Medicine for treating solid tumors and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a medicine for treating solid tumors and application thereof. The medicine provided by the invention simultaneously comprises F1, F3 and 5-FU, and every 100 mu L of the medicine comprises 20 to 40 mu g of F1, 20 to 40 mu g of F3 and 100 to 900 mu g of 5-FU. Experiments prove that in vitro, after the F1 / F3 and the 5-FU are combined for medication, the F1 / F3 and the 5-FU cannot synergistically generate an inhibition effect on tumor cells; however, in vivo, the combination of F1 / F3 and 5-FU can effectively inhibit the growth of solid tumor cells, such as colorectal cancer, melanoma and cervical cancer, and the combined use effect is obviously higher than that of single use of F1 / F3 or 5-FU. Therefore, the invention discloses that F1 / F3 and 5-FU are combined to synergistically inhibit the proliferation of the solid tumor cells for the first time, provides a novel medicine for the treatment of solid tumors, and has important significance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a drug for treating solid tumors and its application. Background Art

[0002] Solid tumors are common types of malignant tumors in clinical practice, including breast cancer, lung cancer, gastric cancer, colorectal cancer, melanoma, etc. Such tumors usually have strong invasiveness, strong early metastasis ability, and high drug resistance to traditional therapeutic drugs, seriously affecting the quality of life and prognosis of patients. The existing treatments for solid tumors mainly include surgical resection, radiotherapy, and chemotherapy with chemical drugs, etc. However, a single therapy usually fails to achieve satisfactory clinical effects, which also increases the treatment difficulty of solid tumors to a certain extent.

[0003] Among chemotherapy drugs, 5-fluorouracil (5-FU), as a classic antimetabolic drug, is widely used in the treatment of various solid tumors such as gastric cancer, colorectal cancer, and breast cancer. Although 5-FU has certain efficacy, it has obvious limitations in clinical applications, including problems such as myelosuppression, gastrointestinal reactions, and the generation of drug resistance, which severely limit the exertion of its long-term efficacy.

[0004] 5-FU (i.e., 5-fluorouracil) is an antitumor drug used for cancer treatment, and it exerts its antitumor effect by affecting the nucleic acid synthesis and metabolism of cells. Specifically, 5-fluorouracil, as a structure similar to purine bases, can be taken up by cells and then inhibit purine synthesis, resulting in DNA damage, etc., to achieve the purpose of inhibiting tumor growth. Currently, the most studied is the ability of 5-FU to reduce MDSCs in tumor patients.

[0005] In recent years, tumor immunotherapy has gradually become a research hotspot. In particular, immune checkpoint inhibitors (such as PD-1, CTLA-4, etc.) have achieved certain clinical progress. These drugs usually have relatively high immune-related adverse reactions and limited patient response rates, resulting in the massive aggregation of immunosuppressive microenvironments such as myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg), and M2 macrophages, which also severely restricts the effect of immunotherapy.

[0006] Caerin 1.1 / 1.9 (also known as F1 / F3) has been proven to inhibit the growth of human and murine cervical cancer cells, human thyroid cancer cells, human breast cancer cells, and murine melanoma cells in vitro and induce their programmed cell death. In addition, F1 / F3 can inhibit the growth of B16 and TC-1 cells in vivo. Its mediated anti-tumor immunity depends on the IFNAR-1 / Stat1 signaling of autocrine IFN in tumor-infiltrating macrophages. F1 / F3 can regulate the heterogeneity of macrophages in tumors, significantly reduce M2 macrophages and increase M1 macrophages, and promote the secretion of IL12 through tumor-infiltrating macrophages. However, these only prove that F1 / F3 can improve the tumor microenvironment. There are very few reports in the prior art on F1 / F3 improving the tumor inhibitory microenvironment dominated by MDSC.

[0007] In summary, in the process of treating tumors in the prior art, it is generally easy to damage the tumor immune microenvironment, resulting in technical problems such as low efficiency of immunotherapy. Summary of the Invention

[0008] In view of the above problems, the present invention provides a drug for treating solid tumors. The present invention proves for the first time that the combination of F1 / F3 and 5-FU can effectively inhibit the proliferation of solid tumor cells, and further improve the tumor immune microenvironment, which is beneficial to improving the efficiency of immunotherapy and provides an important theoretical basis for the clinical treatment of solid tumors.

[0009] The technical solution adopted by the present invention is as follows:

[0010] A drug for treating solid tumors, comprising F1, F3, and 5-FU. In every 100 μL of the drug for treating solid tumors, there are 20-40 μg of F1, 20-40 μg of F3, and 100-900 μg of 5-FU.

[0011] Preferably, the amino acid sequence of F1 is as shown in SEQ ID NO.1; the amino acid sequence of F3 is as shown in SEQ ID NO.2.

[0012] F1: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2 (SEQ ID NO.1);

[0013] F3: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2 (SEQ ID NO.2).

[0014] Preferably, in every 100 μL of the drug for treating solid tumors, there are 30 μg of F1, 30 μg of F3, and 100 μg of 5-FU.

