Use of serine in the preparation of antitumor drugs

Abstract

The present application belongs to the field of biological medicine and tumor immunotherapy, and particularly relates to application of serine or a pharmaceutically acceptable salt or ester thereof in preparation of a medicament for treating tumors, and relates to a treatment strategy for enhancing an anti-tumor immune response by supplementing serine. The present application experimentally proves for the first time that a traditional "serine / glycine starvation therapy" has weak curative effect in an orthotopic liver cancer model, while a serine supplement therapy has a curative effect of more than 3 times that of the starvation therapy in inhibiting orthotopic tumors and reducing metastasis. Moreover, the serine supplement enhances the infiltration of immune cells in the microenvironment, the effector function, and specifically promotes the migration of CD8 + T cells to the core area of the tumor by a mechanism completely dependent on CD8 + T cells, and achieves the effect of immunotherapy. In a PD-1 antibody-resistant advanced liver cancer model, serine can be used in combination with a PD-1 antibody to produce a synergistic effect, reducing the tumor load to 1 / 5 of the control group. Serine can also be used in combination with arginine to produce a synergistic anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and tumor immunotherapy, specifically relating to the use of serine, particularly L-serine or its pharmaceutically acceptable salts or esters, in the preparation of medicaments for treating tumors, and particularly to therapeutic strategies for enhancing anti-tumor immune responses through exogenous serine supplementation, as well as compositions or kits for the combined use of serine with immune checkpoint inhibitors. Background Technology

[0002] Metabolic reprogramming is one of the core characteristics of all malignant tumors. To meet the nutritional demands of rapid proliferation, tumor cells are highly dependent on the uptake of exogenous amino acids. Serine is a conditionally essential amino acid, involved in one-carbon metabolism, nucleotide synthesis, and redox homeostasis regulation, and is crucial for cell proliferation and survival. For a long time, the mainstream strategy for targeting serine metabolism has focused on inhibiting the serine synthesis pathway (such as targeting PHGDH) or depriving serine / glycine through diet, which is a deeply ingrained bias. Based on this understanding, a large amount of research in the field has long focused on "starving" tumor cells through "serine / glycine starvation therapy" (i.e., depriving exogenous serine and glycine intake through dietary restriction) or developing metabolic enzyme inhibitors targeting the serine synthesis pathway (such as PHGDH inhibitors). "Serine / glycine starvation therapy" is achieved through special medical foods or strictly controlled amino acid restriction diets, but compliance and long-term safety are issues. Serine and glycine are also essential for normal physiological functions, and "serine / glycine starvation therapy" is toxic to normal tissues and immune cells. Therefore, how to achieve "selective starvation of tumors" without damaging normal tissues is a core challenge. Furthermore, systemic amino acid levels are difficult to reduce effectively because the liver and kidneys have an active serine synthesis capacity; even with dietary deficiencies, plasma serine levels can only decrease partially. Therefore, long-term strict dietary restrictions are poorly adhered to in humans and may lead to malnutrition.

[0003] However, these strategies all have significant drawbacks: starvation therapy not only has a weak tumor-suppressing effect, but its evaluation system is also based on xenograft models, where the assessment of anti-tumor efficacy is primarily based on direct effects on tumor cells. However, in the real tumor microenvironment, in addition to tumor cells, there are other cells such as immune cells. Therefore, the effects of starvation therapy on immune cells in an immune-intact tumor microenvironment should also be included in the evaluation system. Effector T cells with anti-tumor function are rapidly proliferating cells, requiring a large amount of nutrients to support their anti-tumor function during rapid proliferation and functionalization. In an immune-intact tumor microenvironment, starvation therapy is highly likely to lead to immunosuppression by simultaneously depriving effector T cells of their serine supply, and may even increase the risk of metastasis.

[0004] Immune checkpoint inhibitor therapy has shown remarkable efficacy in many malignant solid tumors, effectively inhibiting tumor growth and prolonging patient survival. The mechanism of action of immune checkpoint inhibitors primarily involves antagonizing immunosuppressive receptors such as CTLA-4, PD-1, or PD-L1, restoring the immune activity of the patient's T cells, thereby enhancing the anti-tumor immune response.

[0005] Immune checkpoint blockade therapy, represented by PD-1 / PD-L1 monoclonal antibodies, is a cutting-edge approach in cancer treatment, achieving breakthroughs in various solid tumors such as melanoma and non-small cell lung cancer. However, clinical practice shows that a large number of solid tumor patients exhibit primary or secondary resistance to immune checkpoint inhibitors. Especially after tumor progression to intermediate or advanced stages, severe metabolic competition and immunosuppression exist within the tumor microenvironment (TME), leading to CD8... + Deep exhaustion of T cell function makes it difficult for PD-1 antibodies to effectively control tumor progression. Therefore, it is crucial to find safe and effective methods to reshape the metabolic conditions of the tumor microenvironment and selectively reactivate CD8. + The anti-tumor function of T cells is a core clinical challenge in reversing resistance to immune checkpoint inhibitors and improving the response rate of immunotherapy.

[0006] Combination immunotherapy can significantly improve tumor response rates, reduce the risk of disease progression, thereby prolonging overall survival and improving patients' quality of life. Common combination immunotherapy regimens currently include: PD-1 inhibitors combined with chemotherapy, PD-1 inhibitors combined with radiotherapy, PD-1 inhibitors combined with targeted drugs, and PD-1 inhibitors combined with other immunotherapies.

[0007] L-Serine is a conditionally essential amino acid that participates in key processes in cellular metabolism, such as one-carbon unit metabolism, nucleotide synthesis, and glutathione production, significantly impacting cell proliferation and survival. For a long time, it has been widely accepted in the field that rapidly proliferating tumor cells are highly dependent on the supply of exogenous serine (e.g., high expression of PHGDH, a key enzyme in the serine synthesis pathway, to maintain endogenous serine synthesis). Therefore, existing technologies have never broken the technological prejudice that "serine supplementation promotes tumor growth," nor have there been any reports on improving T cells in the tumor microenvironment through serine supplementation, thereby enhancing the efficacy of immunotherapy. There are also no reports on combining serine with PD-1 inhibitors to enhance anti-tumor effects. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies such as the stringent clinical implementation requirements of "serine / glycine starvation therapy" (which requires strict restriction of serine and glycine intake in the diet), poor tumor suppression effect, failure of mid-to-late stage tumors to PD-1 antibody therapy, and metabolic depletion of T cells in the tumor microenvironment, this invention provides the application of serine in the preparation of anti-tumor drugs, especially by supplementing serine to achieve anti-tumor effects.

