Tumor immunotherapy use and preparation method of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii

Abstract

The present invention discloses the tumor immunotherapy use and preparation method of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii (NRTUA). The present invention discovers for the first time that the NRTUA-DC vaccine obtained in vitro by activating isolated mouse dendritic cells (DCs) with non-replicating uracil auxotrophic atoxic Toxoplasma gondii (NRTUA) is significantly effective in treating CT-26 mouse colorectal cancer. This overcomes the limitation of conventional dendritic cell (DC) vaccines in terms of poor anti-tumor therapeutic effect, provides a stimulator that better activates the antigen presentation ability of DCs, and reveals its anti-tumor immunoregulatory effect.

Description

Technical Field

[0001] The present invention relates to the field of tumor biotherapy, and in particular to an anti-tumor vaccine and a preparation method thereof. Background Art

[0002] Colorectal cancer is one of the top three cancers in terms of both morbidity and mortality worldwide (Reference: Feng RM, Zong YN, Cao SM, et al. Current cancer situation in China: good or bad news from the 2018 Global Cancer Statistics[J]. Cancer Commun (Lond), 2019, 39(1):22.). Conventional treatments include surgery, chemotherapy, and radiotherapy. The prognosis for patients with advanced colorectal cancer and metastasis remains poor (Reference: Roelands J, Kuppen P, Vermeulen L, et al. Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications[J]. Int J Mol Sci, 2017, 18(10).). Cancer immunotherapy is one of the new options for cancer treatment. Compared with standard treatments such as conventional chemotherapy and radiotherapy, immunotherapy can regulate the patient's own immune system, such as innate and adaptive immune responses, to recognize and kill tumor cells, and has the potential to eradicate tumors (reference: Ganesh K, Stadler ZK, Cercek A, et al. Immunotherapy in colorectal cancer: rationale, challenges and potential [J]. Nat Rev Gastroenterol Hepatol, 2019, 16 (6): 361-375.). However, current immunotherapy methods are basically ineffective for colorectal cancer with microsatellite stable (MSS) or KRAS mutations (such as colorectal cancer inoculated with CT-26 type colorectal cancer cells, the main target of this patent application). Therefore, there is an urgent need to develop new immunotherapy methods.

[0003] Tumor immunotherapy can be mainly divided into active immunotherapy and passive immunotherapy (reference: Roelands J, Kuppen P, Vermeulen L, et al. Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications [J]. Int J Mol Sci, 2017, 18 (10).). Passive immunotherapy mainly uses monoclonal antibodies and adoptive cell therapy targeting tumor antigens and immunosuppressive checkpoints; active immunotherapy is tumor-related vaccines, including tumor antigen vaccines and dendritic cell (DCs) vaccines (reference: Franke AJ, Skelton WP, Starr JS, et al. Immunotherapy for Colorectal Cancer: A Review of Current and Novel Therapeutic Approaches [J]. J Natl Cancer Inst, 2019, 111 (11): 1131-1141.). Existing DCs vaccines primarily utilize single or multiple tumor antigens or other stimulants to stimulate host-derived DCs, which are then returned to the host to achieve the desired tumor treatment effect (Reference: Sarvizadeh M, Ghasemi F, Tavakoli F, et al. Vaccines for colorectal cancer: an update [J]. J Cell Biochem, 2019, 120(6): 8815-8828.). However, this conventional DCs vaccine immunotherapy approach is ineffective for CT-26 colorectal cancer, which is not sensitive to immunotherapy.

[0004] Toxoplasma gondii is a conditionally pathogenic intracellular parasite that can parasitize the cells of various warm-blooded vertebrates, including humans (Blader IJ, Coleman BI, Chen CT, et al. Lytic Cycle of Toxoplasma gondii: 15 Years Later[J]. Annu Rev Microbiol, 2015, 69:463-485). Infection with wild-type Toxoplasma in immunocompetent individuals generally does not result in clinical symptoms (Rommereim LM, Fox BA, Butler KL, et al. Rhoptry and Dense Granule Secreted Effectors Regulate CD8(+) T Cell Recognition of Toxoplasma gondii Infected Host Cells[J]. Front Immunol, 2019, 10:2104). Currently, it is primarily studied as a model pathogen for intracellular parasitism. Toxoplasma mainly invades host cells through secretory proteins secreted by its secretory organs, causing the host to produce a pathogen-clearing immune response (Reference: Rastogi S, Xue Y, Quake SR, et al. Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii[J]. mBio, 2020, 11(3).). The non-replicating uracil auxotrophic atoxic Toxoplasma gondii (NRTUA, see the invention patent with publication number CN107007830A previously authorized by the applicant of the present invention: a new atoxic Toxoplasma was constructed) can induce a strong, tumor-specific immune response in the host without causing damage to the host cells due to replication (reference: Fox BA, Sanders KL, Rommereim LM, et al. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity[J]. PLoS Genet, 2016, 12(7): e1006189.).Toxoplasma lysates, Toxoplasma-specific antigens, inactivated Toxoplasma, and non-toxic Toxoplasma have all been reported to be used for anti-tumor activities, including melanoma (Reference: Baird JR, Byrne KT, Lizotte PH, et al. Immune-mediated regression of established B16F10 melanoma by intratumoralinjection of attenuated Toxoplasma gondii protects against rechallenge[J]. J.