[0015] Preferably, the drug for treating solid tumors further comprises physiological saline and a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutically acceptable carrier includes one or more of a corrosion inhibitor, a filler, a disintegrant, and a binder.

[0017] Preferably, the dosage form of the drug is a carrier drug or a non-carrier drug.

[0018] Preferably, the carrier drug is one of a viral vector, an exogenous cell vector, a liposome, and an exosome.

[0019] Preferably, the non-carrier drug is one of a capsule, a tablet, and an injection.

[0020] The present invention also provides an application of F1 / F3 combined with 5-FU in the preparation of a drug for treating solid tumors.

[0021] Preferably, the solid tumors include, but are not limited to, colorectal cancer, melanoma, and cervical cancer.

[0022] F1 / F3 (i.e., the combination of F1 and F3) of the present invention combined with 5-FU can also be made into a bacterial or cell vector, and the drug can be accurately transported to the target tissue or cell through a specific vector to improve the curative effect and reduce side effects.

[0023] Compared with the prior art, the technical advantages of the present invention are as follows: The present invention proves for the first time that F1 / F3 combined with 5-FU can effectively treat solid tumors, and will not destroy the tumor immune microenvironment, effectively improving the efficiency of immunotherapy, which is an important breakthrough in drugs for treating solid tumors. Description of the Drawings

[0024] Figure 1 It is a graph of cell viability measured by the MTT method when adding different drugs;

[0025] Figure 2 It is a graph of the results of evaluating the inhibitory effect of the combination of F1 / F3 and 5-FU on melanoma cells using the checkerboard method;

[0026] Figure 3 It is a graph of the results of the effect of 5-FU on MDSC in the spleen of tumor-bearing mice;

[0027] Figure 4 It is a graph of the results of the effect of 5-FU on MDSC in the spleen of normal mice;

[0028] Figure 5 It is a graph of the survival period results of mice when different contents of 5-FU are combined with F1 / F3;

[0029] Figure 6 It is a graph of the weight change results of mice when different contents of 5-FU are combined with F1 / F3;

[0030] Figure 7 Summary graph of the survival time of mice when 5-FU with different contents is used in combination with F1 / F3;

[0031] Figure 8 Comparison graph of the results of treating tumors with F1 / F3 combined with 5-FU under different administration routes;

[0032] Figure 9 Comparison graph of the in vivo efficacy of 5-FU or F1 / F3 alone and clinical chemotherapeutic drugs;

[0033] Figure 10 Graph of the results of F1 / F3 combined with 5-FU in improving the tumor microenvironment;

[0034] Figure 11 Graph of the results of the effect of F1 / F3 combined with 5-FU on the spleen immune system of tumor-bearing mice;

[0035] Figure 12 Graph of the results of the effect of F1 / F3 combined with 5-FU on the function of MDSC in the tumor microenvironment;

[0036] Figures 13 to 15 Graph of the results of evaluating the killing of three colorectal cancer cells by F1 / F3 combined with 5-FU using the checkerboard method;

[0037] Figure 16 Graph of the results of the effect of F1 / F3 combined with 5-FU on regulatory T cells in the spleen of TC-1 tumor-bearing mice;

[0038] Figure 17 Graph of the results of the effect of F1 / F3 combined with 5-FU on regulatory T cells in the tumor-draining lymph nodes of B16 tumor-bearing mice. Specific implementation mode

[0039] The present invention will be further explained below in conjunction with specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions identical or similar to the present invention are within the protection scope of the present invention. For those not specified in these embodiments in terms of specific techniques or conditions, operations shall be performed according to the conventional technical methods and the content of the instrument instructions in the art; for those reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] The 5-FU was purchased from Sigma, with the product number F6627-5G. Unless otherwise specified, 100 μL of F1 / F3 was administered to each mouse each time, and the amount of F1 / F3 was 30 μg each; 100 μL of 5-FU was administered to each mouse each time, and the amount of 5-FU was 100 μg.

[0041] Experimental example 1 In vitro detection experiment

[0042] 1. Experimental method:

[0043] First, the cytotoxic effects of F1 / F3 alone and 5-FU alone on B16 cells were determined by the MTT method: B16 cells were seeded at 1×10 4 cells per well in a 96-well plate, and 100 μL of B16 cell suspension was added to each well. According to the experimental design, 4 replicates were set for each group. Administration of 5-fluorouracil (5-FU): For the high-concentration test of 5-FU, the concentration gradients were set as follows: 100 μM, 500 μM, 1000 μM, 1500 μM, 2000 μM. For the low-concentration test of 5-FU, the concentration gradients were set as follows: 10 μM, 20 μM, 40 μM, 80 μM, 160 μM. Administration of F1 / F3: The concentrations of the experimental groups of F1 / F3 were 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 5 μM. The cell viability was detected using an MTT kit (purchased from Beyotime, product number: C0009M), and the IC50 (half-lethal dose) value was calculated by plotting. The results of the latest of two independent experiments were presented.

