Application of sodium bicarbonate in preparation of medicine for enhancing anti-tumor immunity

Abstract

The invention discloses application of sodium bicarbonate in preparation of a medicine for enhancing anti-tumor immunity. The molecular formula of the sodium bicarbonate is NaHCO3. Sodium bicarbonate can enhance natural immunity and acquired immunity of tumor resistance, and is characterized by including but not limited to activation of a cyclic guanylate-adenylate synthetase (cGAS)-interferon gene stimulating factor (cGAS-STING) signal channel in tumor cells, promotion of infiltration of immune cells in tumors and enhancement of the antitumor efficacy of the immune checkpoint inhibitor.

Description

Technical Field

[0001] This invention pertains to pharmaceutical uses, specifically novel pharmaceutical uses of sodium bicarbonate, particularly its application in the preparation of drugs that enhance antitumor immunity. Background Technology

[0002] Sodium bicarbonate has the molecular formula NaHCO3. Its pharmacological effects include: (1) treating metabolic acidosis by increasing the concentration of carbonate ions in plasma, neutralizing hydrogen ions, and thus correcting acidosis; (2) alkalizing urine: the increased carbonate concentration in urine raises the pH value, making it less likely for uric acid, sulfonamides, and hemoglobin to crystallize or aggregate in the urine; (3) neutralizing acid: oral administration can rapidly neutralize or buffer gastric acid without directly affecting gastric acid secretion. Therefore, the rapid increase in gastric pH relieves symptoms caused by high gastric acid.

[0003] Immune checkpoint inhibitors (ICIs) are a class of drugs that enhance the body's ability to attack tumor cells by modulating the immune system. Normally, T cells can recognize and attack tumor cells, but tumor cells can evade the immune system by expressing certain immune checkpoint molecules that suppress T cell activity. The mechanism of action of immune checkpoint inhibitors is to block the binding of these immune checkpoint molecules to their ligands, thereby restoring the ability of T cells to attack tumor cells. Currently, the main targets of immune checkpoint inhibitors include CTLA-4, PD-1, and PD-L1. CTLA-4 inhibitors enhance T cell activity by blocking the binding of CTLA-4 to its ligands CD80 and CD86. PD-1 / PD-L1 inhibitors relieve the suppression of T cells by tumor cells by blocking the binding of PD-1 to its ligand PD-L1. However, the clinical response rate of ICIs is not currently ideal. For example, the objective response rate (ORR) of PD-1 monoclonal antibodies in liver cancer is 15-20%, and in triple-negative breast cancer it is 8%. The ORR of PD-1 / CTLA-4 bispecific antibody in lung cancer was 10%. Summary of the Invention

[0004] In order to address the problems and needs existing in the background art, one object of the present invention is to provide the application of sodium bicarbonate in the preparation of drugs that enhance antitumor immunity.

[0005] The antitumor immune-enhancing drugs include immune checkpoint inhibitors and sodium bicarbonate.

[0006] The immune checkpoint inhibitors include programmed death receptor 1 (PD-1) inhibitors. Sodium bicarbonate is administered locally to the tumor, specifically via injection; the PD-1 inhibitor is administered systemically, specifically via intravenous injection. In other words, sodium bicarbonate and the inhibitor are administered separately. Sodium bicarbonate is used to enhance the efficacy of the PD-1 inhibitor.

[0007] The immune checkpoint inhibitors include programmed death-ligand 1 (PD-L1) inhibitors. Sodium bicarbonate is administered topically, specifically by injection; the PD-L1 inhibitor is administered systemically, specifically by intravenous injection. In other words, sodium bicarbonate and the inhibitor are administered separately. Sodium bicarbonate is used to enhance the efficacy of the PD-L1 inhibitor.

[0008] The immune checkpoint inhibitors include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors. Sodium bicarbonate is administered topically, specifically by injection; the CTLA-4 inhibitor is administered systemically, specifically by intravenous injection. In other words, sodium bicarbonate and the inhibitor are administered separately. Sodium bicarbonate is used to enhance the efficacy of the CTLA-4 inhibitor.

[0009] The programmed death receptor 1 (PD-1) inhibitors include sintilimab, tislelizumab, nivolumab, pembrolizumab, toripalimab, and camrelizumab.

[0010] The programmed death-ligand 1 (PD-L1) inhibitors include atezolizumab and durvalumab.

[0011] The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors include ipilimumab, tremelimumab, and cadonilimab.

[0012] The administration method of the enhanced anti-tumor immune drug is injection.

[0013] The sodium bicarbonate can be administered via intratumoral injection and arterial / venous injection.

[0014] The concentration range of sodium bicarbonate is 60 mM–600 mM. The clinical concentration of sodium bicarbonate injection is 5%, 600 mM.

[0015] Sodium bicarbonate significantly enhances the antitumor effects of immune checkpoint inhibitors. Sodium bicarbonate treatment increases the pH within tumor cells, causing mitochondrial swelling and rupture, leading to the leakage of mitochondrial DNA (mtDNA) into the cytoplasm, activating the cGAS / STING signaling pathway, and promoting the secretion of type-I interferon (IFN-I) by tumor cells. Furthermore, sodium bicarbonate promotes the release of adenosine triphosphate (ATP) and high-mobility group box-1 (HMGB1) from tumor cells, and promotes the exposure of calreticulin (CRT) on the cell surface. These molecular changes promote the infiltration of T cells and dendritic cells (DCs) in tumors. Animal experiments show that the combined use of sodium bicarbonate and immune checkpoint inhibitors significantly inhibits tumor growth and increases the number of T cells and DCs in tumor tissue. Clinical data show that hepatic artery infusion of sodium bicarbonate combined with PD-1 monoclonal antibody achieved an objective response rate of 93.3% and a complete remission rate of 53.3% in patients with liver cancer, significantly higher than that of PD-1 monoclonal antibody monotherapy. Local alkalization with sodium bicarbonate is an effective means to improve the efficacy of immunotherapy.

[0016] The beneficial effects of this invention are: (1) A new use of sodium bicarbonate in the preparation of drugs that enhance antitumor immunity was developed; (2) Administration of drugs within the tumor is non-toxic to the human body. Attached Figure Description

[0017] Figure 1 The concentration of IFN-β in the culture medium after treating tumor cells with different concentrations of sodium bicarbonate was determined.

[0018] Figure 2 120 mM sodium bicarbonate induced mitochondrial swelling in SK-HEP-1 cells.