[0005] Immunol, 2013, 190(1):469-478.), ovarian cancer (Reference: Baird JR, Fox BA, Sanders KL, et al. Avirulent Toxoplasma gondii generates therapeutic antitumorimmunity by reversing immunosuppression in the ovarian cancermicroenvironment[J]. Cancer Res, 2013, 73(13):3842-3851.), pancreatic cancer (Reference: Sanders KL, Fox BA, Bzik D J. Attenuated Toxoplasma gondii therapy ofdisseminated pancreatic cancer generates long-lasting immunity to pancreaticcancer[J].

[0006] Oncoimmunology, 2016, 5(4): e1104447.), Ehrlich ascites carcinoma (Reference: Hafez EN, Moawed F, Abdel-Hamid GR, et al. Gamma Radiation-Attenuated Toxoplasma gondii Provokes Apoptosis in Ehrlich Ascites Carcinoma-Bearing Mice Generating Long-Lasting Immunity[J]. Technol Cancer Res Treat, 2020, 19: 1079194241.), etc. However, to date, there has been no report on the use of avirulent Toxoplasma gondii for immunotherapy of colorectal cancer in CT-26 mice.

[0007] Disadvantages of existing immunotherapy for colorectal cancer in CT-26 mice: Although passive immunotherapy can have significant effects on some tumors, it is not suitable for all colorectal cancer patients due to the different status of the patient's own immune system. Although there is clear clinical evidence that immune checkpoint inhibitors can treat mismatch repair-deficient or high microsatellite instability (MSI-H) metastatic colorectal cancer, the vast majority of patients with mismatch repair-deficient or microsatellite stable (MSS) tumors do not benefit from immunotherapy (Reference: Ciardiello D, Vitiello PP, Cardone C, et al. Immunotherapy of colorectal cancer: Challenges for therapeutic efficacy [J]. Cancer Treat Rev, 2019, 76: 22-32.). The CT-26 mouse colorectal cancer cells containing KRAS mutations that we studied are also of MSS type and are insensitive to immune checkpoint inhibition therapy (Reference: Castle JC, Loewer M, Boegel S, et al. Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma[J]. BMC Genomics, 2014, 15:190.). Existing immune checkpoint inhibitors PD1 / PD-L1 (Reference: Yu G, Wu Y, Wang W, et al. Low-dose decitabine enhances the effect of PD-1 blockade in colorectal cancer with microsatellite stability by re-modulating the tumor microenvironment[J]. Cell Mol Immunol, 2019, 16(4):401-409.) have little inhibitory effect on CT-26.

[0008] Disadvantages of existing DCs vaccines for the treatment of colorectal cancer: The only DCs vaccine currently approved by the FDA is Provenge (Sipuleucel-T) for the treatment of prostate cancer. Currently, there is no DCs vaccine for colorectal cancer in clinical practice (Reference: Jiang S, Good D, Wei M Q. Vaccinations for Colorectal Cancer: Progress, Strategies, and Novel Adjuvants[J]. Int J Mol Sci, 2019, 20(14).). Existing studies on DCs for the treatment of colorectal cancer have shown that neither the use of carcinoembryonic antigen (CEA) to stimulate DCs (Reference: Lesterhuis WJ, de Vries IJ, Schuurhuis DH, et al. Vaccination of colorectal cancer patients with CEA-loaded dendritic cells: antigen-specific T cell responses in DTH skintests [J]. Ann Oncol, 2006, 17 (6): 974-980.) to treat colorectal cancer patients nor the use of colorectal cancer stem cell lysates to stimulate DC maturation (Reference: Szarynska M, Olejniczak A, Kobiela J, et al. Cancer stem cells as targets for DC-based immunotherapy of colorectal cancer [J]. Sci Rep, 2018, 8 (1): 12042.) has been effective, making it difficult for existing basic research on DCs vaccines to enter the next step of clinical experimental research.

[0009] Disadvantages of using avirulent Toxoplasma gondii directly for colorectal cancer treatment: Currently, there are no reports of avirulent Toxoplasma gondii being used directly as a vaccine for colorectal cancer. The lysis products of Toxoplasma gondii can significantly inhibit the growth of CT-26 subcutaneous tumors in athymic mice, but there is no significant effect in normal immune mice (reference: Pyo KH, Jung BK, Xin CF, et al. Prominent IL-12 production and tumor reduction in athymic nude mice after Toxoplasma gondii lysate antigen treatment [J]. Korean J Parasitol, 2014, 52(6): 605-612.). Live, avirulent Toxoplasma gondii elicits stronger anti-tumor CD4+ and CD8+ T cell responses than inactivated Toxoplasma or Toxoplasma lysis products (Reference: Dupont CD, Christian DA, Selleck EM, et al. Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii[J]. PLoS Pathog, 2014, 10(4): e1004047.). Unfortunately, when we used avirulent Toxoplasma NRTUA as a therapeutic vaccine to directly treat immune-normal mice bearing subcutaneous CT-26 tumor cells, there was no significant inhibitory effect.