[0044] Then, the combination effect of F1 / F3 combined with 5-FU in killing melanoma cells was evaluated using the checkerboard method: B16 cells were seeded at 1×10 4 cells per well in a 96-well plate, and 100 μL of B16 cell suspension was added to each well. 5-FU was added horizontally and F1 / F3 was added vertically to form a drug concentration gradient: Horizontally (5-FU): 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, then 0 μL, 1 μL (0.5 mM), 1 μL (1 mM), 2 μL (1 mM), 4 μL (1 mM) were added. Vertically (F1 / F3): 0 μM, 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, then 0 μL, 5 μL (10 μM), 1 μL (100 μM), 1.5 μL (100 μM), 2 μL (100 μM) were added. Similarly, the cell viability was detected using an MTT kit, and the IC50 value was calculated by plotting. The results of the latest of two independent experiments were presented.

[0045] The FICI index is the IC50 of the combination / the IC50 of 5-FU alone + the IC50 of the combination / the IC50 of F1 / F3 alone. The meanings represented by FICI are as follows:

[0046] ① Synergistic effect, that is, the activity after the combination of the two drugs is significantly greater than the sum of the IC50s of their single drugs (FICI < 0.5); ② Additive effect, the activity after the combination is equal to the sum of the activities of their single drugs (0.5 < FICI < 1);

[0047] ③ No interaction, the activity after the combination is equal to the IC50 of their single drugs (1 < FICI < 2);

[0048] ④ Antagonism: the activity of the combined drug is significantly lower than that of a single drug, that is, the activity of one drug is weakened by another drug (FICI>2).

[0049] 2. Test results:

[0050] Cell survival Figure 1 As shown in AC, Figure 1 In the figure, A and B represent the cell survival at different concentrations of 5-FU, the IC50 of 5-FU is 9.2 μM, and 5-FU can effectively inhibit the proliferation of B16 cells. C represents the cell survival at different concentrations of F1 / F3, and the IC50 is 1.605 μM. It can be seen that F1 / F3 can inhibit the proliferation of B16 cells at a lower concentration gradient.

[0051] The checkerboard method was used to evaluate the correlation between F1 / F3 and 5-FU in killing B16 cells. Figure 2 As shown in AC, Figure 2 In the figure, A represents the relationship between drug concentration and OD value after F1 / F3 and 5-FU are used together, B represents the IC50 fitting curve after 5-FU is used alone and in combination with F1 / F3, and C represents the IC50 fitting curve after F1 / F3 is used alone and in combination with 5-FU. According to the IC50 calculation of the drug, 1<FICI=1.28<2, so after F1 / F3 and 5-FU are used together, there is no inhibition on B16 cells through synergistic effect.

[0052] Experimental Example 2 Effect of 5-FU on spleen MDSCs of normal mice and tumor-bearing mice

[0053] 1. Experimental subjects and modeling:

[0054] Animal experiment grouping and drug administration: The groups were non-tumor-bearing group and tumor-bearing group. The non-tumor-bearing group consisted of healthy C57BL / 6 mice, which were also divided into healthy control group and healthy experimental group. 4×10 5 B16 cells were recorded as Day 0. When melanoma was clearly observed on Day 3, the tumor-bearing group was divided into a tumor-bearing control group and a tumor-bearing experimental group. There were 6 mice in each group. The 5-FU dosage for each mouse in the healthy experimental group and the tumor-bearing experimental group was 900 μg / mouse, and the injection volume was 100 μL / mouse. Both were intraperitoneally injected. The administration time was three days / time, and the administration was twice. 5-FU was prepared with sterile PBS and used immediately. The healthy control group and the tumor-bearing control group were injected with the same dose of PBS.

[0055] 2. Detection method:

[0056] On the third day after the end of the drug administration, the spleen was taken to prepare a single cell suspension. The concentration of each sample in each group was adjusted to 1×10 6Flow antibody staining was performed at [X] cells / mL. The flow antibody staining protocol was Fixable Viability Stain 510 (FVS510), FITC-CD45.2, PerCP-Cy5.5-CD11b, APC-Ly6G (Gr1), BV421-Ly6C.

[0057] Add 1 μL of each of the following flow antibodies to both the experimental and control groups of the sample tubes: FVS510, FITC-CD45.2 (clone: 104), PerCP-Cy5.5-CD11b (clone: m1 / 70), APC-Ly6G (Gr1) (clone: 1A8-ly6G), BV421-Ly6C (clone: AI-21). After thoroughly pipetting and mixing, incubate at 4°C for 30 min.

[0058] 3. Test results:

[0059] The results are as Figure 3 and Figure 4 , Figure 3 show the effects of 5-FU on MDSC in the spleens of tumor-bearing mice. A shows the gating method for MDSC, B shows the gating methods for subsets of MDSC and the changes in monocytic myeloid-derived suppressor cells (m-MDSC) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) before and after drug administration, and C shows the statistical analysis of the component differences in the proportions of MDSC and its subsets. Figure 4 show the effects of 5-FU on MDSC in the spleens of normal mice. Figures A - C are the same as above. It can be seen that whether the mice are tumor-bearing or not, 5-FU can inhibit MDSC. After treatment, MDSC basically decreased by nearly 70% or so. And compared with PMN-MDSC, the significant inhibitory effect of 5-FU on the m-MDSC subset is more prominent, with an average inhibition rate of about 85%.

