WO2023010784A1

WO2023010784A1 - 3d-printed tumor vaccine composition, preparation method therefor, and application thereof

Info

Publication number: WO2023010784A1
Application number: PCT/CN2021/141918
Authority: WO
Inventors: 汪超; 张越
Original assignee: 苏州大学
Priority date: 2021-08-03
Filing date: 2021-12-28
Publication date: 2023-02-09
Also published as: CN113521263A

Abstract

Disclosed in the present invention are a 3D-printed tumor vaccine composition, a preparation method therefor, and an application thereof. According to the tumor vaccine composition of the present invention, a vaccine preparation containing a tumor antigen and a biosafety macromolecular material are prepared into 3D printing ink, and the tumor vaccine composition of a porous structure is prepared by means of 3D printing, wherein the porous structure is that a plurality of pores having the pore diameter of 1-10 μm are formed in the surface and the interior of the tumor vaccine composition.

Description

A 3D printed tumor vaccine composition and its preparation method and application

technical field

The invention relates to a 3D printed tumor vaccine composition and its preparation method and application, belonging to the technical field of biomedical materials.

Background technique

As an emerging additive manufacturing technology, 3D printing technology has a wide range of application prospects due to its advantages of rapid manufacturing, low cost, and customization. Compared with the traditional manufacturing process, 3D printing technology calculates through digital models, and uses some adhesive materials to print layer by layer to build objects, which can achieve high-precision manufacturing and effective use of raw materials. In addition, its biggest advantage is that it can Realize high-volume, high-quality rapid manufacturing. 3D printing technology is currently widely used in medical care, cultural education, aerospace and other fields, and is a popular and widely used intelligent rapid prototyping technology. Since 3D printing technology can achieve a high degree of bionic manufacturing, it has broad research and application prospects in the field of clinical personalized customization.

Cancer, as a great problem facing mankind in the 21st century, is seriously threatening human health. As a widely used cancer treatment, immunotherapy aims to suppress and kill tumor cells by stimulating and enhancing the body's immune function, so as to inhibit tumor growth or eliminate tumors. Current immunotherapy includes tumor vaccines, immune checkpoint inhibition and T cell adoptive therapy, which is becoming one of the mainstream therapies for tumor treatment. Tumor vaccines generate sustained anti-tumor immune recognition and stimulate specific immunity by providing multiple tumor antigens. However, research on cancer vaccines is still in its infancy, and most cancer vaccines have poor clinical efficacy. This is mainly due to a lower level of uptake and recognition of tumor antigens by the body and a lower degree of activation of antigen-presenting cells. Based on this, researchers have researched and developed a series of new tumor vaccines, such as the efficient delivery of tumor antigens through nanoparticles and the delivery of antigens through gel carriers. Although these technologies promote the recognition and uptake of antigens by immune cells to a certain extent, they are often accompanied by strong toxic side effects. In addition, due to its own limitations, hydrogel-based tumor vaccines are difficult to manufacture and store stably in batches.

Contents of the invention

In order to solve the above technical problems, the present invention provides a 3D printed tumor vaccine composition to construct an implantable tumor vaccine with a specific structure and realize rapid and stable mass production.

The first object of the present invention is to provide a 3D printed tumor vaccine composition. The tumor vaccine composition is prepared from tumor antigens or vaccine preparations and biosafety macromolecular materials into 3D printing inks. Through 3D printing, A tumor vaccine composition with a porous structure is prepared; wherein, the porous structure is provided with a plurality of pores with a diameter of 1-10 μm on the surface and inside of the tumor vaccine composition.

Further, the biosafety macromolecular material is gelatin, sodium alginate, agar, polylactic acid, polycaprolactone, cellulose, silk fibroin, methacrylic anhydride gelatin, polyethylene glycol diacrylate , polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylamide and its derivatives, hyaluronic acid, carrageenan, etc. one or more.

Further, the tumor antigens are antigenic substances that can be recognized and taken up by antigen-presenting cells, newly appear or overexpressed during tumorigenesis and development, including proteins, polypeptides, nucleic acids, such as carcinoembryonic antigen (CEA), sugar One or more of base antigen, alpha-fetoprotein (AFP), glycoprotein antigen (CA50), cell antigen, tumor lysate, etc.

