence and the expression plasmid containing the CAIX sequence are respectively transformed into competent cells and cultured, two obtained recombinant plasmids are mixed with the H1 nano carrier, and the H1-pIL-24 / pCAIX nano vaccine is obtained. The H1-pIL-24 / pCAIX nano vaccine can significantly inhibit the growth of tumors and the occurrence of pulmonary metastasis, improve DC uptake and antigen presentation, enhance the expression of CD8 cells and IFN-gamma, enhance the expression of central memory T cells, and effectively stimulate the tumor immune response of mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine preparation, and particularly relates to an H1-pIL-24 / pCAIX nano-vaccine, an anti-tumor drug and uses thereof. Background Art

[0002] Carbonic anhydrase 9 (CAIX / CA9), a transmembrane glycoprotein composed of 459 amino acids, plays an important role in regulating cell proliferation and transformation; it is highly expressed in clear cell renal carcinoma and most other types of renal carcinoma, while rarely expressed or not expressed in normal tissues. CAIX can regulate the internal and external acidity of tumor cells, induce the formation of an immunosuppressive tumor microenvironment, promote tumor invasion and metastasis, and resist external intervention and treatment. A large number of studies have shown that the overexpression of CAIX not only greatly promotes tumor metastasis, but also is positively correlated with chemoresistance and radioresistance, which is not conducive to the treatment and prognosis of cancer. Research reports indicate that various vaccines based on the CAIX antigen can effectively stimulate specific immune responses and inhibit the growth of renal carcinoma. Therefore, CAIX, as a specific antigen of renal carcinoma, is an ideal target for renal carcinoma DNA vaccines.

[0003] Vaccine adjuvants can non-specifically change or enhance the body's long-term and highly effective specific immune responses to antigens. For example, some adjuvants encoding cytokines or chemokines can enhance the immune stimulation of DNA vaccines and are used for anti-tumor adjuvant therapy. IL-24 is a novel cytokine that can improve the tumor microenvironment and thus break the body's immune tolerance to antigens. It has broad-spectrum anti-tumor activity and is of great research value for tumor treatment. Therefore, IL-24 can be used as a cytokine adjuvant to enhance the immune induction of tumor vaccines.

[0004] DNA vaccines have been one of the research hotspots in tumor vaccines in recent years. Tumor DNA vaccines use genetic engineering technology to load the genes of tumor-specific antigens onto plasmid vectors. With the help of the vector or the human gene expression system, they can continuously induce specific humoral and cellular immune responses to achieve the purpose of preventing and treating diseases. However, the properties of nucleic acids, such as large molecular weight and negatively charged, result in the difficulty of naked DNA to enter cells autonomously, and it is easily degraded in the body. It cannot effectively break through the barrier formed by endothelial cells in the blood vessel lumen and cannot enter immune cells outside the blood vessels, thus hindering the induction of specific immune responses.

[0005] The nano-carrier H1 (PEI 600 -CyD-FA) is a cationic polymer polyethyleneimine PEI 600A novel nanocomposite obtained by modifying with cyclodextrin CyD and coupling folic acid FA ligand. H1 has the characteristics of low toxicity, low immunogenicity, good water solubility, and good biocompatibility, and can be adsorbed electrostatically and naturally with negatively charged cell membranes or plasmid DNA. It can not only avoid the degradation of the encapsulated antigen by enzymes in body fluids, but also has the functions of enhancing cell adhesion and absorption, promoting intercellular transport, sustained release, and high gene transfection efficiency; and can cause a high level of antigen uptake by DCs and a stronger antigen cross-presentation effect, significantly improving the activity of antigen-specific CTLs and enhancing antigen-specific humoral and cellular immune responses. Due to the strong DNA-binding ability, high vector transfection efficiency, high gene expression ability, safety and low toxicity of H1, it is suitable as an antigen-presenting vector for gene vaccines.