[0060] Experimental Example 3 Concentration Exploration of the Therapeutic Efficacy of F1 / F3 Combined with 5-FU

[0061] 1. Experimental subjects and model establishment:

[0062] Grouping and drug administration for animal experiments: The groups were: PBS + PBS, F1 / F3 + PBS, 5-FU + PBS, F1 / F3 + 5-FU. Drug administration protocol: Prepare 5-FU at different concentrations with sterile PBS. The drug administration volume for each mouse was fixed at 100 μL, and doses of 5-FU containing 900 μg, 600 μg, 300 μg, and 100 μg were prepared. Inject 5-FU intraperitoneally on Day 1, and then intratumorally injection thereafter, 3 times a week for a total of 5 times. Prepare 100 μL of F1 / F3 with PBS, containing 30 μg of F1 / F3, and continuously inject intratumorally for 14 days starting from Day 3.

[0063] 2. Detection method:

[0064] The tumor sizes of mice in each group were observed to draw survival curves, and the body weights of the mice were weighed. PBS was used as the control group for F1 / F3 and / or 5-FU treatment, and the administration time and dose were basically the same as theirs.

[0065] 3. Detection results:

[0066] The results are as Figures 5 to 7 shown. It can be seen therefrom that the median survival time of 300 μg was 26.5 days, the median survival time of 100 μg was 21 days, and the median survival times at the concentrations of 900, 600, 300, and 100 μg after dual therapy were increased to 37.5, 31, 31, and 33 days Figure 5 . Further analysis found that the best cure rate of mice in the 100 μg group after dual therapy was 40%, and the best cure rate in the 900 μg group was 27% ( Figure 7 ). However, in subsequent experiments, for 900 μg of 5-FU treatment, whether used alone or in combination, the body weights of the mice decreased, while the body weights of the mice after 100 μg of 5-FU treatment were similar to those of the control group, and their mental states and activity levels were also the best. Therefore, 100 μg of 5-FU was considered the best concentration for combination with F1 / F3 ( Figure 6 ).

[0067] Experimental Example 4 Exploration of the Administration Route of F1 / F3 Combined with 5-FU Treatment

[0068] 1. Experimental subjects and model establishment:

[0069] The animal experiments were grouped as: blank control group (untreated), F1 / F3 (intratumoral injection), 5-FU (intratumoral injection), F1 / F3 + 5-FU (intratumoral + intratumoral), F1 / F3 + 5-FU (intratumoral + intraperitoneal). Administration plan: The administration volume for each mouse was fixed at 100 μL, and a 5-FU dose containing 100 μg was prepared. 5-FU was administered by intraperitoneal injection or intratumoral injection on Day 3, 3 times a day for a total of 5 times. 100 μL of PBS containing 30 μg of F1 / F3,

[0070] was continuously intratumorally injected for 14 days starting from Day 6. The F1 / F3 group and the 5-FU group were the same as above except without adding the other group of drugs, and the blank control group was given the same dose of PBS.

[0071] 2. Detection method:

[0072] The tumor sizes of the mice were measured using vernier calipers to draw survival curves, and the body weights of the mice were weighed. PBS was used as the control group for F1 / F3 and / or 5-FU treatment, and the administration time and dose were basically the same as theirs.

[0073] 3. Detection results:

[0074] The results are as Figure 8 shown. The median survival time of the Untreated control group was 18 days, the dual survival cycle of intraperitoneal injection of 5-FU was 28 days, and the dual survival cycle of intratumoral injection of 5-FU was 33 days. There was no significant difference in the survival cycle comparison, P = 0.785( Figure 8 A). On Day 27, the tumors were dissected and weighed. It was found that the efficacy of the combination of intratumoral injection of 5-FU was significantly better than that of the combination of intraperitoneal injection, P = 0.0036, and 67% of the tumors in the mice with the combination of intratumoral injection of 5-FU were lower than 0.03 g( Figure 8 B-C). Therefore, intratumoral injection of F1 / F3 combined with 5-FU (100 μg / mouse) can significantly improve the therapeutic effect of F1 / F3 and inhibit the progression of melanoma.

[0075] Comparison of the in vivo efficacy of single 5-FU or F1 / F3 and clinical chemotherapeutic drugs in Test Example 5

[0076] 1. Test subjects and model establishment:

[0077] The grouping is as follows: Untreated, F1 / F3, 5-FU, CDDP (Maclean D807330), DTIC (Maclean D838232). Drug administration plan: Prepare a dose of 5-FU containing 100 μg. The day of tumor inoculation was recorded as Day 0. 5-FU was injected intratumorally on Day 3, 3 times a day for a total of 5 doses. At the same time, F1 / F3 was continuously injected intratumorally for 14 days starting from Day 3. CDDP was prepared with sterile PBS, and the fixed administration volume for each mouse was 100 μL. A dose of CDDP containing 40 μg was prepared and administered by intraperitoneal injection 3 times a day for a total of 5 doses. DTIC was prepared with sterile PBS, and the fixed administration volume for each mouse was 100 μL. A dose of DTIC containing 270 μg was prepared and administered by intraperitoneal injection 3 times a day for a total of 5 doses.