Further, the plurality of pore arrays with a pore diameter of 1-10 μm are arranged on the surface and inside of the tumor vaccine composition.

The second object of the present invention is to provide a method for preparing the tumor vaccine composition, comprising the following steps:

S1. Fully dissolving the biosafety macromolecular material to obtain a biosafety macromolecular material solution;

S2. Prepare the tumor antigen or vaccine preparation into a solution, and filter and sterilize to obtain the tumor antigen or vaccine preparation solution;

S3. Mix the biosafety macromolecular material solution with the tumor antigen or vaccine preparation solution, and remove the air bubbles in the mixed solution to obtain 3D printing ink;

S4. Set the printing parameters, construct the printing model, and then prepare the tumor vaccine scaffold by printing with a 3D printer;

S5. After the printing is completed, the tumor vaccine scaffold is cured and cross-linked to obtain the tumor vaccine composition.

Further, the mass ratio of the biosafety macromolecular material to the tumor antigen or vaccine preparation is 1000:1-200:1.

Further, in the step S3, removing air bubbles in the mixed solution is to ultrasonically treat the mixed solution for more than 3 minutes, and then centrifuge at 1000-3000 rpm.

Further, in step S4, the printing parameters are set as follows: the printing temperature is 20-30° C., the printing speed is 4-20 mm/s, and the layer height is 0.2-04 mm.

The third object of the present invention is to provide the application of said tumor vaccine composition in the preparation of drugs for anti-tumor immunotherapy and prevention.

The present invention uses tumor antigens mixed with biosafety macromolecular materials to construct printable bio-inks, batch, stable, and customizable manufacturing of tumor vaccine inks, and is used for 3D printing of tumor vaccine scaffolds. Through 3D printing technology, implantable tumor vaccines can be constructed to promote the recognition and uptake of antigens by immune cells, induce robust immune responses, and achieve effective anti-tumor immunotherapy and prevention. At the same time, tumor vaccines based on 3D printing technology can also be combined with existing tumor immune preparations to construct a multifunctional immune activation microenvironment to achieve efficient immune system activation.

In biology, a key idea is that structure determines function. We used 3D printing technology to construct an "artificial tertiary lymphatic structure (aTLS)" that can mimic the lymphatic structure in vivo. In our study, the printed scaffolds with porous structures performed similar functions to real lymphoid organs. For example, the tumor vaccine composite 3D scaffold itself can also attract a large number of innate immune cells, including T cells, B cells, macrophages, dendritic cells (DC) and natural killer cells (NK), thereby forming an immune microenvironment, Used to enhance the efficacy of cancer vaccines. The 3D printed scaffolds established a microenvironment conducive to immune cell infiltration. Such precise porous structures are difficult to achieve through conventional hydrogel or chemical engineering methods. In addition, 3D printing technology enables rapid fabrication, high plasticity for personalized medicine, and relatively low cost, which provides new opportunities for the manufacture of vaccine scaffolds.

The beneficial effects of the present invention are:

1. The vaccine carrier of the present invention is based on a biosafety polymer material, has good biosafety, and has few side effects;

2. Compared with the current stage tumor vaccines, the tumor vaccine of the present invention is more convenient to manufacture and store. Compared with the adjuvants of traditional vaccines, the potential adjuvant effect of its biosafety polymer materials has lower Side effects, can effectively promote the recognition and uptake of antigens, and induce a strong anti-tumor immune response;

3. The 3D printed tumor vaccine of the present invention combines 3D printing technology with tumor immunotherapy for the first time, and can simulate the lymphoid structure in the body. Printed scaffolds with porous structures function similarly to real lymphoid organs. For example, the 3D scaffolds compounded by tumor vaccines can also attract a large number of innate immune cells. Using the potential advantages of 3D printing technology, it provides the possibility for clinical application transformation.

4. Tumor vaccines based on 3D printing technology can combine the advantages of traditional vaccines and personalized treatment to provide the basis for precision medicine and personalized prevention.

