Use of anti-pd-1 antibody combined with riboflavine sodium phosphate in the preparation of a drug for treating colon cancer

Abstract

The application relates to the field of biological medicine, in particular to application of an anti-PD-1 antibody combined with riboflavin sodium phosphate in preparation of a drug for treating colon cancer. + Riboflavin sodium phosphate is a metabolic regulator, which can improve CD8 T cell energy metabolism level (such as enhancing mitochondrial function), and the anti-PD-1 antibody can release the inhibition on the T cell; the two jointly act from different ways, realize perfect synergy of'releasing inhibition' and 'providing energy', and thus generate an antitumor effect of 1+1>2. The application can significantly improve the antitumor efficiency by using the anti-PD-1 antibody and riboflavin sodium phosphate in combination, and in an animal model, the tumor volume of the combination treatment group is reduced more obviously.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of anti-PD-1 antibody combined with riboflavin sodium phosphate in the preparation of drugs for treating colon cancer. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract that occurs in the colon. With the continuous advancement of research into tumor immunotherapy and the development of disciplines such as immunology and molecular biology, significant progress has been made in the development of monoclonal antibodies targeting key regulatory targets of cellular immunity, such as PD-1 / PD-L1. Anti-PD-1 antibodies kill tumors by blocking the PD-1 / PD-L1 signaling pathway and activating the patient's own T-cell immune response, representing a breakthrough in cancer treatment. However, the current clinical challenge is the prevalence of primary or acquired drug resistance, meaning a significant proportion of patients cannot benefit from PD-1 / PD-1 antibody therapy.

[0003] Riboflavin and its active derivative, riboflavin sodium phosphate, are key factors in cellular metabolism and redox reactions. Colorectal cancer competitively depletes riboflavin in the microenvironment, leading to CD8... + T cells suffer from impaired function due to nutritional deficiencies. Supplementing with riboflavin can restore T cell activity to some extent and inhibit tumor growth. However, this technology has significant limitations.

[0004] First, its mechanism of action is relatively simple, primarily addressing the "nutrient competition" faced by T cells, but failing to simultaneously alleviate the "signal suppression" mediated by immune checkpoints such as PD-1 / PD-L1. In tumors with a strongly immunosuppressive microenvironment or abnormally activated checkpoint pathways, riboflavin supplementation alone is insufficient to fundamentally reverse the exhaustion state of T cells. Second, the anti-tumor efficacy of this regimen is expected to be limited in complex in vivo environments, especially in tumor models resistant to anti-PD-1 antibodies, where significant breakthroughs are unlikely. Summary of the Invention

[0005] In view of this, the present invention provides the application of anti-PD-1 antibody combined with riboflavin sodium phosphate in the preparation of drugs for treating colorectal cancer. The present invention aims to address the technical shortcomings of existing technologies, such as low efficacy and easy development of drug resistance in anti-PD-1 antibody monotherapy, and the inability of riboflavin monotherapy to reverse immune checkpoint inhibition. It provides a novel combination therapy regimen that significantly enhances anti-tumor immunity through synergistic effects, particularly offering an effective treatment option for cancer patients who are insensitive to or resistant to PD-1 antibodies.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides the application of anti-PD-1 antibody combined with riboflavin sodium phosphate in the preparation of drugs for treating colon cancer.

[0007] In some implementations, the anti-PD-1 antibody is a known monoclonal antibody; riboflavin sodium phosphate is a pharmaceutical grade compound with the chemical name riboflavin 5'-(dihydrophosphate) monosodium salt dihydrate, molecular formula: C 17 H 20 N4NaO9P, calculated on a dried basis, contains riboflavin (C 17 H 20 The content of N4O6 was 74.0%~79.0%.

[0008] Riboflavin sodium phosphate possesses significantly higher water solubility and chemical stability than riboflavin, enabling the formulation of homogeneous and quality-controlled injectable preparations. This perfectly matches the route of administration for anti-PD-1 antibodies. More importantly, as an active precursor, it can be directly utilized by immune cells without conversion, more efficiently reversing T-cell metabolic depletion in the tumor microenvironment. Consequently, it generates a stronger synergistic effect with anti-PD-1 antibodies in overcoming immune resistance. This specific formulation choice fundamentally solves the bottlenecks in formulation, bioavailability, and efficacy of riboflavin in clinical combination anti-tumor therapy.