[0009] This invention is achieved through the following technical solution: This invention provides the application of serine in the preparation of antitumor drugs.

[0010] The tumor is a solid tumor that can be treated by activating the immune system; The tumor is selected from gastric cancer, kidney cancer, bladder cancer, skin cancer, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, thyroid cancer, liver cancer, colorectal cancer, lung cancer, melanoma, breast cancer, etc., preferably liver cancer, colorectal cancer, lung cancer, melanoma or breast cancer.

[0011] The serine mentioned is L-serine or its pharmaceutically acceptable salt or ester.

[0012] Furthermore, the serine can be prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the serine can be combined with other amino acids to form a pharmaceutical composition, preferably, the serine and arginine are combined to form a pharmaceutical composition.

[0014] Experimental results show that exogenous supplementation with serine or its pharmaceutical combinations can significantly inhibit tumor growth, exhibiting a significant inhibitory effect on the growth of colorectal cancer, lung cancer, and hepatocellular carcinoma in situ. It can effectively control lung metastasis, reducing the number of metastatic lesions by half. In contrast, the traditional "serine / glycine starvation therapy" has only a weak and insignificant inhibitory effect on the growth of hepatocellular carcinoma in situ. Therefore, exogenous serine supplementation not only does not accelerate tumor growth but also significantly inhibits it, demonstrating a significantly superior effect compared to traditional starvation therapy.

[0015] Exogenous serine supplementation can effectively promote the growth of CD4+ in liver cancer in situ tumor tissue. + T cells, CD8 + The ratio and absolute number of T cells and natural killer (NK) cells significantly remodel the tumor immune microenvironment. Flow cytometry and tissue immunofluorescence analysis showed that, compared with the control group, CD8+ cells significantly increased in tumor cells. + Unlike other tumors where T cells primarily remain at the tumor periphery, serine supplementation can promote CD8. + Effective infiltration of T cells into the core region of the tumor is crucial for overcoming immune rejection and achieving tumor clearance.

[0016] Furthermore, in vivo CD8 antibody depletion experiments confirmed that when CD8 antibodies were depleted in mice... + Following T-cell therapy, the inhibitory effect of serine supplementation on the growth of in situ liver cancer and lung metastasis completely disappeared, and the tumor burden returned to the control group level. This further demonstrates that the core mechanism by which serine supplementation therapy exerts its anti-tumor effect lies in regulating the immune system, rather than acting directly on tumor cells. Further in vitro experiments showed that adding large amounts of serine during tumor cell culture did not affect tumor growth, migration, survival, or colony formation ability.

[0017] In an in vitro culture system simulating the tumor microenvironment (i.e., T cells cultured in tumor cell culture supernatant TCM), serine supplementation can directly and significantly increase CD8 levels. + T cell activation levels (upregulating CD69 and DNAM-1), proliferation capacity (increasing EdU incorporation and the proportion of Ki67 positive cells), effector function (increasing the secretion of IFN-γ and IL-2), and promotion of their survival (upregulating the expression of the anti-apoptotic molecule BCL-2).

[0018] It is evident that serine increases CD4 levels in tumor tissue. + T cells, CD8 + The number, ratio, and function of T cells and NK cells can enhance the anti-tumor immune response and reshape the tumor immune microenvironment to exert anti-tumor effects.

[0019] The present invention provides a tumor immunotherapy composition comprising serine and an immune checkpoint inhibitor.

[0020] The immune checkpoint inhibitor is a PD-1 antibody inhibitor, a PD-L1 antibody inhibitor, or a CTLA-4 inhibitor, preferably a PD-1 antibody inhibitor, wherein the PD-1 antibody inhibitor is an anti-PD-1 monoclonal antibody.

[0021] Exogenous serine supplementation can effectively promote the growth of CD4+ in liver cancer in situ tumor tissue. + T cells, CD8 + The ratio and absolute number of T cells and natural killer (NK) cells significantly remodel the tumor immune microenvironment. Flow cytometry and tissue immunofluorescence analysis showed that, compared with the control group, CD8+ cells significantly increased in tumor cells. + Unlike other tumors where T cells primarily remain at the tumor periphery, serine supplementation can promote CD8. + Effective infiltration of T cells into the core region of the tumor is crucial for overcoming immune rejection and achieving tumor clearance.

[0022] Furthermore, in vivo CD8 antibody depletion experiments confirmed that when CD8 antibodies were depleted in mice... +Following T-cell therapy, the inhibitory effect of serine supplementation on the growth of in situ liver cancer and lung metastasis completely disappeared, and the tumor burden returned to the control group level. This further demonstrates that the core mechanism by which serine supplementation therapy exerts its anti-tumor effect lies in regulating the immune system, rather than acting directly on tumor cells. Further in vitro experiments showed that adding large amounts of serine during tumor cell culture did not affect tumor growth, migration, survival, or colony formation ability.

[0023] In an in vitro culture system simulating the tumor microenvironment (i.e., T cells cultured in tumor cell culture supernatant TCM), serine supplementation can directly and significantly increase CD8 levels. + T cell activation levels (upregulating CD69 and DNAM-1), proliferation capacity (increasing EdU incorporation and the proportion of Ki67 positive cells), effector function (increasing the secretion of IFN-γ and IL-2), and promotion of their survival (upregulating the expression of the anti-apoptotic molecule BCL-2).

[0024] It is evident that serine increases CD4 levels in tumor tissue. + T cells, CD8 + The number, ratio, and function of T cells and NK cells can enhance the anti-tumor immune response and reshape the tumor immune microenvironment to exert anti-tumor effects.

