A trichosanthin fusion vaccine, its preparation method and use

Abstract

The present application provides a trichosanthin fusion vaccine, a preparation method and application thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a pollen protein fusion vaccine, its preparation method, and its application. Background Technology

[0002] The latest data from 2021 shows that breast cancer ranks first in new cancer cases and remains the second leading cause of cancer-related deaths. Despite the emergence of new treatments, surgery remains a primary means of cancer treatment. However, surgery may increase the risk of tumor cell spread, triggering tumor invasion and migration, and the body possesses immune escape mechanisms that increase the survival rate of circulating tumor cells. These residual tumor cells or circulating tumor cells may lead to tumor recurrence and metastasis months or years after surgery.

[0003] To prevent postoperative tumor recurrence and metastasis, it is necessary to activate the immune microenvironment to eliminate residual tumor cells. Dendritic cells (DCs) are the primary antigen-presenting cells and are key factors in the initiation, regulation, and maintenance of the tumor immune response. Suppression of dendritic cell function is a significant cause of immune tolerance. Therefore, dendritic cells are an important target for tumor immunotherapy. Furthermore, tumor-associated macrophages (TAMs) are a major immunosuppressive component of the tumor microenvironment.

[0004] Tumor antigens are divided into tumor-specific antigens (TBAAAs) and tumor-associated antigens (TAAs). Different types of tumors express different antigens, and even the same type of tumor may have different antigenic components in different organisms. Legumain, an asparagine endopeptidase, is a TAA that is highly expressed in various tumors, especially malignant tumors, promoting tumor cell proliferation, tumor invasion and metastasis, and angiogenesis. Therefore, selecting Legumain as an antigenic component in DC vaccines can mediate the killing of various tumors. Furthermore, Legumain is also highly expressed in tumor-associated macrophages (TAMs), and TAMs promote tumor growth through multiple mechanisms. DC vaccines using Legumain as an antigenic peptide can also help clear TAMs, reshape the tumor immune microenvironment, and synergistically inhibit tumor growth. However, most tumor antigens have weak immunogenicity, and their single application is unlikely to stimulate an effective anti-tumor immune response. Therefore, exploring safe, non-toxic, and highly effective tumor vaccine adjuvants is an urgent problem to be solved in tumor vaccine research. Summary of the Invention

[0005] This invention provides an "all-in-one" integrated vaccine based on trichosanthin (TCS) protein and fused with antigenic peptides. Using mannose as a target, the vaccine targets and activates dendritic cells (DCs) through the immunostimulatory effect of trichosanthin protein, presenting tumor-associated antigenic peptides (e.g., Legumin antigenic peptides) carried by the vaccine to T cells, which then specifically attack and kill tumors.

[0006] Simultaneously, this invention proposes a mannose-recombinant trichosanthes pollen-antigen peptide (e.g., Legumin antigen peptide) vaccine hydrogel prepared using FDA-approved materials (Planick F127) as a treatment strategy for breast cancer. Previous research by the inventors has shown that TCS has excellent immunostimulatory effects, promoting the uptake and presentation of antigens by dendritic cells (DCs) through the activation of STING protein-mediated DC activation and maturation.

[0007] First, the inventors used genetic engineering technology to construct a novel recombinant tumor therapeutic vaccine, TCS-Legumain antigen peptide, based on trichosanthes pollen protein as an immune adjuvant. Then, they used the reaction of isothiocyanate and amino groups to link mannose to prepare an integrated fusion tumor vaccine, Man-TCS-Legumain antigen peptide (MTL). Mass spectrometry results showed that the vaccine was successfully prepared by this invention.

[0008] Stimulating dendritic cell (DC) maturation and activating T cells is fundamental to the anti-tumor effect of tumor vaccines. This invention conducted in vitro immunostimulation experiments using a prepared fusion vaccine. The results showed that the vaccine could stimulate DC maturation and further activate T cells.

[0009] On one hand, the present invention provides a smallpox pollen protein fusion vaccine, which comprises the structure shown in Formula I:

[0010]

[0011] Among them, TCS-antigen peptide refers to a fusion protein containing trichosanthin (TCS) and an antigen peptide.

[0012] In the structure shown in Formula I above, there are no particular restrictions on the linking position of the isothiocyanate to the fusion protein. The isothiocyanate can be linked by reacting with the primary amine (-NH2) on the terminal amino group or other amino acids of the fusion protein.

