Immune combination medicine and application thereof in tumor treatment

Abstract

The invention provides an immune combined medicine and application thereof in tumor treatment. The combined medicine disclosed by the invention can generate obvious synergistic interaction when being used for treating tumors, especially lung cancer and colon cancer, so that the anti-tumor effect is effectively improved, the weight and the volume of tumors are reduced, the growth of far-end tumors can be effectively inhibited, tumor metastasis can be inhibited, and the lifetime can be prolonged. In addition, the combined medicine disclosed by the invention can promote tumor specific immune memory, and has resistance to secondary invasion of same tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to drugs for immune combination and their application in treating tumors. Background Art

[0002] Lung cancer (LC) is one of the most common malignant tumors globally and also a tumor with a relatively high prevalence and fatality rate among cancer patients. Since LC usually has no obvious symptoms in the early stage, most patients have reached the advanced stage at the time of diagnosis, which increases the difficulty and complexity of LC treatment. In addition, LC is characterized by high invasiveness and rapid metastasis, making treatment even more difficult. The 5-year overall survival rate of LC surgical treatment ranges from 41% (stage IIIA) to 92% (stage IAI). Even in combination with traditional chemotherapy regimens and neoadjuvant therapies, the improvement in the prognosis of LC is limited, with only a 5.0%-5.4% increase in the 5-year survival rate.

[0003] Colorectal cancer (CRC) is a colorectal epithelial tumor that penetrates the muscularis mucosa and infiltrates into the submucosa, and can occur in any part of the colon or rectum. This tumor can appear in any part of the colorectum, with the rectum and sigmoid colon being the most common sites, followed by the cecum, ascending colon, descending colon, and transverse colon.

[0004] In recent years, immunotherapy has made certain progress in the treatment of LC and CRC. Immune checkpoint inhibitors (ICIs), such as Atezolizumab, Nivolumab, Lpilimumab, etc., have been approved by the FDA and can extend the survival of patients with advanced LC and CRC, but only about 20% of patients can benefit from them. ICIs and other immunomodulators mainly exert their anti-tumor effects by activating tumor-specific T cell responses. Dendritic cells (DCs), especially cDC1, are crucial for maintaining the T cell anti-tumor response. However, due to the complexity of the tumor microenvironment (TEM), phagocytic antigen DCs often remain in an immature immune tolerance state. In this state, the expression levels of MHC and co-stimulatory molecules on DCs are relatively low, the expression of inhibitory molecules is relatively high, and they secrete immunosuppressive cytokines, thereby inducing T cell tolerance through different signal transduction pathways. Therefore, how to break the immune tolerance state and enable T cells to function normally and produce an anti-tumor response is a key issue in tumor treatment. Summary of the Invention

[0005] Based on this, the object of the present invention is to provide an immunotherapy combination drug and its application in treating tumors. The immunotherapy combination drug can synergistically enhance the efficacy, effectively improve the anti-tumor effect, and inhibit tumor metastasis and recurrence.

[0006] To achieve the above object, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides the application of the combination of HMGN1 and Telratolimod compositions and α-CD137 in the preparation of a drug for treating tumors.

[0008] In some embodiments, the application depends on the activation of anti-tumor responses by DC1s that are transcriptionally dependent on Batf3.

[0009] In some embodiments, the application depends on CD8 + T cell responses.

[0010] In some embodiments, the application includes promoting T cells to secrete cytokines.

[0011] In some embodiments, the cytokines include IFN-γ and Granzyme B.

[0012] The second aspect of the present invention provides the application of the combination of HMGN1 and Telratolimod compositions and α-CD137 in the preparation of a drug for inhibiting tumor metastasis.

[0013] The third aspect of the present invention provides the application of the combination of HMGN1 and Telratolimod compositions and α-CD137 in the preparation of a drug for inhibiting tumor recurrence.

[0014] In some embodiments, the tumors include lung cancer and colon cancer.

[0015] The fourth aspect of the present invention provides a combination drug for anti-tumor, inhibiting tumor metastasis or inhibiting tumor recurrence. The active ingredients of the combination drug include HMGN1, Telratolimod, and α-CD137.

[0016] In some embodiments, the tumors include lung cancer and colon cancer.

[0017] The inventors of the present invention have found through a large number of studies that the combination drug composed of HMGN1 and Telratolimod compositions and α-CD137 can produce obvious synergistic effects in treating tumors, especially in treating lung cancer and colon cancer, effectively improve the anti-tumor effect, reduce the tumor weight and volume, and can effectively inhibit the growth of distal tumors, inhibit tumor metastasis, and prolong the survival period. In addition, the combination drug can promote tumor-specific immune memory and is resistant to the re-invasion of the same type of tumor cells.

[0018] The anti-tumor effect of the combined drugs of the present invention is significantly enhanced, and there are no obvious toxic and side effects, providing new ideas and strategies for the treatment of tumors, especially for the treatment of lung cancer and colon cancer. Description of the Drawings

[0019] Figure 1 It shows the detection results of the tumor progression of mice in each group.

