CN118697740A - Application of hypoxanthine in anti-tumor immunotherapy - Google Patents

Abstract

The invention discloses an application of hypoxanthine in anti-tumor immunotherapy, which can enhance CD8+T activity through verifying in vitro hypoxanthine supplementation, and oral administration of hypoxanthine can promote anti-tumor immune response and can obviously improve the immunotherapy effect of PD-1 monoclonal antibody solid tumor. The mode of combining hypoxanthine with immune checkpoint inhibitor can be used for synergistically improving the anti-tumor immunotherapy effect, and can partially solve the problem of drug resistance of solid tumor immunotherapy.

Description

Application of hypoxanthine in anti-tumor immunotherapy

Technical Field

The present invention relates to the technical field of tumor immunotherapy, and in particular to the application of hypoxanthine in anti-tumor immunotherapy.

Background Art

Malignant tumors have become a major public health problem that threatens the lives and health of the entire population and is one of the most common causes of death worldwide. In recent years, with the approval and clinical application of immune checkpoint inhibitors (ICIs) PD-1/PD-L1 monoclonal antibodies, immunotherapy has developed into an important means of treating malignant tumors. At present, ICIs have been approved for the treatment of at least 14 types of cancer, either alone or in combination. However, most patients still cannot benefit from it. Taking advanced non-small-cell lung cancer (NSCLC) as an example, the objective response rate of anti-PD-1/PD-L1 monotherapy is only about 20%, and there is a risk of drug resistance and super-progression of immunotherapy. Therefore, finding new measures to further improve the therapeutic efficacy of ICIs has become an urgent problem to be solved.

As a nucleoside metabolite, hypoxanthine is a common purine compound that is widely present in the human body and participates in regulating physiological functions. Hypoxanthine can be generated by adenine deamination through the action of adenine deaminase or nitrous acid, and can also be decomposed by phosphorylation of inosine through nucleoside phosphorylase. Studies have shown that hypoxanthine can promote fat decomposition in mice and play a role in reducing body fat mass. After 4 weeks of hypoxanthine treatment of nutritionally obese mice, the body fat mass and total fat mass of obese mice can be effectively reduced, and it has good biosafety and effectiveness. Hypoxanthine is also a potential free radical generator and can be used as an important biochemical indicator of abnormal myocardial energy metabolism. In addition, 6-mercaptopurine, a derivative of hypoxanthine, is also an important anti-tumor drug and plant growth regulator. However, there are no reports on hypoxanthine regulating immunity and enhancing anti-tumor immune function.

Summary of the invention

The present invention verifies that hypoxanthine supplementation in vitro can enhance CD8+T activity, oral hypoxanthine can promote anti-tumor immune response, and can significantly improve the immunotherapy effect of PD-1 monoclonal antibody solid tumors. Therefore, using hypoxanthine as an anti-tumor drug can significantly improve the immunotherapy effect of immune checkpoint inhibitor tumors, and is expected to alleviate the problem of tumor immunotherapy resistance.

In view of this, the scheme of the present invention is:

In one aspect of the present invention, use of hypoxanthine in the preparation of anti-tumor drugs is proposed.

Furthermore, the anti-tumor drug includes at least one of the following uses:

a) Promote the proliferation of CD3+CD8+T cells;

b) Activate CD3+CD8+T cells and enhance the ability of CD3+CD8+T cells to secrete IFN-γ and granzyme B in vitro.

Another aspect of the present invention provides the use of hypoxanthine combined with immune checkpoint inhibitors in the preparation of anti-tumor drugs.

Furthermore, anti-tumor drugs such as hypoxanthine combined with immune checkpoint inhibitors are used to enhance the efficacy of anti-tumor immunotherapy.

Furthermore, the immune checkpoint inhibitors include PD-1 and PD-L1.

Furthermore, the tumor includes tumors that are affected by immune checkpoint inhibitors, but are not limited to melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, cervical squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, and mesothelioma.

Preferably, the tumor is melanoma.

Furthermore, the anti-tumor drug includes one or more pharmaceutically or physiologically acceptable carriers, and/or excipients, and/or diluents.

Furthermore, in the anti-tumor drug, the hypoxanthine is used for oral administration.

Preferably, the hypoxanthine is in the form of an oral liquid or solid preparation.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention verifies that hypoxanthine can enhance CD8+T activity in vitro, promote anti-tumor immune response, and can be used in anti-tumor immunotherapy; in addition, the combination of hypoxanthine and immune checkpoint inhibitors can promote anti-tumor immune response, significantly improve the immunotherapy effect of tumors, and is expected to solve the problem of drug resistance in tumor immunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow cytometric chart showing that hypoxanthine culture medium promotes CD8+ T proliferation in an embodiment of the present invention.

FIG. 2 is a flow cytometric graph showing that hypoxanthine culture medium enhances the secretion of IFN-γ and granzyme B by CD8+T cells in an embodiment of the present invention.