[0006] At present, the treatment of malignant tumors mainly includes surgery, radiotherapy, chemotherapy and targeted therapy. These treatment methods have their own limitations, often causing different degrees of adverse reactions or drug resistance in patients, and are prone to metastasis and recurrence, with poor overall efficacy and low long-term survival rate of patients. Common problems in the research and development of tumor vaccines include insufficient target specificity, weak immunogenicity, and low presentation efficiency. Summary of the Invention

[0007] Based on the above technical problems, the present invention uses CAIX as the renal cancer vaccine target and mIL-24 as the vaccine adjuvant, and uses H1 to encapsulate the adjuvant and target DNA, successfully preparing the H1-pIL-24 / pCAIX nano-vaccine. The prepared H1-pIL-24 / pCAIX nano-vaccine can circulate in the blood for a long time, be recruited to the inflammatory site, and at the same time can penetrate the blood vessel and enter the body tissue, and be taken up by APCs, thereby promoting the antigen to be presented to T cells and inducing specific immunity of the body.

[0008] The specific technical solution provided by the present invention is as follows: The present invention provides an H1-pIL-24 / pCAIX nano-vaccine, which is prepared by the following steps: Construct an expression plasmid containing mIL-24 sequence and an expression plasmid containing CAIX sequence; Transform the expression plasmid containing mIL-24 sequence and the expression plasmid containing CAIX sequence into competent cells respectively, culture, and mix the two obtained recombinant plasmids with the H1 nanocarrier to obtain the H1-pIL-24 / pCAIX nano-vaccine; Among them, mIL-24 The NCBI accession number of is 93672, CAIX The NCBI accession number of is 768.

[0009] As a preferred embodiment of the present invention, construct an expression plasmid containing mIL-24 sequence and an expression plasmid containing CAIX sequence. The mIL-24 sequence and CAIX sequence are respectively ligated to the pcDNA3.1 expression vector.

[0010] As a preferred embodiment of the present invention, the competent cells are DH5a Escherichia coli competent cells.

[0011] As a preferred embodiment of the present invention, the mixing mass ratio of the two recombinant plasmids to the H1 nanocarrier is 1:1:3 - 5.

[0012] As a preferred embodiment of the present invention, the mixing is to dissolve the two recombinant plasmids and the H1 nanocarrier in PBS solution respectively, and then mix them after standing.

[0013] As a preferred embodiment of the present invention, the nanoparticle size of the H1-pIL-24 / pCAIX nanovaccine is about 236 nm.

[0014] In the second aspect of the present invention, there is provided an application of the described H1-pIL-24 / pCAIX nanovaccine in the preparation of an anti-tumor drug.

[0015] As a preferred embodiment of the present invention, the anti-tumor is anti-renal cancer.

[0016] In the third aspect of the present invention, there is provided an anti-tumor drug, and the drug takes the described H1-pIL-24 / pCAIX nanovaccine as the sole active ingredient.

[0017] As a preferred embodiment of the present invention, the drug is compounded from the described H1-pIL-24 / pCAIX nanovaccine and pharmaceutically acceptable excipients.

[0018] As a preferred embodiment of the present invention, the drug is an injection preparation.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses CAIX as a renal cancer vaccine target and mIL-24 as a vaccine adjuvant, and successfully prepares the H1-pIL-24 / pCAIX nano-vaccine by encapsulating the adjuvant and target DNA with H1. The H1-pIL-24 / pCAIX nano-vaccine can have a long circulation in the blood, be recruited to the inflammatory site, and at the same time can penetrate blood vessels and enter the body tissues, being taken up by APCs, thereby promoting the antigen to be presented to T cells and inducing specific immunity of the body. Moreover, the encapsulated antigen can avoid being degraded by enzymes in the body fluid, making it a novel drug delivery system, and thus can be used to prepare anti-tumor drugs, inhibit the proliferation and lung metastasis of tumor cells, effectively stimulate the tumor immune response of mice, enhance the expression of central memory T cells in the spleen, and inhibit the recurrence and metastasis of tumors for a long time.