[0019] Figure 3 Immunofluorescence staining results for mitochondrial swelling in SK-HEP-1 cells.

[0020] Figure 4 Electrophoresis results of PCR products in the cytoplasm of SK-HEP-1 cells after treatment with 120 mM sodium bicarbonate.

[0021] Figure 5Western blotting (WB) was used to detect the phosphorylation levels of STING / TBK1 / IRF3 in SK-HEP-1 cells.

[0022] Figure 6 The ATP concentration in the culture medium after treating tumor cells with different concentrations of sodium bicarbonate was determined.

[0023] Figure 7 To detect the CRT exposed on the surface of SK-HEP-1 cells, 4T1 cells and CT26 cells under different concentrations of sodium bicarbonate treatment by flow cytometry.

[0024] Figure 8 The results of immunofluorescence detection of SK-HEP-1 cells, 4T1 cells and CT26 cells treated with 120 mM sodium bicarbonate are shown.

[0025] Figure 9 Changes in subcutaneous tumor volume in 4T1 patients under different treatment groups.

[0026] Figure 10 Tumor size and mass of 4T1 cells were compared between different treatment groups.

[0027] Figure 11 CD4 in 4T1 tumors of different treatment groups + T-cell infiltration.

[0028] Figure 12 CD8+ in 4T1 tumors of different treatment groups + T-cell infiltration.

[0029] Figure 13 CD11c in 4T1 tumors of different treatment groups + DC immersion.

[0030] Figure 14 Changes in subcutaneous tumor volume in CT26 patients under different treatment groups.

[0031] Figure 15 The size and mass of CT26 tumors in different treatment groups.

[0032] Figure 16 CD4 in CT26 tumors in different treatment groups + T-cell infiltration.

[0033] Figure 17 CD8 in CT26 tumors in different treatment groups + T-cell infiltration.

[0034] Figure 18 CD11c in CT26 tumors of different treatment groups + DC immersion.

[0035] Figure 19 Changes in subcutaneous tumor volume in H22 patients under different treatment groups.

[0036] Figure 20 The size and mass of H22 subcutaneous tumors were compared between different treatment groups.

[0037] Figure 21 CD4 in H22 tumors in different treatment groups + T-cell infiltration.

[0038] Figure 22 CD8 in H22 tumors in different treatment groups + T-cell infiltration.

[0039] Figure 23 CD11c in H22 tumors of different treatment groups + DC immersion.

[0040] Figure 24 Changes in 4T1 tumor volume in different treatment groups.

[0041] Figure 25 Tumor size and mass of 4T1 cells were compared between different treatment groups.

[0042] Figure 26 CD4 in 4T1 tumors of different treatment groups + T-cell infiltration.

[0043] Figure 27 CD8+ in 4T1 tumors of different treatment groups + T-cell infiltration.

[0044] Figure 28 CD11c in 4T1 tumors of different treatment groups + DC immersion.

[0045] Figure 29 Typical MRI image of a patient with hepatic vein tumor thrombus who is aiming for complete remission (CR). The tumor lesion is circled in red, and the tumor thrombus area is circled in yellow.

[0046] Figure 30 Typical MRI image of a patient with portal vein tumor thrombus who is aiming for complete remission (CR). The tumor lesion is circled in red, and the tumor thrombus area is circled in yellow.

[0047] Figure 31 Typical MRI image of a patient with portal vein tumor thrombus who is attempting to achieve partial remission (PR). The tumor lesion is circled in red, and the tumor thrombus area is circled in yellow.

[0048] Figure 32 Typical MRI image of a patient achieving complete remission (CR). The tumor lesion is circled in red.

[0049] Figure 33Typical MRI image of a patient who has achieved SD. The tumor lesion is circled in red.

[0050] Figure 34 Representative images and statistical results of different degrees of mortality in tumor areas of pathological sections.

[0051] Figure 35 Representative images of pathological sections stained with HE, CD3, CD4, and CD8.

[0052] Figure 36 A statistical chart showing the percentage of CD3, CD4, and CD8 positive cells in pathological sections. Detailed Implementation

[0053] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0054] Example 1: Sodium bicarbonate damages the mitochondria of tumor cells, causing mtDNA leakage, activating the cGAS / STING pathway, and inducing the IFN-I response.

[0055] Sodium bicarbonate can raise the pH of cells and mitochondria. Increased pH damages mitochondria. Mitochondrial damage is known to lead to mtDNA leakage. Furthermore, cytoplasmic DNA can bind to cGAS and activate the STING / TBK1 / IRF3 pathway, inducing IFN-α / β secretion and producing a type I interferon (IFN-I) response, thus affecting immune cell function.

[0056] Experimental methods: The liver cancer SK-HEP-1 cells, breast cancer 4T1 cells, and colorectal cancer CT26 cells used in the experiment were obtained from the Cancer Institute of Zhejiang University. All cells were cultured in RPMI-1640 basal medium (containing 10% fetal bovine serum) in a 5% CO2 incubator at 37 ℃. The experimental groups were supplemented with 40 mM, 80 mM, and 120 mM sodium bicarbonate, while the control group was supplemented with 20 mM sodium bicarbonate.

[0057] Cells were cultured in 24-well plates (3 × 10⁻⁶). 5 Cells / well were collected and treated with different concentrations of sodium bicarbonate for the following days (200 mM for 2.5 h, 120 mM for 8 h, 80 mM for 16 h, and 40 mM and 20 mM for 48 h). IFN-β in the cell culture medium was detected using an IFN-β ELISA Kit (Proteintech, Cat# KE00195, China; Yunclone, Cat# SEA222Mu, China).

[0058] To observe mitochondria, SK-HEP-1 cells were cultured in 10 cm dishes. The following day, they were treated with different concentrations of sodium bicarbonate for corresponding time periods (120 mM for 4 h, 20 mM for 48 h). The original culture medium was discarded, and cells were obtained by trypsin digestion. After washing with PBS and centrifugation, cells were fixed with 2.5% glutaraldehyde and incubated overnight at 4 °C. After washing with PBS, 1% osmium tetroxide was added, and the cells were incubated at room temperature for 1 h. After washing with ddH2O, 2% uranium acetate was added, and the cells were incubated at room temperature for 30 min. After dehydration, embedding, and sectioning, mitochondrial images were obtained using uranium acetate-lead citrate double staining and cryo-electron microscopy.