[0078] 2. Test method:

[0079] Observe the tumor size of the mice and draw a survival curve. PBS was used as the control group for the treatment of F1 / F3 and / or 5-FU, and the administration time and dose were basically the same as theirs.

[0080] 3. Test results:

[0081] The results are as Figure 9As shown in the figure, A represents the survival cycle of inhibiting tumor progression in vivo by using 5-FU, F1 / F3, DTIC, and CDDP alone, and B represents their average tumor growth curves. The results show that using 5-FU or F1 / F3 alone is significantly better than the clinical chemotherapy drugs for melanoma. DTIC and CDDP can more effectively extend the survival cycle of tumor-bearing mice and inhibit tumor progression, but there is no significant difference between the two.

[0082] Experimental Example 6 Exploration of F1 / F3 combined with 5-FU in improving the tumor microenvironment and tumor-inhibiting microenvironment 1. Experimental subjects and model establishment:

[0083] After constructing the B16 tumor-bearing model according to Experimental Example 2, the experimental groups were as follows: PBS + PBS, F1 / F3 + PBS, 5-FU + PBS, F1 / F3 + 5-FU. Administration scheme: When tumors were significantly observed on Day 3, 5-FU was injected intratumorally. The administration volume for each mouse was fixed at 100 μL, and a dose containing 100 μg of 5-FU was prepared, administered 3 times a day for a total of 5 times. F1 / F3 was prepared with sterile PBS to obtain 100 μL of F1 / F3, containing 30 μg of F1 and F3, and was continuously injected intratumorally for 7 days starting from Day 6 to observe the tumor size of the mice.

[0084] 2. Experimental method:

[0085] Flow cytometry analysis of the microenvironment was performed on Day 27. The detection method was the same as in Experimental Example 2, and the flow cytometry antibody staining protocol was Fixable Viability Stain 510 (FVS510), FITC-CD45.2, PerCP-Cy5.5-CD11b, APC-Ly6G (Gr1), BV421-Ly6C, APC-cy7-CD3e, PE-CD4, PE-cy7-CD8a.

[0086] 3. Experimental results:

[0087] Results Figure 10 As shown in the figure, Figure 10 A represents the proportion of MDSC in CD45.2+ cells and the flow cytometry scatter plot for each group. The PBS + PBS group was 39.7%, the F1 / F3 + PBS group was 29.1%, the 5-FU + PBS group was 1.84%, and the F1 / F3 + 5-FU group was 11.7%. Figure 10B represents the proportion of subtypes of MDSC, m-MDSC and PMN-MDSC, among CD45.2+ cells in each group and the flow cytometry scatter plots. In the PBS+PBS group, the proportion of m-MDSC was 3.85% and that of PMN-MDSC was 38.1%; in the F1 / F3+PBS group, the proportion of m-MDSC was 10.3% and that of PMN-MDSC was 32.5%; in the 5-FU+PBS group, the proportion of m-MDSC was 16.7% and that of PMN-MDSC was 7.40%; in the F1 / F3+5-FU group, the proportion of m-MDSC was 11.7% and that of PMN-MDSC was 17.0%. Figure 10 C represents the proportion of T cells among CD45.2+ cells in each group and the flow cytometry scatter plots. In the PBS+PBS group, it was 7.16%; in the F1 / F3+PBS group, it was 11.4%; in the 5-FU+PBS group, it was 14.3%; in the F1 / F3+5-FU group, it was 29.6%. Figure 10 D represents the proportion of subtypes of T cells, CD4+ T cells and CD8+ T cells, among CD45.2+ cells in each group and the flow cytometry scatter plots. In the PBS+PBS group, the proportion of CD4+ T cells was 38.9% and that of CD8+ T cells was 27.6%; in the F1 / F3+PBS group, the proportion of CD4+ T cells was 51.9% and that of CD8+ T cells was 17.7%; in the 5-FU+PBS group, the proportion of CD4+ T cells was 51.9% and that of CD8+ T cells was 23.9%; in the F1 / F3+5-FU group, the proportion of CD4+ T cells was 49.5% and that of CD8+ T cells was 42.1%.

[0088] The results show that 5-FU itself has the effect of clearing immunosuppressive cells (especially MDSC), while F1 / F3 can regulate the distribution of MDSC subtypes to a certain extent and may enhance the anti-tumor effect by improving the tumor microenvironment and promoting T cell expansion / infiltration. When the two are combined, it can not only reduce the level of MDSC, but also significantly increase the proportion of T cells (especially CD8+ T cells) in the tumor microenvironment, thus significantly enhancing the immune killing ability of the body against tumor cells. The combined use of F1 / F3 and 5-FU can effectively reduce immunosuppressive MDSC and increase the proportion of effector T cells (CD4+ and CD8+), thereby enhancing the anti-tumor immune effect of the body, demonstrating good immune regulation and therapeutic synergy.

