Preparation method and application of tumor neoantigen DC vaccine based on cytoskeleton

Abstract

The invention relates to a tumor neoantigen DC vaccine based on a cytoskeleton as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The neoantigen DC vaccine comprises a cell scaffold which is formed by a polymer material and has a porous spongy structure, bone marrow-derived dendritic cells BMCD and tumor neoantigen which are dispersed in the cell scaffold, and an active substance which is cross-linked with the cell scaffold and is used for promoting proliferation and differentiation of T cells or B, the active substance comprises CCL21, and the tumor neoantigen is derived from liver cancer cells. According to the novel antigen DC vaccine based on the cytoskeleton, recruitment of T cells is realized in vivo, interaction of DC-T is promoted, and novel antigen specific T cells are efficiently induced and activated; moreover, the cell scaffold can simulate an immune microenvironment of lymph nodes through in-vivo implantation, so that an anti-tumor effect based on neoantigen DC vaccine excitation is realized.

Description

Technical Field

[0001] The present invention relates to the preparation of a cell culture scaffold, which can be used to deliver and activate tumor neoantigen DC vaccines. After being implanted into subcutaneous tissue, it can recruit T cells in vivo, promote the interaction between DC-T, and efficiently induce and activate neoantigen-specific T cells to exert anti-tumor effects. Background Art

[0002] Primary liver cancer is the third leading cause of cancer-related deaths worldwide, of which hepatocellular carcinoma (HCC) accounts for more than 80%. Although screening high-risk populations for liver cancer can increase the chances of early diagnosis of liver cancer, liver cancer is insidious and progresses rapidly, and most patients are already in the advanced stage when they are first diagnosed. For patients with advanced liver cancer, systemic treatment is currently the standard treatment option, and immunotherapy-based combination therapy has become the preferred option for liver cancer. The occurrence and development of liver cancer are closely related to the host's immune imbalance and suppression. Its tumor immune microenvironment is dominated by immunosuppressive cells and related signaling pathways, including regulatory T cells (Treg), tissue-resident macrophages, and monocyte-derived tumor-associated macrophages, which jointly participate in the host's anti-tumor immune disorder. Therefore, the complex immune microenvironment of liver cancer determines that immunotherapy with only a single mechanism, such as programmed death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors, cannot achieve ideal tumor control. Taking the current first-line clinical treatment regimen of atezolizumab combined with bevacizumab as an example, 72.7% of liver cancer patients treated with this regimen still do not respond to immunotherapy. Further exploratory studies on resistance mechanisms have shown that the lack of tumor-specific lymphocyte infiltration is closely related to treatment non-response. Therefore, exploring new immunotherapy combination strategies to further increase the infiltration of liver cancer-specific immune functional cells is of great significance for improving patients' long-term prognosis.

[0003] The occurrence of cancer is a complex process driven by genetic mutations, including DNA mutations, chromosomal translocations, gene fusions and deletions. Mutations in cancer cell DNA may lead to the production of mutated or completely new proteins, which may be recognized by the immune system as foreign substances, thereby stimulating an immune response. Neoantigens are tumor-specific antigens, which are peptides or proteins that are produced by tumor cells due to genetic mutations and exist specifically in tumor cells. The neoantigen epitopes carried on them can be recognized by the immune system, thereby accurately attacking tumor cells. In addition, due to its specific expression based on tumor cells, it can promote specific lymphocyte infiltration in the tumor microenvironment, induce targeted tumor precision treatment, and minimize the "off-target" effect. Therefore, compared with traditional tumor-associated antigens (TAA), immunotherapy targeting neoantigens has the advantages of strong specificity, low toxicity and side effects, low probability of immune tolerance, and personalized precision treatment.

[0004] However, neoantigen peptide vaccines have inherent limitations. First, peptide vaccines have low molecular weight, short half-life, and are easily degraded. Second, neoantigen peptide vaccines are limited by their low immunogenicity and major histocompatibility complex (MHC) restrictions, resulting in the anti-tumor immunity they induce not being sufficient to completely inhibit tumor recurrence or progression. Therefore, how to further improve the anti-tumor immunogenicity induced by neoantigen vaccines is a key strategy to improve neoantigen immunotherapy.

[0005] In order to obtain excellent anti-tumor response, in addition to using new antigens to solve the problem of therapeutic targets, attention should also be paid to reversing the immunosuppression in the patient's body, so as to ensure that the new antigens can be effectively absorbed and presented by the body's dendritic cells (DC). DC is the most powerful antigen-presenting cell in the human immune system. It plays a key role not only in activating innate immunity, but also in inducing adaptive immunity mediated by cytotoxic T lymphocytes (CTL). DC can package, deliver, process and present tumor antigens through MHC-I molecules to activate CTLs. There are obvious DC maturation disorders and functional decline in the tumor microenvironment, which provide a theoretical basis for DC-based immunotherapy. The main characteristics of tumor TIME (Tumor Immune Microenvironment) are that the complex immunosuppressive network can actively inhibit DC differentiation, migration, antigen presentation and activation ability, leading to DC dysfunction and ultimately immune escape. DC vaccines can increase tumor-specific antigen presentation, activate antigen-specific T cells and accelerate T cell homing to induce long-term immunity. Currently, DC vaccines have shown great potential in many clinical trials.

[0006] Neoantigen DC vaccines have specific and efficient anti-tumor immune potential, but how to fully exert the anti-tumor ability of neoantigen DC vaccines in vivo also depends on the lymph node migration ability of DC vaccines. It is generally believed that DC must eventually migrate to or enter the T cell-dependent area of ​​secondary lymphoid organs to effectively activate T cells. The migration of DC to lymph nodes after maturation is crucial for inducing immune responses. Studies have shown that increased migration of DC vaccines is associated with improved overall survival of patients. At present, there is no broad consensus on the best route of administration for DC vaccines. The delivery routes of DC vaccines mainly include: intravenous, subcutaneous, intradermal, lymph node and intratumoral administration. Direct intralymph node administration of DC vaccines may be the best route of administration because active migration of DC is not required. Intralymph node injection ensures that a sufficient number of DCs are directly delivered to the T cell sensitization site and secrete sufficient amounts of interleukin 12 (IL-12) to promote the antigen presentation process. Mouse models have shown that tumor lysate-pulsed DC vaccines can enhance T cell activation and improve anti-tumor responses when injected intralymphically. However, the lymph node administration route has high technical requirements and can easily lead to physical damage to the lymph node structure. Therefore, it is not very feasible in clinical practice and has poor applicability. In addition, when DC vaccines are delivered through conventional intradermal, subcutaneous, and intravenous injection routes, DCs have weak active migration capabilities, which is manifested by the fact that the vast majority (>90%) of DCs quickly lose function and die, and only a few DCs successfully home to the draining lymph nodes to perform antigen presentation functions. A study showed that a large number of DC vaccines loaded with melanoma peptides injected intradermally were retained at the injection site, lost vitality, and were cleared within 48 hours, with only 5% reaching the draining lymph nodes. Therefore, how to increase the DC lymph node homing ability and improve the anti-tumor efficacy of DC vaccines remains a pain point that needs to be solved. Summary of the invention

[0007] Based on the above research, the present invention optimizes the anti-tumor efficacy of DC vaccines through the following scheme.