[0059] To detect mtDNA leakage by immunofluorescence, SK-HEP-1 cells were cultured in confocal microscope glass-bottomed culture dishes (3 × 10⁻⁶). 5 Cells / plate were treated with different concentrations of sodium bicarbonate for the corresponding time periods (120 mM for 4 h, 20 mM for 48 h). The original culture medium was discarded, and the cells were cultured in fresh medium containing Mito-Tracker Red CMXRos (ThermoFisher, USA) for 20 min, then fixed with 4% paraformaldehyde at room temperature for 15 min, and permeabilized with 0.2% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 5% goat serum for 1 h. Then, the cells were incubated overnight at 4 °C with anti-dsDNA antibody (Abcam, Cat # ab27156, China). After washing with PBS, the cells were incubated with coralite488-Goat Anti-Mouse secondary antibody (Proteintech, Cat # Sa00013-1, China) at room temperature in the dark for 1 h. After washing with PBS, images were acquired using a confocal microscope (Zeiss, Germany).

[0060] To detect cytoplasmic mtDNA by PCR, SK-HEP-1 cells were cultured in two 6 cm culture dishes (1 × 10⁻⁶). 6Cells were cultured in medium containing different concentrations of sodium bicarbonate for corresponding time periods (120 mM for 4 h, 20 mM for 48 h). Cells were harvested by trypsin digestion, and each dish was divided into two aliquots: one for cytoplasmic mtDNA extraction and the other for total DNA extraction. Cells for mtDNA extraction were lysed on ice for 20 min with 0.1% NP-40, followed by centrifugation at 14000 × g, 4 °C, and the supernatant was collected. Cells for total DNA extraction were lysed using the lysis buffer from the QIAamp DNA microkit (QIAGEN, USA). All DNA samples were purified using the QIAamp DNA microkit. The PCR primers for mtDNA were as follows: ND1-F: CCTTCGTCTGATCCGTCCT; ND1-R: TGAGGTTGAGGTCTGTTAGT. PCR was performed using PrimeSTAR Max DNAPolymerase (Takara, Japan), and the products were analyzed by agarose gel electrophoresis.

[0061] To detect the phosphorylation levels of STING / TBK1 / IRF3 in cells by Western blotting, SK-HEP-1 cells were cultured in four 6cm culture dishes (1 × 10⁻⁶ cells / ... 6 Cells per dish were treated with different concentrations of sodium bicarbonate for corresponding time periods (120 mM for 4 h, 80 mM for 12 h, and 40 mM and 20 mM for 48 h). Cells were then lysed to obtain proteins. Protein samples were gel electrophoresed, transferred to a membrane, blocked with 5% skim milk, and then incubated overnight at 4 °C with primary antibody. The primary antibodies used were as follows: anti-STING (Proteintech, Cat#19851-1-AP, China), anti-p-STING (CST, Cat#50907, USA), anti-TBK1 (CST, Cat#3504, USA), anti-p-TBK1 (CST, Cat#5483, USA), anti-IRF3 (Proteintech, Cat#11312-1-AP, China), anti-p-IRF3 (CST, Cat#4947, USA), and GAPDH (CST, Cat#2118, USA). The bands were then incubated with secondary antibodies at room temperature for 1 hour. Finally, the bands were incubated with ECL exposure solution and imaged for analysis.

[0062] Experimental results: Treatment with 120 mM and 200 mM sodium bicarbonate significantly increased the secretion of IFN-β by SK-HEP-1 cells, 4T1 cells, and CT26 cells. Figure 1Treatment of SK-HEP-1 cells with 120 mM sodium bicarbonate resulted in significant mitochondrial swelling. Figure 2 Treatment of SK-HEP-1 cells with 120 mM sodium bicarbonate increased the amount of extramitochondrial free mtDNA in the cytoplasm. Figure 3 PCR product electrophoresis results also indicated that 120 mM sodium bicarbonate treatment increased the leakage of mtDNA into the cytoplasm. Figure 4 Treatment of SK-HEP-1 cells with 120 mM sodium bicarbonate resulted in increased phosphorylation levels of STING / TBK1 / IRF3. Figure 5 This indicates that 120 mM sodium bicarbonate treatment activated the cGAS-STING signaling pathway in cells. These experimental results suggest that sodium bicarbonate treatment causes mitochondrial swelling and rupture in tumor cells, leading to the leakage of mitochondrial mtDNA into the cytoplasm, activating the STING / TBK1 / IRF3 signaling pathway, and promoting the expression and secretion of IFN-I.

[0063] Example 2: Sodium bicarbonate induces immunogenic death of tumor cells.

[0064] Principle: Tumor cells release damage-associated molecular patterns (DAMPs) and secrete cytokines to mediate chemotaxis and immunostimulation in response to drug treatment and changes in the cell microenvironment. Tumor cell death is accompanied by the exposure and release of large amounts of DAMPs, a phenomenon known as immunogenic cell death (ICD). Changes in DAMPs during ICD mainly include: secretion of IFN-I; increased exposure of calreticulin (CRT) on the cell membrane surface; release of adenosine triphosphate (ATP) into the extracellular space; and the migration and secretion of high-mobility group box-1 (HMGB1) from the nucleus into the extracellular space. Detecting these changes in DAMPs can indicate whether sodium bicarbonate treatment induces ICD.

[0065] Experimental methods: The cells used in the experiment were obtained from the Cancer Institute of Zhejiang University. Cell culture was performed in the same manner as in Example 1.

[0066] Cells were cultured in 24-well plates (3 × 10⁻⁶). 5Cells / well were collected, and the cells were treated with different concentrations of sodium bicarbonate for the following days (120 mM for 8 h, 80 mM for 16 h, and 40 mM and 20 mM for 48 h). The cell culture supernatant was then collected, and the ATP content in the culture medium was measured using a luciferase-based Enliten ATP Assay (Promega, Cat# FF2000, USA).

[0067] Cells were cultured in 24-well plates (3 × 10⁻⁶). 5 Cells were cultured in multiple wells (120 mM for 4 h, 80 mM for 12 h, and 40 mM and 20 mM for 48 h) the following day. Cells were then digested with trypsin to obtain the cells. For flow cytometry detection of CRT exposure on the cell membrane surface, cells were fixed with 1% paraformaldehyde at room temperature for 15 min, washed with PBS, and blocked with 5% BSA at room temperature for 30 min. Cells were then incubated with anti-CRT antibody (Proteintech, Cat #27298-1-AP, China) at room temperature for 45 min. After washing with PBS, cells were incubated with coralite488-GoatAnti-Rabbit secondary antibody (Proteintech, Cat #Sa00013-2, China) at room temperature in the dark for 45 min. After washing with PBS, the cells were detected using a DxFLEX flow cytometer (Beckman, USA).