Description of drawings:

Fig. 1 is support model and pore structure of the present invention;

Fig. 2 is the relationship between the rheological properties and the shear frequency of the printing ink of the present invention;

Fig. 3 is the relationship between the rheological properties and the temperature of the printing ink of the present invention;

Fig. 4 is the XRD analysis of the printing material before and after antigen loading of the present invention, wherein A is simple printing ink, and B is printing ink loaded with tumor antigen;

Fig. 5 is the relevant mechanical properties of the printable ink of the present invention;

Fig. 6 is the scanning electron microscope picture of printing scaffold vaccine of the present invention;

Fig. 7 is the proof of the mass production and printing performance of the printed scaffold vaccine of the present invention;

Fig. 8 is the in vivo degradation and related characterization of the 3D printing scaffold vaccine of the present invention;

Fig. 9 is a HE stained section picture of tissue skin near the inoculation area after the 3D printing scaffold vaccine of the present invention is implanted;

Figure 10 shows that the 3D printing scaffold vaccine of the present invention induces the maturation of bone marrow-derived dendritic cells in vitro;

Figure 11 shows that the 3D printing scaffold vaccine of the present invention induces the secretion of cytokines related to bone marrow-derived dendritic cells in vitro;

Figure 12 is a HE stained slice picture of the main organs of the mouse seven days after the 3D printing immunization vaccine of the present invention;

Fig. 13 is the change of relevant blood routine parameters of mice after vaccination with the 3D printing scaffold of the present invention;

Figure 14 is the recruitment of cells in vivo by the 3D printing scaffold vaccine of the present invention;

Figure 15 is the 3D printing scaffold vaccine of the present invention recruiting DC cells in vivo to promote DC cell maturation and antigen presentation;

Figure 16 shows that the 3D printing scaffold vaccine of the present invention recruits macrophages in vivo to promote macrophage differentiation and antigen presentation;

Figure 17 is the tumor growth curve after vaccination with the 3D printed scaffold of the present invention;

Figure 18 is the survival curve and body weight changes of mice vaccinated with 3D printed scaffolds of the present invention;

Figure 19 shows the immune cell infiltration and immune activation of the tumor tissue of mice vaccinated with the 3D printed scaffold of the present invention;

Figure 20 shows the mathematical simulation and bionic manufacturing of the resected tumor through the 3D laser scanning system before the surgical resection of the tumor;

Figure 21 is the tumor growth curve of the 3D printed scaffold vaccine of the present invention inoculated at the resection site after surgical resection;

Figure 22 is the mouse survival curve and mouse body weight diagram of the 3D printed scaffold vaccine of the present invention inoculated at the resection site after surgical resection to prevent tumor metastasis;

Figure 23 shows the secretion of related cytokines in mouse serum after inoculation of the 3D printing scaffold vaccine of the present invention at the resection site after surgical resection;

Fig. 24 is a HE stained slice of a tumor in a mouse vaccinated with a printed scaffold vaccine after surgical resection.

Detailed ways

The present invention will be further described below in conjunction with specific examples, so that those skilled in the art can better understand the present invention and implement it, but the given examples are not intended to limit the present invention.

C57BL/6 female mice aged 6-8 weeks were purchased from Changzhou Cavens Experimental Animal Co., Ltd. All mouse experiments were carried out in accordance with the animal experiment protocol approved by the Experimental Animal Center of Soochow University.

Mouse melanoma B16-OVA tumor cells were purchased from the Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Bone marrow-derived dendritic cells (BMDCs) were extracted from the bone marrow cavity of 7-8-week-old C57BL/6 mice according to established methods. Cells subcultured in the third or fourth generation were used in the embodiments of the present invention.

Example 1: Preparation of 3D scaffold immune vaccine printing ink and construction of scaffold vaccine

Weigh sodium alginate and dissolve it in 10mL deionized water, and fully mix and dissolve under the mixer. After obtaining a uniform sodium alginate solution, weigh a certain amount of gelatin and add it to the sodium alginate solution, and place it in an oil bath at 60° C. for 30 minutes to fully dissolve the gelatin to obtain a printable ink. Weigh the tumor-associated antigen, dissolve it in PBS, add the prepared sterile antigen solution into the printing ink, and mix the antigen solution and the printing ink fully through physical mixing. The prepared tumor vaccine printing ink was sonicated for 1 minute, and then centrifuged at 1500-3000 rpm for 3 minutes to remove air bubbles in the solution, so as to avoid affecting the stability of subsequent printing and reducing the mechanical properties of the material. After removing air bubbles, draw the prepared solution into a sterile syringe and store in the refrigerator until use.