FIG. 3 is a statistical diagram of tumor volume in the mouse B16-F10 subcutaneous tumor model treated with hypoxanthine combined with PD-1 monoclonal antibody in an embodiment of the present invention.

4 is a flow cytometric chart showing that hypoxanthine combined with PD-1 monoclonal antibody promotes CD8+T cell infiltration and activation in the tumor microenvironment in a mouse subcutaneous tumor model in an embodiment of the present invention.

FIG. 5 is a flow cytometric graph showing that hypoxanthine combined with PD-1 monoclonal antibody promotes splenic CD8+T cell activation in a mouse subcutaneous tumor model in an embodiment of the present invention.

6 is a flow cytometric chart showing that hypoxanthine combined with PD-1 monoclonal antibody promotes CD8+T cell infiltration and activation in tumor-draining lymph nodes in a mouse subcutaneous tumor model in an embodiment of the present invention.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

In one embodiment, it was verified that hypoxanthine treatment in vitro can promote CD8+T proliferation and promote its ability to secrete IFN-γ and granzyme B; in the mouse B16-F10 melanoma subcutaneous tumor model, normal saline, hypoxanthine, PD-1 monoclonal antibody or hypoxanthine combined with PD-1 monoclonal antibody were given respectively, and the growth of subcutaneous tumors was detected. It was found that the tumor growth of mice in the combined treatment group was significantly inhibited, and the effect was better than that of PD-1 monoclonal antibody alone. The subcutaneous tumors, spleens and tumor-draining lymph nodes of each group of mice were collected, and the IFN-γ secretion ability of T cells in different tissue microenvironments was detected by flow cytometry. The results showed that the number of T cell infiltration in the tumor microenvironment and tumor-draining lymph nodes in the combined treatment group increased significantly, and the IFN-γ secretion ability of T cells was significantly enhanced; the number of T cells secreting IFN-γ and granzyme B in the spleen and tumor-draining lymph nodes also increased significantly. The above results show that hypoxanthine can be used to enhance the anti-tumor efficacy of PD-1 monoclonal antibody.

In a preferred embodiment, an oral hypoxanthine preparation is proposed, which can effectively enhance the anti-tumor efficacy of PD-1 monoclonal antibody and activate anti-tumor immunity by combining it with immunotherapy using PD-1 monoclonal antibody, thereby hopefully solving the problem of drug resistance in PD-1 monoclonal antibody immunotherapy.

In the above embodiments, the tumors affected by immune checkpoint inhibitors, in addition to melanoma, can also be lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, cervical squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, and mesothelioma.

The following are specific examples for verifying the effect of hypoxanthine and its combination with PD-1 monoclonal antibody for enhancing tumor immunotherapy. Unless otherwise specified, the materials or reagents used are commonly used in the field, and the experimental methods used are familiar to those skilled in the art.

1. Prepare T cell culture medium containing 1 mM hypoxanthine

Hypoxanthine was added to the T cell culture medium according to the standard of 136.11 ug/ml, and after dissolution was promoted in a 37°C water bath, the culture medium was filtered using a sterilizing filter device to obtain a T cell culture medium containing 1 mM hypoxanthine.

2. Hypoxanthine promotes CD8+ T cell proliferation in vitro

CD8 ⁺ T cells were sorted from the spleen of C57BL/6 mice. After activation by incubation with CD3/CD28 antibodies for 48 hours, the cells were labeled with CFSE and treated with a medium containing 1mM hypoxanthine. After 72 hours, samples were collected to prepare single-cell suspensions, and the proliferation efficiency of CD3 ⁺ CD8 ⁺ T cells in the samples was detected by flow cytometry. The flow cytometry antibodies used included ZombieBV421, Per CP/Cy5.5-CD45, and APC/Cy7-CD3. As shown in Figure 1, 1mM hypoxanthine treatment can promote the proliferation of CD3 ⁺ CD8 ⁺ T cells, and *** indicates statistical P < 0.001.

3. Hypoxanthine promotes CD8+ T activation in vitro

CD8 ⁺ T cells sorted from the spleen of C57BL/6 mice were prepared, activated by incubation with CD3/CD28 antibodies for 48 hours, and then treated with medium containing 1mM hypoxanthine. Samples were collected 24 hours later to prepare single cell suspensions, and the activation of CD3 ⁺ CD8 ⁺ T cells in the samples was detected by flow cytometry. The flow cytometry antibodies used included Zombie NIR-APC/Cy7, Per CP/Cy5.5-CD45, FITC-CD3, BV510-CD8, BV421-IFN-γ, and PE-Granzyme B. As shown in Figure 2, medium containing 1mM hypoxanthine enhanced the ability of CD3+CD8+T cells to secrete IFN-γ and granzyme B in vitro, and *** indicates statistical P < 0.001.