[0089] Experimental Example 7 Exploration of the Effect of F1 / F3 Combined with 5-FU on the Spleen Immune System of Tumor-Bearing Mice

[0090] 1. Experimental Subjects and Modeling:

[0091] After successfully establishing the B16 tumor-bearing model, it was then evenly divided into each group according to tumor size: PBS+PBS, F1 / F3+PBS, 5-FU+PBS, F1 / F3+5-FU. Before drug treatment, the tumor size data was measured with a vernier caliper every other day to observe the survival cycle of the mice. Drug administration plan: Intratumoral injection of 5-FU started on Day 8, with a fixed administration volume of 100 μL per mouse, preparing a dose of 5-FU containing 100 μg, 3 times a day for a total of 5 administrations. F1 / F3 was prepared with sterile PBS at 100 μL, containing 30 μg of F1 and F3, and was continuously intratumorally injected for 7 days starting from Day 8 to observe the tumor size of the mice.

[0092] 2. Experimental method:

[0093] On Day 27, the spleens of the mice in each group were collected for flow cytometry analysis of the microenvironment. The flow cytometry antibody staining protocol was Fixable Viability Stain 510 (FVS510), FITC-CD45.2, PerCP-Cy5.5-CD11b, APC-Ly6G (Gr1), BV421-Ly6C.

[0094] 3. Experimental results:

[0095] The results are as Figure 11 shown, Figure 11 A represents the proportion of MDSC in CD45.2+ cells in each group and the flow cytometry scatter plot. The PBS+PBS group was 6.71%, the F1 / F3+PBS group was 6.31%, the 5-FU+PBS group was 6.17%, and the F1 / F3+5-FU group was 4.11%. Figure 11 B represents the proportion of the subtypes m-MDSC and PMN-MDSC of MDSC in CD45.2+ cells in each group and the flow cytometry scatter plot. The m-MDSC in the PBS+PBS group was 18.1% and the PMN-MDSC was 46.8%; the m-MDSC in the F1 / F3+PBS group was 17.0% and the PMN-MDSC was 41.9%; the m-MDSC in the 5-FU+PBS group was 14.3% and the PMN-MDSC was 41.8%; the m-MDSC in the F1 / F3+5-FU group was 11.3% and the PMN-MDSC was 47.2%.

[0096] In the spleen, an peripheral immune organ, the basal level of MDSC (proportion of CD45.2+ cells) is far lower than that in the tumor microenvironment; the change amplitude brought by single use of F1 / F3 or 5-FU is small. However, when the two are combined, the decrease effect of MDSC in the spleen is still obvious (4.11%), especially the proportion of the m-MDSC subset (11.3%) is significantly lower than that of other groups, which indicates that the combined regimen also has the effect of clearing or inhibiting MDSC at the peripheral level. Although the proportion of PMN-MDSC in the spleen does not show a large change, the down-regulation of the overall MDSC level is beneficial to reducing systemic immunosuppression and providing a more favorable environment for the activation of anti-tumor immune effector cells such as T cells subsequently.

[0097] Therefore, these results suggest that the combined treatment of F1 / F3 and 5-FU can not only significantly reduce the level of MDSC locally in the tumor and improve the immunosuppressive microenvironment, but also reduce MDSC in the spleen to a certain extent, especially the m-MDSC subtype, thus further supporting the anti-tumor immune response at the peripheral immune level.

[0098] Experimental Example 8 Exploration of the Functional Changes of MDSC in the Tumor Microenvironment by F1 / F3 Combined with 5-FU

[0099] 1. Experimental Subjects and Modeling:

[0100] Construct an F1 / F3 combined with 5-FU treatment model according to Experimental Example 7, collect tumor tissues, use magnetic beads to sort out MDSC, after counting, adjust the concentration to 2.8×10 5 cells / mL and inoculate them in a 24-well plate, and add LPS (final concentration of 100 ng / mL) to stimulate and culture overnight.

[0101] 2. Experimental Methods:

[0102] Centrifuge the MDSC cells cultured overnight at 1000 rpm for 20 min, collect the supernatant and use ELISA to detect the secretion of cytokines such as IL-10, IL-6, TNF-α, and IL-1β.

[0103] 3. Experimental Results:

[0104] The results are as Figure 12As shown, taking the PBS + PBS control group as a reference, the secretion levels of IL-10, IL-6, and TNF-α in the F1 / F3 + PBS treatment group were significantly increased. The secretion level of IL-10 in the 5-FU treatment group was more significant than that in the F1 / F3 + PBS group, and there was a statistical difference. It is worth noting that the F1 / F3 + 5-FU combination treatment group showed an obvious inhibitory effect on IL-10, IL-6, and TNF-α. There was basically no obvious change in the secretion level of IL-1β in different treatment groups of MDSC. These results provide valuable information for us to further explore the function of F1 / F3 + 5-FU in inhibiting MDSC.