[0020] Figure 2 It shows the detection results of the body weight, liver and kidney function indexes of mice in each group.

[0021] Figure 3 It shows the tumor progression results of the treatment group and the non-treatment group of wild-type and Batf3 - / - mice.

[0022] Figure 4 It shows the tumor progression results of mice given α-CD4, α-CD8, α-NK1.1 monoclonal antibodies during the treatment process.

[0023] Figure 5 It shows the results of the activation of antigen-specific T cells in mice in each group.

[0024] Figure 6 It shows the results of IFN-γ secretion of antigen-specific T cells in mice in the treatment groups of wild-type and Batf3 - / - mice.

[0025] Figure 7 It shows the detection results of the inhibition of distal tumor growth in mice in each group.

[0026] Figure 8 It shows that the combination therapy can stimulate the immune protection ability of the body. Detailed Embodiments

[0027] In the following embodiments of the present invention, the experimental methods without specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers. All kinds of commonly used chemical reagents used in the embodiments are commercially available products.

[0028] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] The terms "comprise" and "include" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products or devices.

[0030] DCs

[0031] DCs are currently considered the most powerful antigen-presenting cells (APCs). Their antigen-presenting ability far exceeds that of other APCs. They can efficiently process and present exogenous antigens to MHC I and MHC II molecules and present them to CD4 + T and CD8 + T cells, thereby initiating an adaptive immune response. However, inhibitory immune cells present in TEM and the cytokines they release can cause DCs to be in an immature or tolerogenic state. These immature DCs cannot effectively activate T cells to produce an anti-tumor immune response. On the contrary, they may promote angiogenesis by producing angiogenic factors and enhancing endothelial cell migration, thus ultimately promoting tumor growth. Therefore, in the process of combating tumors, it is crucial to generate a large number of phenotypically and functionally mature DCs.

[0032] Immunotherapy strategies using DCs as an entry point are mainly based on the ability of DCs to coordinate innate and adaptive immune responses. The goal of DC cancer vaccines is to induce and enhance tumor-specific cellular and humoral immune responses, delay tumor growth, induce tumor immune cell memory, and thereby inhibit tumor recurrence. The key to successfully preparing DC cancer vaccines lies in providing relatively optimal specific tumor antigens for mature DCs. DC cancer vaccines are generally divided into two types: (1) Loading antigens after culturing DCs in vitro and then reinfusing: In this method, DCs are obtained from the patient, cultured and processed in vitro, loaded with tumor antigens (activated), and then reinfused into the patient to induce a tumor-specific immune response; (2) Inducing DCs to mature, capture and present antigens in vivo (inducing the activation of endogenous DCs): This method involves using various DC proliferation and activation induction preparations in the patient's body to promote DCs to mature, capture and present tumor antigens, thereby triggering a tumor-specific immune response. Compared with the former, the latter is relatively simple, easy to implement, safe, and its effect has systemic and integrity in vivo.

[0033] High mobility group nucleosomal protein 1

[0034] In 2005, Joost J. Oppenheim and Professor Yang De first proposed the concept of "alarmin". Alarmin refers to a class of endogenous mediators with diverse structures. When the body is stimulated by danger signals, these alarmins can be rapidly released extracellularly, and then play the role of chemotaxing and activating DCs, monocytes and macrophages, thereby stimulating and promoting the body's immune response. The release and action of alarmins play an important regulatory role in the immune response, helping the body to promptly make an immune reaction to counter external threats.

[0035] High-mobility group nucleosome-binding protein 1 (HMGN1; TLR4 agonist), a new alarmin discovered in recent years, has the ability to induce the phenotypic and functional maturation of DCs and can recruit DCs in vivo. HMGN can activate and recruit DCs from humans and mice, and promote the development of Th1 polarized immune responses on the basis of antigen co-action. HMGN1 induces the phenotypic and functional maturation of DCs through the TLR4 - myeloid differentiation primary response gene 88 (MyD88) - TIR domain-containing adapter-inducing interferon-β pathway. Therefore, as a new alarmin, the ability of HMGN1 in DC recruitment and activation and enhancing adaptive immune responses makes it play an important role in the human immune system.

[0036] Telratolimod

[0037] Telratolimod (tilotolimod; 3M-052; TLR7 / 8 agonist) is a novel TLR7 / 8 agonist that activates immune cells through the TLR7 / TLR8 - MyD88-dependent signaling pathway. Its lipid-modified physical property enables it to stay at the vaccination site, form a depot and release slowly, thereby enhancing the local Th1 cell immune response while reducing the systemic drug level and cytokine release without the release of systemic cytokines. This property helps to prolong the immune activation time in the tumor and tumor-draining lymph nodes, which is crucial for promoting the anti-tumor immune response, because the rapid diffusion of the drug in the body may cause systemic immune activation without effectively activating anti-tumor activity.