[0020] 2. The H1-pIL-24 / pCAIX nano-vaccine prepared in the present invention can be used to prepare drugs for preventing and treating tumors such as renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the particle size of the H1-DNA nanoparticle; Figure 2 is the prevention and treatment effect of the H1-pIL-24 / pCAIX nano-vaccine prepared in the present invention on renal cancer Renca; Figure 3 is the tumor metastasis situation of the lung pathological sections of tumor-bearing mice in the control group and the experimental group detected by HE staining in the present invention; Figure 4 is the CD8 staining situation of the spleen sections of tumor-bearing mice in the control group and the experimental group detected by immunohistochemistry experiment in the present invention; Figure 5 is the expression of IFN-γ secreted by spleen lymphocytes of immunized mice detected by ELISPOT experiment in the present invention; Figure 6 is the maturity of DC cells in the spleens of immunized control group and experimental group mice detected by flow cytometry in the present invention (A) and the statistical chart (B).

[0022] Figure 7 is the expression of CD4 central memory T cells in the spleens of immunized control group and experimental group mice detected by flow cytometry in the present invention (A) and the statistical chart (B).

[0023] Figure 8 is the expression of CD8 central memory T cells in the spleens of immunized control group and experimental group mice detected by flow cytometry in the present invention (A) and the statistical chart (B). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the described embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0025] At present, the treatment of malignant tumors mainly includes surgery, radiotherapy, chemotherapy and targeted therapy. These treatment methods each have their limitations, often causing varying degrees of adverse reactions or drug resistance in patients, and are also prone to metastasis and recurrence. The overall therapeutic effect is poor, and the long-term survival rate of patients is low. Common problems in the research and development of tumor vaccines include insufficient target specificity, weak immunogenicity, and low presentation efficiency.

[0026] Based on this, the present invention provides an H1-pIL-24 / pCAIX nano-vaccine, which uses the H1 nano-carrier to encapsulate the expression plasmid containing the mIL-24 sequence and the expression plasmid containing the CAIX sequence to form H1-pIL-24 / pCAIX nanoparticles, making it more effectively absorbed by the body, enhancing its immunogenicity. The prepared H1-pIL-24 / pCAIX nano-vaccine can be used to prepare anti-tumor drugs, can inhibit the proliferation and metastasis of tumor cells, enhance the expression of central memory T cells in the spleen, and effectively stimulate the tumor immune response of mice.

[0027] Example 1 1. Obtaining and detection of H1-pIL-24 / pCAIX vaccine 1.1 Construction of pIL-24 plasmid According to the mIL-24 sequence (the NCBI accession number in the international gene bank is 93672), primers were designed. The forward primer is: 5'-GAT AAGCTT ATG CTA ACA GAA CCA GCA CAA CTA TTC-3', SEQ ID NO.1, and the underlined part is the Hind III restriction enzyme site; the reverse primer is: 5'-TGC TCTAGA TTA TTA TAG GTG GTA GAA CTT CTG CAT CCA-3', SEQ ID NO.2, and the underlined part is the Xba I restriction enzyme site. The mIL-24 sequence was amplified using these primers.

[0028] The amplification reaction system is: plasmid template 0.5 μL, upstream and downstream primers each 1 μL, 2x Master Mix 10 μL, water 7.5 μL, and the total volume is 20 μL.

[0029] The amplification program is as follows: 98°C for 3 min; 98°C for 15 s, 55°C for 5 s, 72°C for 8 min, for 30 cycles; 72°C for 10 min; store at 4°C.

[0030] Using pcDNA3.1 as the eukaryotic expression vector, the amplified mIL-24 sequence was inserted at the multiple cloning sites Hind Ⅲ and Xba Ⅰ to construct the pcDNA3.1-mIL-24 plasmid (denoted as pIL-24) and verified by sequencing. The specific process is as follows:

[0031] After purification of the PCR amplification product, it was double digested with the endonucleases Hind Ⅲ and Xba Ⅰ. The reaction was carried out in a water bath at 37°C for 4 h. A sample was taken for 2% agarose gel electrophoresis detection. Under ultraviolet irradiation, the target DNA fragment was excised, and the digested product was recovered from the excised gel using a DNA recovery kit.

[0032] The double digestion reaction system is as follows: 50 μl of DNA, Hind 3 μl of Xba Ⅰ, 14 μl of 2×Tango, with a total volume of 70 μl.