[0105] Experimental Example 9 Evaluation of the Associative Effect of F1 / F3 Combined with 5-FU in Killing Three Kinds of Colorectal Cancer Cells 1. Experimental Method:

[0106] MC38 cells were seeded at 1×10 4 cells per well in a 96-well plate, and 100 μL of MC38 cell suspension was added to each well. 5-FU was added horizontally and F1 / F3 was added vertically to form a drug concentration gradient: Horizontally (5-FU): 0, 10, 20, 40, 80, 160 μM, then 0 μL, 2 μL (0.5 mM), 4 μL (0.5 mM), 8 μL (0.5 mM), 1.6 μL (5 mM), 3.2 μL (5 mM) were added. Vertically (F1 / F3): 0, 0.5, 1.0, 1.5, 2.0, 2.5 μM, then 0 μL, 5 μL (10 μM), 1 μL (100 μM), 1.5 μL (100 μM), 2 μL (100 μM), 2.5 μL (100 μM) were added. The cell viability was detected using an MTT kit (Product No.: C0009M), and the IC50 value was calculated by plotting. The results of the latest of two independent experiments were shown.

[0107] SW480 cells were seeded at 1×10 4Inoculate each well with the cells at a density of 1×10 per well into a 96-well plate, and add 100 μL of SW480 cell suspension to each well. Add 5-FU horizontally and F1 / F3 vertically to form a drug concentration gradient: Horizontally (5-FU): 0, 125, 250, 500, 1000, 2000 μM, then add 0 μL, 2.5 μL (5 mM), 5 μL (5 mM), 10 μL (5 mM), 10 μL (10 mM), 20 μL (10 mM). Vertically (F1 / F3): 0, 1.0, 2.0, 3.0, 4.0, 5.0 μM, then add 0 μL, 1 μL (100 μM), 2 μL (100 μM), 3 μL (100 μM), 4 μL (100 μM), 5 μL (100 μM). Use the MTT kit (product number: C0009M) to detect cell viability and plot a graph to calculate the IC50 value. The results of the latest of two independent experiments are presented.

[0108] HT29 cells were inoculated into a 96-well plate at a density of 1×10 4 per well, and 100 μL of HT29 cell suspension was added to each well. Add 5-FU horizontally and F1 / F3 vertically to form a drug concentration gradient: Horizontally (5-FU): 0, 125, 250, 500, 1000, 2000 μM, then add 0 μL, 2.5 μL (5 mM), 5 μL (5 mM), 10 μL (5 mM), 10 μL (10 mM), 20 μL (10 mM). Vertically (F1 / F3): 0, 2.0, 4.0, 6.0, 8.0, 10 μM, then add 0 μL, 2 μL (100 μM), 4 μL (100 μM), 6 μL (100 μM), 8 μL (100 μM), 10 μL (100 μM). Use the MTT kit (product number: C0009M) to detect cell viability and plot a graph to calculate the IC50 value. The results of the latest of two independent experiments are presented.

[0109] 2. Test results:

[0110] The checkerboard method was used to evaluate the combined effect of F1 / F3 and 5-FU on killing MC38 cells as Figure 13 shown. According to the IC50 of the drugs, FICI = 0.9 < 1 was calculated. Therefore, after the combined use of F1 / F3 and 5-FU, the combination of F1 / F3 and 5-FU has an additive effect on the inhibition of MC38.

[0111] The checkerboard method was used to evaluate the combined effect of F1 / F3 and 5-FU on killing SW480 cells as Figure 14 shown. According to the IC50 of the drugs, FICI = 1.0 was calculated. Therefore, after the combined use of F1 / F3 and 5-FU, the combination of F1 / F3 and 5-FU has an additive effect on the inhibition of SW480.

[0112] Evaluation of the associative effect of F1 / F3 and 5-FU on killing HT29 cells by the checkerboard method is as Figure 15 shown. According to the IC50 of the drugs, FICI = 0.74 < 1 is calculated. Therefore, after the combination of F1 / F3 and 5-FU, the combination of F1 / F3 and 5-FU has an additive effect on the inhibition of HT29.

[0113] Experimental example 10. Effect of F1 / F3 combined with 5-FU on regulatory T cells in the spleen of TC-1 tumor-bearing mice 1. Experimental subjects and modeling:

[0114] Grouping and administration of animal experiments: 5×10 5 TC-1 cells were inoculated into the lateral abdomen and recorded as Day0. After obvious tumors were observed on Day3, they were evenly divided into four groups: UN group, F1 / F3 group, 5-FU group, and F1 / F3 + 5-FU group according to the tumor size. The administration method was that the UN group was given PBS once a day for 14 consecutive days, the F1 / F3 group was given F1 / F3 polypeptide once a day for 14 consecutive days, the 5-FU group was given 5-FU once every three days (i.e., Day3, Day6, Day9, Day12, Day15), and the F1 / F3 + 5-FU group was given F1 / F3 polypeptide once a day for 14 consecutive days and 5-FU once every three days (i.e., Day3, Day6, Day9, Day12, Day15). 2. Experimental method:

[0115] According to the method of Experimental example 2, the flow antibody staining protocol was Fixable Viability Stain 510 (FVS510), FITC-CD45.2, PerCP-Cy5.5-CD4, APC-cy7-CD3e, PE-Foxp3.