[0038] α-CD137

[0039] CD137 (also known as 4-1BB or TNFRSF9) is a target that has received much attention. It belongs to the tumor necrosis factor receptor (TNFR) superfamily and is an inducible co-stimulatory receptor. It is mainly expressed on activated T cells and natural killer cells and mediates co-stimulation of these two types of lymphocytes. In addition, CD137 is also expressed on activated macrophages, B cells and DCs.

[0040] On T cells, CD137 ligation can trigger a signaling cascade that leads to upregulation of anti-apoptotic molecules, cytokine secretion, and enhanced effector functions. In dysfunctional T cells, CD137 ligation shows the ability to restore their effector functions. In activated NK cells, CD137 is also significantly expressed, and its signaling can increase antibody-dependent cell-mediated cytotoxicity. Based on the understanding of CD137 signaling, specific antibodies targeting CD137 have been developed as agonists, namely anti-CD137 (α-CD137), for cancer immunotherapy.

[0041] The following is an illustration with specific examples.

[0042] Since the research involved in the present invention uses mice, the α-CD137 used is an anti-CD137 antibody against mice. For example, the anti-mouse CD137 antibody (also known as anti-mouse 4-1BB antibody) with the catalog number BE0239 purchased from BioxCell was used in the following examples. The monoclonal antibody with the clone number 3H3 can react with mouse CD137, which is a member of the TNF receptor superfamily and is also known as CD137. CD137 is a 39 kDa transmembrane protein expressed by T lymphocytes, NK cells, dendritic cells, granulocytes, and mast cells. After binding to its ligand CD137L, CD137 provides co-stimulatory signals to CD4 and CD8 T cells by activating the downstream pathways of NF-κB, c-Jun, and p38.

[0043] Currently, multiple CD137 monoclonal antibodies have entered clinical research. Among them, Urelumab (BMS-663513, IgG4) from Bristol-Myers Squibb Company and Utomilumab (PF-05082566, IgG2) from Pfizer Company have the fastest progress and are both in clinical phase II.

[0044] In the following examples, the composition of HMGN1 and telotristat ethyl (3M-052) is abbreviated as CisVac, and CisVac combined with α-CD137 forms a combined drug.

[0045] Example 1

[0046] I. Experimental methods

[0047] 1.1 Experimental reagents and materials

[0048] 1.1.1 Cell lines and their cultivation

[0049] The mouse lung cancer cell line LLC was purchased from Wuhan Procell Life Science & Technology Co., Ltd. The mouse MC38 colon cancer and KP13 lung cancer cell lines were respectively from the research groups of Professor Xu Chenqi and Professor Ji Hongbin of the Shanghai Institute of Biochemistry and Cell Biology. The MC38-OVA colon cancer cell line was from the research group of Professor Deng Liufu of Shanghai Jiao Tong University. The PANC02 pancreatic cancer cell line was from the research group of Professor Liu Hui of the Fifth Affiliated Hospital of Guangzhou Medical University.

[0050] 1.1.2 Experimental animals

[0051] The C57BL / 6J mice (6 - 8 weeks old, female, over 18 g) and OT-1 mice used in this experiment were purchased from Zhaoqing Resyuan Biotechnology Co., Ltd. and Jacksonlab in the United States respectively. The Batf3 - / - mice were gifted by the laboratory of Professor Leng Qibin. The Batf3 - / - and OT-1 mice were bred in the SPF animal experiment center of Resyuan Biotechnology Co., Ltd. and then transported to the animal center of Yuexiu Campus of Guangzhou Medical University through a sealed process for experiments. During the experiment, the mice were 6 - 10 weeks old and weighed over 18 g, regardless of gender. The ethical review number for animal experiments in this paper is 20230055, which was approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.

[0052] 1.1.3 Main experimental reagents and consumables

[0053] The main experimental reagents and consumables are shown in Tables 1 - 3.

[0054] Table 1 Main experimental reagents

[0055]

[0056]

[0057] Table 2 Main antibodies

[0058]

[0059]

[0060] Table 3 Main experimental consumables

[0061]

[0062] 1.2 Preparation of main reagents

[0063] (1) Preparation of DMEM cell culture medium

[0064] The serum was thawed from a -80°C freezer, inactivated in a 37°C water bath, and aliquoted for storage. After filtration through a 45μm filter, the serum was added to DMEM at a ratio of 10% FBS and 1% P / S antibody, and stored in a 4°C refrigerator until use.

[0065] (2) Preparation of HMGN1 protein solution

[0066] Gently shake the reagent bottle to allow the 50ng protein to precipitate to the bottom. Then add 100μL of sterile PBS buffer (concentration of 2ng / μL) to each bottle. If a white precipitate appears, let it stand at room temperature for 30 minutes or refrigerate at 4°C overnight before processing. Aliquot each tube into 10μL portions and store in a -80°C refrigerator for three months. When using, remove the sample and dilute it to a concentration of 0.01ng / μL with sterile PBS buffer. All operations must be performed in a clean bench.