[0033] The double digestion reaction system and conditions of the vector plasmid are the same as above. After detection by 1% agarose gel electrophoresis, the gel excision and purification process is the same as above.

[0034] Take the above PCR double digestion product and the vector digestion fragment for ligation reaction, and ligate overnight at 4°C. The ligation reaction system is as follows: 2 μl of 10×buffer, 15 μl of PCR product, 2 μl of vector, 1 μl of T4 DNA ligase, with a total volume of 20 μl.

[0035] The ligation product was transformed into Escherichia coli DH5α: The DH5α competent bacteria were taken out from the -80°C refrigerator and placed on ice to melt for 5 minutes; the ligation product was added to the competent bacteria, the EP tube lid was covered, and incubated on ice for 30 minutes; heat shocked at 42°C for 90 seconds; incubated on ice for 2 minutes; 500 μl of LB liquid medium was added, and cultured with shaking at 37°C for 1 hour; 100 μl was taken and evenly spread on a sterile LB agar plate. The plate was placed in a 37°C incubator and incubated upside down overnight. The next day, observe the LB plate, pick a monoclonal colony into LB liquid medium, culture with shaking at 37°C for 16 hours, collect the bacterial solution, centrifuge, pour off the supernatant medium, retain the bacteria, extract the plasmid using a plasmid kit, detect the purity by 1% agarose gel electrophoresis, detect the DNA concentration using a nanodrop instrument, and take a sample for sequencing verification.

[0036] 1.2. Construction of pCAIX plasmid According toCAIX Primers were designed for the sequence (GenBank accession number: 768). The forward primer is: 5’-TT AAGCTT ATG GCT CCC CTG TGC CCC AGC-3’, SEQ ID NO.3. The underlined part is the Hind restriction site of enzyme III; the reverse primer is: 5’-TA CTCGAG CTA GGC TCC AGT CTC GGC TAC CTC TGC -3’, SEQ ID NO.4. The underlined part is the Xho restriction site of enzyme I. The CAIX sequence was amplified using these primers. The reaction system and procedure were the same as above.

[0037] Using pcDNA3.1 as the eukaryotic expression vector, the amplified CAIX sequence was inserted at the multiple cloning sites of HindⅢ and XhoⅠ. The reaction systems and conditions for digestion and ligation were the same as those in the construction process of the pIL-24 plasmid. The pcDNA3.1-CAIX plasmid (denoted as pCAIX) was constructed. After detecting the purity and concentration, sequencing verification was performed.

[0038] 2. Preparation of H1 nanocarrier (1) Cyclodextrin (CyD) was dissolved in dimethyl sulfoxide (DMSO) solution, and triethylamine was added as a catalyst; separately, carbonyldiimidazole (CDI) was dissolved in DMSO and stirred for 3 h.

[0039] (2) Polyethyleneimine 600 (PEI600) was dissolved in DMSO. Under the condition of nitrogen protection and light avoidance, it was added dropwise to the reaction solution obtained in step (1). After adding the catalyst, the reaction continued for 5 h to obtain PEI600-CyD.

[0040] (3) Folic acid (FA) was dissolved in DMSO. After dissolving by water bath heating, an activator and a catalyst were added, and the reaction was carried out at room temperature under nitrogen for 3 h to obtain a folic acid reaction solution.

[0041] (4) The above folic acid reaction solution was added dropwise to the PEI600-CyD solution, the catalyst was added, and the reaction was carried out overnight at room temperature under nitrogen. The reaction solution was dialyzed with pure water and freeze-dried to obtain PEI600-CyD-FA (denoted as H1).

[0042] 3. Preparation of nano-vaccine The extracted pIL-24 and pCAIX plasmids were mixed in equal amounts and diluted with PBS. The H1 nanocarrier (PEI600-CyD-FA) was dissolved in PBS and allowed to stand for 5 minutes. The two were slowly mixed in equal volumes and then allowed to stand for 10 minutes to obtain the H1-pIL-24 / pCAIX nanoparticle solution (denoted as H1-DNA).