[0008] First, the present invention constructs a cell scaffold with cell loading and T cell chemotaxis functions. The cell scaffold is prepared by functionally modifying alginate with anti-TIM3 antibody (α-TIM3), CCL21, IL-2, IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). α-TIM3 can enhance the activity of DC and improve its antigen presentation ability by blocking the TIM3 pathway, and maintain DC-activated T cells in a low exhaustion state, thereby enhancing their ability to kill tumor cells. CCL21 is an important chemokine that recruits naive T cells into the cell scaffold, which helps the cell scaffold mimic the lymph node environment. IL-4 and GM-CSF help maintain the differentiation and activation of DC in the cell scaffold, and IL-2 helps maintain the growth and proliferation of T cells that chemotaxis enters the cell scaffold.

[0009] Furthermore, a cell scaffold loaded with DC and an adenovirus containing a nucleotide sequence for co-expressing CD40L protein and one or more polypeptides as active ingredients is implanted subcutaneously into the tumor patient, aiming to achieve spontaneous T cell recruitment and promote co-localization of DC and T cells, thereby improving the efficiency of DC antigen presentation and enhancing the anti-liver cancer therapeutic effect of the new antigen DC.

[0010] One or more polypeptides as active ingredients are screened according to patent CN114601923A. A sequencing library is constructed using DNA and RNA from tumor tissue and somatic cells of the tumor patient himself, and sequenced to screen out tumor neoantigen polypeptides. Then, the nucleotide sequences of one or more polypeptides as active ingredients are synthesized.

[0011] The purpose of the present invention is to design a multifunctional cell scaffold based on alginate with the function of loading DC cells and T cell chemotaxis. The cell scaffold can provide a three-dimensional microstructure when implanted in the body, recruit T cells and promote the co-localization and interaction of DC and T cells in the scaffold, thereby overcoming the shortcomings of limited antigen presentation and poor clinical efficacy caused by the low homing ability of DC vaccines in traditional DC vaccine treatment pathways.

[0012] The technical solution adopted by the present invention is as follows:

[0013] First, α-TIM3 was coupled with hyaluronic acid (HA) and then added to alginate hydrogel together with CCL21, IL-2, IL-4 and GM-CSF. A cross-linking reaction occurred under the action of calcium ions, and a cell scaffold with a porous sponge-like structure was formed after casting and vacuum freeze-drying.

[0014] The HA is a natural polysaccharide in the extracellular matrix with high biocompatibility and good safety. Coupling α-TIM3 with HA helps to reduce the dispersion and degradation of α-TIM3 and maintain the local concentration of α-TIM3 in the cell scaffold, thereby ensuring that α-TIM3 in the cell scaffold can interact more effectively with DC and T cells.

[0015] The alginate hydrogel is a natural biopolymer from brown algae with good biocompatibility. The porous scaffold structure formed after cross-linking is conducive to imitating the extracellular matrix and supporting cell adhesion, proliferation and differentiation.

[0016] The cell scaffold is cross-linked with CCL21, IL-2, IL-4 and GM-CSF, wherein CCL21 is an important chemokine that recruits naive T cells into the cell scaffold, and helps the cell scaffold to mimic the lymph node environment. IL-4 and GM-CSF help maintain the differentiation and activation of DCs in the cell scaffold, and IL-2 helps maintain the growth and proliferation of T cells that chemotactically enter the cell scaffold.

[0017] Specifically, the cell scaffold is constructed by the following method:

[0018] (1) Hyaluronic acid-conjugated anti-TIM-3 antibody:

[0019] 1) ICG (Indocyanine Green) labeled anti-TIM3 antibody: 5 μL ICG-NHS solution (10 mg / ml) was mixed with 2 mg anti-TIM-3 antibody, fixed to 2 mL with PBS, and shaken at 4°C overnight.

[0020] 2) The mixed solution was added to an ultrafiltration tube (molecular weight cutoff 10 kDa), and ultrafiltration was performed at 4°C and 14000 rpm for 30 minutes, and repeated twice to remove unreacted ICG and other small molecule impurities.

[0021] 3) Use the fluorescence spectrophotometer to measure the characteristic absorption peak of ICG at 800-840nm and draw a fluorescence standard curve.

[0022] 4) Mix 1 ml of 1% hyaluronic acid solution with ICG-anti-TIM3 antibody (1 mg / mL), add 10 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 10 mg NHS (N-hydroxysuccinimide), incubate overnight at 4°C, and control the pH at 6.5;

[0023] 5) Transfer the coupling reaction mixture to a dialysis bag with a molecular weight cutoff of 8000Da and dialyze at 4°C, changing deionized water every 2-3 hours for 1 day to remove unreacted impurities such as EDC and NHS;

[0024] 6) The characteristic absorption peak of the coupling mixture ICG at 800-840 nm was detected by fluorescence spectrophotometer, and the coupling rate was calculated according to the fluorescence standard curve.

[0025] (2) Cell scaffold construction:

[0026] 1) Solution preparation: Prepare a 2% (w / v) alginate solution, add 2 ng GM-CSF, 1 ng IL-4, 20 ng IL-2, 70 ng CCL21 and 2.6 μg hyaluronic acid-conjugated anti-TIM3 antibody per mg alginate, and stir for 5 minutes to ensure sufficient mixing;

[0027] 2) Hydrogel: The alginate solution was mixed with an equal volume of 0.4% calcium gluconate solution and stirred vigorously for 15 minutes to form a hydrogel;

[0028] 3) Casting: Add the hydrogel dropwise into a 24-well plate and a 48-well plate for casting, 1 mL per well of the 24-well plate and 300 μL per well of the 48-well plate;

[0029] 4) Freeze drying: The hydrogels cast in 24-well plates and 48-well plates were placed in a -20°C refrigerator overnight, and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain cell scaffolds of corresponding sizes. The cell scaffold materials were routinely stored in a 4°C refrigerator.