[0067] (3) Preparation of 3M-052 solution

[0068] Centrifuge the sample at 10,000 rpm for 30 seconds. Then, in a clean bench, add 20 μL of DMSO solution. Ultrasonicate the sample in a 37°C ultrasonic bath for 5 minutes. Repeat this step once. Then, add 2500 μL of sterile PBS buffer and aliquot the mixture into 500 μL tubes. All steps were performed in a clean bench.

[0069] (4) Tissue digestive fluid

[0070] Preparation of collagenase IV solution: Dissolve 100 mg of collagenase I in 5 mL of RPMI 1640 medium (20 mg / mL), store in aliquots at -20°C. Dilute to 1 mg / mL in RPMI 1640 medium before use.

[0071] Preparation of DNase I solution: Dissolve 25 mg of DNase powder in 5 mL of RPMI 1640 (5 mg / mL). Aliquot and store in a -20°C refrigerator. Dilute to 0.1 mg / mL before use.

[0072] Preparation of tumor tissue digestion solution: 1 mg / mL collagenase IV + 0.1 mg / mL DNaseI + RPMI 1640.

[0073] (5) 1× red blood cell lysis buffer

[0074] Mix 10× red blood cell lysis buffer and ddH2O at a ratio of 1:9 and shake well.

[0075] (6) 1×Perm Buffer PB

[0076] Mix 10× Perm Buffer PB with ddH2O in a ratio of 1:9 and shake well.

[0077] (7) FACS Buffer

[0078] Fully dissolve 2% BSA bovine serum in PBS and mix well.

[0079] 1.3 Experimental methods

[0080] 1.3.1 Cell culture

[0081] (1) Cell resuscitation: Before the experiment, preheat the DMEM complete medium, and place the required reagents and consumables in the ultra-clean workbench for 30 minutes of ultraviolet disinfection. After taking out the cells from the -80°C refrigerator, quickly thaw the cells with the preheated complete medium in the ultra-clean workbench. Transfer the cells to a 15 mL centrifuge tube, centrifuge at 1200 rpm at room temperature for 5 minutes, remove the supernatant, resuspend the cells with 2 mL of medium, and then transfer them to a T25 culture flask. Add 3 mL of preheated DMEM complete medium. Place the culture flask in an incubator with 5% CO2 at 37°C for culture.

[0082] (2) Cell passage: During the experiment, observe the cell morphology every day. When the cell density in the culture flask reaches about 80%, passage the cells. The passage operation includes removing the original medium, washing with PBS and discarding it, and adding 0.25% EDTA. After observing that the cell volume shrinks under the microscope, add 3 times the volume of the complete medium to terminate the reaction. Centrifuge to remove the supernatant, resuspend the cells, and then transfer them to a new culture flask and add an appropriate amount of complete medium for culture. After the cells are passaged three times, collect the supernatant to check for mycoplasma. After confirming that the cells are free of mycoplasma infection, continue the culture.

[0083] (3) Cell cryopreservation: When the cell density reaches 90%, cryopreserve the cells. According to the cell passage method, digest the cells and resuspend them with serum-free cryopreservation medium. Subsequently, aspirate 1 mL of the suspension into a cryopreservation tube, label it, and store it at -80°C.

[0084] 1.3.2 Primary mouse tumor treatment protocol

[0085] (1) Establishment of tumor model and treatment protocol: For LLC, KP13, PANC2, MC38, and MC38-OVA tumor cells without mycoplasma contamination after detection, when the cells are in exponential growth, digest the cells with trypsin and wash the cells twice with sterile PBS. Inoculate 1×10 6For each cell, tumor cells will be inoculated subcutaneously (s.c.) into the right dorsal side of wild-type C57BL / 6J or Batf3 mice using an insulin syringe. The length and width of the tumor will be measured every two days using vernier calipers until the end of the experiment. Mice will be sacrificed when the tumor volume reaches 1500 mm - / - . The tumor calculation formula is: length × width × width / 2. Before inoculation, the hair on the back of the mice will be removed to facilitate inoculation and subsequent tumor observation. 3 When the tumor diameter grows to 8 mm - 10 mm (LLC tumor cells inoculated for 12 days, KP13 tumor cells inoculated for 10 days, MC38 tumor cells inoculated for 9 days), treatment of tumor-bearing mice will begin. At this time, the tumor-bearing mice will be randomly divided into four groups: PBS group (as a control), CisVac group (containing HMGN1 and 3M-052): intratumoral injection of CisVac (HMGN1: 0.5 ng; 3M-052: 20 μg; 0.1 mL) on days 0, 4, and 8 after the start of treatment, α-CD137 group: intraperitoneal injection of α-CD137 (200 μg; 0.2 mL) on days 1 and 6 after the start of treatment, CisVac + α-CD137 group: intratumoral injection of CisVac (HMGN1: 0.5 ng; 3M-052: 20 μg; 0.1 mL) on days 0, 4, and 8 after the start of treatment, and intraperitoneal injection of α-CD137 (200 μg; 0.2 mL) on days 1 and 6 after treatment. For the immune cell depletion experiment, mice will be intraperitoneally injected with 200 μg of isotype control IgG, α-CD4, α-CD8, α-NK1.1 monoclonal antibodies and treated simultaneously with CisVac and α-CD137. For mice without obvious tumor foci, tumor re-inoculation will be performed after 100 days. The treatment experiments for the three types of tumor-bearing mice will be repeated two or three times.