[0116] 3. Experimental results:

[0117] As Figure 16 shown, the gating strategy for regulatory T cells (Treg) was that the proportion of the Treg population in the Untreated group was 1.25%, the proportion of the Treg population in the F1 / F3 polypeptide group was 2.3%, the proportion of the Treg population in the 5-FU group was 2.34%, and the proportion of the Treg population in the F1 / F3 + 5-FU group was 1.46%. The results showed that the combination of F1 / F3 and 5-FU could significantly reduce the proportion of Treg in spleen cells compared with the single use of F1 / F3, and improve the immunosuppressive microenvironment.

[0118] Experimental example 11 Effect of F1 / F3 combined with 5-FU on regulatory T cells in the tumor-draining lymph nodes of B16 tumor-bearing mice

[0119] 1. Experimental subjects and modeling:

[0120] Animal experiment grouping and drug administration: 4×10 5 B16 cells were recorded as Day 0. After the tumor was obviously observed on Day 3, the mice were evenly divided into four groups according to the tumor size: UN group, F1 / F3 group, 5-FU group, and F1 / F3+5-FU group. The UN group was given PBS once a day for 14 consecutive days, the F1 / F3 group was given F1 / F3 polypeptide once a day for 14 consecutive days, the 5-FU group was given 5-FU once every three days (i.e. Day 3, Day 6, Day 9, Day 12, Day 15), the F1 / F3+5-FU group was given F1 / F3 polypeptide once a day for 14 consecutive days, and 5-FU was given once every three days (i.e. Day 3, Day 6, Day 9, Day 12, Day 15).

[0121] 2. Test methods:

[0122] The flow cytometry antibody staining protocol was Fixable Viability Stain 510 (FVS510), FITC-CD45.2, PerCP-Cy5.5-CD4, APC-cy7-CD3e, and PE-Foxp3.

[0123] 3. Test results:

[0124] like Figure 17 As shown, the results showed that the proportion of Treg in the Untreated group was 11.5%, the proportion of Treg in the Caerin 1.1 / 1.9 peptide group was 12.1%, the proportion of Treg in the 5-FU group was 10.3%, and the proportion of Treg in the Caerin 1.1 / 1.9 peptide + 5-FU combined treatment group was 7.78%. Statistical analysis showed that there was no significant difference between the Untreated group and the Caerin 1.1 / 1.9 peptide group, while the 5-FU group had a significant difference compared with the Untreated group (P<0.01), and the Caerin 1.1 / 1.9 peptide + 5-FU combined treatment group showed a very significant difference compared with the Untreated group (P<0.0001). The results showed that in the B16 tumor-bearing mouse model, 5-FU alone could significantly reduce the proportion of Treg in TDLN of B16 tumor-bearing mice, while the combined use of Caerin 1.1 / 1.9 peptide and 5-FU had a more significant inhibitory effect on the proportion of Treg in TDLN of B16 tumor-bearing mice, further revealing the potential synergistic effect of combined therapy in improving the tumor microenvironment.

Claims

1. A drug for treating solid tumors, characterized in that, It includes F1, F3, and 5-FU. In every 100 μL of the therapeutic drug for solid tumors, there is 20 - 40 μg of F1, 20 - 40 μg of F3, and 100 - 900 μg of 5-FU.

2. The drug for treating solid tumors as claimed in claim 1, wherein The amino acid sequence of the said F1 is as shown in SEQ ID NO.1; the amino acid sequence of the said F3 is as shown in SEQ ID NO.

2.

3. The drug for treating solid tumors according to claim 1, characterized in that, In every 100 μL of the therapeutic drug for solid tumors, it includes 30 μg of F1, 30 μg of F3, and 100 μg of 5-FU.

4. The drug for treating solid tumors as claimed in claim 1, wherein, The therapeutic drug for solid tumors further includes normal saline and a pharmaceutically acceptable carrier.

5. The drug for treating solid tumors according to claim 1, wherein, The pharmaceutically acceptable carrier includes one or more of corrosion inhibitors, fillers, disintegrants, and binders.

6. The drug for treating solid tumors according to claim 1, wherein, The dosage form of the said drug is a carrier drug or a non-carrier drug.

7. The drug for treating solid tumors according to claim 6, wherein The carrier drug is one of a viral vector, an exogenous cell vector, a liposome, and an exosome.

8. The drug for treating solid tumors according to claim 6, wherein, The non-carrier drug is one of a capsule, a tablet, and an injection.

9. Use of F1 / F3 combined with 5-FU in the preparation of a therapeutic drug for solid tumors.

10. The application according to claim 1, characterized in that, The said solid tumors include colorectal cancer, melanoma, and cervical cancer.

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