[0025] The present invention provides a tumor immunotherapy composition comprising serine and an immune checkpoint inhibitor.

[0026] The immune checkpoint inhibitor is a PD-1 antibody inhibitor, a PD-L1 antibody inhibitor, or a CTLA-4 inhibitor, preferably a PD-1 antibody inhibitor, wherein the PD-1 antibody inhibitor is an anti-PD-1 monoclonal antibody.

[0027] The serine mentioned is L-serine or its pharmaceutically acceptable salt or ester.

[0028] The composition may also contain a pharmaceutically acceptable carrier or excipient.

[0029] Furthermore, the composition comprises serine and a PD-1 antibody inhibitor, wherein the mass ratio of the PD-1 antibody inhibitor to serine is 1:80-800, preferably 1:80-200.

[0030] In the tumor immunotherapy composition, serine combined with a PD-1 antibody inhibitor can effectively overcome PD-1 antibody resistance and significantly enhance the anti-tumor efficacy of immune checkpoint inhibitors.

[0031] The PD-1 antibody inhibitor is a PD-1 antibody.

[0032] The present invention provides a tumor immunotherapy composition comprising serine and arginine, wherein the mass ratio of serine to arginine is 1:1-10, preferably 1:2.5-5.

[0033] This invention provides the use of the tumor immunotherapy composition in the preparation of antitumor drugs, especially drugs for treating liver cancer.

[0034] This invention provides the application of serine in the preparation of sensitizers or synergists for tumor immunotherapy.

[0035] Furthermore, the present invention provides the use of serine in the preparation of sensitizers or synergists that enhance the treatment of liver cancer with PD-1 antibody inhibitors.

[0036] This invention provides the application of serine combined with PD-1 antibody inhibitors in the preparation of antitumor drugs.

[0037] The ratio of PD-1 antibody inhibitor to serine is 1:80-800, preferably 1:80-200.

[0038] The tumors are unresponsive, resistant, or poorly treated with PD-1 antibody inhibitors alone.

[0039] This invention provides the application of serine combined with arginine in the preparation of antitumor drugs.

[0040] The dosage ratio of serine to arginine is 1:1-10, preferably 1:2.5-5.

[0041] The aforementioned combination refers to administering at least one dose of L-serine and / or at least one dose of PD-1 antibody within a certain time period. The time period can be within a dosing cycle, preferably within 4 weeks, 3 weeks, 2 weeks, 1 week, or within 24 hours.

[0042] Combined administration methods are selected from simultaneous administration, independently prepared and co-administered, or independently prepared and sequentially administered.

[0043] Preferably, the combined administration method is to administer PD-1 antibody and L-serine simultaneously or sequentially; or to administer arginine and L-serine simultaneously or sequentially.

[0044] Results showed that in an orthotopic model of advanced hepatocellular carcinoma unresponsive to PD-1 antibody monotherapy, PD-1 antibody alone was ineffective, but its combination with serine supplementation therapy produced a significant synergistic effect. After combination therapy, the number of tumor nodules in the orthotopic hepatocellular carcinoma decreased to one-fifth of the control level. Tissue analysis showed that combination therapy significantly increased CD8+ in tumor tissue. +The total number of T cells and their infiltration rate in the tumor core region. Therefore, serine can synergistically enhance and reverse resistance to PD-1 antibodies.

[0045] This invention provides the application of serine in the preparation of immune cells that enhance tumor-killing ability.

[0046] Furthermore, the present invention provides a method for preparing or expanding immune cells with anti-tumor activity, wherein the immune cells are CD8+. + T cells, the immune cells described herein, are prepared by adding serine to the culture medium for culturing immune cells. The cultured immune cells can enhance their ability to secrete IFN-γ and granzyme B.

[0047] Furthermore, the amount of serine added to the system is 150~800 μM, preferably 400~800 μM.

[0048] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention breaks with the common understanding that "serine supplementation promotes cancer, while serine deficiency inhibits cancer." This invention's experiments directly demonstrate for the first time that the traditional "serine / glycine starvation therapy" has weak efficacy in an orthotopic liver cancer model. This indiscriminate deprivation strategy, while attempting to "starve" tumor cells, also severely damages CD8 cells, which are also highly dependent on exogenous serine for proliferation and effector function. + T cells, on the contrary, may cause systemic immunosuppression, leading to a decline in tumor immune surveillance function. Serine supplementation therapy, however, is more than three times more effective than starvation therapy in inhibiting in situ tumors and reducing metastasis. It effectively inhibits tumor growth in various solid tumors.

[0049] 2. In vitro experiments showed that serine supplementation did not promote the proliferation of tumor cells such as liver cancer and colorectal cancer. In vivo experiments showed that long-term high-dose intake had no negative impact on mouse body weight, major organs, or liver function, demonstrating high safety, and significantly inhibited tumor cell growth. The minimum effective dose of serine is 3 mg / day.

[0050] 3. The combination of serine and PD-1 antibody reduced the tumor burden of advanced liver cancer to 1 / 5 of the control group and significantly promoted CD8. + T cell infiltration in the tumor core. This provides a novel treatment strategy for achieving potent reversal of immune resistance in patients with intermediate-to-advanced tumors who are unresponsive to or resistant to PD-1 antibodies.

[0051] 4. The combined use of serine and arginine can synergistically inhibit the growth of in situ liver cancer, reducing the number of liver nodules to 1 / 4 of the original number and the number of lung metastases to 1 / 4 of the original number.

[0052] 5. The anti-tumor effects of the present invention have been verified in tumor models from various tissue sources, such as colon cancer, lung cancer, and liver cancer, demonstrating its great potential as a universal immunometabolic adjuvant across tumor types.

[0053] 6. Mechanistic studies have shown that the addition of serine enhances CD8... + T cell effector function is enhanced, promoting their infiltration into the tumor core, thereby achieving tumor immunotherapy.