[0086] 1.3.3 Distal effect

[0087] 1 × 10

[0088] and 0.5 × 10 6 MC38 tumor cells will be injected into the right (primary tumor) and left (secondary tumor) sides of C57BL / 6J female mice respectively. On the tenth day, when the tumor on the right grows to approximately 8 mm and the tumor on the left can be palpated, these mice will be randomly divided into 4 groups: PBS group, CisVac group, α-CD137 group, CisVac + α-CD137 group. The administration methods and doses for each group are the same as in 1.3.2. CisVac will be injected into the tumor on the right on days 10, 14, and 18, and the tumor on the left will not receive treatment. α-CD137 will be intraperitoneally injected on days 11 and 16 with or without combination. The tumor size will be detected every two days. 6

[0089] ​1.3.4 Detection by Flow Cytometry

[0090] (1) Preparation of Single-Cell Suspension

[0091] After sacrificing the mice, use surgical instruments to dissect the spleens, and then place them in a six-well plate containing 2 mL of pre-cooled PBS medium. Gently grind the spleens and filter them into a 15 mL centrifuge tube. After centrifugation to remove the supernatant, resuspend the cells with 1×RBC and let it stand for 5 minutes to fully lyse the red blood cells in the spleens. Add 5 volumes of PBS to terminate the reaction. After centrifugation to remove the supernatant, resuspend to obtain a single-cell suspension of the spleens.

[0092] (2) Surface Cell Staining

[0093] For the single-cell suspensions obtained from different tissues, pipette an appropriate amount of the cell suspension and transfer it to a flow tube. Add 100 μL of Fc block to each tube, mix well and incubate in a 4°C refrigerator for 30 minutes. Then add 1 mL of PBS and centrifuge to remove the supernatant. Next, perform cell surface staining. Add the required flow antibodies to 0.1 mL of the cell suspension, including Viability, TCR, CD4, CD8, and staining antibodies. Among them, the Viability antibody is diluted with PBS at a ratio of 1:2000, and the other antibodies are diluted at a ratio of 1:200. After incubating in a 4°C refrigerator for 30 minutes, add 1 mL of PBS to terminate the reaction. After centrifugation to remove the supernatant, add 0.3 mL of PBS to resuspend, and then flow cytometry analysis can be performed.

[0094] (3) Intracellular Staining

[0095] For the detection of secreted cytokines by intracellular staining, after surface staining, add 0.5 mL of 1× Fixation / Permeabilization buffer, incubate in the dark at 4°C for 25 minutes, then centrifuge to remove the supernatant. Then use PB and let it stand at 4°C for 30 minutes. Subsequently, perform intracellular staining. Stain with IFN-γ and Granzyme B antibodies and PB at a ratio of 1:200 for half an hour at 4°C. Then terminate the reaction with PB, centrifuge to remove the supernatant, and resuspend with PB, and then it can be detected using a flow cytometer.

[0096] 1.3.5 Detection of Cytokines

[0097] Using the MC38-OVA tumor as a model, on the third day after the end of treatment of tumor-bearing mice, the mice were sacrificed, the spleens of the mice were removed to make single-cell suspensions, and the cells were stimulated with the MC38-OVA cell lysate. One day before the experiment, the cultured MC38-OVA tumor cells were stimulated with mouse IFN-γ overnight, the tumor cells were irradiated with 60 Gy, and the splenocytes and the irradiated tumor cells were added to a 96-well round-bottom plate at a ratio of 10:1, stimulated for 16 hours, and BFA was added and cultured for another 3 hours, followed by cytokine detection.

[0098] 1.3.6 ELISpot

[0099] Pre-coat the PVDF membrane culture plate with the AN18 antibody and wash off the antibody the next day. Add 200 μL of 1640 medium containing 10% FBS to each well and incubate at room temperature for 30 minutes. After washing off the medium, add 400,000 splenocytes to each well, and stimulate with the irradiated MC38-OVA cell lysate in a cell culture incubator at 37 °C and 5% CO2 for 28 hours. Set non-stimulation as the negative control and ConA as the positive control. After the stimulation, wash off the remaining cells in the wells, dilute R4-6A2-biotin to 1 μg / mL with PBS containing 0.5% FBS, add 100 μL to each well, and incubate at room temperature for 2 hours. Wash off the remaining liquid, dilute streptavidin 1000-fold with PBS containing 0.5% FBS and add 100 μL to each well, and incubate at room temperature for 1 hour. Wash off the streptavidin, add 100 μL of BCIP / NB chromogenic substrate to each well and develop until obvious spots appear. All the above processes are carried out under sterile conditions. Finally, rinse the culture plate repeatedly with tap water to terminate the color development. After drying the culture plate in a dark place, it can be counted using an enzyme-linked immunospot analyzer.