[0043] Dissolve the H1-DNA nanoparticles in PBS solution, dilute with pure water and add to a clean cuvette, and measure the particle size of the H1-DNA nanoparticles using a particle size analyzer. The results show that the particle size of the nanoparticles is approximately 236 nm ( Figure 1 ).

[0044] Example 2 Functional verification of the prepared H1-pIL-24 / pCAIX nano-vaccine 1. Anti-tumor effect of the H1-pIL-24 / pCAIX nano-vaccine Randomly divide female BALB / c mice aged 6 - 8 weeks into groups: control group, H1-pIL-24 (i.e., a mixture of pIL-24 and H1 nanocarrier), H1-pCAIX (i.e., a mixture of pCAIX and H1 nanocarrier), and H1-pIL-24 / pCAIX experimental group. Each mouse is injected with 50 μg of pIL-24 or pCAIX plasmid in the nano-vaccine each time. The H1-pIL-24 / pCAIX experimental group contains 50 μg of pIL-24 and 50 μg of pCAIX plasmid. Subcutaneous immunotherapy is performed once every 2 weeks for a total of 4 times. After the immunization, collect Renca cells in the logarithmic growth phase and inject 5×10 5 cells / mouse into the ventral subcutaneous area of the mouse to establish a mouse subcutaneous xenograft tumor model, and observe the anti-tumor effect of the vaccine. Sacrifice the mice 3 weeks after tumor inoculation, and collect blood, tumors, and internal organs.

[0045] As Figure 2 shown, compared with the control group, the tumors of the mice in the H1-pIL-24 / pCAIX immunized group are significantly smaller, indicating that the H1-pIL-24 / pCAIX vaccine is beneficial to inhibiting the proliferation of mouse tumor cells.

[0046] 2. Detection of tumor lung metastasis in pathological sections of tumor-bearing mice by HE staining Sacrifice the mice, take the lungs and fix them in 10% formaldehyde solution, embed them in paraffin, cut 4 μm thick sections, perform HE staining, and observe and photograph under an optical microscope.

[0047] As Figure 3 shown, there are a large number of tumor metastases in the alveoli of the control group, and there are fewer tumor metastases in the lungs of the mice in the H1-pIL-24 / pCAIX experimental group, indicating that the H1-pIL-24 / pCAIX vaccine is beneficial to inhibiting the metastasis of mouse tumor cells and reducing the spread.

[0048] 3. Detection of CD8 infiltration in spleen sections of tumor-bearing mice by immunohistochemistry Sacrifice the mice, take the spleens of the mice in each experimental group and fix them in 10% formaldehyde solution, section them as above, and detect the CD8 staining of each group of sections by immunohistochemistry.

[0049] As Figure 4 shown, compared with the control group, significant CD8 cell infiltration (the brown part in the figure) was observed in the spleen tissue sections of mice in the H1-pIL-24 / pCAIX immunization group, indicating that H1-pIL-24 / pCAIX immunization successfully activated the CD8 cell activity in the spleen of mice.

[0050] 4. Detection of functional T cells secreting IFN-γ in spleen lymphocytes by ELISPOT assay Under sterile conditions, open the 96-well plate pre-coated with IFN-γ antibody in the ELISPOT detection kit (Daiyou mouse IFN-γ 2210005, Dakaiwei), add 200 μL / well of RPMI-1640 medium and incubate for 5 - 10 min for blocking; isolate the spleen cells of mice in each experimental group and seed them at 10 5 cells / well on the ELISPOT plate; add stimulants respectively. Add 10 μL of the working solution (the 10 μL working solution contains 500 ng / mL of phorbol ester PMA and 10 μg / mL of ionomycin) to the positive control group, and add 10 μg / mL of CAIX to the experimental group. Incubate in a 37°C, 5% CO2 incubator for 3 d. Pour out the cells and medium in the wells, add 200 μL / well of pre-cooled deionized water, and place at 4°C for 10 min; flick off the liquid in the wells and wash the plate 6 times, blotting dry on absorbent paper each time; add 100 μL / well of the diluted biotin-labeled IFN-γ detection antibody working solution and incubate at 37°C for 1 h; wash the plate 6 times; add the diluted streptavidin-HRP enzyme-labeled avidin working solution and incubate at 37°C for 1 h; wash the plate 5 times; discard and blot dry the liquid in the wells; add the AEC substrate chromogenic solution and let it stand at room temperature in the dark for 30 min; pour out the liquid in the wells, uncover the base, and rinse with tap water to terminate the reaction; after drying at room temperature in a cool place, count and read the plate with an immunoblot imaging analyzer within one week.