[0068] To detect HMGB1 cell migration out of the nucleus, cells were cultured in confocal microscope glass-bottomed culture dishes (3 × 10⁻⁶). 5Cells per dish were treated with different concentrations of sodium bicarbonate for the corresponding time periods (120 mM for 4 h, 20 mM for 48 h). The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with 0.2% Triton X-100 for 15 min. After washing with PBS, the cells were blocked with 5% goat serum for 1 h. Then, the cells were incubated overnight at 4 °C with anti-HMGB1 antibody (Proteintech, Cat #10829-1-AP, China). After washing with PBS, the cells were incubated at room temperature in the dark with coralite488-Goat anti-Rabbit secondary antibody (Proteintech, Cat #Sa00013-2, China). After washing with PBS, the cells were incubated at room temperature for 1 h with Hoechst 33342 (Beyotime, China). After washing with PBS, the cells were incubated at room temperature for 15 min with a confocal microscope (Zeiss, Germany).

[0069] Experimental results: Treatment with 80 mM and 120 mM sodium bicarbonate significantly increased the release of extracellular ATP from SK-HEP-1, 4T1, and CT26 cells. Figure 6 Treatment with 120 mM sodium bicarbonate increased CRT on the surface of SK-HEP-1 cells, 4T1 cells, and CT26 cells. Figure 7 Treatment with 120 mM sodium bicarbonate decreased intranuclear HMGB1 levels and increased extranuclear HMGB1 levels in SK-HEP-1, 4T1, and CT26 cells. Figure 8 This indicates that treatment with 120 mM sodium bicarbonate promoted HMGB1 migration. These experimental results demonstrate that sodium bicarbonate treatment significantly alters the DAMPs in tumor cells, leading to ICD in tumor cells.

[0070] Example 3: Sodium bicarbonate enhances the efficacy of the immune checkpoint inhibitor PD-1 monoclonal antibody in the treatment of breast cancer.

[0071] Principle: PD-1 is an important immunosuppressive molecule expressed on the surface of immune cells. PD-1 binds to PD-L1 on the surface of tumor cells, inhibiting the immune-killing effect of T cells. AntiPD-1 monoclonal antibodies are important drugs in tumor immunotherapy, exerting their therapeutic effect by relieving the inhibition of T cells by PD-1 / PD-L1. Although antiPD-1 monoclonal antibodies can relieve the inhibition of T cells by PD-1 / PD-L1 signaling, they do not solve the problem of immune cell infiltration in tumors. The aforementioned in vitro cell experiments have shown that sodium bicarbonate can induce ICD in tumor cells. IFN-I, ATP, HMGB1, and CRT have the effect of promoting the maturation and infiltration of dendritic cells (DCs) and T cells.

[0072] Experimental methods: Twenty-eight 6-week-old female BALB / c mice used in the experiment were purchased from Shanghai Slack Biotechnology Co., Ltd., and housed in the SPF environment of the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. The mice were fed normally for 3 days after receipt to allow them to acclimatize. During this period, the water was changed daily, and feed was added every three days. The mouse breast cancer cell line 4T1 (derived from the Cancer Institute of Zhejiang University) was cultured in RPMI-1640 medium (containing 10% fetal bovine serum) in a 5% CO2 incubator at 37 ℃.

[0073] Culture 4T1 cells until they reach the logarithmic growth phase. When the cell density is approximately 80%, trypsin digestion is performed to obtain cells. Wash the cells twice with pre-chilled PBS solution to completely remove serum components from the culture medium. Resuspend the cells in pre-chilled serum-free medium, count them, and adjust the cell density to 1 × 10⁶ cells / year. 6 / mL. Place the cell suspension on ice and inoculate at the animal experimental center. Inject 100 μL of cell suspension (containing 1×10⁶ cells / mL) using a 1 mL syringe. 5 A mouse breast cancer model was constructed by inoculating mice with cells into the second mammary pad. The inoculated mice were randomly divided into 4 groups of 7 mice each.

[0074] One week later, the tumors in the four groups of mice were measured with calipers to be approximately 4-6 mm in length. Starting every other day, the mice were injected with the corresponding drugs according to the following regimens: The control group received an intratumoral injection of 100 µl of PBS buffer and an intraperitoneal injection of 100 µl of PBS buffer; the antiPD-1 monoclonal antibody group received an intratumoral injection of 100 µl of PBS buffer and an intraperitoneal injection of 10 mg / kg antiPD-1 monoclonal antibody (BioX Cell, Cat#be0146, USA); the sodium bicarbonate group received an intratumoral injection of 100 µl of 200 mM sodium bicarbonate (Sigma, USA, prepared in PBS buffer) and an intraperitoneal injection of 100 µl of PBS buffer; and the sodium bicarbonate + antiPD-1 monoclonal antibody group received an intratumoral injection of 100 µl of 200 mM sodium bicarbonate and an intraperitoneal injection of 10 mg / kg antiPD-1 monoclonal antibody. As the tumors grew, when the length of the tumor was approximately 9-10 mm, the intratumoral injection volume increased to 300 µl.

[0075] The size of the tumor was measured using calipers the following day, including its maximum diameter (L) and minimum diameter (W). The tumor volume was calculated as V = L × W × W / 2. The experiment was terminated after 2 weeks. Mice were euthanized after the experiment, the tumors were isolated, weighed, and photographed. The tumors were then fixed in 4% paraformaldehyde solution. Immunohistochemical analysis was performed on the paraformaldehyde-fixed tumor tissue.

[0076] Immunohistochemical analysis was performed on paraformaldehyde-fixed tumor tissue. First, the tumor tissue was dehydrated and paraffin-impregnated to form paraffin blocks, which were then cut into 3-5 μm thick sections. The sections were dewaxed with xylene and hydrated with graded-concentration ethanol, followed by high-temperature, high-pressure antigen retrieval. After blocking with 3% H2O2 and 10% goat serum, the sections were covered with anti-CD4, anti-CD8, and anti-CD11c primary antibodies (Abcam, USA, diluted 1:2000 in PBS containing 1% BSA) and incubated overnight at 4 °C. After washing away the primary antibodies with PBS buffer, the sections were incubated with enzyme-labeled goat anti-rabbit IgG polymer (Zhongshan Jinqiao, Cat#PV6001, China) at room temperature for 30 minutes. After washing with PBS, DAB staining was performed for 1 minute. Then, the sections were soaked in hematoxylin staining solution for 3 minutes. After washing with water, the sections were briefly soaked in hematoxylin differentiation solution and blueing solution. After dehydration with graded-concentration ethanol, clearing with xylene, and finally mounting, the sections were air-dried and observed under a microscope.