The scaffold vaccine is modeled by slicing software, by constructing a 10*10*4mm hexagonal scaffold vaccine, a 10mm cylindrical scaffold vaccine, and a personalized model prepared by customization. By adjusting the filling rate, the external pore distribution of the scaffold vaccine is adjusted. The higher the filling rate, the smaller the pore interval. Taking a layer height of 0.25 mm as an example, a 16-layer stacked scaffold node structure is constructed by printing and stacking. When the filling rate is 20%, the external scaffold pores are 500 μm, and the internal pore diameter distribution is about 10 μm, as shown in Figure 1. Based on different models, the printing speed is 4-20mm/min, the printing layer height is 0.2-0.4mm, the printing temperature is 20-30 degrees Celsius, the filling rate is 20-100%, and the printing G code is output. A 3D printer is used to construct the scaffold vaccine. After the printing is completed, the scaffold vaccine is placed in a 2% CaCl ₂ solution for 3 minutes to improve the stability and mechanical properties of the scaffold vaccine through calcium ion cross-linking.

Example 2: Relevant characterization of 3D printed scaffold vaccines

First, we verified the rheological properties of tumor vaccines through rheological experiments. The results are shown in Figure 2 and Figure 3. With the change of shear frequency, tumor vaccine printing inks still have good rheological properties. Furthermore, a gel-sol transition occurs with increasing temperature. XRD analysis proved that before and after adding the antigen, the material had an amorphous structure, as shown in FIG. 4 . A mechanical testing system was used to study the mechanical properties of the printing ink before and after adding the antigen. As shown in Figure 5, after adding the antigen, the mechanical properties of the material improved to a certain extent, and its tensile stress, fracture strain and compressive stress All were effectively improved, proving that the antigen can act as a component in the system. The prepared scaffold vaccine was characterized by scanning electron microscopy. As shown in Figure 6, the scaffold structure was complete without obvious nodules. In addition, the scaffold vaccine had a good pore structure with an average pore size of about 5 μm. The printing system and batch Wendy manufacturing were proved. The results are shown in Figure 7. The stent vaccine can be stably manufactured in batches through the 3D printer. In addition, the printing system also has good reducibility and can better manufacture the required model.

Example 3: Biosafety Analysis of 3D Printed Preparation Vaccines

Mice were subcutaneously inoculated with stent vaccines, and the in vivo degradation of the stent vaccines was studied. The results are shown in Figure 8. As the implantation time prolongs, the stent vaccines gradually degrade. On the seventh day, most of the stent vaccines degrade. In addition, the scanning electron microscope pictures also studied the morphology and structure of the scaffold vaccine at different time points, which also proved the degradation of the scaffold vaccine in vivo. At the same time, the skin tissue in the vaccinated area was collected to study the toxic and side effects of the relevant area after vaccination. It can be found through the HE stained section of the skin tissue that there is no obvious damage to the skin in the vaccinated area, which proves that the scaffold vaccine has good Biological safety and low side effects, the experimental results are shown in Figure 9.

Example 4: 3D printed scaffold vaccine induces DC cell maturation and related cytokine secretion in vitro

Extract mouse bone marrow-derived dendritic cells according to existing methods, and when the maturity reaches about 10%, incubate them with LPS, PBS, blank scaffolds, and scaffold vaccines loaded with tumor antigens for 24 hours, and collect the supernatant for storage For subsequent detection of cytokines at -80°C, BMDCs were collected by centrifugation at 1,200 rpm for 3 minutes, and the expression of co-stimulatory factors (CD80, CD86) was analyzed by flow cytometry. The results are shown in Figure 10, the scaffold vaccine can effectively promote the maturation of DC cells, in addition, the blank scaffold can also improve the maturity of DC to a certain extent, which proves the potential adjuvant effect of the blank scaffold. The relevant cytokines secreted by DC cells after stimulation were analyzed by ELISA. The results are shown in Figure 11. After being stimulated by blank scaffolds and scaffold vaccines, BMDCs effectively secreted TNF-α, IL-1β, IL-6 and other cytokines , which provides a basis for the subsequent induction of a strong anti-tumor immune response in vivo.