[0030] (3) Preparation of new antigen DC vaccine in cell scaffold:

[0031] Flow cytometry was used to analyze the expression level of BMDC CD40L and evaluate the efficiency of Adv-CD40L-Neo infection of BMDC in the cell scaffold. The specific steps are as follows: Cell scaffold group: 3×10 5 BMDCs were mixed with Adv-CD40L-Neo (MOI = 400) adenovirus solution, and the volume was adjusted to 200 μL with complete culture medium and dripped into the cell scaffold; traditional infection group: 3×10 5 BMDCs were mixed with Adv-CD40L-Neo adenovirus solution (MOI = 400) and added to 24-well plates for infection; 1 mL of RPMI-1640 complete medium containing GM-CSF and IL-4 was added to the cell scaffold and 24-well plates. BMDCs from each group were collected 48 hours after infection, and CD11c in BMDCs was analyzed by flow cytometry. + Cell CD40L expression.

[0032] The present invention adopts a multifunctional cell scaffold with good biocompatibility, aiming to optimize the treatment path of DC vaccine to improve the immune response of DC vaccine. In the design of the cell scaffold, we selected alginate, which has been widely used in clinical practice, as the basic skeleton material. The selection of alginate is mainly based on the following reasons: 1. The gelation conditions of alginate are mild, and the pore size and mechanical strength are adjustable, which is conducive to supporting cell adhesion, proliferation and differentiation; 2. Alginate helps to encapsulate bioactive molecules, including cytokines and chemokines, to ensure their protective delivery and sustained release at the target site; 3. Alginate has good biocompatibility and good clinical transformation potential. Electron microscopy characterization shows that the cell scaffold has a good three-dimensional pore structure, which provides an entry and exit channel for DC, T cell migration and nutrient exchange.

[0033] The cell scaffold optimizes the DC vaccine treatment pathway mainly through its spontaneous recruitment of T cells. CCL21 plays an important role in the lymph node co-localization of naive T cells and DC cells presenting antigens, mainly through the chemokine receptor 7 (CCR7) on the surface of naive T cells or DC cells. In view of the role of CCL21 in coordinating the spatial co-localization of mature DC and naive T cells, we added CCL21 to the cell scaffold. Alginate helps to encapsulate and protect CCL21, avoiding the rapid degradation of CCL21 in the body, thereby achieving the protective delivery and sustained release of CCL21, which provides a good basis for the recruitment of T cells by the cell scaffold and can simulate the lymph node environment to achieve T cell recruitment.

[0034] The present invention prepares Adv-CD40L-Neo adenovirus carrying CD40L expression function and neoantigen expression function, and proves that the cell scaffold can improve the efficiency of Adv-CD40L-Neo infection and activation of DC, and effectively prepares neoantigen DC vaccine.

[0035] The present invention evaluates the in vivo anti-tumor efficacy of cell scaffolds loaded with DC and Adv-CD40L-Neo through the Hepa 1-6 liver tumor in situ model, and also compares the anti-tumor efficacy of the back and axillary implantation routes, indicating that the implantation site does not affect the therapeutic effect of the cell scaffold.

[0036] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0037] 1. The cell scaffold designed by the present invention can be used for the preparation of new antigen DC vaccines, and at the same time realize the recruitment of T cells in vivo, promote DC-T interaction, and efficiently induce and activate new antigen-specific T cells;

[0038] 2. The cell scaffold prepared by the present invention can be implanted in vivo to simulate the immune microenvironment of lymph nodes and achieve anti-tumor effects stimulated by new antigen DC vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 To prepare the method and material characterization of alginate cell scaffolds; Figure 1 A is the construction of cell scaffold by coupling α-TIM3 with hyaluronic acid and adding chemokines; Figure 1 B is alginate hydrogel and cell scaffold formed after freeze-drying; Figure 1 C is an electron microscope photograph of the cell scaffold; Figure 1 D is the image of DC2.4 cells cultured in the cell scaffold for 12 hours.

[0040] Figure 2 The efficiency of adenovirus co-expressing CD40L and neoantigens in infecting DCs and activating T cells; Figure 2 (AB) Flow cytometry analysis of the CD40L expression efficiency of BMDCs in each group 48 hours after adenovirus infection; Figure 2 (CD) is the flow cytometry analysis of CD8 + CD69 + T cell ratio; Figure 2 E is a confocal microscope showing T cells recruited into the cell scaffold in the T cell chemotaxis experiment; Figure 2 F is the cumulative release curve of CCL21 from the cell scaffold; Figure 2 G is the chemotaxis index of cytoscaffold-attracted T cells; Figure 2 (HI) is a comparison of the efficiency of chemotactic activated T cells killing Hepa1-6 cells in each experimental group.

[0041] Figure 3 Flow cytometry was used to evaluate the effects of CCL21 and α-TIM3 in the cell scaffolds on T cell recruitment and activation after subcutaneous implantation of the cell scaffolds. Figure 3 (AB) CD8 cell scaffold recruitment + T cell ratio; Figure 3 (CD) is CD8 in the cell scaffold + CD69 + T cell ratio; Figure 3 (EF) shows the activation of the neoantigen Ptpn2-specific CD8 induced by cell scaffold implantation + The proportion of T cells in the blood.

[0042] Figure 4 The anti-tumor efficacy of the cell scaffold in the Hepa1-6 orthotopic tumor model of C57BL / 6 mice; Figure 4A is a schematic diagram of the experimental timeline, showing the therapeutic interventions at designated time points after tumor inoculation; Figure 4 (BC) shows the changes in tumor size of mice in each treatment group within 60 days after treatment; Figure 4 D is the Kaplan-Meier survival curve for all treatment groups; Figure 4 E is the display of liver tumors and HE sections of each group after treatment; Figure 4 F is the expression of DC costimulatory molecules CD80 and CD86 in each treatment group analyzed by flow cytometry; Figure 4 G: Flow cytometry analysis of the MHC-I expression levels of DCs in each treatment group.