[0077] Experimental results: Of the four groups, the sodium bicarbonate + antiPD-1 monoclonal antibody group had the slowest tumor growth rate. Both the sodium bicarbonate group and the antiPD-1 monoclonal antibody group had slower tumor growth rates than the PBS group, but faster than the combination group. Figure 9The sodium bicarbonate + antiPD-1 monoclonal antibody group had the smallest tumors, and one mouse showed complete tumor regression; the size and mass of tumors in the sodium bicarbonate group were also smaller than those in the PBS group. Figure 10 The above results indicate that treatment with 200 mM sodium bicarbonate can inhibit the growth of breast cancer, and that sodium bicarbonate combined with antiPD-1 monoclonal antibody treatment is the most effective for breast cancer, as sodium bicarbonate can enhance the efficacy of antiPD-1 monoclonal antibody.

[0078] Figure 11 and Figure 12 These are CD4+ in different groups of 4T1 tumors. + and CD8 + T-cell infiltration. Figure 11 and Figure 12 The results showed that CD4 was present in the tumors of the sodium bicarbonate group. + and CD8 + The number of T cells was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and CD4 counts were higher in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group. + and CD8 + The highest number of cells was observed in the sodium bicarbonate group, indicating that sodium bicarbonate promotes T cell infiltration into the tumor. The tumors in the sodium bicarbonate group also showed the highest CD11c count. + The number of dendritic cells (DCs) was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and CD11c was also higher in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group. + The number of DCs is the highest among the four groups. Figure 13 This indicates that 200 mM sodium bicarbonate promotes the infiltration of dendritic cells (DCs) into the tumor. DCs are antigen-presenting cells, CD4+. + T cells include helper T cells, CD8 + T cells mainly include effector T cells that play a role in cellular immunity. The above results suggest that sodium bicarbonate's inhibition of breast cancer growth is partly due to its promotion of immune cell infiltration, thus exerting an anti-tumor immune effect. Sodium bicarbonate can enhance the efficacy of PD-1 monoclonal antibody therapy for breast cancer by promoting T cell infiltration.

[0079] Example 4: Sodium bicarbonate enhances the efficacy of PD-1 monoclonal antibody therapy for colorectal cancer.

[0080] Experimental methods: The strain, source, and feeding of the mice used in the experiment were the same as in Example 3. The mouse colorectal cancer cell line CT26 (Zhejiang University Cancer Institute) was cultured in RPMI-1640 medium (containing 10% fetal bovine serum) in a 5% CO2 incubator at 37 ℃.

[0081] The preparation method of the cell suspension used to construct the mouse tumor model is the same as in Example 3. Inject 100 μL of cell suspension (containing 5 × 10⁻⁶ cells) using a 1 mL syringe. 5A mouse subcutaneous colorectal cancer tumor model was constructed by inserting cells into the axilla of the upper limb of mice. Subsequent mouse grouping and treatment were the same as in Example 3.

[0082] Experimental results: The tumor growth rate was slowest in the sodium bicarbonate + antiPD-1 monoclonal antibody group. Both the sodium bicarbonate group and the antiPD-1 monoclonal antibody group had slower tumor growth rates than the PBS group, but faster tumor growth rates than the combination group. Figure 14 The sodium bicarbonate + antiPD-1 monoclonal antibody group had the smallest tumors; the size and mass of tumors in both the sodium bicarbonate group and the antiPD-1 monoclonal antibody group were also smaller than those in the PBS group. Figure 15 The above results indicate that treatment with 200 mM sodium bicarbonate can inhibit the growth of colorectal cancer, and that the combination of 200 mM sodium bicarbonate and antiPD-1 monoclonal antibody is the most effective treatment for colorectal cancer, with sodium bicarbonate enhancing the effect of antiPD-1 monoclonal antibody.

[0083] Figure 16 and Figure 17 CD4 in different groups of tumors + and CD8 + T-cell infiltration. Figure 16 and Figure 17 The tumors in the sodium bicarbonate group showed CD4 levels. + and CD8 + The number of T cells was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and CD4 counts were higher in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group. + and CD8 + The highest cell count was observed in the sodium bicarbonate-treated group, indicating that sodium bicarbonate promotes T cell infiltration into colorectal cancer. The sodium bicarbonate-treated group also showed a higher CD11c count in their tumors. + The number of dendritic cells (DCs) was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and CD11c was also higher in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group. + The number of DCs is the largest. Figure 18 This indicates that sodium bicarbonate treatment promotes the infiltration of dendritic cells (DCs) into colorectal cancer tissue. These results suggest that sodium bicarbonate promotes the infiltration of immune cells into colorectal cancer to exert its anti-tumor immune effect, and that sodium bicarbonate can enhance the efficacy of antiPD-1 monoclonal antibody therapy for colorectal cancer by promoting T cell infiltration.

[0084] Example 5: Sodium bicarbonate enhances the efficacy of PD-1 monoclonal antibody therapy for liver cancer.

[0085] Experimental methods: The strain, source, and feeding of the mice used in the experiment were the same as in Example 3. The mouse hepatocellular carcinoma cell line H22 (Zhejiang University Cancer Institute) was cultured in RPMI-1640 medium (containing 10% fetal bovine serum) in a 5% CO2, 37°C incubator.

[0086] The preparation method of the cell suspension used to construct the mouse tumor model is the same as in Example 3. Inject 100 μL of cell suspension (containing 5 × 10⁻⁶ cells) using a 1 mL syringe. 5 A mouse subcutaneous hepatocellular carcinoma model was constructed by inserting cells into the axilla of the upper limb of mice. Subsequent mouse grouping and treatment were the same as in Example 3.

[0087] Experimental results: The tumor growth rate was slowest in the sodium bicarbonate + antiPD-1 monoclonal antibody group. Both the sodium bicarbonate group and the antiPD-1 monoclonal antibody group had slower tumor growth rates than the PBS group, but faster tumor growth rates than the combination group. Figure 19 The sodium bicarbonate + antiPD-1 monoclonal antibody group had the smallest tumors; the size and mass of tumors in both the sodium bicarbonate group and the antiPD-1 monoclonal antibody group were also smaller than those in the control group. Figure 20 The above results indicate that sodium bicarbonate can inhibit the growth of liver cancer, and that sodium bicarbonate combined with antiPD-1 monoclonal antibody treatment has the best effect on liver cancer, with sodium bicarbonate enhancing the efficacy of antiPD-1 monoclonal antibody.