[0013] In this invention, the antigenic peptide may refer to a tumor-associated antigenic peptide, examples of which include, but are not limited to, Legumain antigenic peptide, CA125 antigenic peptide, 72-4 antigenic peptide, etc.

[0014] In one embodiment, the amino acid sequence of the pollen protein can be DVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYADETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIALLLNRNNMA (SEQ ID NO:1).

[0015] In one embodiment, the antigenic peptide may be a Legumain antigenic peptide. In one embodiment, the amino acid sequence of the Legumain antigenic peptide may be EDVTPENFLAVLR (SEQ ID NO:2).

[0016] In this invention, the TCS-antigen peptide may have a linker peptide between pollen protein (TCS) and the antigen peptide. In some embodiments, the linker peptide may be 3 to 8 Gs (glycine). In one embodiment, the linker peptide may be GGGG (SEQ ID NO:3).

[0017] In one embodiment, the amino acid sequence of the TCS-antigen peptide can be DVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYADETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIALLLNRNNMAGGGGEDVTPENFLAVLR (SEQ ID NO:4).

[0018] Furthermore, the present invention provides a method for preparing the above-mentioned smallpox pollen protein fusion vaccine, comprising the following steps:

[0019] S1. Construct an expression vector containing a fusion protein gene of TCS-antigen peptide, and express it to obtain a purified fusion protein containing TCS-antigen peptide.

[0020] S2, by adding 4-isothiocyanate phenyl-α-mannopyranoside and stirring, yields the structure shown in Formula I.

[0021] On the other hand, the present invention provides a pollen protein fusion vaccine hydrogel, which is a thermosensitive hydrogel formed by adding the above-mentioned pollen protein fusion vaccine to 18-22% by weight of Pronic F127. This hydrogel is liquid at low temperatures (e.g., 4°C) and transforms into a gel state at 27-33°C.

[0022] In another aspect, the present invention provides a method for preparing the above-mentioned pollen protein fusion vaccine hydrogel, comprising the step of adding the above-mentioned pollen protein fusion vaccine to 18-22% by weight of Prönnick F127 to form a thermosensitive hydrogel.

[0023] In another aspect, the present invention provides the application of the above-mentioned pollen protein fusion vaccine or the above-mentioned pollen protein fusion vaccine hydrogel in the preparation of antitumor drugs.

[0024] In some embodiments, the antitumor activity may be tumor-associated antigen peptides, particularly Legumain antigen peptides, which target tumors including, but not limited to, breast cancer, prostate cancer, colon cancer, non-small cell lung cancer, glioma, and other solid tumors.

[0025] In one embodiment, the antitumor drug may be a drug that inhibits the growth and metastasis of breast cancer.

[0026] Despite decades of laboratory, epidemiological, and clinical research, the incidence of breast cancer continues to rise, remaining a leading cause of disease burden for women. While new treatments are constantly emerging, the incidence of breast cancer is trending upward, and the mortality rate is declining slowly, resulting in stagnant progress in breast cancer treatment. Against this backdrop, this invention addresses the recurrence and metastasis of breast cancer by utilizing dendritic cells (DCs) as the target site to design a DC vaccine, MTL. Once in vivo, this vaccine is effectively taken up by DCs through the targeting action of mannose, thereby stimulating DC maturation. This leads to the uptake and presentation of the tumor antigen Legumain to T lymphocytes. The activated T lymphocytes in the lymph nodes then migrate to the solid tumor site to specifically kill the tumor, thereby reactivating the body's immune system. Furthermore, MTL reduces the proportion of M2 macrophages in the tumor immune microenvironment, relieving immunosuppression and reshaping the tumor immune microenvironment.

[0027] MTL thermosensitive hydrogel, prepared using FDA-approved Pluronic F127, is used for implantation therapy at the resection site of breast cancer. This reduces the number of immunizations and slowly releases the vaccine to achieve long-term inhibition of tumor recurrence and metastasis. MTL can inhibit the growth of recurrent breast cancer tumors in situ and reduce the risk of distant metastasis.

[0028] This study combines genetic engineering and chemical methods to construct a DC vaccine. The vaccine targets the mannose receptor and uses the natural toxic protein peptide TCS as an immunostimulant to activate DCs. The Legumain antigen peptide used is a tumor-associated antigen that is highly expressed in various malignant tumors such as prostate cancer and colon cancer. Therefore, the constructed DC vaccine may be effective against a variety of tumors and has certain clinical potential.