[0043] Figure 5 Flow cytometry was used to analyze T cell infiltrating subsets in tumor tissues; Figure 5 A is the CD3 + CD4 + T cell infiltration; Figure 5 B is the CD3 + CD8 + T cell infiltration. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0045] The present invention uses the following experimental materials:

[0046] α-TIM3 refers to anti-TIM3 antibodies. TIM3 is the abbreviation of T cell immunoglobulin mucin 3, which is a protein expressed on the surface of immune cells (such as T cells and dendritic cells) and is involved in regulating immune responses. α-TIM3 antibodies can block its inhibitory signals by binding to TIM3, thereby enhancing the activity of immune cells.

[0047] CCL21 is a chemokine, the full name of which is Chemokine (CC motif) ligand 21. It plays a role in attracting immune cells, especially T cells, to migrate to specific areas during immune response, and helps immune cells to gather in areas of inflammation or infection.

[0048] IL-2, short for interleukin-2, is a cytokine that is mainly produced by activated T cells. IL-2 plays a key role in immune response, promoting the growth, proliferation and survival of T cells and enhancing immune response.

[0049] IL-4, short for Interleukin-4, is also a cytokine that participates in regulating the growth and differentiation of B cells and T cells, and has a regulatory effect on immune responses, especially in promoting Th2 immune responses.

[0050] GM-CSF, short for Granulocyte-macrophage colony-stimulating factor, is a cytokine that stimulates the production of granulocytes and macrophages and is essential for the development and function of the immune system.

[0051] DiI is a commonly used fluorescent dye used to label cell membranes. It is a lipophilic dye with a long lipophilic hydrocarbon chain that can be embedded in the cell membrane and emit strong red fluorescence.

[0052] DC2.4 is a mouse dendritic cell line.

[0053] CD40L, also known as gp39, TNF associated activation protein (TRAP) or T cell-B cell-activating molecule (T-BAM), is a 33kD type II transmembrane glycoprotein with a total of 266 amino acids, including 214 amino acids in the extracellular region, which has a high homology with TNF at the amino acid level. The main function of CD40L is to enhance antigen presentation of T cells by activating DCs.

[0054] pDC315-CMV is a mammalian adenovirus expression vector used to express target genes in mammalian cells.

[0055] T2A self-cleaving peptide is a short peptide sequence derived from Thosea asigna virus, usually composed of 18-22 amino acids. The self-cleavage mechanism of T2A peptide occurs between glycine (G) and proline (P) at its C-terminus. This feature allows the ribosome to skip the peptide bond synthesis between GP when translating to the 2A peptide sequence, resulting in the separation of proteins connected to both sides of the T2A peptide segment. The uniqueness of this type of peptide segment is that they can self-cleave during the translation process, thereby separating the two proteins connected to the T2A peptide segment to form independent protein molecules.

[0056] Escherichia coli Top10 is a commonly used molecular cloning strain. The Top10 strain is suitable for efficient DNA cloning and plasmid amplification, and can ensure the stable inheritance of high-copy plasmids. Top10 competent cells are resistant to streptomycin (StrR) and can be used for cloning and blue-white screening.

[0057] MOI, multiplicity of infection, MOI=400 means that each cell will be infected with 400 virus particles.

[0058] Example 1: Preparation of a cell scaffold with cell loading and T cell chemotaxis functions

[0059] 1. Hyaluronic acid-coupled anti-TIM-3

[0060] (1) ICG (Indocyanine Green) labeling of anti-TIM3 antibody: 5 μL of ICG-NHS (indocyanine green-N-hydroxysuccinimide ester) solution (10 mg / ml) was mixed with 2 mg of anti-TIM-3 antibody, fixed to 2 mL with PBS, and shaken overnight at 4°C to promote the covalent reaction between ICG-NHS and the amino group on the anti-TIM3 antibody.

[0061] (2) The mixed solution was added to an ultrafiltration tube (molecular weight cutoff 10 kDa) and ultrafiltered at 4°C and 14,000 rpm for 30 minutes. This was repeated twice to remove unreacted ICG and other small molecule impurities.

[0062] (3) Use a fluorescence spectrophotometer to measure the characteristic absorption peak of ICG at 800-840 nm and draw a fluorescence standard curve.

[0063] (4) 1 ml of 1% hyaluronic acid (HA) solution was mixed with ICG-anti-TIM3 antibody (1 mg / mL), 10 mg EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 10 mg NHS (N-hydroxysuccinimide, N-hydroxysuccinimide) were added, and the mixture was incubated at 4°C overnight, and pH was controlled to be 6. In this step, hyaluronic acid is a polysaccharide rich in carboxylic acid, and its carboxyl group is activated by EDC / NHS, and then reacts with the amine group of the anti-TIM3 antibody to form an amide bond, thereby coupling the anti-TIM3 antibody to the hyaluronic acid. This coupling can increase the solubility and stability of the anti-TIM3 antibody, and can regulate the biodistribution and half-life of the anti-TIM3 antibody.

[0064] (5) The coupling reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed at 4 °C with deionized water replaced every 2-3 hours for 1 day to remove unreacted EDC, NHS and other impurities;

[0065] (6) The characteristic absorption peak of ICG at 800-840 nm in the dialyzed coupling reaction mixture is detected by a fluorescence spectrophotometer, and the coupling rate is calculated based on the fluorescence standard curve. The coupling rate refers to the proportion of ICG molecules successfully coupled to the antibody.

[0066] 2. Construction of cell scaffold

[0067] (1) Solution preparation: A 2% (w / v) alginate solution was prepared by adding 2 ng GM-CSF, 1 ng IL-4, 20 ng IL-2, 70 ng CCL21 and 2.6 μg hyaluronic acid-conjugated anti-TIM3 antibody (HA-α-TIM3) to each 1 mg alginate solution and stirring for 5 minutes to ensure adequate mixing.

[0068] (2) Hydrogel: The solution prepared in step (1) was mixed with an equal volume of 0.4% (w / v) calcium gluconate solution and stirred vigorously for 15 minutes to form a hydrogel;

[0069] (3) Casting: The hydrogel prepared in step (2) was added dropwise into a 24-well plate and a 48-well plate for casting, with 1 mL added to each well of the 24-well plate and 300 μL added to each well of the 48-well plate;

[0070] (4) Freeze drying: The hydrogels cast in the 24-well plate and 48-well plate were placed in a -20°C refrigerator and then freeze-dried for 48 hours using a vacuum freeze dryer to obtain cell scaffolds of corresponding sizes. Figure 1 B, Electron microscopic characterization of cell scaffolds containing HA-α-TIM3 Figure 1 C. Cell scaffold materials are routinely stored in a 4°C refrigerator.