[0054] 7. It provides the application of serine in the preparation of T cells that enhance tumor-killing ability. Attached Figure Description

[0055] Figure 1 Dietary serine supplementation effectively inhibits tumor growth in various mouse models.

[0056] AE: Effects of serine supplementation on mean tumor size, weight, and spleen weight in a subcutaneous xenograft model of colon cancer.

[0057] FL: Effects of serine supplementation on body weight, spleen weight, liver weight, liver weight / body weight ratio, number of orthotopic nodules, size of the largest orthotopic hepatocellular carcinoma nodule, and tumor morphology in an orthotopic hepatocellular carcinoma xenograft model.

[0058] MP: Effects of serine supplementation on mean tumor size, tumor weight, and spleen weight in a subcutaneous xenograft model of lung cancer.

[0059] Figure 2 Head-to-head parallel comparison of serine supplementation therapy and serine / glycine starvation therapy.

[0060] AH: Comparison of liver weight, maximum nodule size, number of in situ nodules, lung weight, and number of spontaneous lung metastases between serine supplementation therapy and serine / glycine starvation therapy in an orthotopic liver cancer model.

[0061] IK: Comparison of splenic lymphocyte function effects of serine supplementation therapy versus serine / glycine starvation therapy in an orthotopic model of liver cancer.

[0062] Figure 3 Effects of serine / glycine deprivation on the activity, proliferation, and colony-forming ability of various tumor cells.

[0063] AC: Effects of serine / glycine deprivation on the proliferation of HepG2, Hepa1-6BL, and HTC116 cells.

[0064] DF: Effects of serine / glycine deprivation on colony formation ability of HepG2, Hepa1-6BL, and HTC116 cells.

[0065] Figure 4The effect of adding serine on tumor cell proliferation and cell viability.

[0066] A, B, C: Effects of serine on the viability of HepG2, Hepa1-6BL, and HTC116 cells.

[0067] D, E, F: Effects of serine on the proliferation of HepG2, Hepa1-6BL, and HTC116 cells.

[0068] GJ: Effects of serine on migration, cloning, and colony formation in HTC116 cells.

[0069] Figure 5 CD8 + T cell exhaustion assay validates CD8 + The role of T cells in serine replacement therapy.

[0070] A: Schematic diagram of the experiment B: Liver weight C: Size of the largest hepatocellular carcinoma nodule D: Number of in situ hepatocellular carcinoma nodules E: Liver tumor morphology; F: Number of spontaneous lung metastases; G: Representative image of lung metastatic nodules.

[0071] Figure 6 The effects of serine supplementation on CD8 were investigated using flow cytometry and immunofluorescence analysis. + CD4 + The influence of NK cell number and proportion.

[0072] A: CD4⁺ T cells B: CD8⁺ T cells + C: T cells; D: NK cells; E: Immunofluorescence representation of CD8 positive cells in tumor tissue; E: Immunofluorescence statistical analysis of CD8. + The proportion of T cells in tumor tissue.

[0073] Figure 7 Supplementing with serine for CD8 + The effects of T cell activation, proliferation, effector function, and survival.

[0074] AI represents the addition of 400 uM serine to TCM to treat CD8 T cells. A: Expression of IFNγCD8 + The percentage of T cells (B): expressing IL-2CD8 + The percentage of T cells C: Expression activation indicators CD69 CD8 + percentage of T cells D: Expression activation marker DNAM-1 CD8 + percentage of T cells E: The proportion of cells replicating DNA during the EdU reaction. F: Ki67 reflects the proportion of cells currently in the cell cycle (i.e., those with proliferative potential). G: Mean fluorescence intensity of Ki67; H: Positive CD8 for anti-apoptotic protein BCL-2. + T cell ratio I: Average fluorescence intensity of BCL-2.

[0075] JN represents CD8 T cells treated with 150μM, 300μM, 400μM, 600μM, and 800μM serine, respectively, in TCM.

[0076] J: Expression of IFNγCD8 + The percentage of T cells (K): expressing IL-2 and CD8 + The percentage of T cells: L: The percentage of CD69-expressing CD8T cells; M: The percentage of Ki67-positive CD8T cells; N: Statistical analysis and representative images of the average fluorescence intensity of Ki67.

[0077] Figure 8 Effects of serine supplementation combined with PD-1 antibody on tumor cells in an advanced PD-1 antibody-resistant liver cancer model.

[0078] A: Schematic diagram of the model construction for combined serine supplementation and PD1 antibody therapy for hepatocellular carcinoma in situ. B: Size and representative image of the largest in situ hepatocellular carcinoma nodule. C: Statistical analysis of the total number of in situ hepatocellular carcinoma nodules and the number of nodules of different sizes D: Immunofluorescence staining analysis of the number and distribution of CD8 T cells in tumor tissue after different treatments. E: Immunofluorescence analysis of the proportion of CD8 T cells in tumor tissue after different treatments.

[0079] Figure 9 Effects of serine supplementation on body weight, organs, histology, and liver function.

[0080] AI represents the changes in parameters in mice without established tumor models after the treatment group was given 1500 mg / L of serine in drinking water.

[0081] A: Body weight of mice at different time points after serine supplementation B: Liver weight and representative image of mice after serine supplementation C: Body weight D: Liver-to-body weight ratio E: Spleen weight F: Kidney weight and representative image G: Colon length H: HE staining analysis of colon, liver, kidney, etc. I: Serum ALT and AST levels.

[0082] JL represents the treatment group mice that were provided with 6 g / L of serine in drinking water in the establishment of orthotopic liver cancer.

[0083] J: Number of in situ hepatocellular carcinoma nodules; K: Liver / body weight ratio; L: Spleen weight.

[0084] Figure 10 Serine and L-arginine, when combined, can synergistically inhibit tumor growth and metastasis.