[0029] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the synthesis and immune activation mechanism of MTL.

[0031] Figure 2 Characterization of MTL is shown. Among them: (A) Separation map of MTL and unreacted mannoside; (B) SDS-PAGE gel map of TL and MTL; (C) ESI-MS map of MTL.

[0032] Figure 3 The cytotoxic effects of MTL were demonstrated. Among them: (A) cytotoxicity of MTL on 4T1 cells; (B) cytotoxicity of MTL on BMDCs.

[0033] Figure 4 The images show cellular uptake of MTL. Among them: (A) and (B) MTL uptake in 4T1 cells; (C) and (D) MTL uptake in BMDCs.

[0034] Figure 5 The results showed that MTL promotes BMDC activation, maturation, and antigen presentation. Specifically: (A) and (B) MTL promotes BMDC maturation, which in turn promotes CD8... + T cell activation and proliferation (C)(D). TNF-α (E) and IL-12 (F) ELISA assays of BMDCs. All data are presented as mean ± SD (n = 3 per group). Significance was determined by one-way ANOVA using the Tukey test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant (n = 3).

[0035] Figure 6The study demonstrated the inhibitory effect of MTL on the growth of breast cancer in situ. (A) Experimental flowchart; (B) Tumor volume; (C) Organ coefficients. All data are presented as mean ± SD (n=3 per group). Significance was determined by one-way ANOVA using the Tukey test: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant (n=3).

[0036] Figure 7 This study demonstrates the activating effect of MTL on immune cells in lymph node tissue (LN). Among them: (A)(B) maturation of dendritic cells (DCs) in mouse lymph nodes; (C)(D) CD8+ in mouse lymph node tissue. + With CD4 + Changes in T cell proportions. All data are presented as mean ± SD (n=3 per group). Significance was determined by one-way ANOVA using the Tukey test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns not significant (n=3).

[0037] Figure 8 This demonstrates the activating effect of MTL on immune cells in tumor tissue. Among them: (A)(B)(C) Granzyme B + (Granzyme B + ) and IFN-γ + CD8 + T cells and regulatory T cells (FoxP3) + CD4 + Changes in the proportion of T cells; (D)(E)(F) M1 macrophages (F4 / 80) + CD80 + ) and M2 macrophages (F4 / 80 + CD206 + Proportional changes. All data are presented as mean ± SD (n=3 per group). Significance was determined by one-way ANOVA using the Tukey test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns not significant (n=3).

[0038] Figure 9 These are pathological sections of the major organs of BALB / c mice.

[0039] Figure 10The effects of MTL hydrogel on tumor recurrence and lung metastasis were shown. (A)(B) Effect of MTL hydrogel on recurrent tumor volume; (C)(D) Effect of MTL hydrogel on lung metastasis of 4T1 breast cancer. All data are presented as mean ± SD (n=6 per group). Significance was determined by one-way ANOVA using the Tukey test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns not significant (n=3). Detailed Implementation

[0040] The following embodiments will further illustrate the present invention with reference to the accompanying drawings.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example

[0044] Example 1: Preparation and Characterization of MTL

[0045] 1 Materials and Instruments

[0046] 1.1 Materials

[0047] The TCS gene sequence (ATGGATGTTAGCTTCCGTTTATCAGGTGCAACAAGCAGTTCCTATG, SEQ ID NO:5) was donated by Professor Pang-Chui Shaw of the Chinese University of Hong Kong; pTXB1 vector (New England Biolabs, USA); Legumin antigen peptide (sequence: EDVTPENFLAVLR, SEQ ID NO:2) (Nanjing Peptide Biotechnology Co., Ltd., Nanjing, China); LB medium (Oxoid, UK); isopropyl β-D-1-thiogalactoside (IPTG), ampicillin, and L-cysteine ​​were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China);4-Isothiocyanatophen yla-α-mannopyranoside (Beijing Kailaisen Technology Co., Ltd., Beijing, China); Chitin resin (New England Biolabs, Massachusetts, USA).