[0071] (5) Analysis of cell permeability of cell scaffolds: DiI-labeled DC2.4 cells were loaded into the cell scaffolds and cultured for 12 hours. Microscopic and electron microscopic observations at both the planar and stereoscopic levels showed that DC2.4 cells penetrated into the cell scaffolds and maintained intact cell morphology ( Figure 1 D).

[0072] Example 2: Preparation of adenovirus co-expressing CD40L and neoantigens

[0073] 1. Preparation of pDC315-CMV-mCherry plasmid

[0074] PCR was performed on the nucleotide sequence expressing the mCherry protein, and the double restriction site EcoR I / Xho I was added. After double restriction digestion, it was ligated to the pDC315-CMV vector containing the restriction site Bgl II / Xho I using T4 ligase.

[0075] The above ligation products were transformed into competent cells, E. coli Top10, and single clones were picked and sequenced 16 hours after plating. The plasmids were extracted after amplification of E. coli containing the correct plasmids and stored at -20℃ for future use.

[0076] 2. Prepare pDC315-CMV-CD40L-T2A-Neoantigens-EF1α-mCherry (Adv-CD40L-Neo) plasmid to co-express CD40L and neoantigen fusion protein.

[0077] The 7 nucleotide sequences expressing Hepa 1-6 liver cancer cell neoantigens screened in the early stage are published in Example 1 of CN114601923A: a nucleotide sequence for expressing Mpk3_S284F, a nucleotide sequence for expressing Lmf1_F523V, a nucleotide sequence for expressing Samd91_K752M, a nucleotide sequence for expressing Ttaf_C403W, a nucleotide sequence for expressing Dtnb_K40T, a nucleotide sequence for expressing Lbr_A341P, and a nucleotide sequence for expressing Ptpn2_I383T, which are respectively connected to SEQ ID NO: 1 (a nucleotide sequence for expressing CD40L) through SEQ ID NO: 2 (a nucleotide sequence for expressing a T2A self-cleaving peptide). Specifically, the nucleotide sequence expressing CD40L is amplified by PCR to add a Bgl II / EcoR I double restriction site, and the nucleotide sequence expressing Hepa1-6 neoantigens is amplified by PCR to add an EcoR I / Xho I double restriction site, the digested fragments were then connected to the pDC315-CMV vector with Bgl II / Xho I double restriction sites, where a nucleotide sequence expressing T2A self-cleaving peptide was introduced between CD40L and Neoantigens to construct pDC315-CMV-CD40L-T2A-Neoantigens-EF1α-mCherry plasmid, and the above ligation products were transformed into competent cells: Escherichia coli Top10, and single clones were picked and sequenced 16 hours after plating. After amplification of Escherichia coli containing the correct plasmid, the shuttle plasmid was extracted and stored at -20°C for later use.

[0078] When the cell scaffold-based tumor neoantigen DC vaccine of the present invention is used to treat tumors in tumor patients, liver cancer cell neoantigens are screened according to the method disclosed in Example 2 of CN114601923A, and the nucleotide sequence of liver cancer cell neoantigens is synthesized.

[0079] 3. Adenovirus packaging and purification

[0080] (1) The shuttle plasmid and backbone plasmid were co-transfected into the adenovirus packaging cell line HEK-293A using the liposome transfection reagent Lipofectamine 3000. 20 μg of shuttle plasmid (containing the target gene) and backbone plasmid (containing adenovirus packaging protein) were added to a 10 cm culture dish at a ratio of 1:1.

[0081] (2) After 24 hours, the HEK-293A cells were observed using a fluorescence microscope to see whether they carried the mCherry fluorescent protein label on the shuttle plasmid, to evaluate the transfection status and subsequently determine whether the virus was successfully packaged.

[0082] (3) After about 7-15 days, when HEK-293A cells show obvious plaques, collect the cells and supernatant.

[0083] (4) Lyse the cells by repeated freeze-thaw method (liquid nitrogen or -80°C refrigerator can be used), thaw at 37°C for 10 min and shake for 1 min, and repeat freeze-thaw for more than three times to allow the virus to be fully released from the cells into the culture medium.

[0084] (5) Centrifuge at 5000 rpm and 4°C for 10 min, discard the cell debris precipitate, collect the supernatant containing the virus, and obtain the P0 generation adenovirus after passing it through a 0.22 μm filter (operated in a clean bench). Store it at -80°C for later use or directly add it to HEK-293A cells and repeat the amplification three times.

[0085] (6) The collected virus solution was taken out from -80°C, thawed in a 37°C water bath, centrifuged at 7000 g for 10 min at 4°C to precipitate cell debris, and the supernatant containing the virus was collected and filtered through a 0.22 μm filter;

[0086] (7) PEG8000 (polyethylene glycol 8000 is used to precipitate proteins) precipitation: add 50 mL of PEG8000 to every 100 mL of virus supernatant, place on ice for 2 h (the time can be extended appropriately), centrifuge at 7000g for 20 min at 4°C, discard the supernatant, and resuspend the virus precipitate with 10 mL of 1.1 g / mL CsCl solution to prepare for the next step of density gradient centrifugation. The density of CsCl solution is slightly lower than that of virus particles, which helps the virus to form bands in the subsequent density gradient centrifugation.

[0087] (8) CsCl density gradient centrifugation: In a 38 mL centrifuge tube, add 10 mL of 1.4 g / mL CsCl solution, 10 mL of 1.3 g / mL CsCl solution, and 10 mL of virus resuspension solution in sequence. Use 1.1 g / mL CsCl solution to make up to 38 mL; centrifuge at 26,000 rpm for 2 hours at 4°C using a Beckman SW28 rotor. Using the gradient formed by CsCl solutions of different densities, the virus particles form different bands in the gradient according to their density.

[0088] (9) Virus collection: Virus particles were separated by collecting liquids of different density layers. Virus bands with a density between 1.3 g / mL and 1.4 g / mL CdCl were accurately collected from the centrifuge tube using a syringe. The collected viruses were transferred to a dialysis bag (3500 kDa) that had been boiled in 10 mM EDTA for 10 minutes and cooled to room temperature for dialysis.