[0088] Figure 21 and Figure 22 CD4 in tumors of different treatment groups + and CD8 + T-cell infiltration. Figure 21 and Figure 22 The tumors in the sodium bicarbonate group showed CD4 levels. + and CD8 + The number of T cells was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and the number of CD4 cells in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group was higher. + and CD8 + The sodium bicarbonate group showed the highest number of T cells, indicating that sodium bicarbonate promotes T cell infiltration into liver cancer and also increases the number of T cells in liver cancer treated with antiPD-1 monoclonal antibodies. + The number of dendritic cells (DCs) was higher in the sodium bicarbonate + antiPD-1 monoclonal antibody group than in the PBS group, and CD11c was also higher in the tumors of the sodium bicarbonate + antiPD-1 monoclonal antibody group. + The number of DCs is the largest. Figure 23 This indicates that sodium bicarbonate treatment promotes dendritic cell (DC) infiltration into liver cancer. These results suggest that sodium bicarbonate promotes the infiltration of immune cells into liver cancer to exert its anti-tumor immune effect, and that sodium bicarbonate can enhance the efficacy of antiPD-1 monoclonal antibody therapy for liver cancer by promoting T cell infiltration.

[0089] Example 6: Sodium bicarbonate enhances the efficacy of the immune checkpoint inhibitor CTLA-4 monoclonal antibody in the treatment of breast cancer.

[0090] Principle: Like PD-1, CTLA-4 is an immunosuppressive molecule, but it binds to different ligands. CTLA-4 competitively binds to B7 molecules (CD80 / CD86), negatively regulating signal transduction between T cells and antigen-presenting cells, and inhibiting T cell activation. CTLA-4 monoclonal antibodies have been developed for tumor immunotherapy. The above examples demonstrate the good efficacy of sodium bicarbonate combined with PD-1 monoclonal antibody in treating tumors; this treatment regimen promotes immune cell infiltration.

[0091] Experimental methods: The strain, source, and feeding of the mice used in the experiment were the same as in Example 3. The source and culture of the 4T1 cells used in the experiment were the same as in Example 3.

[0092] The method for constructing an orthotopic mouse model of breast cancer was the same as in Example 3. Mice with tumors were randomly divided into 4 groups of 7 mice each and labeled. When the long diameter of the tumors reached approximately 4-6 mm, the mice in the 4 groups were injected with the corresponding drugs every other day, as follows: the control group received 100 µl of PBS; the antiCTLA-4 monoclonal antibody group received intratumoral injection of PBS buffer and intraperitoneal injection of 10 mg / kg antiCTLA-4 monoclonal antibody (BioX Cell, Cat#be0164, USA); the sodium bicarbonate group received intratumoral injection of 200 mM sodium bicarbonate and intraperitoneal injection of PBS buffer; and the sodium bicarbonate combined with antiCTLA-4 monoclonal antibody group received intratumoral injection of 200 mM sodium bicarbonate and intraperitoneal injection of the monoclonal antibody. The dosage and administration method were the same as in Example 3. The observation indicators and tumor sample processing were the same as in Example 3.

[0093] Experimental results: The tumor growth rate was slowest in the sodium bicarbonate + antiCTLA-4 monoclonal antibody group. Both the sodium bicarbonate group and the antiCTLA-4 monoclonal antibody group had slower tumor growth rates than the PBS group, but faster than the combination group. Figure 24 The sodium bicarbonate + antiCTLA-4 monoclonal antibody group had the smallest tumors, and two mice showed complete tumor regression; the size and mass of tumors in the sodium bicarbonate group were also smaller than those in the PBS group. Figure 25 The above results indicate that sodium bicarbonate combined with antiCTLA-4 monoclonal antibody treatment is the most effective for breast cancer, and sodium bicarbonate can enhance the efficacy of antiCTLA-4 monoclonal antibody.

[0094] Figure 26 and Figure 27 CD4 in different groups of 4T1 tumors + and CD8 + T-cell infiltration. Figure 26 and Figure 27 The results showed that CD4 levels in tumors of the sodium bicarbonate + antiCTLA-4 monoclonal antibody group were higher. + and CD8+ The highest number of cells was observed in the sodium bicarbonate group, indicating that sodium bicarbonate promotes T cell infiltration into the tumor. The tumors in the sodium bicarbonate group also showed the highest CD11c count. + The number of dendritic cells (DCs) was higher in the sodium bicarbonate + antiCTLA-4 monoclonal antibody group than in the PBS group, and CD11c was higher in the tumors of the sodium bicarbonate + antiCTLA-4 monoclonal antibody group. + The number of DCs is the largest. Figure 28 This indicates that sodium bicarbonate promotes the infiltration of dendritic cells (DCs) into the tumor. These results suggest that sodium bicarbonate's inhibition of breast cancer growth is partly due to its promotion of immune cell infiltration, thus exerting an anti-tumor immune effect. Sodium bicarbonate enhances the efficacy of CTLA-4 monoclonal antibody therapy for breast cancer by promoting DC and T cell infiltration.

[0095] Example 7: Clinical study of hepatic artery infusion of sodium bicarbonate combined with PD-1 monoclonal antibody for the treatment of liver cancer method: 1. Patient This prospective study was conducted in accordance with the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee for Human Research at the Second Affiliated Hospital of Zhejiang University School of Medicine (ChiCTR registration number: ChiCTR2100053537). Thirty patients with advanced hepatocellular carcinoma who received treatment at the Second Affiliated Hospital of Zhejiang University School of Medicine between November 2021 and May 2023 were included in this prospective study.

[0096] Patients were included according to the following specific criteria: (a) patients were diagnosed with HCC by imaging or pathology according to the EASL Clinical Practice Guidelines; (b) patients had received at least one cycle of hepatic artery catheterization with 5% sodium bicarbonate injection and anti-PD-1 monoclonal antibody immunotherapy; (c) patients had not received any cancer-related treatment prior to or during enrollment; (d) patients were stage B or C of the Barcelona Clinical Hepatocellular Carcinoma (BCLC) stage; (e) patients had a Child-Pugh score of A; (f) patients were older than 18 years of age; (g) patients had no other diagnosed malignancies; and (h) patients had complete medical and follow-up data. All laboratory serum test data were collected within 7 days prior to initial treatment. Imaging evaluation included dynamic contrast-enhanced magnetic resonance imaging (MRI) within one week prior to initial treatment.