[0010] In summary, the prior art has the following shortcomings:

[0011] (1) Existing passive immunotherapy is not suitable for all colorectal cancer patients;

[0012] (2) Currently, there is no DCs vaccine for colorectal cancer in the clinic, and there is no research report on the direct use of non-toxic Toxoplasma as a vaccine to treat colorectal cancer. The applicant's research found that the non-toxic Toxoplasma NRTUA as a therapeutic vaccine to treat normal immune mice subcutaneously loaded with CT-26 tumor cells had no significant inhibitory effect. Summary of the Invention

[0013] In order to overcome the deficiencies of the prior art, the present invention provides a tumor immunotherapy application and preparation method of NRTUA-DC obtained by stimulating DCs in vitro with non-toxic Toxoplasma gondii.

[0014] The technical solution adopted by the present invention to solve its technical problem is:

[0015] The use of NRTUA-DCs obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating tumors.

[0016] Use of NRTUA-DCs obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating colorectal cancer.

[0017] The invention relates to the use of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating CT-26 colorectal cancer.

[0018] The invention relates to the use of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii for producing biological drugs for treating tumors.

[0019] The invention relates to the use of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii for manufacturing biological drugs for treating colorectal cancer.

[0020] The invention relates to the use of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii for manufacturing biological drugs for treating CT-26 colorectal cancer.

[0021] The invention relates to the use of NRTUA-DC vaccine obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating tumors.

[0022] Use of NRTUA-DC vaccine obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating colorectal cancer.

[0023] The invention relates to the use of NRTUA-DC vaccine obtained by stimulating DCs with non-toxic Toxoplasma gondii for treating CT-26 colorectal cancer.

[0024] The method for preparing NRTUA-DCs by stimulating DCs with non-toxic Toxoplasma gondii is as follows:

[0025] (1) Take a mouse and kill it, then soak the mouse in alcohol.

[0026] (2) Remove the mouse skin and cut off the hind limbs near the hip and distal to the ankle.

[0027] (3) Mouse femurs were dissected in a 60 mm culture dish with PBS, the muscles surrounding the bones were removed, and the bone marrow was exposed.

[0028] (4) Soak the peeled bone tissue in pre-cooled PBS until all the bone tissue is collected.

[0029] (5) Soak all collected bone tissues in alcohol first, and then wash them with sterile PBS.

[0030] (6) Pour pre-cooled PBS into a 100 cm culture dish, transfer the bone tissue into the culture dish containing PBS, and place the culture dish in a biosafety cabinet.

[0031] (7) Cut the femur with sterile scissors, fix the bone with forceps, and use a syringe to draw pre-cooled PBS to flush the bone. Insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush out the bone marrow into the culture dish until the femur turns completely white.

[0032] (8) Transfer the bone marrow into a centrifuge tube and centrifuge it at 1500 rpm for 3 min. Discard the supernatant after centrifugation.

[0033] (9) Add ACK lysis buffer to remove red blood cells, resuspend for 15 seconds, and then centrifuge at 1500 rpm for 3 minutes to remove the ACK lysis buffer.

[0034] (10) Resuspend the cells in the basic culture medium of RMPI1640+10% FBS+1% PS (penicillin, streptomycin)+55 μM 2-β-mercaptoethanol, and filter the cells using a cell strainer.

[0035] (11) Adjust the cell concentration to 1-1.6×10 6 / ml, seeded in 6-well plates.

[0036] (12) Cytokines GM-CSF and IL-4 were added to the culture medium, and then the cells were transferred to a 37°C, 5% CO2 incubator for culture.

[0037] (13) Perform semi-quantitative medium change by aspirating the old culture medium and replacing it with new culture medium.

[0038] (14) Observe the cells. DCs cells grow rapidly on the 4th day, and the culture medium needs to be replaced and cytokines added in time.

[0039] (15) On the 5th day, immature DCs were collected and resuspended to 1×10 6 pcs / ml.

[0040] (16) HFF cells were revived and cultured in a 37°C, 5% CO2 incubator using 10% DMEM+10% FBS+1% PS maintenance medium.

[0041] (17) When the confluence of HFF cells reaches more than 90%, the non-toxic Toxoplasma NRTUA is revived, and the culture medium of HFF cells is replaced with a maintenance medium containing 250 μM uracil, and NRTUA is added to the HFF cell culture flask.

[0042] (18) The HFF cell culture flask inoculated with NRTUA was placed in an incubator at 37°C and 5% CO2. After 4-5 days, the NRTUA completely broke through the cell wall and could be passaged.

[0043] (19) After cell wall destruction, the NRTUA was filtered out with a filter to remove cell debris, and the filtered NRTUA was collected and resuspended in PBS to a concentration of 1-2×10 7 pcs / ml.

[0044] (20) The collected immature bone marrow dendritic cells (BMDC) were resuspended to 1×10 using DCs differentiation medium (basic medium containing GM-CSF and IL-4). 6 / ml, and re-plated into 6-well plates.

[0045] (21) Collect the NRTUA filtered by the filter and resuspend it in PBS to a concentration of 2×10 7 500 μl of NRTUA was added to each well, and the mixture was gently mixed. The cells were then placed in a 37°C, 5% CO2 incubator for 48 h.

[0046] (22) After the culture, NRTUA-DCs or unstimulated DCs were collected and resuspended in PBS to a concentration of 1×10 7 The concentration was 100 μg / ml and stored on ice.