[0048] 1.2 Instruments

[0049] Constant temperature water bath (Shanghai Titan Technology Co., Ltd., Shanghai, China); Constant temperature shaker (Taicang Experimental Equipment Factory, Taicang, China); Clean bench (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory, Shanghai, China); FPLC protein rapid purification system ( Purifier 10 (GE Healthcare, Stockholm, Sweden); HiTrap Desalting column (5ml, GE Healthcare, Stockholm, Sweden); Gel size exclusion chromatography column (Superdex 200, GE Healthcare, Stockholm, Sweden); Ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China); High-speed refrigerated centrifuge (Multifuge™ X1, Thermo Scientific, Massachusetts, USA); MALDI-TOF mass spectrometer (MALDI-TOF / TOF 5800 analyzer, AB Sciex, Massachusetts, USA); Gel imaging system (ChemiDoc MP™ Imaging). System, BIO-RAD, California, USA); Magnetic stirrer (85-2A, Shanghai Hengqin Instrument Equipment Co., Ltd., Shanghai, China); Manual pipette (Eppendorf, Hamburg, Germany); Electronic balance (BT25S, Sartorius). Germany).

[0050] 2 Experimental Methods

[0051] 2.1 Reagent Preparation

[0052] A sterile ampicillin solution (Amp solution, 50 mg / mL) was prepared using ultrapure water.

[0053] Sterile glycerin (50%), diluted with ultrapure water.

[0054] A sterile isopropyl β-D-1-thiogalactoside (IPTG) solution (500 mM) was prepared with ultrapure water.

[0055] Column binding buffer (elution) formulation: HEPES 4.76 g / L, NaCl 29.22 g / L, disodium EDTA 0.372 g / L. Weigh the above powder, add 800 mL of ultrapure water, dissolve thoroughly, and finally adjust the pH of the solution to 8.5 with 2M sodium hydroxide. Then add 0.5‰ Tween 20, mix well, and bring the volume to 1 L with ultrapure water. Store at 4℃.

[0056] Column cleavage buffer: Cysteine ​​(50mM), weigh 0.302g of powder, prepare with column binding buffer, prepare fresh and use immediately, adjust pH to 8.5.

[0057] 2.2 Expression and purification of TCS-Legumain (TL) protein

[0058] BL21(DE3) competent cells and the pTXB1 plasmid vector containing the TL gene sequence (SEQ ID NO:6) were thawed on ice. The competent cells were divided into two 50 μL portions, and 1 μL of plasmid (containing 50 ng) was added to each portion. After thorough mixing, the cells were incubated on ice for 30 min, then heat-shocked in a 42°C water bath for 90 s. The cells were then quickly placed on ice and allowed to stand for 5 min. LB medium was added and the cells were cultured in a shaker (37°C, 180 rpm) for 45 min. This process is called "small shake". The cells were collected by centrifugation (5000 g, 5 min), and the cells were resuspended in the remaining 0.1 mL of medium. The cells were spread on LB solid medium and incubated upside down overnight at 37°C to obtain single colonies. Single colonies were picked and placed in LB medium (containing 50 μg / mL ampicillin sodium), and amplified at 37℃ and 250 rpm for 5-6 h until the cells became turbid (absorbance at 600 nm reached 0.5-0.6). IPTG solution was added to a final concentration of 0.3 mM, and expression was induced at 25℃ and 150 rpm for 14-16 h.

[0059] Bacterial cells were collected by refrigerated centrifugation (9000g, 4℃), resuspended in 40mL binding buffer, and sonicated (ice-water bath, 180W, 3s sonication, 3s pause) for 30min. The supernatant was then obtained by refrigerated centrifugation at 10000rpm for 30min. After filtering through a 0.45μm filter membrane, the filtrate was added to a chitin column at a flow rate of 1mL / min. Non-specifically bound proteins were then washed with 25 volumes of binding buffer at a flow rate of 5mL / min. The column was then rapidly flow-through with 3 volumes of cysteine ​​cleavage buffer until approximately 2cm of the chitin packing was submerged, and the column was sealed. The column was then cleaved at 4℃ for 14h. The buffer containing the target protein was collected, and 30mL of binding buffer was added to completely elute the target protein. The target protein was concentrated to approximately 3mL using a 10kDa ultrafiltration centrifuge tube. The protein buffer was replaced with PBS using rapid protein liquid chromatography (FPLC) to obtain the TL solution.