[0097] 2. Method for perfusing sodium bicarbonate solution through hepatic artery cannulation The general principle is to locate all tumor-feeding arteries and thoroughly infuse them with 5% sodium bicarbonate solution to alkalize the local tumor microenvironment. The simplified procedure is as follows: First, locate all tumor-feeding arteries. Then, embolize the extrahepatic arteries to completely switch tumor blood supply to the hepatic artery. Afterward, infuse with 5% sodium bicarbonate. If the tumor burden is large (diameter > 8 cm) or the tumor response is unsatisfactory after the previous sodium bicarbonate infusion, a second infusion of 5% sodium bicarbonate can be administered via a microinfusion pump the following day. Arterial blood gas analysis before and after treatment ensures that the patient does not develop metabolic alkalosis.

[0098] 3. Immunotherapy Receive anti-PD-1 monoclonal antibody therapy within 3 days prior to the start of hepatic artery infusion of sodium bicarbonate injection. The anti-PD-1 monoclonal antibody is administered intravenously at the standard dose (tislelizumab injection, 200 mg, every three weeks).

[0099] 4. Treatment cessation and other treatments Treatment discontinuation depends on disease progression, intolerable toxicity, patient withdrawal of consent, or changes in the treatment plan. For patients who do not respond well to the treatment regimen in this study, to maximize patient benefit, local treatments such as TACE, HAIC, SBRT, or other systemic therapies may be added; however, efficacy will only be counted after the first three treatments in the efficacy analysis.

[0100] 5. Follow-up plan Enhanced MRI scans are performed every 4–6 weeks after treatment begins. Routine follow-up intervals are 1–2 months. Tumor survival is assessed using MRI scans / dynamic contrast-enhanced CT, and tumor markers, liver and kidney function tests, and routine blood tests are performed to determine if further treatment is needed.

[0101] 6. Efficacy evaluation Efficacy assessments were conducted independently by two radiology experts. On day 30 after initial treatment and during subsequent follow-ups, the target tumor response to treatment was assessed according to the modified Real-Time Evaluation Criteria in Solid Tumors (mRECIST), specifically defined as follows: complete response (CR) refers to complete tumor necrosis; partial response (PR) refers to a reduction of ≥30% in the surviving portion of the tumor lesion; stable disease (SD) refers to any case that does not meet the criteria for partial response or disease progression; and progressive disease (PD) refers to an increase of ≥20% in surviving tumor lesions or the appearance of new lesions.

[0102] 7. Overall survival (OS) Overall survival (OS) was measured from the start of initial treatment until death from any cause or the last follow-up. The median follow-up time was 24.5 months, ranging from 5 to 41 months (calculated from the start of initial treatment until death from any cause or the last follow-up).

[0103] 8. Study endpoint Objective response rate (ORR) was the primary endpoint, and treatment efficacy was assessed according to mRECIST. ORR was defined as CR + PR, and disease control rate (DCR) was defined as ORR + SD. Secondary endpoints were progression-free survival (PFS) and overall survival (OS).

[0104] 9. Treatment-related adverse events The adverse events monitored included fever, abdominal pain, nausea and vomiting, skin ulceration, abnormal liver enzymes, bilirubin-related abnormalities, and immune-related adverse events such as immune-mediated pneumococcal disease and immune-mediated dermatitis. These adverse events were scored according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, and all adverse events received timely symptomatic treatment.

[0105] 10. Statistical Analysis Statistical analyses were performed using R software and GraphPad Prism 8. 95% confidence intervals were calculated using the Wilson method. Survival curves were calculated using the Kaplan-Meier method, and the Log-rank test was used for comparison of survival curves. In all tests, a p-value less than 0.05 was considered statistically significant.

[0106] result: 1. Baseline characteristics. From November 2021 to May 2023, a total of 30 patients (76.7% male) were enrolled. 66.7% of patients had AFP levels exceeding 400 ng / mL. Most tumors were large (100% ≥5 cm, 63.3% >10 cm). 93.3% of patients had BCLC stage C, 93.3% had large vessel invasion, and 13.3% had extrahepatic metastases (see Table 1 for details).

[0107] Table 1 Baseline Characteristics 2. Objective response rate: The ORR for 30 patients was 93.3%, CR was 53.3%, PR was 40.0%, SD was 6.7%, and DCR was 100% (Table 2). Six patients underwent conversion surgery. Typical MRI images are shown below. Figures 29-33 As shown. Figure 29 Typical MRI image of a patient with hepatic vein tumor thrombosis who is aiming for complete remission (CR). Figure 30 Typical MRI image of a patient with portal vein tumor thrombus in order to achieve complete remission (CR). Figure 31 Typical MRI image of a patient with portal vein tumor thrombus in order to achieve partial remission (PR). Figure 32 Typical MRI images of patients who have achieved complete remission (CR). Figure 33 Typical MRI image of a patient who has achieved SD (stable disease). The red circle indicates the tumor lesion, and the yellow circle indicates the tumor embolus area.

[0108] Table 2 Treatment Response 3. Survival Outcomes: By the end of the follow-up period (April 2025), the median overall survival (mOS) had not been reached. The estimated OS rates at 1, 2, and 3 years were 82%, 60%, and 53%, respectively (Table 3). The median progression-free survival (mPFS) was 31 months, and the estimated PFS rates at 1 and 2 years were both 55% (Table 3).

[0109] Table 3 Survival Outcomes 4. Safety: Adverse events occurred in all patients, but most were related to tumor death, such as abdominal pain, fever, and transient elevation of liver enzymes. A few were related to PD-1 monoclonal antibody. These adverse events were all manageable and tolerable to patients (Table 4). No serious adverse events of CTCAE grade 3 or higher occurred. The patients' liver function classification remained stable before and after treatment.

[0110] Table 4 Adverse Reactions Example 8: Hepatic artery infusion of sodium bicarbonate combined with PD-1 monoclonal antibody therapy promotes T cell infiltration method: 1. Source of patient samples: Patient data, MRI images, and pathological sections in this study were obtained from liver cancer patients who visited the Second Affiliated Hospital of Zhejiang University School of Medicine in China between 2021 and 2024. This study was supervised and approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (Ethics Approval No.: (2024) Lun Shen Yan No. (1593)).