[0047] The beneficial effects of the present invention are:

[0048] 1. The present invention utilizes non-replicating uracil auxotrophic atoxic Toxoplasma gondii (NRTUA) to stimulate dendritic cells (DCs) in vitro, activating DCs without the need for specific tumor antigens to form a DCs vaccine with anti-colorectal cancer function. This is a dendritic cell (DCs) vaccine that can broadly kill tumor cells, overcoming the limitations of conventional dendritic cell (DCs) vaccines, which have poor anti-tumor therapeutic effects, and providing a stimulant that better activates the antigen presentation ability of DCs.

[0049] 2. The avirulent Toxoplasma gondii (NRTUA) used in this patent application is the avirulent Toxoplasma gondii constructed by the same first inventor in the invention patent publication number CN107007830A. This avirulent Toxoplasma gondii is a living intracellular parasite that can retain the ability to stimulate the host's immune system while being harmless to the host. The NRTUA-stimulated DC vaccine (NRTUA-DC) can significantly inhibit colorectal cancer in microsatellite-stable and KRAS-mutant CT-26 mice, and the tumor-suppressing effect of the NRTUA-DC vaccine is closely related to changes in the infiltration of immune cells and immune factors in the tumor microenvironment of tumor-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further described below with reference to the accompanying drawings and examples.

[0051] Figure 1 It is the technical roadmap of the present invention;

[0052] Figure 2 This is a schematic diagram of the experimental results of the effect of NRTUA-DC vaccine on the growth of subcutaneous tumors in CT-26 colorectal cancer mice;

[0053] Figure 3 This is a schematic diagram of the effects of NRTUA-DC vaccine on the infiltration of immune cells and immune factors in the tumor microenvironment of CT-26 colorectal cancer mice. DETAILED DESCRIPTION

[0054] Reference Figure 1 , wherein DCs, dendritic cells; NRTUA, non-replicating uracil auxotrophic atoxic Toxoplasma gondii, is an atoxic Toxoplasma gondii constructed by the first inventor of the same invention patent with publication number CN107007830A previously applied for by the same first inventor as the patent in this application. The detailed method of its preparation can be found in the authorized invention patent; NRTUA-DC, NRTUA and DCs are co-cultured at a ratio of 1:1 for 48 hours to obtain a DCs vaccine containing NRTUA; iDC, immature dendritic cells; PBS, phosphate buffered saline; IL-4, interleukin-4; GM-CSF, granulocyte macrophage stimulating factor; CT-26, mouse colorectal cancer cell line.

[0055] The detailed experimental steps are as follows:

[0056] First, isolation and culture of dendritic cells (DCs):

[0057] (1) On day 0, mice (>6 weeks, male mice 8-10 weeks) were killed and soaked in 75% alcohol for 10 seconds.

[0058] (2) Remove the skin and cut off the hind limbs near the hip and distal to the ankle.

[0059] (3) Mouse femurs were dissected in a 60 mm culture dish with PBS, the muscles surrounding the bones were removed, and the bone marrow was exposed.

[0060] (4) Soak the peeled bone tissue in pre-cooled (4°C) PBS until all the bone tissue is collected.

[0061] (5) Soak the bone tissue in 75% alcohol for 15 seconds and wash it twice with sterile PBS.

[0062] (6) Pour pre-cooled PBS into a 100 cm culture dish, transfer the bone tissue into the culture dish containing PBS, and place the culture dish in a biosafety cabinet.

[0063] (7) Cut the femur with sterile scissors, fix the bone with forceps, and flush the bone with a 27g syringe needle (5ml syringe with pre-cooled PBS). Insert the needle into the bone marrow cavity from both ends of the bone and repeatedly flush the bone marrow into the culture dish until the femur turns completely white.

[0064] (8) Transfer the bone marrow to a centrifuge tube, centrifuge the cells at 1500 rpm for 3 min, and discard the supernatant.

[0065] (9) Add ACK lysis buffer (3-5 ml) to remove red blood cells, resuspend for 15 seconds, and centrifuge at 1500 rpm for 3 minutes to remove the ACK lysis solution.

[0066] (10) Resuspend the cells in the basic culture medium of RMPI1640+10% FBS+1% PS (penicillin, streptomycin)+55 μM 2-β-mercaptoethanol, and filter the cells using a cell sieve with a diameter of 70 μm.

[0067] (11) Adjust the cell concentration to 1-1.6×10 6 / ml, seeded in 6-well plates (4-5ml culture medium / well).

[0068] (12) Cytokines GM-CSF (20 ng / ml) and IL-4 (20 ng / ml) were added to the culture medium and cultured in a 37°C, 5% CO2 incubator.

[0069] (13) Change the medium semi-quantitatively every day, carefully aspirating the old medium and replacing it with fresh medium. If the cells are overcrowded, gently pipette to separate the cells. Epithelial cells and macrophages are firmly attached to the wall, while DCs may be able to float, so epithelial cells and macrophages can be distinguished.

[0070] (14) Observe the cells every day. DCs cells grow rapidly on the 4th day, and the culture medium needs to be replaced and cytokines added in time.