4. Anti-tumor therapeutic effect of hypoxanthine combined with PD-1 monoclonal antibody in subcutaneous tumor model

Wild-type C57BL/6 mice aged 6 to 8 weeks were prepared, and 100 μL of tumor cell suspension (3×10 ⁵ B16-F10 melanoma cells) was subcutaneously inoculated at the root of the right thigh to construct a subcutaneous tumor model of B16-F10 melanoma in C57BL/6 mice. When the tumor volume reached 100 mm ³ , the mice were divided into four groups according to the following treatments: ① oral saline; ② oral hypoxanthine; ③ PD-1 monoclonal antibody; ④ oral hypoxanthine + PD-1 monoclonal antibody. PD-1 monoclonal antibody was intraperitoneally injected at a dose of 10 mg/kg, twice a week; hypoxanthine was dissolved in saline and administered orally every other day at 50 mg/kg. After the start of treatment, the long and short diameters of the mouse tumors were measured every other day and the tumor growth curve was drawn. As shown in Figure 3, compared with the control group, hypoxanthine combined with PD-1 monoclonal antibody treatment significantly inhibited tumor growth in the mouse melanoma subcutaneous tumor model, and the effect was better than that of PD-1 monoclonal antibody alone. Among them, *** indicates statistical P <0.001; ** indicates statistical P <0.01; ns indicates no statistically significant difference.

5. Hypoxanthine combined with PD-1 monoclonal antibody enhances CD8 ⁺ T cell anti-tumor immune response in subcutaneous tumor model

As described in 4, a mouse B16-F10 subcutaneous tumor model was constructed and treated in groups. 24 hours after the last treatment, the mouse tumor tissue, spleen, and tumor-draining lymph nodes were taken to make single cell suspensions, and the infiltration and activation markers of CD8 ⁺ T cells in the above were detected by flow cytometry. The flow antibodies used included Zombie NIR-APC/Cy7, PerCP/Cy5.5-CD45, FITC-CD3, BV510-CD8, and BV421-IFN-γ. As shown in Figure 4, the ratio of CD3+CD8+T cells to CD3+CD4+T cells in the tumor microenvironment, as well as the proportion of cytotoxic T cells (IFN-γ+CD8+) in CD8+T cells were detected by flow cytometry. Compared with other groups, hypoxanthine combined with PD-1 monoclonal antibody treatment can significantly promote CD8+T cell infiltration in the tumor microenvironment and increase the proportion of cytotoxic T cells. Among them, *** indicates statistical P < 0.001. As shown in Figure 5, the proportion of cytotoxic T cells (IFN-γ+CD8+) in the spleen of mice accounted for CD8+T cells by flow cytometry. Compared with other groups, hypoxanthine combined with PD-1 monoclonal antibody treatment can significantly increase the proportion of cytotoxic T cells in the spleen of tumor-bearing hours. Among them, *** indicates statistical P < 0.001. As shown in Figure 6, the ratio of CD3 ⁺ CD8 ⁺ T cells to CD3 ⁺ CD4 ⁺ T cells in the tumor-draining lymph nodes of mice, as well as the proportion of cytotoxic T cells (IFN-γ ⁺ CD8 ⁺ ) in CD8 ⁺ T cells were detected by flow cytometry. Compared with other groups, hypoxanthine combined with PD-1 monoclonal antibody treatment can significantly promote CD8 ⁺ T cell infiltration in the tumor microenvironment and increase the proportion of cytotoxic T cells. Among them, *** indicates statistical P <0.001; * indicates ns statistical P <0.05; ns indicates no significant statistical difference. The above results showed that compared with the control group, hypoxanthine combined with PD-1 monoclonal antibody treatment can significantly promote the infiltration of CD8 ⁺ T cells in the tumor microenvironment, spleen and tumor-draining lymph nodes, and increase the proportion of cytotoxic T cells (CD8 ⁺ IFN-γ ⁺ ).

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. The application of hypoxanthine in preparing antitumor drugs.

2. The use according to claim 1, wherein the antitumor drug comprises at least one of the following uses:

a) Promote proliferation of CD3 ⁺CD8⁺ T cells;

b) Activating CD3 ⁺CD8⁺ T cells, and enhancing the capability of the CD3+CD8+T cells to secrete IFN-gamma and granzyme B in vitro.

3. The application of hypoxanthine combined immune checkpoint inhibitor in preparing antitumor medicine is provided.

4. The use according to claim 3, wherein the antitumor drug is for enhancing the efficacy of antitumor immunotherapy.

5. The use according to claim 3, wherein the immune checkpoint inhibitor comprises PD-1, PD-L1.

6. The use according to claim 3, wherein the tumour comprises melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, renal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, cervical squamous carcinoma, esophageal squamous carcinoma, hodgkin's lymphoma, mesothelioma.

7. The use according to claim 6, wherein the tumour is melanoma.

8. The use according to claim 3, wherein the antitumor drug comprises one or more pharmaceutically or physiologically acceptable carriers, and/or excipients, and/or diluents.

9. The use according to claim 3, wherein in the antineoplastic agent, the hypoxanthine is for oral administration.

10. The use according to claim 9, wherein the hypoxanthine is an oral liquid or a solid preparation.