[0085] A: Schematic diagram of tumor model establishment B: Mouse liver weight C: Number of orthotopic hepatocellular carcinoma nodules D: Size of the largest in situ hepatocellular carcinoma nodule; E: Number of spontaneous lung metastases. Detailed Implementation

[0086] The present invention will be further described in detail below through examples, but the content of the present invention is not limited to these examples. L-serine (purity ≥99%) used in the experiment was purchased from Sigma-Aldrich, anti-PD-1 monoclonal antibody (clone number RMP1-14) and anti-CD8β monoclonal antibody (clone number 53-5.8) were purchased from Bio X Cell, mouse liver cancer cells Hepa1-6BL, colon cancer cells MC38, lung cancer cells 3LL and various human tumor cells were all purchased from ATCC, and C57BL / 6J wild-type mice (6–8 weeks old, female) were purchased from Vital River Pharmaceuticals, Beijing.

[0087] Example 1: Broad-spectrum antitumor effects of serine supplementation in various tumor models Colorectal cancer model: according to Figure 1 Method A, 5×10 5 A colon cancer model was established by subcutaneously inoculating MC38 cells into C57BL / 6 mice. The day of tumor cell injection was designated as day 0.

[0088] Serine supplementation: On the day of injection, mice were given serine in their drinking water at a concentration of 600 mg / L to ensure they had free access to drinking water.

[0089] Tumor measurement: During this period, the size of the subcutaneous tumor was measured using calipers. After euthanizing the mice at 18 days, the tumor weight and spleen weight were measured.

[0090] In situ model of liver cancer: according to Figure 1 Method F involves injecting 5 × 10⁵ cells via a high-pressure tail vein into C57BL / 6 mice. 5 An orthotopic liver cancer model was established using Hepa1-6BL cells.

[0091] 1) Day 0 is the day the tumor cells are injected. On this day, mice are given serine in their drinking water at a concentration of 600 mg / L to ensure that they have free access to drinking water throughout the experiment.

[0092] 2) On day 21, (a) the mice were euthanized, and their body weight, spleen weight, and liver weight were measured. (b) The largest tumor nodule was measured using calipers. (c) The number of nodules of different sizes on the liver of the mice was counted.

[0093] Lung cancer model: according to Figure 1 Method M involves subcutaneous inoculation of C57BL / 6 mice with 3 × 10⁻⁶ cells. 5 A lung cancer treatment model was established by injecting 3LL cells and starting intervention on day 8. The day of tumor cell injection is designated as day 0.

[0094] Serine supplementation: Serine was added to the mice's drinking water at a concentration of 600 mg / L for 8-16 days, ensuring that the mice had free access to drinking water.

[0095] Tumor measurement: During this period, the size of the subcutaneous tumor was measured using calipers. After the mice were sacrificed on day 22, the tumor weight and spleen weight were measured.

[0096] Each mouse consumed an average of 5 ml of drinking water containing 600 mg / L serine per day, which means that each mouse consumed approximately 3 mg of serine per day.

[0097] Tumor-bearing mice given normal drinking water were used as the control group.

[0098] The results are as follows Figure 1 As shown: In a colon cancer model, as the number of days increased, the tumors in the control group gradually increased in size, while the tumors in the serine-supplemented group grew slowly. By day 18, tumor growth was slow, and the tumor size was approximately half that of the control group. Tumor weight was also approximately half that of the control group. Spleen weight showed no significant change. Figure 1 BE) In the orthotopic liver cancer model, the body weight and spleen weight of the serine-supplemented group did not change significantly and were comparable to those of the control group. However, the total weight of the tumor-bearing liver, the liver weight / body weight ratio, the area of ​​the largest tumor nodule, and the total number of tumor nodules were all significantly reduced compared to the control group. Figure 1 GL) In a lung cancer model, as the number of days increased, the tumors in the control group gradually increased in size, while the tumors in the serine-supplemented group grew slowly. By day 22, tumor growth was slow, and the tumor size was approximately two-thirds that of the control group. The tumor weight was approximately half that of the control group. Spleen weight showed no significant change. Figure 1 NP) The experimental results above demonstrate that exogenous serine supplementation via the addition of serine to the drinking water of mice significantly inhibits tumor growth in various solid tumor models originating from different tissues, including colon cancer (MC38 model), liver cancer (Hepa1-6BL orthotopic model), and lung cancer (3LL model). Specifically, tumor volume and weight are significantly reduced. In the orthotopic liver cancer model, the weight of the tumor-bearing liver, the liver weight / body weight ratio, and the number of orthotopic tumor nodules are all significantly decreased. This indicates that serine supplementation, as a single intervention, possesses broad-spectrum tumor-suppressing potential across tumor types. It not only exhibits a high tumor inhibition rate but also demonstrates good safety.

[0099] Example 2: Effects of serine supplementation therapy and serine / glycine "starvation therapy" on tumor growth and metastasis A C57BL / 6 mouse model of orthotopic hepatocellular carcinoma was established by high-pressure tail vein injection of Hepa1-6BL cells. Mice were randomly divided into three groups (n=7 per group): (1) a normal diet control group; (2) a serine-supplemented group (normal diet + 600 mg / L serine-containing drinking water); and (3) a serine / glycine starvation group (fed a customized diet devoid of serine and glycine). Mice were sacrificed at the end of the experiment, and the orthotopic tumor burden and spontaneous lung metastasis were comprehensively analyzed.

[0100] Tumor injection is considered day 0.

[0101] (1) Normal diet control group: Throughout the experiment, the control group was provided with normal drinking water and normal feed.

[0102] (2) Serine supplementation group: Normal drinking water and normal feed were given to mice 7 days before tumor cell injection. From day 0 to day 21 after tumor injection, mice were given normal feed and drinking water containing 600 mg / L of serine to ensure that mice could ingest it freely during this period.

[0103] (3) Serine / glycine starvation group: Starting 7 days before tumor cell injection, mice were fed a diet without serine and glycine until the end of the experiment (i.e. day 21).

[0104] The mice in the serine-supplemented group consumed an average of 5 ml of drinking water containing 600 mg / L serine per day, which means that each mouse consumed approximately 3 mg of serine per day.

[0105] On day 21, all mice were euthanized, and liver, lung, and spleen tissues were collected for the following tests: Weight measurement: Measure the weight of the liver, lungs, and body weight, and calculate the ratio of liver to body weight.