[0111] The inclusion criteria for patients in this study were: (1) patients diagnosed with hepatocellular carcinoma by pathological histology or meeting the clinical diagnostic criteria for hepatocellular carcinoma; (2) at least one measurable intrahepatic lesion; and (3) adult patients aged 20 years or older. The exclusion criteria for patients in this study were: (1) incomplete important clinical information; (2) failure to be followed up at the Second Affiliated Hospital of Zhejiang University School of Medicine; (3) participation in other clinical trials; (4) having an autoimmune disease or immunodeficiency disease; (5) a history of organ transplantation or preparation for organ transplantation; (6) using corticosteroids or other immunomodulatory drugs during treatment; (7) other untreated cancer diseases; (8) having received radiotherapy, chemotherapy, radioactive particle implantation, or other molecular targeted therapies during treatment; and (9) other factors that were not suitable for inclusion.

[0112] 2. Patient grouping: Data and MRI images of patients treated at the Second Affiliated Hospital of Zhejiang University School of Medicine between November 2021 and May 2023 under three different treatment regimens were collected: sodium bicarbonate + antiPD-1 group; antiPD-1 monoclonal antibody group; and surgery group. The sodium bicarbonate + antiPD-1 group consisted of patients who underwent surgical resection after hepatic artery infusion of sodium bicarbonate combined with PD-1 monoclonal antibody therapy. The antiPD-1 group consisted of patients who received only PD-1 monoclonal antibody immunotherapy. The surgery group consisted of patients who had not received any other treatment before surgery.

[0113] 3. Response rate assessment: Treatment response is assessed by a specialist physician based on MRI images and mRECIST criteria.

[0114] 4. Pathological sections: The patient's pathological sections and staining were provided and stained by the Department of Pathology, Second Affiliated Hospital of Zhejiang University School of Medicine.

[0115] 5. Statistical Analysis: Data are presented as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. Statistical analysis was performed using GraphPad Prism 8 software. The experimental group was considered an independent factor. Statistical significance was defined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0116] result: Treatment response to tumor lesions was assessed according to the mRECIST criteria. All patients receiving hepatic artery infusion of sodium bicarbonate plus antiPD-1 immunotherapy achieved a treatment response, with 4 patients (66.7%) achieving complete remission (CR) and 2 patients (33.3%) achieving partial remission (PR). Among the 8 patients receiving antiPD-1 immunotherapy alone, 2 patients (25%) achieved CR, 4 patients (50%) achieved PR, and 2 patients (25%) experienced stable disease (SD).

[0117] Subsequently, tumor tissue sections from the patients who underwent surgery were collected from the pathology department (sodium bicarbonate + antiPD-1 group, n = 6; antiPD-1 group, n = 8; surgery group, n = 11). After HE staining, cell death in the tumor area was analyzed. Based on the area of ​​cell death in the tumor area, sections were categorized as complete cell death (≥ 95%), partial cell death (5% < cell death < 95%), and no cell death (≤ 5%). In the sodium bicarbonate + antiPD-1 group, significant cell death was observed in the tumor area of ​​all patients' sections, with 1 patient (16.7%) showing complete tumor death and 5 patients (83.3%) showing partial tumor death. In the antiPD-1 group, 4 patients (50%) had no significant cell death in their sections, 3 patients (37.5%) had partial tumor death, and 1 patient (12.5%) had complete tumor death. In the surgery group, no significant cell death was observed in the tumor area of ​​any patient's sections. Figure 34 ).

[0118] The above data demonstrate that local sodium bicarbonate infusion combined with PD-1 monoclonal antibody immunotherapy has achieved a good therapeutic response in liver cancer patients.

[0119] IHC staining analysis was performed on surgically removed liver cancer tissue sections from patients. Since complete tumor death prevented cell counting and specific staining, patients with completely dead tumor areas were excluded. A total of 23 patients underwent IHC staining analysis (Surgery group n = 11; antiPD-1 group n = 7; sodium bicarbonate + antiPD-1 group n = 5). The IHC staining results are shown in the figure. The tumor tissue sections from patients in the sodium bicarbonate + antiPD-1 group showed the highest numbers of CD3+, CD4+, and CD8+ cells. This indicates that local perfusion of sodium bicarbonate promotes T cell infiltration in the tumor tissue of liver cancer patients. Figure 35 and Figure 36 ).

[0120] Based on the above results, it is believed that hepatic artery infusion of sodium bicarbonate enhances immune cell infiltration and improves the clinical efficacy of PD-1 monoclonal antibody therapy for liver cancer.

Claims

1. Application of sodium bicarbonate in the preparation of drugs that enhance antitumor immunity.

2. The application of sodium bicarbonate according to claim 1 in the preparation of drugs that enhance antitumor immunity, characterized in that: The antitumor immune-enhancing drugs include immune checkpoint inhibitors and sodium bicarbonate.

3. The application of sodium bicarbonate according to claim 2 in the preparation of drugs that enhance antitumor immunity, characterized in that, The immune checkpoint inhibitors include programmed death receptor 1 (PD-1) inhibitors.

4. The application of sodium bicarbonate according to claim 2 in the preparation of drugs that enhance antitumor immunity, characterized in that, The immune checkpoint inhibitors include programmed death-ligand 1 (PD-L1) inhibitors.

5. The application of sodium bicarbonate according to claim 2 in the preparation of drugs that enhance antitumor immunity, characterized in that, The immune checkpoint inhibitors include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors.

6. The use of sodium bicarbonate according to claim 3 in the preparation of drugs that enhance antitumor immunity, characterized in that, The programmed death receptor 1 (PD-1) inhibitors include sintilimab, tislelizumab, nivolumab, pembrolizumab, toripalimab, and camrelizumab.

7. The use of sodium bicarbonate according to claim 4 in the preparation of drugs that enhance antitumor immunity, characterized in that, The programmed death-ligand 1 (PD-L1) inhibitors include atezolizumab and durvalumab.

8. The use of sodium bicarbonate according to claim 5 in the preparation of drugs that enhance antitumor immunity, characterized in that, The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors include ipilimumab, tremelimumab, and cadonilimab.

9. The use of sodium bicarbonate according to any one of claims 1-8 in the preparation of drugs that enhance antitumor immunity, characterized in that, The administration method of the enhanced anti-tumor immune drug is injection.

10. The use of sodium bicarbonate according to claim 9 in the preparation of drugs that enhance antitumor immunity, characterized in that, The sodium bicarbonate can be administered via intratumoral injection and arterial / venous injection.

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