[0089] (10) Dialysis: Immerse the dialysis bag containing the virus in 1L of dialysis solution and gently stir overnight at 4°C. The dialysis solution needs to be replaced once in the middle to remove CsCl and other small molecules that may affect the stability of the virus. (Dialysis solution configuration: 50g sucrose, 10ml 1M Tris-HCl, PH 8.0, 2mL 1M MgCl to 1L). Collect the virus after 24 hours, divide it into aliquots and store it at -80°C for later use.

[0090] Example 3: Preparation of new antigen DC vaccine

[0091] 1. Culture of BMDC (bone marrow-derived dendritic cells)

[0092] Male C57BL / 6 mice aged 6 to 8 weeks were selected and killed by cervical dislocation. The femur and tibia were separated in an ultra-clean workbench, the surface muscles and ligaments were shaved off, and the bones were cut at both ends. The rinsing fluid was collected after flushing the bone marrow cavity with PBS and filtered through a 40μm cell filter to remove large pieces of tissue. The filtered cell suspension was centrifuged at 800g for 15 minutes, the supernatant was discarded, and the cell pellet was suspended with red blood cell lysis buffer and allowed to stand for 3 minutes to remove red blood cells. Centrifuge at 800g for 5 minutes, remove the supernatant, and then wash with PBS (repeat twice). Resuspend and count the cells at 1×10 6The cells were distributed into 6-well culture plates at a density of 10 cells / well, and 2 mL of complete culture medium (RPMI-1640 culture medium with 10% FBS, 100 IU / mL penicillin-streptomycin mixture, 20 ng / mL GM-CSF and 10 ng / mL IL-4) was added to each well, and cultured in a constant temperature incubator at 37°C and 5% CO2. On the 2nd and 5th days, an equal amount of fresh RPMI-1640 culture medium was replaced to maintain cell growth conditions. Finally, primary BMDCs, i.e., bone marrow-derived dendritic cells, were obtained on the 7th day.

[0093] 2. Analysis of the efficiency of adenovirus infection of BMDC in cell scaffold environment

[0094] Cell scaffold group: 3×10 5 BMDCs were mixed with the Adv-CD40L-Neo (MOI=400) adenovirus solution in Example 2, and the volume was adjusted to 200 μL with complete culture medium and dripped into the cell scaffold;

[0095] Traditional infection group: 3×10 5 BMDCs were mixed with Adv-CD40L-Neo adenovirus solution (MOI=400) and added to 24-well plates for infection.

[0096] The cell scaffold and 24-well plate were supplemented with 1 mL of RPMI-1640 complete medium containing GM-CSF and IL-4. BMDCs from each group were collected 48 hours after infection, and CD11c in BMDCs was analyzed by flow cytometry. + Cells (expressing CD11c + The expression of CD40L in dendritic cells of Figure 2 Compared with the traditional infection group (in plate) and the cell scaffold group (in cell scaffold), the dendritic cells expressing CD11c (i.e., CD11c + The expression level of CD40L is higher in WT cells.

[0097] Flow cytometric analysis of CD8 + T cell CD69 expression, the results are shown in Figure 2 Compared with the traditional infection group (in plate) and the cell scaffold group (in cell scaffold), CD8 + T cells expressed higher levels of CD69.

[0098] Example 4: Cell scaffold chemotaxis activates T cells to kill Hep1-6:

[0099] 1. Verification of the efficiency of cell scaffold chemotaxis of T cells

[0100] First, the release kinetics of CCL21 from the cell scaffold was analyzed. Figure 2 F in the figure. The results showed that the cell scaffold could effectively and continuously release CCL21 under physiological conditions at 37°C.

[0101] The T cell Transwell assay was used to evaluate the T cell recruitment ability of the cell scaffold. The specific steps are as follows:

[0102] (1) Grouping: The experimental group was a fully functional cell scaffold containing CCL21, and the control group was a cell scaffold without CCL21.

[0103] (2) The pore size of the Transwell is 3 μm, and 5×10 5 Mouse primary T cells labeled with DiI dye were placed on the lower layer of the chamber with cell scaffolds for the experimental group and the control group, respectively, and cultured at 37° C. and 5% CO 2 for 4 hours.

[0104] (3) Remove the material and observe the distribution of T cells in the cell scaffold using a confocal microscope. Figure 2 E in;

[0105] (4) Collect and measure the T cells that have migrated into the material and the lower layer of the chamber, and then count the T cells.

[0106] 2. In vitro killing experiment of Hepa1-6 liver cancer cells

[0107] The mice were sensitized 3 times with a new antigen peptide adjuvant vaccine (new antigen peptide: 100 μg of each new antigen peptide per mouse + adjuvant Poly (I:C): 50 μl per mouse) at 4-day intervals (the new antigen peptide was consistent with the new antigen expressed by Adv-CD40L-Neo, and all of them were 7 Hepa1-6 new antigen peptides with strong immunogenicity screened by our team in the early stage). The mice were killed on the 14th day, and the spleen was taken to extract T cells for later use.

[0108] 2×10 6 BMDCs were mixed with Adv-CD40L-Neo (MOI=400) and added to the cell scaffold.

[0109] Specific operation sequence:

[0110] (1) The cell scaffold was placed in a constant temperature incubator at 37°C and 5% CO2 for 24 hours to allow the BMDC and Adv-CD40L-Neo mixture to permeate the cell scaffold.

[0111] (2) 24 hours later, 1×10 6 T cells were co-cultured for 24 h to activate T cells. Figure 2 H,

[0112] (3) 24-well plate pre-seeded with 1×10 5 Hepa 1-6 cells were placed in the lower chamber of the chamber after adhesion based on different cell scaffolds, including the cell scaffold group without using Adv to activate DC and adding CCL21, the cell scaffold group with using Adv to activate DC but not adding CCL21, and the cell scaffold group with Adv to activate DC and adding CCL21.

[0113] Microscopic observation after 48 hours of co-culture confirmed the cell death observed in the study. Figure 2 The H in the cell scaffold proved that the addition of CCL21 to the cell scaffold can effectively attract T cells, and the constructed adenovirus can effectively activate DC cells, thereby enhancing the activation of effector T cells against Hepa 1-6 cells. The supernatant of each group was collected for LDH release detection to evaluate the degree of cell damage. Figure 2 I in.