[0100] 1.3.7 Statistical analysis

[0101] Use GraphPad Prism 9.0 software to perform statistical analysis and graphing on the data. One-way ANOVA is used for single-factor comparison, two-way ANOVA is used for the tumor growth curve, and Log-rank analysis is used for the difference in the survival curve of mice. When the P value < 0.05, it is judged that there is a statistical difference between the two groups. In the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0102] II. Experimental results

[0103] 2.1 CisVac and α-CD137 synergistically inhibit the progression of multiple mouse tumors

[0104] The treatment regimen is as Figure 1As shown in A. In LLC lung cancer-bearing mice, administration of CisVac or α-CD137 could slow down the tumor growth rate, while combination therapy showed a significant synergistic effect and effectively inhibited tumor growth ( Figure 1 in B). In LLC tumor-bearing mice receiving combination therapy, 2 out of 15 mice had tumor regression and survived for more than three months ( Figure 1 in E). However, in the KP13 lung cancer tumor model, although the treatment regimen could slow down tumor growth, tumor regression was not observed ( Figure 1 in C, Figure 1 in F). In the MC38 colon cancer tumor model, due to the high immunogenicity of this model, compared with the control group, CisVac or α-CD137 could effectively inhibit tumor growth, while 8 / 15 tumor-bearing mice in the combination therapy group had complete tumor regression and a significantly increased survival period ( Figure 1 in D, Figure 1 in G). Therefore, in these tumors, the treatment regimen of CisVac combined with α-CD137 effectively alleviated tumor growth. However, due to the differences in tumor immunogenicity among different tumor-bearing mice, tumors with low immunogenicity may not regress.

[0105] Figure 1 In, (A) Schematic diagram of the mouse treatment regimen. (B, C, D) Show the average tumor growth volume of different treatment groups in LLC (n = 15), KP13 (PBS n = 8, others n = 10), and MC38 (n = 15) tumor-bearing mice respectively. (E, F, G) Are the survival days of tumor-bearing mice in different treatment groups of LLC, KP13, and MC38 respectively (***P < 0.001, ****P < 0.0001; mean ± SEM).

[0106] Next, we analyzed the synergistic effect between CisVac and α-CD137 and calculated their interaction index CI. The calculation method is as follows: CI = AB / (A × B), where T is the tumor inhibition rate of the experimental group, C is the tumor inhibition rate of the control group, AB is the T / C value of the combination therapy group, and A and B are the T / C values of CisVac and α-CD137 alone, respectively. When CI < 1, it indicates that combination drug use produces a synergistic effect.

[0107] After calculation, the CI values of CisVac and α-CD137 are shown in Table 4.

[0108] Table 4

[0109]

[0110] As can be seen from Table 4, CisVac and α-CD137 showed significant synergistic enhancement.

[0111] 2.2 There were no obvious toxic or side effects in combination therapy

[0112] During the treatment of LLC, KP13, and MC38 tumor-bearing mice, we closely monitored their body weight changes. The results showed that compared with the PBS control group, there were no obvious body weight changes in the mice treated with combination therapy or single-agent therapy, indicating that the treatment regimen had no obvious adverse effects on the overall health status of the mice ( Figure 2 in A- Figure 2 in C). In addition, at the end of the treatment, HE staining was performed on the liver, kidneys, and lungs of MC38 tumor-bearing mice, and no obvious organ damage was found, further indicating the safety of combination therapy ( Figure 2 in D).

[0113] Figure 2 in, (A-C) are the average body weight changes of LLC (n = 15), KP13 (PBS n = 8; others n = 10), and MC38 (n = 15) tumor-bearing mice during different treatment processes, respectively. Representative images of HE staining of the organs of MC38 tumor-bearing mice after treatment (n = 5) (ns P>0.05; mean±SEM).

[0114] In summary, compared with the PBS control group, neither combination therapy nor single-agent therapy caused obvious abnormalities in body weight, liver, and kidney functions, indicating the safety of the treatment. This provides a beneficial reference for the further clinical application of this treatment regimen and also provides a safety basis for the development and clinical transformation of drugs.

[0115] 2.3 Combination therapy depends on Batf3-transcribed DC1

[0116] The Batf3 gene encodes a transcription factor that plays an important role in the development and function of DCs. Batf3 - / - mice lack certain types of DCs, especially cDC1. To explore the role of DCs in combination therapy in depth, we established a tumor model using Batf3 - / - mice, with wild-type mice as the control. When the tumor diameter reached 8-10 mm, we divided different types of mice into two groups: the PBS untreated group and the combination therapy group, and observed tumor growth and mouse survival. The research results showed that compared with the wild-type mouse treatment group, the tumors in the Batf3 - / - mouse treatment group were not effectively inhibited, resulting in the death of all mice ( Figure 3 in A- Figure 3 in B). These results indicate that cDC1 plays a key role in the process of combination therapy.