[0051] As Figure 5 shown, H1-pIL-24 / pCAIX immunization can effectively activate the Th1 cell immune response in mice and promote the expression of IFN-γ.

[0052] 5. Detection of the maturity of DC cells in the spleen of immunized mice Detection of the expression of CD11c+ in spleen cells of mice in each group by flow cytometry: Take the spleen of mice, wash it with physiological saline, and obtain a single-cell suspension of the spleen after grinding and filtering; add APC-conjugated anti-mouse CD11c antibody; stain at 4°C in the dark for 30 min, wash away the unbound antibody with PBS, and detect the expression of CD11c+ by flow cytometry.

[0053] As Figure 6As shown, the CD11c+ expression of spleen DC cells in the H1-pIL-24 / pCAIX immunized group of mice was significantly higher than that of other control groups.

[0054] 6. Detection of central memory T cells in spleen lymphocytes by flow cytometry Detection of the expression of CD44 and CD62L in CD4 and CD8 cells of the spleen of mice in each group by flow cytometry: The spleens of mice were taken, rinsed with physiological saline, ground and filtered to obtain single-cell suspensions of the spleen; AF488-conjugated anti-mouse CD4, APC-conjugated anti-CD44, PE-Cy7-conjugated anti-mouse CD8, and PE-conjugated anti-mouse CD62L antibodies were added; stained at 4°C in the dark for 30 min, and the unbound antibodies were washed away with PBS, and then detected by flow cytometry.

[0055] As Figure 7 and Figure 8 shown, the expression of central memory T cells in CD4 and CD8 cells of the spleen of mice in the H1-pIL-24 / pCAIX immunized group was significantly higher than that of other control groups.

[0056] In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A H1-pIL-24 / pCAIX nano-vaccine, characterized in that, It is prepared through the following steps: Construct an expression plasmid containing mIL-24 sequence and an expression plasmid containing CAIX sequence; Transform the expression plasmid containing mIL-24 sequence and the expression plasmid containing CAIX sequence into competent cells respectively, culture them, and mix the two resulting recombinant plasmids with the H1 nanocarrier to obtain the H1-pIL-24 / pCAIX nano-vaccine; Among them, mIL-24 The NCBI accession number of mIL-24 is 93672, CAIX The NCBI accession number of CAIX is 768.

2. The H1-pIL-24 / pCAIX nano-vaccine according to claim 1, wherein Construct expression plasmids containing mIL-24 sequences and expression plasmids containing CAIX sequences are obtained by ligating the mIL-24 sequences, CAIX sequences to the pcDNA3.1 expression vector respectively.

3. The H1-pIL-24 / pCAIX nano-vaccine according to claim 1, wherein The competent cells are DH5α Escherichia coli competent cells.

4. The H1-pIL-24 / pCAIX nano-vaccine according to claim 1, wherein The mixing mass ratio of the two recombinant plasmids to the H1 nanocarrier is 1:1:3 - 5.

5. The H1-pIL-24 / pCAIX nano-vaccine according to claim 1, characterized in that, The mixing is to dissolve the two recombinant plasmids and the H1 nanocarrier in PBS solution respectively, and then mix them after standing.

6. Use of the H1-pIL-24 / pCAIX nanovaccine according to claim 1 in the preparation of an anti-tumor drug.

7. The application according to claim 6, characterized in that, The anti-tumor effect is against renal cancer.

8. An anti-tumor drug, characterized in that, The drug uses the H1-pIL-24 / pCAIX nanovaccine according to claim 1 as the only active ingredient.

9. The anti-tumor drug according to claim 8, wherein The drug is compounded from the H1-pIL-24 / pCAIX nanovaccine according to claim 1 and pharmaceutically acceptable excipients.

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