[0060] TL gene sequence (SEQ ID NO:6):

[0061] ATGTTAGCTTCCGTTTATCAGGTGCAACAAGCAGTTCCTATGGAGTTTTCATTTCAAATCTGAGAAAAGCTCTTCCAAATGAAAGGAAACTGTACGATATCCCTCTGTTACGTTCCTCTTCCAGGTTCTCAACGCTACGCATTGATCCATCTCACAAATTACGCCGATGAAACCATTTCAGTGGCCATAGACGTAAC GAACGTCTATATTATGGGATATCGCGCTGGCGATACATCCTATTTTTTCAACGAGGCTTCTGCAACAGAAGCTGCAAAATATGTATTCAAAGACGCTATGCGAAAAGTTACGCTTCCATATTCTGGCAATTACGAAAGGCTTCAAACTGCTGCAGGCAAAATAAGGGAAAATATTCCGCTTGGACTCCCTGCTTTGGAC AGTGCCATTACCACTTTGTTTTACTACAACGCCAATTCTGCTGCGTCGGCACTTATGGTACTCATTCAGTCGACGTCTGAGGCTGCGAGGTATAAATTTATTGAGCAACAAATTGGGAAGCGTGTTGACAAAACCTTCCTACCAAGTTTAGCAATTATAAGTTTGGAAAATAGTTGGTCTGCTCTCTCCAAGCAAATTC AGATAGCGAGTACTAATAATGGACAGTTTGAAAGTCCTGTTGTGCTTATAAATGCTCAAAACCAACGAGTCACGATAACCAAGTTGATGCTGGAGTTGTAACCTCCAACATCGCGTTGCTGCTGAATAGAAACAATATGGCACTCGAGGCGGTGGTGGCGAAGATGTGACACCTGAAAATTTTCTGGCAGTGCTGCGT

[0062] 2.3 Preparation of MTL

[0063] 4-Phenylan-α-mannopyranoside 4-isothiocyanate (5 mg / mL in PBS, pH 7.4) was added dropwise to TL solution (20 mg / mL in PBS, pH 7.4) at a molar ratio of 3:1. The mixture was stirred at room temperature for 4 h, and then purified using a desalting column to remove excess 4-phenyl-α-mannopyranoside 4-isothiocyanate.

[0064] 2.4 Characterization of MTL

[0065] 2.5. Characterization of MTL using SDS-PAGE and MALDI-TOF-MS methods

[0066] Take 40 μL of purified MTL and TL, add 10 μL of 5× loading buffer, mix well, boil in water for 15 min, perform SDS-PAGE electrophoresis, then stain with Coomassie Brilliant Blue for 6 h, decolorize with 10% glacial acetic acid until the colloid is clear, and take pictures for recording.

[0067] MTL was desalted using a gravity desalting column. After mixing 1 μL of the sample with an equal volume of sinapic acid solution (10 mg / ml), 1 μL of the mixture was then analyzed by mass spectrometry.

[0068] 3 Results and Discussion

[0069] MTL is obtained by reacting isothiocyanate from mannoside with the terminal amino group on TL protein. The reaction mechanism is as follows: Figure 1 As shown:

[0070] The reaction product MTL was first purified using a desalting column to remove unreacted mannosides, such as... Figure 2 As shown in Figure A, MTL and unreacted mannoside can be largely separated, yielding purified MTL; then, its molecular weight is compared with that of TL protein, as shown in Figure A. Figure 2 As shown in B and C, the molecular weight of MTL is larger than that of TL, indicating that mannose was successfully linked.

[0071] Example 2: In vitro evaluation of the effects of MTL stimulation on DC maturation

[0072] 1 Materials and Instruments

[0073] 1.1 Materials

[0074] DMEM and RPMI 1640 cell culture medium, fetal bovine serum (FBS), and 0.25% trypsin-EDTA were purchased from Gibco (New York, USA); BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China); 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protease inhibitors, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich Co., Ltd., Missouri, USA; FITC-PEG-NHS (MW5000) (Guangzhou Carbohydrate Technology Co., Ltd., Guangzhou, China); mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and mouse interleukin-4 (IL-4) were purchased from Novus Biologicals (Centennial, Nevada, USA).

[0075] 1.2 Instruments

[0076] Cell culture incubator (MCO-18AIC, Sanyo, Osaka, Japan); flow cytometer (ACEA NovoCyte3000, California, USA); PCR instrument (Applied Biosystems, California, USA); microplate reader (Biotek Synergy 4, Vermont, USA).