[0071] (15) On the 5th day, immature DCs were collected and resuspended in DCs differentiation medium (basic medium containing GM-CSF and IL-4) to 1×10 6 Note: The purity of immature DCs can be detected by flow cytometry using CD11c antibodies. Immature DCs (iDCs) with a purity >80% are ideal.

[0072] Second, the cultivation and collection of avirulent Toxoplasma NRTUA:

[0073] (1) Resuscitate HFF cells and culture them in a 37°C, 5% CO2 incubator using 10% DMEM+10% FBS+1% PS maintenance medium.

[0074] (2) When the confluence of HFF cells reaches more than 90%, the non-replicating uracil auxotrophic atoxic Toxoplasma gondii (NRTUA, the construction method of which can be found in the invention patent with publication number CN107007830A authorized by the applicant of this patent on July 4, 2020: Use, vaccine and preparation method of a composition of a non-toxic Toxoplasma gondii and traditional Chinese medicine polysaccharide adjuvant) is revived, and at the same time, the culture medium of the HFF cells is replaced with a maintenance medium containing 250 μM uracil, and NRTUA is added to the HFF cell culture flask.

[0075] (3) The HFF cell culture flask inoculated with NRTUA was placed in a 37°C, 5% CO2 incubator for culture. After 4-5 days, the NRTUA completely broke through the cell wall and could be passaged.

[0076] (4) After cell wall destruction, NRTUA was filtered out using a 3 μm diameter filter to remove cell debris. The filtered NRTUA was collected and resuspended in PBS to 1-2×10 7 The concentration of cells / ml was used for subsequent experiments.

[0077] Third, culture of CT-26 colorectal cancer cells and subcutaneous tumor formation in mice:

[0078] (1) Resuscitate mouse colorectal cancer cells CT-26 and culture them in RPMI1640+10% FBS+1% PS complete medium at 37°C in a 5% CO2 incubator.

[0079] (2) When the cells grew to the logarithmic phase, they were digested with 0.25% trypsin-EDTA and resuspended in PBS to a volume of 1.5×10 6 pcs / ml.

[0080] (3) Twenty-four female BALB / c mice aged 6-8 weeks were selected and injected subcutaneously into the right flank of each mouse with 1.5×10 5 CT-26 cells / 100 μl.

[0081] (4) The growth of subcutaneous tumors in mice was observed every 3 days, and the volume of tumors in mice was measured with a vernier caliper. The formula for calculating the volume of tumors in mice was: tumor volume (mm 3 )=0.5×long diameter (mm)×(short diameter (mm)) 2 .

[0082] (5) About 7 days after the mice were inoculated with CT-26 cells, a hard mass could be palpated at the injection site, and the tumor volume reached 30 mm. 3About, start the subsequent experiments.

[0083] Fourth, collection of non-toxic Toxoplasma gondii stimulated DCs vaccine (NRTUA-DC):

[0084] (1) The collected immature bone marrow dendritic cells (BMDC) were resuspended to 1×10 6 cells / ml, and re-plated into 6-well plates, with 5×10 cells / well 6 cells / 5 ml of culture medium.

[0085] (2) Collect NRTUA filtered through a 3 μm filter and resuspend it in PBS to 2×10 7 The concentration of NRTUA was 1:1, and it was added to DCs at a ratio of NRTUA:DC=1:1. 500 μl of NRTUA was added to each well, and the mixture was gently mixed. The cells were cultured in a 37°C, 5% CO2 incubator for 48 h.

[0086] (3) After the co-culture, DCs stimulated with NRTUA (NRTUA-DC) or unstimulated DCs (iDC) were collected and resuspended in PBS to 1×10 7 The concentration of 100 μg / ml was kept on ice for subsequent treatment.

[0087] Fifth, mouse experimental grouping and treatment: 24 CT-26 tumor-bearing mice were divided into groups. When the tumor volume reached 30 mm, 3 They were randomly divided into 4 groups and started to receive medication. The grouping is as follows:

[0088] (1) PBS: Intraperitoneal injection of 200 μl of PBS.

[0089] (2) NRTUA: intraperitoneal injection of 2×10 6 NTUA Toxoplasma / 200μl.

[0090] (3) iDC: intraperitoneal injection of 2×10 6 Unstimulated DCs / 200 μl.

[0091] (4) NRTUA-DC: intraperitoneal injection of 2×10 6 DCs were stimulated for 48 h with NRTUA:DC=1:1 per 200 μl.

[0092] (5) The body weight of each mouse was monitored every 2 days, and the tumor volume was measured. The drug was administered once every 3 days, for a total of 3 times. On the 3rd day after the last administration, the mice were killed by cervical dislocation, the tumor volume of the mice was measured, and the tumor tissue was collected for subsequent experiments.

[0093] Sixth, tumor tissue is prepared into single cells:

[0094] (1) After killing the mice by cervical dislocation, soak them in 75% alcohol for 10 seconds and separate the tumor tissues of the mice using two sets of scissors and forceps (one set for cutting the skin and the other for removing the tumor tissue).

[0095] (2) Cut the mouse tumor tissue into 1mm pieces as much as possible. 3 For small pieces, add HBSS solution containing 200U / ml collagenase IV and digest at 37℃ for 2h. Stir several times during the process, or digest with slow stirring using a blender.