[0117] Figure 3 in, 1×106 MC38 tumor cells were separately inoculated subcutaneously into the right dorsal side of wild-type mice and Batf3 - / - mice, and the tumor growth curves of the control group (PBS) and the treatment group (CisVac / α-CD137) were observed ( Figure 3 in A) and the survival curves of the mice ( Figure 3 in B) (n = 5) (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM).

[0118] 2.4 The combination therapy mainly relies on CD8 + T cell responses

[0119] To further determine which type of lymphocyte mainly plays an important role during the combination therapy, we used anti-CD4, CD8, and NK1.1 monoclonal antibodies to deplete the corresponding immune cells in mice and observed the tumor growth during the treatment ( Figure 4 in A). Compared with IgG, after administration of α-CD8 monoclonal antibody in the combination therapy group, the average tumor growth in mice was inhibited to a certain extent. However, since the depletion of CD8 + T cells in the body was not sufficient to generate an effective anti-tumor immune response, the inhibited tumors would relapse later, resulting in the complete death of mice due to tumor burden, with a survival rate of 0 / 5, while 3 / 5 survived in the combination therapy group (Therapy). After the combination therapy, when α-CD4 monoclonal antibody was given, the overall survival of the mice was significantly different compared with the IgG control group. However, compared with the combination therapy group, only 2 mice were cured. After administration of α-NK1.1 monoclonal antibody, compared with the combination therapy group, there was no significant difference in the average tumor growth and overall survival of the mice, and 3 mice were cured in both groups ( Figure 4 in B - Figure 4 in C). These data indicate that the combination therapy mainly relies on the response of CD8 + T cells in the body.

[0120] Figure 4 In, (A) Schematic diagram of drug administration to tumor-bearing mice. (B) Average tumor growth curves of 5 treatment groups: IgG, α-CD8 / Therapy, α-CD4 / Therapy, α-NK1.1 / Therapy, Therapy (CisVac / α-CD137) and (C) Survival curves of tumor-bearing mice in each treatment group (n = 5, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM).

[0121] 2.5 The combination therapy enhances the efficacy through cytokines secreted by T cells

[0122] Antigen-specific T cells play an important role in tumor immune responses. They can recognize specific antigens on the surface of tumor cells and release cytotoxins such as granzymes and perforins, directly leading to apoptosis of tumor cells. At the same time, activated antigen-specific T cells can also release cytokines such as IFN-γ, regulate the immune environment, activate other immune cells in the body, and further enhance the immune response. To comprehensively evaluate the response of T cells to tumor antigens, we used irradiated tumor cell preparations as antigens because they contain a variety of antigens released by tumor cells, which can promote the activation of various cells in the body and activate the responses of multiple antigen-specific T cells. Studies have shown that the activated specific CD8 + T cells secreted significantly more IFN-γ cytokine and Granzyme B ([[]] Figure 5 in A- Figure 5 in B), and at the same time, the secretion of IFN-γ cytokine and Granzyme B by CD4 + T cells also increased ([[]] Figure 5 in C- Figure 5 in D). These results indicate that combination therapy enhanced the function of specific T cells in the body and improved the effect of anti-tumor immune responses.

[0123] Figure 5 In, the statistics of IFN-γ (A) and Granmyze B (B) secreted by activated CD8 + T cells, and the statistics of IFN-γ (C) and Granmyze B (D) secreted by activated CD4 + T cells are shown. The left shows the flow cytometry representative diagram, and the right is the statistical chart.

[0124] 2.6 Combination therapy exerts its efficacy by activating T cells through Batf3-transcribed DC1

[0125] The levels of IFN-γ secreted by T cells in different treatment groups on the 2nd day after treatment were detected by ELISpot. Compared with the PBS group, the IFN-γ level in the CisVac / α-CD137 group was significantly increased, highlighting the enhanced functional activity of activated tumor antigen-specific T cells ([[]] Figure 6 in A). To clarify the role of the antigen cross-presentation function of Batf3-dependent classical type 1 dendritic cells (cDC1) in T cell activation, we used wild-type (WT) and Batf3 gene knockout (Batf3 - / -) Mice were subcutaneously inoculated with MC38 tumor cells and were treated with PBS control or CisVac / α-CD137 combination therapy, respectively. The level of IFN-γ secreted by T cells in the spleen on the 2nd day after treatment was detected by ELISpot. The results showed that in WT mice, the CisVac / α-CD137 treatment group significantly increased the IFN-γ + T cell frequency compared with the PBS control group, while in Batf3 - / - mice, this treatment only caused a weak increase in IFN-γ without statistical significance ([ Figure 6 in B). This indicates that cDC1-mediated antigen cross-presentation is the main pathway for functional activation of T cells. Although Batf3 - / - mice completely lack cDC1, low levels of IFN-γ secretion can still be detected in the treatment group, suggesting that α-CD137 may trigger partial activation through non-classical pathways (such as directly binding to the CD137 receptor on the surface of T cells). However, the intensity of this activation is significantly weaker than the cDC1-dependent antigen presentation pathway.