[0077] 1.3 Cell lines

[0078] The mouse breast cancer cell line 4T1 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0079] 1.4 Laboratory Animals

[0080] Female BALB / c mice (SPF grade) aged 4-5 weeks were purchased from Shanghai Slack Laboratory Animal Co., Ltd. They were housed at 25°C room temperature with a 12-hour day / night cycle, with regular replenishment of water and sterile food, and periodic cage changes. All animal experimental procedures followed the standards and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0081] 2 Experimental Methods

[0082] 2.1 In vitro cytotoxic effects of MTL

[0083] 2.1.1 Extraction and culture of bone marrow-derived dendritic cells (BMDC)

[0084] BALB / c mice, aged 4-5 weeks, were used as the source of bone marrow mononuclear cells. Before the experiment, surgical scissors, ophthalmic forceps, filter gauze, and beakers were disinfected by soaking in 75% ethanol for 15 minutes. After euthanasia, the mice were soaked in 75% ethanol for 10 minutes. The tibia and femur of the lower limbs and the humerus of the upper limbs were separated, and all muscle was removed. The cells were then washed twice with sterile PBS after soaking in 75% ethanol for 5 minutes. The ends of the tibia, femur, and humerus were removed with sterile scissors. Serum-free DMEM culture medium was drawn into a 1mL syringe, and the bone marrow was flushed out, mixed thoroughly, and transferred to a 15mL centrifuge tube. The cells were centrifuged at 1800g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 3-5mL of sterile erythrocyte lysis buffer. The cells were incubated at room temperature for 5 minutes, the reaction was stopped with sterile PBS, and the cells were centrifuged at 1800g for 5 minutes after passing through a sterile filter. The supernatant was discarded, and the cells were collected; these were the bone marrow-derived cells. Resuspend cells in DMEM containing 20% ​​serum and 1% penicillin-streptomycin, add GM-CSF to a final concentration of 20 ng / mL and IL-4 to a final concentration of 10 ng / mL, and incubate at 37°C in a 5% CO2 incubator for 3 days. On the third day, perform a half medium change (discard half and add half of the BMDC stimulation medium), and incubate for another 2 days to obtain immature BMDCs.

[0085] 2.1.2 CCK8 assay for the cytotoxic effect of MTL

[0086] Collect BMDCs and 4T1 cells at a ratio of 5 × 10⁻⁶. 3 Add one sample per well to a 96-well plate and incubate for 16 h, then administer the drug and incubate for 18 h, followed by incubation at 37°C for 1 h with CCK8 reagent. Detect absorbance at 450 nm and calculate IC50. 50 It is evident that TL and MTL have relatively low toxicity to immune cells BMDCs. Figure 3 B, IC 50 The concentrations were 44.54 μM (TL) and 179.6 μM (MTL), respectively, but they showed greater cytotoxicity against 4T1 tumor cells. Figure 3 A, IC 50 The values ​​were 13.16 μM (TL) and 14.67 μM (MTL), respectively.

[0087] 2.2 The promoting effect of MTL on BMDC uptake

[0088] To verify the targeting effect of mannose on the mannose receptor, a comparative study was conducted on the uptake effects of TL and MTL in mannose-high expression cells (BMDCs) and 4T1 cells. The study included a normal control group, a PBS group, a TL group, an MTL group, and a group treated with mannose followed by MTL (M+MTL). The uptake time was 4 hours. The results showed that mannose could promote uptake in 4T1 cells (BMDCs and 4T1 cells). Figure 4 (A and B) and BMDCs ( Figure 4The uptake of MTL by C and D was significantly inhibited when mannose was added beforehand to block it, which also verifies the promoting effect of mannose on MTL uptake.

[0089] 2.3 In vitro efficacy validation of MTL as a DC vaccine

[0090] Induced BMDCs were seeded into cell culture plates and stimulated with antigen. Four groups were established: a PBS negative control group, a LEG group, a TCS group, a TL group, and an MTL group. Cells were collected for flow cytometry analysis 18 hours after in vitro stimulation. Using the maturation markers CD80, CD86, and MHC II of BMDCs as evaluation indicators, the in vitro stimulation effect of the vaccine on BMDCs was studied. The results showed that, compared with the LEG, TCS, and TL groups, MTL had the most significant stimulatory effect on BMDCs, showing a significant difference compared with the normal group. Figure 5 (A and B) and can stimulate BMDCs to increase the secretion of IL-12 and TNF-α (E and F in the figure). Co-culturing well-stimulated BMDCs with T cells isolated from the spleen showed a significant promotion of CD8. + T cell proliferation and differentiation, increasing Granzyme B + The percentage of T cells ( Figure 5 The results suggest that MTLs may be a promising DC vaccine and could potentially become an effective therapeutic vaccine against Legumain antigen-positive tumors. (The text also mentions C and D in the formula, but the connection to the main sentence is unclear.)