[0106] Tumor statistics: The size of the largest nodule of in situ hepatocellular carcinoma is recorded, and the number of in situ hepatocellular carcinoma nodules and spontaneous lung metastases are counted.

[0107] Immune cell analysis: The function of immune cells in the spleen of mice from different groups was analyzed by flow cytometry. The analysis was mainly based on the expression levels of interferon-gamma (IFN-γ) and granzyme B in CD4, CD8, and NK cells, which play important regulatory roles in tumor growth.

[0108] The results are as follows Figure 2 As shown, compared with the control group, the traditional "serine / glycine starvation therapy" had only a weak and insignificant inhibitory effect on the growth of in situ hepatocellular carcinoma. Unexpectedly, the serine supplementation therapy of this invention significantly inhibited the growth of in situ hepatocellular carcinoma, reducing the largest tumor nodule area by more than three times and the total number of all tumor nodules by more than three times, demonstrating a significantly better tumor-suppressing effect than starvation therapy. More importantly, the number of spontaneous lung metastatic nodules in the serine supplementation group was only half that of the control group, while the starvation group did not show any metastasis-inhibiting advantage. Splenic lymphocyte analysis showed that serine supplementation significantly increased CD4+. + T, CD8 + The ability of T and NK cells to produce IFNγ and granzyme B. This experiment fundamentally overturns the traditional understanding of "depriving serine of cancer treatment".

[0109] Example 3: Removing the inhibitory effect of serine / glycine on tumor cells in cell culture medium The complete culture medium for culturing tumor cells is RPMI 1640.

[0110] The culture medium was supplemented with 10% fetal bovine serum, 1% L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin, 133 μM glycine and 300 μM serine.

[0111] To investigate the inhibitory effect of removing serine / glycine on tumor cells, this embodiment used a culture medium lacking serine and glycine as described above to culture human hepatocellular carcinoma cells HepG2, mouse hepatocellular carcinoma cells Hepa1-6BL, and human colorectal carcinoma cells HCT116 in vitro according to conventional methods, and performed cell proliferation and colony formation experiments.

[0112] To detect tumor cell proliferation, tumor cells were seeded in 12-well plates with three replicates per group, and the specified culture medium was added. Cell proliferation was assessed by counting on the second, fourth, and sixth days after culture.

[0113] Colony formation assay: Tumor cells were seeded at a density of 800 cells per well in 6-well plates containing complete culture medium and allowed to adhere overnight. Afterward, the old culture medium was completely aspirated and replaced with the specified conditioned medium. After 10 days of cell culture, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The culture wells were photographed, and the colonies were analyzed using ImageJ software.

[0114] The proliferation and colony formation of tumor cells are as follows Figure 3 show.

[0115] Experimental results showed that culturing tumor cells in a medium lacking serine / glycine reduced their proliferation and colony-forming abilities, indicating that serine / glycine deprivation directly and significantly inhibited the proliferation and colony formation of these tumor cells. This further confirms that tumor cell growth and proliferation depend on serine / glycine.

[0116] Example 4: Serine supplementation has no effect on the proliferation and growth of tumor cells. In the complete culture medium RPMI 1640 described in Example 3, an additional 400 μM of serine was added to culture liver cancer cells (HepG2, Hepa1-6BL). Colorectal cancer cells (HCT116) were cultured in the complete culture medium RPMI 1640 described in Example 3 with 400 μM, 600 μM, and 800 μM serine, respectively.

[0117] The cultured tumor cells were subjected to tumor cell viability assays, proliferation experiments, wound healing experiments, and colony formation experiments.

[0118] Tumor cell viability: Tumor cell viability was detected using the Cell Counting Kit-8 (CCK-8) assay, following the kit instructions. Tumor cells were seeded into 96-well plates at a density of 2 × 10⁶ cells / well. 3 Cells were selected and treated with or without 400 µM serine. After incubation with CCK-8 reagent for 1 hour, cell viability was assessed by measuring absorbance at 450 nm using a microplate reader.

[0119] Proliferation assay: To detect tumor cell proliferation, tumor cells were seeded in 12-well plates with three replicates per group, and the specified culture medium was added. Cell proliferation was assessed by counting at 24 and 48 hours after culture.

[0120] Wound healing assay: The scratch assay was used to detect the migration ability of tumor cells. The simplified procedure is as follows: 12-well plates were coated with poly-L-lysine (prepared as a 0.1 mg / mL working solution with sterile distilled water) and dried at room temperature for 2 hours. Subsequently, 3 × 10⁶ cells were seeded into each well.5 Tumor cells were cultured for 24 hours to achieve suitable confluence. A linear scratch was made on the cell monolayer using a sterile 200 μL pipette tip. The cells were washed twice with serum-free medium, and then 2 mL of serum-free medium was added. Images were taken at different time points, and the degree of cell migration was quantitatively analyzed using ImageJ software.

[0121] Colony formation assay: Tumor cells were seeded at a density of 800 cells per well in 6-well plates containing complete culture medium and allowed to adhere overnight. Afterward, the old culture medium was completely aspirated and replaced with the specified conditioned medium. After 10 days of cell culture, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The culture wells were photographed, and the colonies were analyzed using ImageJ software.

[0122] Experimental results are as follows Figure 4 As shown.

[0123] The experimental results showed that, compared with the normal culture medium, the viability, migration ability and colony formation ability of tumor cells cultured in the serine-added culture medium did not change significantly.

[0124] A comparison with Example 3 revealed that adding serine to the normal culture medium had no promoting effect on the proliferation and colony formation of human and mouse liver cancer and colorectal cancer cell lines. Although depriving serine inhibits tumor cells, simply supplementing it does not "feed" tumor cells, breaking the technical prejudice that "serine supplementation must nourish tumors" and demonstrating relative safety. The experimental results also showed that the addition of serine did not inhibit tumor cell growth, further illustrating that the addition of serine cannot directly kill tumor cells.