Example 5: In vivo biosafety and cell recruitment of 3D printed scaffold vaccines

(1) Biosafety in vivo

Mice were inoculated with blank scaffolds, scaffold vaccines, and PBS to study the biosafety of printed scaffold immune vaccines in vivo. On the seventh day after inoculation, the main organs of the mice were collected and analyzed by HE staining section. The results are shown in Figure 12. After the blank scaffold and scaffold vaccine in the section, the major organs of the mice did not appear obvious damage, which confirmed the scaffold Biosafety of vaccines. In addition, the blood of the mice was collected and anticoagulant was added for routine blood tests. The results are shown in Figure 13. Compared with the control group, the main parameters of the blood routine in the experimental treatment group were significantly changed, and they were all within the normal index range. It further proves the safety and rationality of scaffold vaccine application in vivo.

(2) In vivo immune cell recruitment

Blank scaffolds and scaffold immunized vaccines were respectively implanted in mice, and the immune cell recruitment and related immune cell activation of scaffold vaccines at different time points were studied. The results are shown in Figure 14. The scaffold vaccine can effectively recruit T cells, B cells, NK cells, macrophages and DC cells. In addition, compared with the blank scaffold, the immune cell recruitment effect of the scaffold vaccine is also better. Among the recruited immune cells, DC cells and macrophages accounted for the largest proportion, therefore, DC cell activation and macrophage polarization in two different scaffolds were investigated. As shown in Figure 15, the maturity of DC cells in the scaffold vaccine is higher than that of the blank scaffold, indicating that the scaffold vaccine can activate DC cells more effectively after antigen loading, which is consistent with the aforementioned activation of DC cells in vitro. In addition, the antigen presentation level of DC cells in the scaffold vaccine is also more obvious. On the ninth day, the expression level of antigen-presenting molecule MHC II is twice that of the blank scaffold, indicating that the scaffold vaccine can more effectively promote the antigen expression of DC cells. identification and ingestion. The polarization of macrophages also confirmed that the scaffold vaccine had a better immune activation effect than the blank scaffold. In the scaffold vaccine, more macrophages showed M1 type, and the antigen presentation level of macrophages also decreased. Consistent with DC cells, the results are shown in Figure 16. It is further proved that the scaffold vaccine can more effectively promote the recognition and uptake of antigens, and improve the immune activation level of the body.

Example 6: 3D printing scaffold vaccine in vivo tumor prevention and anti-tumor efficacy

The in vivo tumor prevention efficacy of the scaffold vaccine was verified by constructing a prevention model. After the mice were inoculated with the scaffold vaccine, B16-OVA tumor cells were used to verify the preventive efficacy of the scaffold vaccine, and the tumor prevention efficacy was judged by detecting the tumor growth. As shown in Figure 17, the scaffold vaccine can significantly inhibit the growth of tumors, and after combined anti-PD-L1 treatment, synergistic treatment can be achieved, thereby effectively inhibiting the growth of tumors. The survival time of the mice in the scaffold vaccine group and the combined treatment group was also effectively prolonged, indicating that the scaffold vaccine has a good preventive effect. In addition, the body weight of the mice did not change significantly during the experiment, which also indicates that the treatment method has relatively low side effects, and the results are shown in FIG. 18 . Through the analysis of immune cells in tumor tissue, the intrinsic mechanism of scaffold vaccine to inhibit tumor growth was studied. As shown in Figure 19, CD8 ⁺ T cells were significantly increased in the scaffold vaccine treatment group and the combined treatment group. After scaffold vaccine treatment, the ratio of CD8 ⁺ T cells to Treg was also significantly higher than that of the control group. At the same time, the expression levels of Ki67, IFN-γ and Granmezy B also increased significantly, confirming that after scaffold vaccination, Effectively activate the immune response of the system and form a robust anti-tumor immune microenvironment.

Example 7: Surgical resection personalized customized 3D printing scaffold vaccine and tumor prevention