[0009] In some implementations, anti-PD-1 antibodies and riboflavin sodium can be administered simultaneously, sequentially, or alternately.

[0010] In some implementations, the anti-PD-1 antibody and riboflavin sodium are administered by injection.

[0011] In some implementations, the anti-PD-1 antibody and riboflavin sodium are administered via intraperitoneal or intravenous injection.

[0012] In some implementations, colon cancer is defined as colon cancer that is insensitive to PD-1 / PD-L1 antibody therapy or has developed acquired resistance.

[0013] In some embodiments, the concentration of riboflavin sodium phosphate is 1-50 mM; preferably 5-20 mM.

[0014] In some implementations, the anti-PD-1 antibody is administered every 2-4 days, and flavin phosphate is administered every 1-3 days.

[0015] In some implementations, the anti-PD-1 antibody is administered every 3 days, and flavin phosphate is administered every 2 days.

[0016] In the technical solution of this invention, riboflavin sodium phosphate acts as a metabolic regulator, by increasing CD8... +The energy metabolism level within T cells (such as enhancing mitochondrial function) restores their vitality from a state of "exhaustion"; while the anti-PD-1 antibody relieves the inhibition of T cells. The two work together through different pathways to achieve a perfect synergy between "relieving inhibition" and "providing energy", thus producing an anti-tumor effect of 1+1>2.

[0017] In a second aspect, the present invention provides a pharmaceutical composition for treating colon cancer, comprising an anti-PD-1 antibody and riboflavin sodium phosphate.

[0018] In some implementations, the anti-PD-1 antibody is administered every 2-4 days, and flavin phosphate is administered every 1-3 days.

[0019] In some implementations, the anti-PD-1 antibody is administered every 3 days, and flavin phosphate is administered every 2 days.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention significantly improves anti-tumor efficacy through the combined use of anti-PD-1 antibody and riboflavin sodium phosphate. In animal models, the tumor volume reduction was more pronounced in the combined treatment group. This invention also reduces drug resistance through the combined use of anti-PD-1 antibody and riboflavin sodium phosphate. The combined treatment can reduce adaptive resistance of tumor cells to PD-1 antibody, and both anti-PD-1 antibody and riboflavin sodium phosphate have high safety profiles. Within the experimental dosage range, no additional toxic side effects were observed with the combined use of anti-PD-1 antibody and riboflavin sodium phosphate. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a schematic diagram illustrating the antitumor effects of different concentrations of riboflavin sodium phosphate; Figure 2 This is a comparison chart of tumor sizes at different concentrations of riboflavin sodium phosphate; Figure 3 This is a statistical chart of tumor weights at different concentrations of riboflavin sodium phosphate; Figure 4 This is a comparison chart of tumor growth curves at different concentrations of riboflavin sodium phosphate. Figure 5 Riboflavin phosphate sodium is for depleted CD8 + A diagram illustrating the impact of T cell effector factors; Figure 6 Riboflavin phosphate sodium is for depleted CD8 + Statistical chart showing the impact of T cell effector factors on production; Figure 7It is riboflavin sodium phosphate for CD8 + Figure showing the effect of T cell oxidative phosphorylation capacity; Figure 8 This is a schematic diagram illustrating the anti-tumor effect of anti-PD-1 antibody combined with riboflavin sodium phosphate; Figure 9 This is a comparison chart of tumor size between the combination therapy group and the single-drug group; Figure 10 This is a statistical chart of tumor weight in the combination therapy group and the single-drug group; Figure 11 This is a comparison chart of tumor growth curves between the combination therapy group and the single-drug group. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0025] Example 1 1. Group settings: Six- to eight-week-old male C57BL / 6 mice were subcutaneously inoculated with mouse colon cancer cells (MC38 cells) and randomly divided into four groups: Control group: Intraperitoneal injection of phosphate-buffered saline (PBS); Riboflavin sodium phosphate 1 mM group: 0.1 mL of 1 mM riboflavin sodium phosphate solution was injected intraperitoneally; Riboflavin sodium phosphate 10 mM group: 0.1 mL of 10 mM riboflavin sodium phosphate solution was injected intraperitoneally; Riboflavin sodium phosphate 50 mM group: 0.1 mL of 50 mM riboflavin sodium phosphate solution was injected intraperitoneally.