[0125] Example 5: CD8 + The role of T cells in fighting tumors with serine replacement therapy An orthotopic hepatocellular carcinoma model was established, with the following groups included: a control group, a serine supplementation group (600 mg / L drinking water), an anti-CD8β antibody depletion group, and a combined serine and anti-CD8β antibody intervention group. Anti-CD8β antibodies (100 μg / animal) were administered intraperitoneally to eliminate CD8β cells in the animal. + T cells. In CD8 + In the T cell exhaustion experiment, a mouse orthotopic liver cancer model was first established using Hepa1-6BL tumor cells according to the aforementioned method (same as Example 1).

[0126] Dosage regimen: Control group: Throughout the experiment, the control group was provided with normal drinking water and received intraperitoneal injections of 100 μL of PBS on days 1, 6, 12, and 18.

[0127] Serine supplementation group: Starting from day 0 of the modeling day, mice were given 600 mg / L of drinking water containing serine to ensure that they could ingest it freely during this period.

[0128] Anti-CD8β antibody depletion group: Day 0 was the day of modeling, and then 100 μL of anti-CD8β antibody solution (PBS solution containing 100 μg of anti-CD8β antibody) was injected intraperitoneally on days 1, 6, 12 and 18.

[0129] Serine and anti-CD8β antibody combined intervention group: Starting from day 0 of modeling, mice were given drinking water containing 600 mg / L serine to ensure free access during this period. At the same time, 100 μL of anti-CD8β antibody solution (PBS solution containing 100 μg of anti-CD8β antibody) was injected intraperitoneally on days 1, 6, 12 and 18.

[0130] In the serine supplementation group and the combined intervention group, each mouse consumed an average of 5 ml of drinking water containing 600 mg / L serine per day, which means that each mouse consumed approximately 3 mg of serine per day.

[0131] Mice were euthanized on day 19, and liver and lung tissues were collected. The weight of the liver, lungs, and mice, as well as the number and size of in situ hepatocellular carcinoma nodules and spontaneous lung metastases, were measured.

[0132] like Figure 5 As shown, in the case of unexhausted CD8 + In mice with T cells, serine supplementation effectively inhibited the growth of in situ hepatocellular carcinoma and lung metastasis (serine supplementation group). However, when CD8... + After T cells were depleted, the anti-tumor effect of serine supplementation completely disappeared, and the liver tumor burden was no different from that of the control group (combined intervention group). This conclusively proves that serine supplementation works by acting on CD8. + T cells mediate anti-tumor immunity.

[0133] Example 6: Serine supplementation reshapes the tumor immune microenvironment and promotes CD8. + T cell core infiltration The orthotopic liver cancer model established in Example 1 was used to analyze tumor-infiltrating immune cells by flow cytometry. The results are as follows: Figure 6 As shown.

[0134] Figure 6 AC results indicate that serine supplementation can effectively increase CD4 levels in tumor tissue. + T cells, CD8 + The ratio and absolute number of T cells and NK cells.

[0135] Figure 6 Immunofluorescence staining of DE showed that CD8 in the control group+ T cells mainly accumulate at the boundary between tumor and normal tissue, making it difficult for them to penetrate the tumor core; however, serine supplementation therapy successfully promoted CD8 cell activity. + The infiltration and survival of T cells into the tumor center significantly increased their proportion. This indicates that serine improves the chemotaxis and infiltration capabilities of immune cells.

[0136] Example 7: Serine directly enhances T cell function under in vitro tumor microenvironment conditions In vitro, CD8 + T cells (CTLL-2) were cultured in conditioned medium (TCM) derived from Hepa1-6BL tumor cells to mimic an immunosuppressive tumor microenvironment, with or without supplementation of 150-800 μM serine. Results were as follows: Figure 7 As shown.

[0137] Experimental results show that after supplementing TCM with serine, CD8 + The expression of T cell activation markers (CD69, DNAM-1) was upregulated, and the expression of effector cytokines (IFN-γ, IL-2) was increased. EdU incorporation and the proportion of Ki67-positive cells, reflecting proliferative capacity, were significantly increased. Simultaneously, the proportion of T cells positive for the anti-apoptotic protein BCL-2 also increased significantly. This indicates that in a highly inhibitory microenvironment, serine supplementation can directly act as an "immune energizer" to restore and enhance T cell proliferation, activation, effector function, and survival. The effect is more pronounced when supplementing with 400-800 μM serine.

[0138] Example 8: Serine supplementation combined with PD-1 antibody reverses immunosuppressive disorder in advanced liver cancer like Figure 8 As shown in Figure A, an orthotopic liver cancer model was established by injecting Hepa1-6BL cells via the tail vein.

[0139] Mice were randomly divided into four groups: (1) control, (2) PD-1 antibody monotherapy (100 μg / mouse), (3) serine monotherapy (600 mg / L drinking water), and (4) serine + PD-1 antibody combined therapy.

[0140] Control group: Throughout the experiment, the control group was provided with normal drinking water, and each mouse was injected intraperitoneally with 100 μL of PBS on days 11, 14 and 17.

[0141] The day of tumor cell injection is designated as day 0. After tumor inoculation, mice are given normal drinking water until day 10.

[0142] PD-1 antibody monotherapy group: Starting from day 11 after tumor cell inoculation, PD-1 antibody was administered every 3 days, that is, on days 11, 14 and 17, each mouse was injected intraperitoneally with 100 μL of anti-PD-1 antibody solution (clone number 29F.1A12, PBS solution containing 100 μg of PD-1 antibody).

[0143] Serine monotherapy group: From day 11 to day 18 after tumor cell inoculation, mice were given drinking water containing L-serine (600 mg / L).

[0144] Serine + PD-1 antibody combination therapy group: The drugs were administered in the same manner as the serine monotherapy group and the PD-1 monotherapy group (at the same time and dosage as the monotherapy group).

[0145] In both the serine monotherapy group and the combination therapy group, each mouse consumed an average of 5 ml of drinking water containing 600 mg / L serine per day, which means that each mouse consumed approximately 3 mg of serine per day.