Since 3D printing technology can achieve good biomimicry and manufacturing, based on this, combined with clinical practice, we combined surgical resection with scaffold vaccine to construct a personalized 3D printed scaffold vaccine. First, we used laser scanning 3D modeling technology to mathematically simulate the tumor to be resected in mice to build a personalized tumor vaccine model. The results are shown in Figure 20. 3D printing technology can effectively realize the personalized customization of surgical wounds . After the mouse tumor was surgically removed, the customized scaffold vaccine was implanted in the surgical site for wound filling and secondary immunization. The mice were subsequently studied for tumor migration and recurrence after customized surgical filling and immunization. The results are shown in FIG. 21 , the personalized tumor vaccine can effectively inhibit the growth of the tumor, and at the same time effectively prolong the survival time of the mice. Combining anti-PD-L1 can more effectively inhibit tumor recurrence and migration, and at the same time effectively ensure the survival of mice after surgery. The results are shown in Figure 22. After inoculation of personalized vaccines, mice serum was collected, and the relevant cytokines in mouse serum were studied. The results are shown in Figure 23. In the scaffold vaccine and combined treatment groups, TNF-α and IFN-γ in mouse serum Both were significantly up-regulated, revealing the activation of the intrinsic immune system in mice. The mouse tumors were collected, and analyzed by HE staining sections. It was found that individualized tumor vaccination after surgical resection could effectively inhibit the growth of tumors, and the results are shown in FIG. 24 .

To sum up, the 3D printing-based tumor immune vaccine of the present invention can not only effectively prevent tumors, but also can make full use of the characteristics of 3D printing technology to realize batch stable and customized manufacturing of tumor vaccines. At the same time, tumor antigens can be used as the internal components of the scaffold vaccine to promote the improvement of the mechanical properties of the scaffold vaccine. The potential adjuvant effect of the scaffold component can also effectively recruit immune cells to promote the recognition and uptake of immune cells to antigens, thereby Realize the construction of "artificial tertiary lymph nodes", and then realize effective immune activation and tumor prevention.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims

A 3D printed tumor vaccine composition, characterized in that the tumor vaccine composition is prepared from tumor antigens or vaccine preparations and biosafety macromolecular materials into 3D printing ink, and the porous structure of the tumor is prepared by 3D printing Vaccine composition; wherein, the porous structure is provided with several pores with a diameter of 1-10 μm on the surface and inside of the tumor vaccine composition.
The tumor vaccine composition according to claim 1, wherein the biosafety macromolecular material is gelatin, sodium alginate, agar, polylactic acid, polycaprolactone, cellulose, silk fibroin, formazan One or more of acrylic anhydride-based gelatin, polyethylene glycol diacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylamide and its derivatives, hyaluronic acid, and carrageenan.
The tumor vaccine composition according to claim 1, characterized in that, the tumor antigen is an antigen substance that can be recognized and taken up by antigen-presenting cells, and newly appears or is overexpressed during tumorigenesis and development.
The tumor vaccine composition according to claim 3, wherein the tumor antigen is one or more of carcinoembryonic antigen, glycosyl antigen, alpha-fetoprotein, glycoprotein antigen, cell antigen, tumor lysate kind.
The tumor vaccine composition according to claim 1, wherein the plurality of pore arrays with a pore diameter of 1-10 μm are arranged on the surface and inside of the tumor vaccine composition.
A preparation method of the tumor vaccine composition according to any one of claims 1 to 5, characterized in that it comprises the steps of:

S1. Fully dissolving the biosafety macromolecular material to obtain a biosafety macromolecular material solution;

S2. Prepare the tumor antigen or vaccine preparation into a solution, and filter and sterilize to obtain the tumor antigen or vaccine preparation solution;

S3. Mix the biosafety macromolecular material solution with the tumor antigen or vaccine preparation solution, and remove the air bubbles in the mixed solution to obtain 3D printing ink;

S4. Set the printing parameters, construct the printing model, and then prepare the tumor vaccine scaffold by printing with a 3D printer;

S5. After the printing is completed, the tumor vaccine scaffold is cured and cross-linked to obtain the tumor vaccine composition.
The preparation method according to claim 6, characterized in that the mass ratio of the biosafety macromolecular material to the tumor antigen or vaccine preparation is 1000:1-200:1.
The preparation method according to claim 6, characterized in that, in step S3, removing the air bubbles in the mixed solution is to ultrasonically treat the mixed solution for more than 3 minutes, and then centrifuge at 1000-3000 rpm.
The preparation method according to claim 6, characterized in that, in the step S4, the printing parameters are set as follows: the printing temperature is 20-30°C, the printing speed is 4-20mm/s, and the layer height is 0.2-04mm.
The application of the tumor vaccine composition described in any one of claims 1 to 5 in the preparation of anti-tumor immunotherapy and prevention drugs.