[0091] Example 3: In vivo evaluation of the inhibitory effect of MTL on breast cancer growth

[0092] 1 Materials and Instruments

[0093] 1.1 Materials

[0094] DMEM and RPMI 1640 cell culture media, fetal bovine serum (FBS), 0.25% trypsin-EDTA, collagenase, and hyaluronidase were purchased from Gibco (New York, USA); BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China); Pronek F127 and bovine serum albumin were purchased from Sigma-Aldrich Co., Ltd. (Missouri, USA); flow cytometry antibodies were purchased from Shanghai Dako Biotechnology Co., Ltd. (Shanghai, China).

[0095] 1.2 Instruments

[0096] Cell culture incubator (MCO-18AIC, Sanyo, Osaka, Japan); flow cytometer (ACEA NovoCyte3000, California, USA); PCR instrument (Applied Biosystems, California, USA); ELISA reader (Biotek Synergy 4, Vermont, USA); high-throughput tissue homogenizer (Shanghai Biheng Biotechnology Co., Ltd., Shanghai, China).

[0097] 1.3 Cell lines

[0098] The mouse breast cancer cell line 4T1 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0099] 1.4 Laboratory Animals

[0100] Female BALB / c mice (SPF grade) aged 4-5 weeks were purchased from Shanghai Slack Laboratory Animal Co., Ltd. They were housed at 25°C room temperature with a 12-hour day / night cycle, with regular replenishment of water and sterile food, and periodic cage changes. All animal experimental procedures followed the standards and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0101] 2 Experimental Methods

[0102] 2.1 Inhibitory effect of MTL on the growth of in situ breast cancer

[0103] After a week of acclimatization feeding, BALB / c mice aged 4-6 weeks were given 5×10 5 Inject 4 T1 cells per tumor on the right side of the second pair of nipples. When the tumor volume reaches approximately 30 mm... 3 Mice were randomly divided into 5 groups: PBS (control), LEG, TCS, TL, and MTL. Every 3 days, mice were injected subaxillarily with 100 μL of the drug / vaccine (containing 10 μg of the drug), for a total of 3 injections. The experiment ended when the tumor volume exceeded 1500 mmHg. 3 Mice were euthanized (as the experimental endpoint). Lymph nodes and tumors were removed from the mice, and the ratio of dendritic cells (DCs) to T cells was analyzed by flow cytometry. Major organs (including liver, heart, spleen, lungs, and kidneys) of the mice were weighed, and organ coefficients were calculated using the following formula. Major organs were immersed in 4% paraformaldehyde for histopathological examination to assess side effects.

[0104]

[0105] Studies on the efficacy of MTL as a therapeutic vaccine have found that after three subcutaneous injections of MTL into BALB / c mice, it can significantly inhibit the growth of 4T1 in situ tumors compared to other treatments. This is reflected in a significant reduction in individual tumor volume and average tumor volume in tumor-bearing mice, with no significant difference in organ coefficients between groups, which preliminarily indicates that the in vivo biosafety of MTL administered subcutaneously is good.

[0106] At the end of the experiment, inguinal lymph nodes from mice were collected for flow cytometry analysis. The results showed that MTL effectively activated antigen-presenting dendritic cells (DCs) in the lymph nodes, promoting DC maturation and antigen presentation. Figure 7 In studies involving A and B, the types and proliferative capacity of T cells in lymph nodes were analyzed. MTL was found to significantly increase CD8+ in lymph nodes. + The number of T cells is reduced, and CD4 is decreased. + The proportion of T cells enhances the body's anti-tumor immunity. Figure 7 The flow cytometry antibodies and staining protocols used for flow cytometry experiments on lymph nodes C and D are shown in the table below:

[0107]