[0146] All mice were sacrificed on day 19. Changes in tumor nodules and CD8+ levels in tumor cells were examined in each group of mice. + T cell abundance. Results are shown in [link to table]. Figure 8 .

[0147] The results showed that, when PD-1 antibody monotherapy had no effect, the combination therapy group exhibited a strong synergistic anti-tumor effect, reducing the total number of in situ liver cancer tumor nodules to 1 / 5 of the control level. Figure 8 (B, C). Immunofluorescence analysis further confirmed that the combined regimen not only increased CD8 levels in tumor tissue. + The overall abundance of T cells also strongly promoted CD8. + Distribution of T cells in the tumor core region Figure 8 D, E).

[0148] Example 9: In vivo safety verification of high-dose serine supplementation Normal C57BL / 6 mice were given high-concentration (1500 mg / L) serine in drinking water for 15 consecutive days to simulate a long-term, high-dose drug administration scenario. Results were as follows... Figure 9 As shown.

[0149] The results showed that all mice gained weight normally, and the weight and morphology of the liver, spleen, and kidneys were not different from the control group. Histopathological examination of the colon, liver, and kidneys using hematoxylin and eosin (HE) staining revealed no organic damage. Serum liver function indicators ALT and AST levels were not different from the control group. This verified the extremely high in vivo safety of serine supplementation therapy. Furthermore, increasing the serine concentration in drinking water to 6 g / L still effectively inhibited the growth of in situ hepatocellular carcinoma. This indicates that serine supplementation strategies can effectively inhibit tumor growth within the range of 600 mg / L to 6 g / L of serine concentration in drinking water.

[0150] Each mouse ingested an average of 5 ml of drinking water containing serine per day. Even when each mouse ingested approximately 30 mg of serine per day, tumor growth was still effectively inhibited.

[0151] Example 10: The combined use of serine and L-arginine effectively inhibits tumor growth and metastasis. like Figure 10 As shown in A, an orthotopic liver cancer model was established by injecting Hepa1-6BL cells into the tail vein. Mice were randomly divided into 4 groups: (1) control, (2) serine monotherapy (600 mg / L drinking water), (3) L-arginine monotherapy (1.5 g / L drinking water), and (4) serine + L-arginine combined therapy.

[0152] Control group: Normal drinking water was provided to the control group throughout the experiment.

[0153] The day of tumor cell injection is designated as day 0. After tumor inoculation, mice are given normal drinking water until day 10.

[0154] Serine monotherapy group: Starting from day 11 after tumor cell inoculation, drinking water containing L-serine (600 mg / L) was administered.

[0155] L-arginine monotherapy group: Starting from day 11 after tumor cell inoculation, drinking water containing L-arginine (1.5 g / L) was administered.

[0156] Serine + L-arginine combined treatment group: Starting from the 11th day after tumor cell inoculation, drinking water containing 600 mg / L L-serine and 1.5 g / L L-arginine was administered.

[0157] Each mouse consumed an average of 5 ml of drinking water containing serine and / or arginine per day, compared to 3 mg of L-serine and 7.5 mg of L-arginine per day.

[0158] On day 19, all mice were euthanized. Liver and lung tissues were collected to evaluate tumor burden and metastasis.

[0159] L-arginine treatment alone showed a slight inhibitory effect on tumor growth, but the overall effect was weaker than that of serine treatment alone. Combined treatment with serine and L-arginine synergistically inhibited the growth of in situ hepatocellular carcinoma, reducing the number of liver nodules and lung metastases to one-quarter of their original levels. This result suggests that serine combined with arginine can synergistically inhibit tumor growth and metastasis, representing a potential combination therapy. Increasing the composition and intake of these two amino acids in the diet can enhance their anti-tumor effects.

Claims

1. The use of serine or its pharmaceutically acceptable salts or esters in the preparation of medicaments for the treatment of tumors.

2. The application according to claim 1, characterized in that, The tumor is selected from stomach cancer, kidney cancer, bladder cancer, skin cancer, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, thyroid cancer, liver cancer, colorectal cancer, lung cancer, melanoma, or breast cancer.

3. The application according to claim 1 or 2, characterized in that, The serine or its pharmaceutically acceptable salts or esters exert their antitumor effects by enhancing the antitumor immune response and remodeling the tumor immune microenvironment.

4. A tumor immunotherapy composition, characterized in that, It contains the active ingredient serine or a pharmaceutically acceptable salt or ester thereof and an oncology treatment drug; preferably, the oncology treatment drug is an immune checkpoint inhibitor.

5. The tumor immunotherapy composition according to claim 4, characterized in that, The immune checkpoint inhibitor is one or more of PD-1 inhibitors or PD-L1 inhibitors; preferably, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody; the mass ratio of the immune checkpoint inhibitor to serine or its pharmaceutically acceptable salt or ester is 1:80-800, preferably 1:80-200.

6. A tumor immunotherapy composition, characterized in that, It contains serine or a pharmaceutically acceptable salt or ester thereof and arginine, preferably in a mass ratio of serine or a pharmaceutically acceptable salt or ester thereof to arginine of 1:1-10, more preferably 1:2.5-5.

7. Use of the tumor immunotherapy composition according to claim 5 or 6 in the preparation of a medicament for treating tumors.

8. The use of serine or its pharmaceutically acceptable salts or esters in the preparation of sensitizers or synergists for tumor immunotherapy.

9. The use of serine or its pharmaceutically acceptable salts or esters in combination with PD-1 antibodies in the preparation of antitumor drugs.

10. The use of serine or its pharmaceutically acceptable salts or esters in combination with arginine in the preparation of antitumor drugs.

11. The use of serine or a pharmaceutically acceptable salt or ester thereof in the preparation of immune cells with enhanced tumor-killing ability, wherein the immune cells are preferably CD8+. + T cells.

12. A method for preparing immune cells with enhanced tumor-killing ability, characterized in that, Add serine to the culture medium for culturing immune cells, wherein the immune cells are CD8. + For T cells, the concentration of serine is 150-800 μM, preferably 400-800 μM.

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