[0026] 2. Experimental Methods 2.1. Subcutaneous tumorigenesis experiment using MC38 cells from C57BL / 6 mice Hair was shaved from the injection site in C57BL / 6 mice before cell injection. MC38 cells were digested, collected, and counted. They were resuspended in PBS and adjusted to a concentration of 5 × 10⁻⁶. 6 Prepare a cell suspension of 5 × 10⁶ cells / mL and place it on ice. Before injection, gently pipette the cells to mix them thoroughly. Transfer the cell suspension to a syringe, expel all air, and administer subcutaneously at a dose of 0.1 mL per mouse, i.e., 5 × 10⁶ cells per mouse. 5 Each cell.

[0027] 2.2 Riboflavin Sodium Therapy A schematic diagram illustrating the antitumor effects of riboflavin sodium phosphate is shown below. Figure 1 As shown. Starting from the fifth day after tumor inoculation, different concentrations of riboflavin sodium were injected intraperitoneally every two days.

[0028] 2.3 Observe tumor growth Observe the tumor growth. After a subcutaneous mass appears, measure the long diameter (a) and short diameter (b) of the tumor every two days. Calculate the tumor volume as 1 / 2 × a × b. 2 The tumor growth curves were recorded and plotted. Nineteen days after inoculation, the mice were sacrificed, and the tumors were photographed and weighed.

[0029] 3. Experimental Results Figure 2 This is a comparison chart of tumor sizes at different concentrations of riboflavin sodium phosphate. Figure 3 This is a statistical chart of tumor weight at different concentrations of riboflavin sodium phosphate. Figure 4 This is a comparison chart of tumor growth curves at different concentrations of riboflavin sodium phosphate. Figure 3 The asterisk (*) in the middle represents P <0.05, Figure 4 The "**" in the middle is P <0.01. From Figure 2 , Figure 3 , Figure 4 It can be seen that, compared with the control group, intraperitoneal injection of riboflavin sodium phosphate showed better therapeutic effects, with smaller tumor volume and weight. Compared with the 1 mM and 50 mM groups, the 10 mM group had smaller tumor volume and weight, and better anti-tumor effect.

[0030] Example 2: Effects of riboflavin sodium phosphate on exhausted cytotoxic T lymphocytes (CD8) + The influence of T cell effector factors 1. Primary CD8 in mice + T cell extraction and culture (1) The C57BL / 6 mice were euthanized, and the axillary, neck, groin, mesenteric lymph nodes and spleen were dissected and removed. The cells were washed three times in pre-cooled PBS, ground in 5 mL PBS and the cell suspension was filtered through a 70 μM filter. (2) Centrifuge at 400 × g for 5 min, pour out the liquid, add 1 mL of red blood cell lysis buffer (Beyotime, cat#C3702), let stand for 2 min, and add 5 mL of PBS to terminate the lysis; (3) Centrifuge at 400 × g for 5 min, pour out the liquid, and use the mouse's initial CD8a + T-cell isolation kit (NaiveCD8a) +T Cell Isolation Kit, mouse (miltenyi, cat#130-096-543) extracts CD8 + T cells; (4) The product that flows out naturally from the sorting column is CD8. + T cells were used with 50 μM β-cells containing 10% fetal bovine serum (FBS). IMDM medium (Beijing Maichen, cat#CM10016) containing mercaptoethanol (Gibico, cat#31350010) and 10 ng / mL interleukin-2 (IL-2) (R&D Systems, 402-ML-020) was added to 24-well plates coated one day in advance with 1 μg / mL CD3e (Biolegend, cat#100340) and 0.5 μg / mL CD28 (Biolegend, cat#102116) and cultured for 2 days. The plates were divided into PBS group and 10 mM riboflavin sodium group. (5) Replace the culture plate with the same conditions every two days, for a total of three times.