[0108] 100 mg of tumor tissue was weighed, surgically minced, and added to DMEM medium containing digestive enzymes (1 mg / mL collagenase and 1 mg / mL hyaluronidase). The mixture was digested at 37°C and 220 rpm until turbidity was achieved without yellowing. Blocking and flow cytometry staining were then performed. The prepared sample was added to a 96-well plate and placed on the flow cytometer stage for experiments. Flow cytometry results of the tumor tissue showed that LEG, TCS, TL, and MTL could all effectively stimulate intratumoral immune cells and upregulate the proportion of cytotoxic T cells in the tumor tissue. Figure 8 (A and B) effectively reduce Fox P3 + The percentage of Treg cells ( Figure 8 (C), while increasing the proportion of intratumoral M1 macrophages and decreasing the proportion of intratumoral M2 macrophages (C). Figure 8 The drugs (D, E, F) mobilize the immune system to exert a synergistic anti-tumor effect, with MTL showing the most significant therapeutic effect. The flow cytometry antibodies and staining protocols used for flow cytometry experiments on tumor tissues are shown in the table below:

[0109]

[0110]

[0111] The pathological section results of the major organs of mice in each group are as follows: Figure 9 As shown, no significant lesions were observed in the heart, liver, spleen, lungs, and kidneys in any of the treatment groups, especially the MTL group, which demonstrates the in vivo biocompatibility of MTL.

[0112] 2.2 Inhibitory effect of MTL hydrogel on in situ breast cancer

[0113] Using 22% Pronic F127 as a raw material, MTL vaccine or the chemotherapy drug methotrexate (MTX) was added and stirred at 4°C to form a thermosensitive hydrogel, thereby achieving sustained release and long-lasting effect of the vaccine. Subcutaneous xenografts in BALB / c mice were surgically removed and sutured, followed by injection of the thermosensitive hydrogel into the surgical site. The mice were divided into a blank hydrogel group, an MTX hydrogel group, and an MTL hydrogel group. The size of recurrent tumors at the affected site was observed, with tumors exceeding 1500 mm² being considered the target. 3 As the experimental endpoint, mouse lungs were collected for pathological sectioning and H&E staining. It was observed that, compared to the MTX and PBS groups, the MTL hydrogel group significantly reduced the volume of recurrent tumors. Figure 10 The study found that the tumors in groups A and B effectively inhibited lung metastasis, with the number of lung metastatic lesions significantly lower than that in the normal group. Figure 10 (C and D in the middle).

Claims

1. A smallpox pollen protein fusion vaccine comprising the structure shown in Formula I: Formula I in, TCS-antigen peptide indicates a fusion protein containing trichosanthes pollen protein TCS and an antigen peptide. The amino acid sequence of the pollen protein is shown in SEQ ID NO:

1. The antigenic peptide is a tumor-associated antigenic peptide. The antigenic peptide is the Legumain antigenic peptide. The amino acid sequence of the Legumain antigen peptide is shown in SEQ ID NO:

2.

2. The smallpox pollen protein fusion vaccine according to claim 1, wherein, In the TCS-antigen peptide, there is a linker peptide between the pollen protein TCS and the antigen peptide.

3. The smallpox pollen protein fusion vaccine according to claim 2, wherein, The linker peptide is 3 to 8 G (glycine).

4. The smallpox pollen protein fusion vaccine according to claim 2, wherein, The linker peptide is GGGG (SEQ ID NO:3).

5. The smallpox pollen protein fusion vaccine according to claim 1, wherein, The amino acid sequence of the TCS-antigen peptide is shown in SEQ ID NO:

4.

6. A method for preparing a smallpox pollen protein fusion vaccine according to any one of claims 1-5, comprising the following steps: S1. Construct an expression vector containing a fusion protein gene of TCS-antigen peptide, and express and purify it to obtain a fusion protein containing TCS-antigen peptide. S2, by adding 4-isothiocyanate phenyl-α-mannopyranoside and stirring, yields the structure shown in Formula I.

7. A pollen protein fusion vaccine hydrogel, which is a thermosensitive hydrogel formed by adding the pollen protein fusion vaccine of any one of claims 1-5 to 18-22% by weight of Pronek F127.

8. The method for preparing the pollen protein fusion vaccine hydrogel as described in claim 7, comprising the step of adding the pollen protein fusion vaccine to 18-22% by weight of Prönkel F127 to form a thermosensitive hydrogel.

9. The use of the pollen protein fusion vaccine as described in any one of claims 1-5 or the pollen protein fusion vaccine hydrogel as described in claim 7 in the preparation of antitumor drugs, wherein, The tumor is breast cancer.

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