[0096] (3) At the end of incubation, use a 10 ml syringe with a large-caliber needle (18-20G) to aspirate the tissue block 5-7 times to break up the remaining tissue and obtain a single cell suspension.

[0097] (4) Transfer the digested tumor tissue to a 50 ml centrifuge tube and a cell filter with a diameter of 70 μm. Gently grind it with a 5 ml syringe plunger tip, and transfer the collected cell suspension to a 15 ml centrifuge tube.

[0098] (5) Wash the cells with PBS, centrifuge at 500 g for 5 min, and discard the supernatant.

[0099] (6) Add 10 ml of ACK red blood cell lysis buffer, vortex for 3 seconds, incubate at room temperature for 3-5 minutes, centrifuge at 500g for 5 minutes, discard the supernatant, and repeat this step if the cell pellet is still red.

[0100] (7) Wash the cells with PBS, centrifuge at 500 g for 5 min, and discard the supernatant.

[0101] (8) After the separated tumor cells are fully resuspended in an appropriate amount of cell separation buffer (PBS, RPMI1640, flow cytometry buffer), they are placed on ice for later use.

[0102] Seventh, flow cytometry detection of immune cells in tumor tissue:

[0103] (1) Collect single tumor cells: Resuspend the collected single cells in PBS into a 15 ml centrifuge tube, centrifuge at 500 g for 3 min, and resuspend the cells in 1 ml flow cytometry staining buffer (PBS + 2% FBS).

[0104] (2) Cell washing: resuspend the cells, centrifuge at 500g, 4℃ for 5 minutes, discard the supernatant, and wash 1-2 times.

[0105] (3) Blocking before staining: Resuspend the cells in 100 μl flow cytometry buffer, then add 0.5 μl of CD16 / CD32 monoclonal antibody at a concentration of 0.25 μg, incubate at 4°C for 15-30 minutes, then centrifuge at 4°C, 500g for 5 minutes, and discard the supernatant. Resuspend the cells in 50-100 μl flow cytometry staining buffer and add the corresponding antibodies.

[0106] (4) Cell flow cytometry antibody staining: Prepare 1.5ml EP tubes and set up 3 replicates for each group. Resuspend the cells in 50-100μl flow cytometry staining buffer for each tube, add the corresponding surface factor antibody MIX, and the working concentration of each fluorescent antibody is 0.2μg / 100μl. The information of immune cell surface factors and their corresponding antibodies is shown in Table 1 (immunological detection indicators) and Table 2 (flow cytometry antibody information) below:

[0107] immune cells Cell surface factors CD8+ T cells CD45+,CD3+,CD8+ CD4+ T cells CD45+,CD3+,CD4+ NK cells CD45+,CD49b DCs CD45+,CD11b+,CD11c+ macrophages CD45+,CD11b+,F4 / 80+

[0108] Table 1

[0109]

[0110]

[0111] Table 2

[0112] (5) Flow cytometry antibody incubation: Incubate at 4°C for 30-60 minutes in the dark.

[0113] (6) Wash away excess antibody: Add 900-950 μl of flow cytometry buffer to each tube, centrifuge at 4°C, 500 g for 5 min, and discard the supernatant. Wash 1-2 times. Finally, add 200 μl of flow cytometry buffer to each EP tube to resuspend the washed cells.

[0114] (7) On-machine testing: Prepare an ice box and flow cytometry tubes, mark the flow cytometry tubes with corresponding labels, transfer 200μl lymphocytes to the flow cytometry tubes with corresponding labels, place them in the ice box, and wait for the test to be performed on the machine.

[0115] Eighth, enzyme-linked immunosorbent assay (ELISA) detection of immune cytokines in tumor tissue:

[0116] (1) Tissue protein extraction (operated on ice): ① Place approximately 50 mg of tumor tissue in a 1.5 ml EP tube, add 500 μl of tissue lysis buffer, and mince the tissue with scissors. The tissue lysis buffer composition is: 10 μl protease inhibitor cocktail (100×) + 990 μl RIPA protein lysis buffer; ② Use an ultrasonic disruptor to break up the tissue and let it stand for 5-10 minutes; ③ Centrifuge at 4°C, 120,000 rpm for 10 minutes, collect the supernatant, and transfer it to a new 1.5 ml EP tube.