[0031] 2. Flow cytometry staining Cells were collected for staining with the following antibodies: anti-CD8a-APC-Cy7 (BD, cat#557654), anti-PD1-PE-cy7 (Biolegend, cat#135216), anti-Tim3-BV605 (Biolegend, cat#119721), FixableViability Stain 700 (BD, cat#564997), anti-IFN-γ-BV711 (BD, cat#564336), anti-TNF-α-PerCP-e710 (Invitrogen, cat#46-7321-82), and anti-Granzyme B-Pacific Blue (Biolegend, cat#515408).

[0032] 3. Experimental Results Figure 5 Riboflavin phosphate sodium is for depleted CD8 + A diagram illustrating the impact of T cell effector factors. Figure 6 Riboflavin phosphate sodium is for depleted CD8 + A statistical graph showing the impact of T cell effector factors, in which... Figure 6 The "**" in the middle is P <0.01, "***" represents P <0.001. For example... Figure 5 , 6As shown, compared with the control group, the 10 μM riboflavin sodium phosphate treatment group depleted CD8+. + The proportions of effector factors perforin, granzyme B (GZMB), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in T cells were significantly increased.

[0033] Example 3: Detection of the effect of riboflavin sodium phosphate on CD8 + Effects of T cell oxidative phosphorylation 1. Preparations the day before the experiment 1.1 Turn on and preheat the detection system Press the switch on the back of the Agilent Cellular Energy Metabolism Analyzer (Seahorse XF Pro Analyzer) to turn on the main unit; press the switch on the right side of the Seahorse XF Pro control computer.

[0034] 1.2. Turn on the controller Open the Wave Pro Controller software and wait for the controller to connect successfully to the instrument host and for the temperature to rise to 37°C.

[0035] 1.3 Hydration Probe Plate-1 (1) Open the mitochondrial stress test kit (XF Cell Mito Stress Test Kit (Agilent, cat#103015-100)) and remove its contents; (2) Place the probe plate upside down next to the tool plate; (3) Add 200 µL of XF calibration solution to each well of the tool plate; (4) Place the XF hydration auxiliary plate on top of the tool plate and press the XF hydration auxiliary plate down to ensure tight assembly with the tool plate; (5) Lower the sensor probe plate through the opening of the XF hydration auxiliary plate onto the tool plate, so that the probe is immersed in the XF calibration solution; (6) Determine whether the XF calibration liquid level is sufficient to keep the probe submerged; (7) Place it in a CO2-free incubator at 37 °C overnight. To prevent the XF calibration solution from evaporating, the incubator should be humidified.

[0036] 1.4. Poly-DLysine coating of Seahorse cell culture plates (1) Add 25 µL of the solution to each well at room temperature and incubate for 20 min.

[0037] (2) Rinse each well twice with 200 µL of sterile water.

[0038] (3) The coated Seahorse cell culture plates were dried overnight in a clean bench.

[0039] 2. On the day of the experiment, the machine will be used for testing. CD8 + T extraction is the same as in Example 2.

[0040] 2.1 Hydration Probe Plate-2 (1) Remove the probe plate assembled with the XF hydration aid plate and tool plate from the incubator; (2) Place the probe plate upside down next to the tool plate; (3) Hold the tool board with one hand and lift the XF hydration auxiliary board from one corner with the other hand to remove it; (4) Place the probe plate back on the tool plate and load the compound onto the dosing port as required, and place it on the XF Pro analyzer for calibration.

[0041] 2.2 Preparation of Seahorse detection solution (1) Dispense 97 mL of Seahorse XF DMEM medium (in a clean bench), add 1 mL of glucose, 1 mL of pyruvate and 1 mL of glutamine to it in sequence and mix well to prepare the detection solution (the volume of substrate added can be adjusted according to the concentration required for the experiment). (2) Place the prepared test solution in a cell culture incubator at 37 °C without CO2 for later use (or use directly after a 37 °C water bath).