[0126] Figure 6 In, (A) Enzyme-linked immunosorbent spot (ELISpot) analysis of IFN-γ spot-forming cells in splenocytes of WT mice stimulated with irradiated MC38-OVA tumor cells. (B) Detection of IFN-γ secretion in PBS and treatment groups (CisVac / α-CD137) of WT and Batf3 - / - MC38-OVA tumor-bearing mice. Left: Mean count; Right figure: Representative pictures of each group (ns: not significant, **P < 0.01, ****P < 0.0001; mean ± SEM).

[0127] 2.7 Combination therapy effectively inhibits the growth of distant tumors

[0128] We further explored whether the combination of CisVac and α-CD137 in the treatment of primary tumors could inhibit the growth of distant tumors and thus trigger a systemic anti-tumor immune response. To verify this conjecture, we established a bilateral tumor model of MC38 colon cancer and observed the growth of bilateral tumors ( Figure 7 in A). The research results showed that compared with the PBS control group, α-CD137 treatment group and CisVac treatment group, the CisVac / α-CD137 treatment group not only effectively inhibited the growth of primary tumors, but also significantly inhibited the growth of distant tumors CD137 ( Figure 7 in B), indicating that combination therapy can induce a systemic anti-tumor immune response. This systemic anti-tumor effect may be closely related to mechanisms such as immune cell activation, T cell proliferation, cytokine release, and immune memory formation induced by the combination therapy of CisVac and α-CD137.

[0129] Figure 7 Among them, (A) Schematic diagram of establishing bilateral tumor model. (B) Right side: Average growth curve of primary tumor; Left side: Average growth curve of distal tumor (n = 5, ns P > 0.05, **P < 0.01, ****P < 0.0001; mean ± SEM).

[0130] 2.8 Combination therapy can activate anti-tumor immune protection ability

[0131] After observing tumor-bearing mice with significant inhibitory effects for up to 100 days, in order to observe whether the cured MC38 tumor-bearing mice have specific immune protection against the same tumor antigen, we re-inoculated MC38 tumors at the primary site and inoculated PANC2 mouse pancreatic cancer on the contralateral side as a control ( Figure 8 as shown in A). The study showed that the cured mice were resistant to the re-invasion of the same tumor cells, but could not inhibit the invasion of another unrelated tumor ( Figure 8 as shown in B). To exclude the influence of the inoculation site, we inoculated PANC2 as a control at the primary site and inoculated MC38 tumors on the contralateral side ( Figure 8 as shown in C). The results showed that the tumors of 5 / 7 mice did not grow ( Figure 8 as shown in D). It indicated that the CiSVac / α-CD137 combination therapy could promote tumor-specific immune memory.

[0132] Figure 8 Among them, (A) Schematic diagram of ipsilateral inoculation of MC38 and contralateral inoculation of PANC02 tumor cells. (B) Average tumor growth curves of tumor regression (red line) and naive (black line) mice of contralateral PANC02 (right) and primary site MC38 tumor (left) (n = 5). (C) Schematic diagram of contralateral inoculation of MC38 and ipsilateral inoculation of PANC02 cells. (D) Average growth curves of contralateral MC38 (right) and ipsilateral PANC02 tumor (left) (n = 7).

[0133] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0134] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. Use of a combination of HMGN1 and Telratolimod compositions with α-CD137 in the preparation of a medicament for treating tumors.

2. The application according to claim 1, characterized in that The said use depends on the activation of anti-tumor responses by DC1 transcribed by Batf3.

3. The application according to claim 1, wherein The said use depends on CD8+ T cell responses.

4. The application according to claim 1, characterized in that, The said use includes promoting the secretion of cytokines by T cells.

5. The application according to claim 4, wherein, The said cytokines include IFN-γ and granzyme B.

6. Use of a combination of HMGN1 and Telratolimod compositions with α-CD137 in the preparation of a medicament for inhibiting tumor metastasis.

7. Use of a combination of HMGN1 and Telratolimod compositions with α-CD137 in the preparation of a medicament for inhibiting tumor recurrence.

8. The application according to any one of claims 1 to 7, characterized in that, The said tumors include lung cancer and colon cancer.

9. A combined drug for anti-tumor, inhibiting tumor metastasis or inhibiting tumor recurrence, characterized in that, The active ingredients of the said medicament include HMGN1, Telratolimod and α-CD137.

10. The combined medicament according to claim 9, wherein The said tumors include lung cancer and colon cancer.

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