[0117] (2) BAC method for protein content determination: ① Preparation of protein standard: Take an appropriate amount of 25 mg / ml protein standard and dilute it with PBS to a final concentration of 0.5 mg / ml; ② Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. BCA working solution is stable at room temperature for 24 hours. ③ Protein concentration determination: Add 0, 1, 2, 4, 8, 12, 16, or 20 μl of the standard to the standard wells of a 96-well plate. Add standard diluent to make up to 20 μl, which corresponds to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. Add an appropriate volume of sample to the sample wells of a 96-well plate (if the sample is less than 20 μl, add standard diluent to make up to 20 μl). Please note the sample volume. Add 200 μl of BCA working solution to each sample well and standard well, incubate at 37°C for 20-30 min, and measure the absorbance at A562 or other wavelengths between 540-595 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0118] (3) ELISA detection of the expression of immune factors in the target sample (adjust according to the instructions of the ELISA kit for each cytokine): ① Coating capture antibody: dilute the capture antibody with coating solution according to the dilution multiple in the instructions, mix well, 100 μl / well, spread into a 96-well plate, cover with tin foil, incubate in a humidified box at 4°C overnight, discard the solution, wash 3 times with washing solution (washing solution volume > 250 μl), shake and wash for 1 min / time, and try to dry the residual liquid for the last time; ② Blocking: reagent diluent, 200 μl / well, in a humidified box, incubate at room temperature for 1 hour, discard the solution, wash 3 times with washing solution, shake and wash for 1 min / time, and try to dry the residual liquid for the last time; ③ Preparation of standards and samples (during blocking): Standards: prepare 2 replicate wells for each standard concentration, and dilute the standard with reagent diluent at a 1:1 gradient for gradient selection. Take 500μl of standard solution from well 1, add 250μl of reagent diluent to wells 2-8 respectively, take 250μl of standard solution from well 1 and add it to well 2, mix well, take 250μl of standard solution from well 2 to well 3, and repeat the same operation to well 7. The concentration of standard solution is diluted 2 times from well 1 to well 7. Sample: prepare 3 replicate wells for each sample. For tumor tissue protein, dilute the sample with reagent diluent at an appropriate ratio as needed to ensure that the concentration of immune factors in the sample is within the detection range; ④ Add standard and sample: according to the layout of 96-well plate, add standard and sample to the corresponding wells, 100μl / well, mix well on a shaker, incubate at room temperature for 2h in a wet box, discard the liquid, wash 5 times with wash solution, shake and wash for 1min / time, and try to pat dry the residual liquid for the last time; ⑤ Add detection antibody (Detection ⑥ Addition of enzyme (Streptavidin-HRP): Dilute the Streptavidin-HRP with the reagent diluent according to the dilution factor specified in the instructions, 100 μl / well, incubate in a wet box at room temperature for 30 min, discard the solution, wash 7 times with the wash solution, wash 1 min / time on a shaker, and try to dry the residual liquid for the last time; ⑦ Color development: substrate TMB, 100 μl / well, in a wet box at room temperature, protected from light, color development time no more than 30 min; ⑧ Stop: stop solution (2N H2SO4), 50 μl / well, mix on a shaker, read the plate at wavelength λ = 450 nm; ⑨ Data processing and graphing.

[0119] Reference Figure 2 , the above experimental results are as follows Figure 2 As shown, Figure 2(A) Photos of tumors collected from BALB / c mice after subcutaneous inoculation of CT-26 cells and different treatments with NRTUA-DC vaccine and corresponding control groups; Figure 2 (B) BALB / c mice were subcutaneously inoculated with CT-26 cells and the tumors grew to 30 mm. 3 , Statistical graph of tumor volume change trend in patients treated with NRTUA-DC vaccine and corresponding control group; Figure 2 (C) Tumor body weights were collected from BALB / c mice subcutaneously inoculated with CT-26 cells and treated with the NRTUA-DC vaccine and the corresponding control groups. The control group was treated with PBS; the NRTUA group was treated with non-replicating uracil auxotrophic avirulent Toxoplasma gondii (NRTUA); the iDC group was treated with immature DCs isolated from bone marrow cells cultured in vitro for 7 days in DC differentiation medium; and the NRTUA-DC group was treated with DCs containing NRTUA after 5 days of in vitro culture in DC differentiation medium followed by the addition of NRTUA at a 1:1 ratio for 48 hours. Each group consisted of six mice, and each statistical data was replicated in triplicate. Quantitative data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the PBS group, or as a comparison between two groups as indicated.

[0120] like Figure 2 The experimental results showed that compared with the control group and the NRTUA and iDC treatment groups, the NRTUA-DC vaccine treatment group was able to significantly inhibit the growth of tumors in CT-26 tumor-bearing mice, and the tumor volume and weight were also significantly reduced.

[0121] Reference Figure 3 NRTUA-DC vaccine treatment changed the infiltration of immune cells and immune factors in the tumor microenvironment of CT-26 tumor-bearing mice, including Figure 3 (A) Tumor tissues were collected from BALB / c mice after subcutaneous inoculation of CT-26 cells and different treatments. Single cells were isolated from the tumor tissues, and the infiltration of CD3+ T cells, CD4+ T cells, CD8+ T cells, macrophages, NK cells, and DC cells was detected by flow cytometry. Figure 3(B) Tumor tissues were collected from BALB / c mice subcutaneously inoculated with CT-26 cells and treated with various methods. Proteins were extracted and enzyme-linked immunosorbent assay (ELISA) was used to assess the expression of IFN-γ, IL-12, TNF-α, IL-4, and IL-10. The control group was treated with PBS; the NRTUA group was treated with non-replicating uracil auxotrophic avirulent Toxoplasma gondii (NRTUA); the iDC group was treated with immature DCs isolated from bone marrow cells cultured in vitro with DC differentiation medium for 7 days; and the NRTUA-DC group was treated with DCs containing NRTUA after 5 days of in vitro culture with DC differentiation medium, followed by the addition of NRTUA at a 1:1 ratio for 48 hours. Each statistical analysis was performed with three replicates. Quantitative data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the PBS group, or as comparisons between two groups as indicated.