[0114] Example 5: Cell scaffolds recruit and activate neoantigen-specific T cells in vivo

[0115] 1. The cell scaffold was implanted subcutaneously on the back of mice. After 3 days, the cell scaffold was recovered, the cells in the cell scaffold were lysed and collected, and the chemotaxis of the cell scaffold and the specific activation of T cells by new antigens were analyzed by flow cytometry.

[0116] (1) Experimental groups were divided into groups according to whether the cell scaffolds contained α-TIM3 and CCL21: cell scaffolds lacking CCL21 and α-TIM3 (Group 1), cell scaffolds lacking α-TIM3 (Group 2), and complete cell scaffolds (Group 3).

[0117] (2) Intervention: 2×10 6 BMDCs were mixed with Adv-CD40L-Neo (MOI=400) adenovirus, adjusted to a total volume of 200 μL with RPMI-1640 complete medium, added to each group of cell scaffolds, and placed at 37°C, 5% CO2 for 1 hour to allow the mixture to penetrate completely.

[0118] (3) Mice were anesthetized with 1% sodium pentobarbital, and the cell scaffolds of each group loaded with BMDC and Adv-CD40L-Neo were implanted subcutaneously on the back of the mice.

[0119] (4) After the cell scaffold of step (1) was implanted subcutaneously in mice for 3 days, the mouse was anesthetized and the cell scaffold was removed. The cells in the cell scaffold were obtained by enzymatic hydrolysis, washed with PBS, blocked with 5% BSA, and stained with anti-CD3, anti-CD8, and anti-CD69 flow cytometry antibodies. The proportion of CD8 T cells and the expression of CD69 in CD8 T cells were analyzed by flow cytometry. Figure 3In A to D, compared with the control group and the blank group, the number of CD8 T cells in the complete cell scaffold group (Group 3) is greater, and the expression level of CD69 in these cells is higher.

[0120] (5) At the same time, blood was collected from the mouse's eye sockets and collected in anticoagulant tubes.

[0121] (6) Add 3 ml of Ficoll solution to a 15 ml centrifuge tube. Dilute the blood to 1 ml with PBS and slowly add the upper layer of Ficoll separation solution. Centrifuge for 20 min in a horizontal centrifuge density gradient centrifuge (24°C, 800 g, 1:10).

[0122] (7) Pipette the middle cloud layer cells into a new 15 mL centrifuge tube, add 10 mL PBS, centrifuge at 800 g for 5 minutes, and discard the supernatant.

[0123] (8) Add 1 mL of red blood cell lysis buffer, lyse for 5 min, add 3 mL of PBS to terminate the reaction, centrifuge at 800 g for 5 min, and remove the supernatant;

[0124] (9) The cell pellet was resuspended in 200 μl of 5% BSA solution and blocked for 20 minutes. Then, 1 mL of PBS was added to wash the cells and centrifuged at 800 g for 5 minutes to discard the supernatant.

[0125] (10) The cell pellet was resuspended in a 0.5% BSA solution containing Ptpn2 tetramer flow cytometry antibody, the total volume was adjusted to 100 μL, and incubated in the dark for 30 min;

[0126] (11) Add 1 μL of anti-CD8 flow cytometry antibody and incubate in the dark for 30 min;

[0127] (12) Wash the cells with 1 mL of PBS, discard the supernatant after centrifugation, resuspend the cells in 300 μL of PBS, and analyze the results by flow cytometry. Figure 3 In Figures E to F, compared with the control group and the blank group, the expression level of CD8 in CD8 T cells in the complete cell scaffold group (Group 3) was higher.

[0128] Example 6: Construction of a mouse liver cancer orthotopic tumor model

[0129] 1. Take Hepa1-6-luc cells in logarithmic growth phase, count them, and resuspend them in PBS to 1×10 7 / mL of cell suspension was mixed with matrix gel at a volume ratio of 1:1 and placed on ice for later use; the abdominal hair of C57BL / 6 mice was removed, and the mice were anesthetized with 1% sodium pentobarbital (80 mg / kg). After anesthesia, a 0.5-1 cm incision was made downward from the xiphoid process of the mice to enter the abdominal cavity, and the cell suspension was aspirated with an insulin syringe. 25 μL of tumor cells were slowly injected under the capsule of the left lobe of the liver; after injection, stay for 5 seconds, rotate and withdraw the needle to avoid cell reflux, and white protrusions can be seen in the liver after successful injection. After the injection, the incision was sutured and disinfected.

[0130] 2. The treatment of this experiment is divided into four groups. The specific groups and corresponding treatment plans are shown in Figure 4 A and B in.

[0131] (1) PBS group (Ctrl): On day 0 and day 14 of the experiment, 200 μl of PBS solution was injected subcutaneously near the axillary lymph nodes of mice.

[0132] (2) Blank scaffold group: On day 0 and day 14, the cell scaffolds were subcutaneously implanted into the back of mice. (3) DC vaccine group: First, 2×10 6 BMDCs were infected with Adv-CD40L-Neo adenovirus for 48 hours to prepare DC vaccine. On day 0 and day 14 of the experiment, the neoantigen DC vaccine was injected subcutaneously into the axillary lymph nodes of mice.

[0133] (4) Fully functional cell scaffold group (aLN): 2×10 6 BMDCs were mixed with Adv-CD40L-Neo (MOI = 400) and added to the cell scaffold. The cell scaffold was placed in a constant temperature incubator at 37°C and 5% CO2 for 1 hour to allow the BMDC and Adv-CD40L-Neo mixture to penetrate the cell scaffold. Afterwards, it was implanted subcutaneously into the back of the mouse. This treatment process was repeated twice on day 0 and day 14, respectively.

[0134] 3. Observe the changes in the fluorescence value of the orthotopic tumor and the survival of the mice after treatment. After 7 days of in situ implantation of Hepa1-6-luc in mice, they were randomly divided into groups for intervention. The intervention start day was defined as day 0, and then on days 10, 20, 30, 40, 50, and 60, each group of mice was intraperitoneally injected with 100 μL of luciferase catalytic substrate (concentration of 30 mg / mL). 8 minutes after injection, the mice were imaged using an animal in vivo imaging system to observe their bioluminescence. Figure 4 In addition, the survival of the mice was followed up and the Kaplan-Meier survival curve was drawn as shown in Figure 4In D, mice treated with the fully functional cell scaffold group had fewer liver tumor cells and improved survival rates.