[0042] 2.3, CD8 + T cell platelet formation (1) Prepare the test solution incubated at 37 °C; (2) Take a 15 mL centrifuge tube and collect CD8 pretreated with PBS and 10 μM riboflavin sodium phosphate. + T cells; (3) Centrifuge the cells at 200 × g for 5 min at room temperature; (4) When centrifuging the cells, add 50 μL of detection solution to the background correction well of the cell culture plate that has been placed at room temperature and coated; (5) Discard the cell supernatant after centrifugation; (6) Add 2 mL of incubation test solution to the centrifuge tube to resuspend the cells for counting, and adjust the cell concentration to 1×10⁻⁶. 5 / 50μL; (7) Change the centrifuge setting to zero braking; (8) Transfer the cell suspension to a sterile filling tank and aspirate it using a multi-channel pipette, or aspirate it directly from a centrifuge tube; (9) Add 50 μL / well to the cell culture plate along the sidewall of each well into the 96-well plate. Do not seed cells in the background correction well. (10) Centrifuge the cell plate at 200 × g (zero braking) for 1 min. Ensure the centrifuge is properly equilibrated; (11) Place the cell culture plate in a cell culture incubator without CO2 supplementation and incubate at 37 ℃ for 25-30 min. Observe under a microscope to ensure that the cells are completely attached to the wall. (12) Slowly add 130 µL of preheated detection solution along the sidewall of each well to the cell well, being careful not to disturb the cells; (13) Observe the cells under a microscope to ensure that the cells adhere to the wall; (14) Place the cell plate back into the incubator and incubate for 15-25 min; (15) After 15-25 min, the cell plate can be used for analysis. To obtain the best results, the total time after centrifugation should not exceed 60 min.

[0043] 2.4 Prepare the medication and add it to the dosing well of the probe plate. 2.4.1 Medication preparation process (1) Open the aluminum foil bag and take out the three tubes containing oligomycin (blue cap), FCCP (yellow cap) and Rot / AA (red cap). Place the three tubes on the appropriate tube rack. (2) Take out the prepared 37 ℃ test solution, add it to the corresponding drug tubes according to the volume shown in Table 1, and gently mix it by pipetting (piping about 10 times or vortexing) to fully dissolve the drug; Table 1. Compound volume concentration table

[0044] (3) Use the test solution to dilute the resuspended drug to the required concentration (1.5 μM for oligomycin; 1 μM for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP); and 0.5 μM for rotenone and antimycin A (Rot / AA)). Prepare about 2-3 mL of each drug working solution. After the drug working solution is prepared, wait for the drug to be added.

[0045] 2.4.2. Dosing process (1) Take out the hydrated probe plate from the 37 ℃ CO2-free cell culture incubator, remove the hydration auxiliary plate and probe plate cover, and ensure that the probe plate is placed on the tool plate; (2) Take the corresponding dosing aid plate and place it on the green probe plate; (3) Take out the prepared drug working solution (37 ℃) and add the corresponding drugs to the corresponding wells of the probe plate in the corresponding volumes (the volumes added to each series of wells are: 20 μL for well A, 22 μL for well B, 25 μL for well C, and 27 μL for well D). (4) Remove the dosing aid plate after the drug loading is complete; (5) Check the drug injection status in each dosing hole.

[0046] 3. Experimental Results Figure 7 It is riboflavin sodium phosphate for CD8 + The effect of T cell oxidative phosphorylation capacity is shown in the figure. Mitochondrial function was assessed by measuring cellular oxygen consumption rate (OCR), revealing that treatment with 10 μM riboflavin sodium phosphate significantly enhanced CD8+. + The basal respiration, maximum respiratory capacity, adenine triphosphate (ATP) production, and reserve respiratory capacity of T cells indicate a significant increase in oxidative phosphorylation.

[0047] Example 4: MC38 subcutaneous tumorigenesis model in C57BL / 6 mice in anti-PD 1. Growth curves and tumor weight determination under combined antibody and riboflavin sodium therapy 1. Group settings: Six- to eight-week-old male C57BL / 6 mice were subcutaneously inoculated with colon cancer cells from MC38 mice and randomly divided into four groups.

[0048] IgG: Intraperitoneal injection of IgG, n=6; anti PD-1: Intraperitoneal injection of PD-1 antibody, n=6; IgG + 10 mM riboflavin sodium phosphate: Intraperitoneal injection of IgG and riboflavin sodium phosphate, n=6; anti PD-1 + 10 mM riboflavin sodium phosphate: Intraperitoneal injection of PD-1 and riboflavin sodium phosphate, n=6.