[0122] like Figure 3 Results showed that compared with the control group and the NRTUA and iDC-treated groups, the NRTUA-DC vaccine group significantly promoted the infiltration of CD4+ and CD8+ T cells into tumor tissues, as well as the infiltration of dendritic cells (DCs) and natural killer (NK) cells, while inhibiting the infiltration of tumor-associated macrophages. Furthermore, the expression of anti-tumor immune factors IFN-γ, IL-12, and TNF-α in tumor tissues of mice in the NRTUA-DC group was significantly increased, while the expression of IL-4 and IL-10, which promote tumor immune escape, was significantly decreased.

[0123] The applicant has conducted detailed research and experiments primarily on colorectal cancer, particularly CT-26 colorectal cancer, and obtained detailed experimental data, including the detailed and complete experimental data described in this application. Preliminary research has also been conducted on other tumors, such as liver cancer, gastric cancer, and intestinal cancer, but detailed and complete experimental data have not yet been generated. Existing data indicate that NRTUA-DC also has a certain effect on the treatment of digestive tract tumors such as liver cancer, gastric cancer, and intestinal cancer. The above embodiments are not intended to limit the scope of protection of the present invention. Equivalent modifications and variations made by those skilled in the art without departing from the overall concept of the present invention are still within the scope of the present invention.

Claims

1. Use of NRTUA-DCs obtained by stimulating DCs with non-toxic Toxoplasma gondii for the preparation of biopharmaceuticals for treating CT-26 colorectal cancer, wherein: The avirulent Toxoplasma is the non-replicating uracil auxotrophic avirulent Toxoplasma NRTUA, DCs are dendritic cells, and NRTUA-DCs are DCs containing NRTUA collected after isolated bone marrow dendritic cells were cultured in vitro for 5 days using DCs differentiation induction medium and then NRTUA was added, with NRTUA:DCs = 1:1 and co-cultured for 48 hours.

2. The use of NRTUA-DC obtained by stimulating DCs with non-toxic Toxoplasma gondii according to claim 1 for preparing a biopharmaceutical for treating CT-26 colorectal cancer, characterized in that The steps for preparing NRTUA-DCs by stimulating DCs with non-toxic Toxoplasma gondii are as follows: (1) Take a mouse and kill it, then soak the mouse in alcohol; (2) Remove the mouse skin and cut off the hind limbs near the hip and distal to the ankle; (3) Dissect the mouse femur in a 60 mm culture dish with PBS, remove the muscle around the bone, and expose the bone marrow; (4) Soak the peeled bone tissue in pre-cooled PBS until all the bone tissue is collected; (5) Soak all collected bone tissues in alcohol and then wash them with sterile PBS; (6) Pour pre-cooled PBS into a 100 cm culture dish, transfer the bone tissue into the culture dish containing PBS, and place the culture dish in a biosafety cabinet; (7) Cut the femur with sterile scissors, fix the bone with forceps, and use a syringe to draw pre-cooled PBS to flush the bone. Insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush out the bone marrow into the culture dish until the femur turns completely white. (8) Transfer the bone marrow to a centrifuge tube and centrifuge it at 1500 rpm for 3 min. Discard the supernatant after centrifugation. (9) Add ACK lysis buffer to remove red blood cells, resuspend for 15 seconds, and then centrifuge at 1500 rpm for 3 minutes to remove the ACK lysis buffer; (10) Resuspend the cells in RMPI1640 basic culture medium containing 10% FBS, 1% PS, and 55 μM 2-β-mercaptoethanol, and filter the cells using a cell strainer; (11) Adjust the cell concentration to 1-1.6×10 6 / ml, seeded in 6-well plates; (12) Add cytokines GM-CSF and IL-4 to the culture medium, and then transfer to a 37°C, 5% CO2 incubator for culture; (13) Semi-quantitative medium replacement: aspirate the old medium and replace it with new medium; (14) Observe the cells. DCs cells grow rapidly on the 4th day, and the culture medium needs to be replaced and cytokines added in time; (15) On the 5th day, immature DCs were collected and induced to differentiate using a medium containing GM-CSF and IL-4 and the cells were resuspended to 1×10 6 / ml, and re-plated into 6-well plates; (16) HFF cells were revived and cultured in 10% DMEM + 10% FBS + 1% PS maintenance medium at 37°C in a 5% CO2 incubator; (17) When the confluence of HFF cells reaches more than 90%, resuscitate the non-toxic Toxoplasma NRTUA, and replace the HFF cell culture medium with a maintenance medium containing 250 μM uracil, and add NRTUA to the HFF cell culture flask; (18) The HFF cell culture flask inoculated with NRTUA was placed in an incubator at 37°C and 5% CO2. After 4-5 days, the cell wall of the HFF cell was completely broken and the cell wall could be passaged. (19) The cell debris was filtered out by filtering the broken NRTUA with a filter, and the filtered NRTUA was collected and resuspended in PBS to a concentration of 2×10 7 / ml; add NRTUA to DCs at a ratio of NRTUA:DCs = 1:1, add 500μl of NRTUA to each well, mix gently, and then place in a 37℃, 5% CO2 incubator for 48h; (20) After the culture was completed, NRTUA-DCs were collected and resuspended in PBS to a concentration of 1×10 7 The concentration was 100 μg / ml and stored on ice.

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