[0135] 4. Liver tumor anatomy and HE staining

[0136] (1) After reaching the end point of treatment, mice were killed by cervical dislocation and the liver was completely dissected. Figure 4 E in

[0137] (2) Dewaxing of paraffin sections: Dewaxing with xylene I, xylene II, and xylene III in sequence, 10 minutes each time; soaking with gradient alcohols 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and 55% ethanol, 10 minutes each time; washing with tap water

[0138] (3) Hematoxylin staining: Stain the sections with hematoxylin for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water;

[0139] (4) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 minutes each, and then stained in eosin solution for 5 minutes;

[0140] (5) Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to make them transparent, and then sealed with neutral gum;

[0141] (6) Microscopic examination, image acquisition and analysis see Figure 4 In Figure E, there are significantly fewer tumor cells in the liver tissue of mice implanted with the fully functional cell scaffold group, indicating that the fully functional cell scaffold group has a better therapeutic effect on mouse tumor cells.

[0142] 5. Analysis of lymph node DC maturation and activation: Axillary lymph nodes were separated and washed 3 times with PBS; placed in a filter, which was placed in a 6-well plate filled with PBS, and gently ground to obtain grinding solution; centrifuged at 800g for 5 minutes, discarded the supernatant, and resuspended in PBS to obtain lymph node single cell suspension; blocked with 5% BSA for 15 minutes, washed once with PBS, and analyzed by flow cytometry after staining with anti-CD11, CD80, CD86, and MHC-II flow cytometry antibodies. Figure 4 F~G in.

[0143] 6. Flow cytometry analysis of T cell infiltration in tumor tissue: separate the tumor tissue and cut it into 1-2 mm pieces 3After the fragments were removed, digestive enzymes (1 mg / mL type IV collagenase, 0.2 mg / mL hyaluronidase, 0.0.2 mg / mL deoxyribonuclease I) were added and digested at 37°C for 2 hours. The cells were collected by centrifugation at 800g for 5 minutes, and the large fragments that were not completely digested were removed by a 40μm filter. The cells were collected by centrifugation at 800g for 5 minutes, and the cells in the middle cloud layer were collected by density gradient centrifugation with Ficoll separation solution; after blocking with 5% BSA for 15 minutes, the cells were washed once with PBS, and the cells were analyzed by flow cytometry after staining with anti-CD3, CD4, and CD8 flow cytometry antibodies. Figure 5 A~B in.

Claims

1. A tumor neoantigen DC vaccine based on a cell scaffold, characterized in that: The vaccine includes a cell scaffold with a porous sponge-like structure formed by a polymer material, bone marrow-derived dendritic cells BMCD dispersed in the cell scaffold, an adenovirus containing a nucleotide sequence that co-expresses CD40L protein and one or more polypeptides as active ingredients, and an immune cell activity regulator that promotes T cell or B proliferation and differentiation and is cross-linked with the cell scaffold; the immune cell activity regulator includes a chemokine CCL21 and an anti-TIM3 antibody, the polypeptide is a mutated neoantigen expressed by the cancer cells of the tumor patient, and each polypeptide can specifically bind to a T cell antigen receptor on the surface of the T cell of the tumor patient.

2. The cell scaffold-based tumor neoantigen DC vaccine according to claim 1, characterized in that: The polymer material for preparing the cell scaffold is selected from one or more of sodium alginate, carboxymethyl chitosan and carboxymethyl cellulose.

3. The cell scaffold-based tumor neoantigen DC vaccine according to claim 1, characterized in that: The active substances also include interleukins IL-2, IL-4 and colony stimulating factor GM-CSF.

4. The cell scaffold-based tumor neoantigen DC vaccine according to claim 1, characterized in that: The polymer material is a hydrogel generated by mixing sodium alginate and hyaluronic acid coupled with anti-TIM3 antibody with a calcium gluconate solution.

5. The cell scaffold-based tumor neoantigen DC vaccine according to claim 4, characterized in that: The anti-TIM3 antibody-coupled hyaluronic acid is formed by reacting a hyaluronic acid solution activated by EDC and NHS with the anti-TIM3 antibody.

6. The cell scaffold-based tumor neoantigen DC vaccine according to claim 1, characterized in that: The nucleotide sequence includes a nucleotide sequence 1 expressing CD40L protein as shown in SEQ ID No.01, a nucleotide sequence 2 expressing one or more polypeptides, and a nucleotide sequence 3 expressing a T2A self-cleaving peptide as shown in SEQ ID No.02, and the nucleotide sequence 3 connects the nucleotide sequence 1 and the nucleotide sequence 2.

7. The method for preparing a tumor neoantigen DC vaccine based on a cell scaffold according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, mixing the hyaluronic acid solution with the anti-TIM3 antibody to obtain hyaluronic acid coupled to the anti-TIM3 antibody; S2, preparing a cell scaffold, adding 2ng GM-CSF, 1ng IL-4, 20ng IL-2, 70ng CCL21 and 2.6μg hyaluronic acid coupled to anti-TIM3 antibody to an alginate solution at a ratio of 1mg alginate, mixing and stirring thoroughly, and then reacting with a solution containing calcium ions to form a cell scaffold with a porous sponge-like structure after casting and vacuum freeze-drying; S3. Prepare a neoantigen DC vaccine. Prepare an adenovirus containing a nucleotide sequence that co-expresses CD40L protein and one or more polypeptides as active ingredients. Mix the adenovirus venom with bone marrow-derived dendritic cells (BMCD). Adjust the volume with complete culture medium and then drip into the cell scaffold prepared in step S2 to obtain a tumor neoantigen DC vaccine.

8. The preparation method according to claim 7, characterized in that: The specific steps of step S1 are as follows: mixing 1% hyaluronic acid solution with 1 mg / mL anti-TIM3 antibody, adding equal amounts of EDC and NHS, incubating overnight at 4°C, controlling pH=6, and then dialyzing at low temperature to remove impurities.

9. The preparation method according to claim 7, characterized in that: In step S2, the added amounts of GM-CSF, IL-4, IL-2, and CCL21 are 2 ng, 1 ng, 20 ng, and 70 ng, the added amount of the hyaluronic acid-coupled anti-TIM-3 antibody mixture is 2.6 μg, and the concentration of the alginate solution is 2% (w / v).

10. Use of the cell scaffold-based tumor neoantigen DC vaccine according to any one of claims 1 to 3 in the preparation of liver cancer drugs, wherein the tumor is a tumor of the tumor patient.

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