[0049] 2. Experimental Methods 2.1 Subcutaneous tumor formation assay in C57BL / 6 mice (MC38) Hair was shaved from the injection site in C57BL / 6 mice before cell injection. MC38 cells were digested, collected, and counted. They were resuspended in PBS and adjusted to a concentration of 5 × 10⁻⁶. 6 Prepare a cell suspension of 5 × 10⁶ cells / mL and place it on ice. Before injection, gently pipette the cells to mix them thoroughly. Transfer the cell suspension to a syringe, expel all air, and administer subcutaneously at a dose of 0.1 mL per mouse, i.e., 5 × 10⁶ cells per mouse. 5Each cell.

[0050] 2.2 Anti-PD-1 and riboflavin sodium therapy Figure 8 This is a schematic diagram illustrating the anti-tumor effect of anti-PD-1 antibody combined with riboflavin sodium phosphate. Starting from the third day after tumor inoculation, IgG and anti-PD-1 antibodies are injected intraperitoneally every three days. PD-1 antibody, 100 μg per mouse, once.

[0051] Starting from the fifth day after tumor inoculation, each mouse was injected intraperitoneally with 10 mM riboflavin sodium every two days.

[0052] 2.3 Observe tumor growth Observe the tumor growth. After a subcutaneous mass appears, measure the long diameter (a) and short diameter (b) of the tumor every 2 days, and calculate the tumor volume = 1 / 2 × a × b. 2 The tumor growth curves were recorded and plotted. Nineteen days after inoculation, the mice were sacrificed, and the tumors were photographed and weighed.

[0053] 3. Experimental Results Figure 9 This is a comparison chart of tumor size between the combination therapy group and the single-drug group. Figure 10 This is a statistical chart of tumor weight in the combination therapy group and the single-drug group. Figure 11 This is a comparison chart of tumor growth curves between the combination therapy group and the single-drug group. Figure 10 The asterisk (*) in the middle represents P <0.05, "**" means P <0.01, "****" represents P <0.0001; Figure 11 The asterisk (*) in the middle represents P <0.05, "***" represents P <0.001, "****" is P <0.0001.

[0054] Figure 9 , Figure 10 , Figure 11 The comparison charts of tumor size, tumor weight, and tumor growth curves in mice showed that, compared with the IgG treatment group, the anti-tumor... PD Group 1 showed better therapeutic effects, confirming the efficacy of anti-inflammatory drugs. PD 1. Treatment has shown good results in cancer treatment. Based on this, anti- PD 1 treatment group and anti PD In the 1 + 10mM riboflavin sodium phosphate treatment group, we found that the combination therapy group significantly enhanced anti- PD 1. The therapeutic effect of using it alone.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of anti-PD-1 antibody combined with riboflavin sodium phosphate in the preparation of drugs for treating colon cancer.

2. The application as described in claim 1, characterized in that, Riboflavin sodium phosphate is a pharmaceutical grade compound containing 74.0% to 79.0% riboflavin on a dried basis.

3. The application as described in claim 1, characterized in that, Anti-PD-1 antibodies and riboflavin sodium can be administered simultaneously, sequentially, or alternately.

4. The application as described in claim 1, characterized in that, Anti-PD-1 antibody and riboflavin sodium phosphate were administered by injection.

5. The application as described in claim 4, characterized in that, Anti-PD-1 antibody and riboflavin sodium phosphate are administered via intraperitoneal or intravenous injection.

6. The application as described in claim 1, characterized in that, The colon cancer referred to is colon cancer that is insensitive to PD-1 / PD-L1 antibody therapy or has developed acquired resistance.

7. The application as described in claim 1, characterized in that, The concentration of riboflavin sodium phosphate is 1-50 mM.

8. The application as described in claim 1, characterized in that, Anti-PD-1 antibody is administered every 2-4 days, and flavin phosphate is administered every 1-3 days.

9. The application as described in claim 8, characterized in that, Anti-PD-1 antibody was administered every 3 days, and flavin phosphate was administered every 2 days.

10. A pharmaceutical composition for treating colon cancer, characterized in that, The pharmaceutical composition includes an anti-PD-1 antibody and riboflavin sodium phosphate.

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