Use of cordyceps and PD1 inhibitor in preparation of drug for non-small cell lung cancer

Abstract

Provided in the present disclosure is a therapeutic agent for preventing or treating non-small cell lung cancer. The therapeutic agent contains Cordyceps and a PD1 inhibitor as the therapeutic agent, specifically against non-small cell lung cancer with high expression of proteins such as VEGF and Ki67.

Description

Use of Cordyceps sinensis and PD1 inhibitors in the preparation of non-small cell lung cancer drugs Technical Field

[0001] The present disclosure relates to the use of a Cordyceps sinensis fermentation product and a PD1 inhibitor in the preparation of cancer drugs. Background Art

[0002] Lung cancer ranks first among all cancers due to its high morbidity and mortality, with non-small cell lung cancer (NSCLC) accounting for 80%-85% of lung cancer cases. Currently, drugs used to treat NSCLC primarily modulate immune responses, target specific molecules, or have direct cytotoxic effects on lung cancer cells.

[0003] Blocking programmed cell death (PD1) has been shown to be a successful treatment for various forms of cancer and has been widely used as a standard treatment for non-small cell lung cancer. However, it has significant immune side effects, including pneumonia, adverse skin reactions, and gastrointestinal toxicity. Moreover, when treated with anti-PD1 agents alone, most patients still do not initiate a sustained anti-cancer response. Increasing the number of patients who respond to PD1 antibody therapy is a major challenge. Moreover, patients with advanced NSCLC have impaired immune function and relatively poor physical condition, and the adverse reactions of combination therapy also increase, seriously affecting the quality of life of patients. Summary of the Invention

[0004] On the one hand, the present disclosure provides a therapeutic agent for treating non-small cell lung cancer. On the other hand, the present disclosure also provides a therapeutic drug combination for treating non-small cell lung cancer in a subject in need thereof. It also provides a treatment method for non-small cell lung cancer, and the use of a PD1 inhibitor and Cordyceps sinensis as drugs for treating non-small cell lung cancer.

[0005] Specifically, the present disclosure first provides a therapeutic agent for non-small cell lung cancer, comprising a PD1 inhibitor and a second agent for use with the PD1 inhibitor, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products. The therapeutic agent for non-small cell lung cancer of the present disclosure can be used to administer an effective amount of a PD1 inhibitor and a second agent for use with the PD1 inhibitor, wherein the second agent is selected from Cordyceps sinensis and its fermentation products, to a patient with non-small cell lung cancer.

[0006] The second agent is provided in the form of fermented Cordyceps sinensis powder;

[0007] The PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof;

[0008] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0009] Furthermore, the non-small cell lung refers to non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein.

[0010] Furthermore, the therapeutically effective amount of the PD1 antibody and the second agent used in combination with the PD1 antibody can effectively reduce the expression of any one of the highly expressed proteins VEGF, Ki67, RhoA, Raf-1, and c-fos. Preferably, it can effectively reduce the expression of any one of the proteins VEGF, Ki67, RhoA, Raf-1, and c-fos in non-small cell lung cancer, preferably, it can simultaneously effectively reduce the expression of VEGF and Ki67 proteins in non-small cell lung cancer, more preferably, it can simultaneously effectively reduce the expression of RhoA, Raf-1, and c-fos proteins in non-small cell lung cancer, preferably, it can simultaneously effectively reduce the expression of VEGF, Ki67, RhoA, Raf-1, and c-fos proteins in non-small cell lung cancer.

[0011] Furthermore, the therapeutically effective amount refers to the subject's dosage of fermented bacteria powder of approximately 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d, 91-910 mg / Kg / d, and the subject's dosage of the PD1 inhibitor is approximately 0.1-0.2 mg / Kg / d.

[0012] In another aspect, the present disclosure provides a method for treating non-small cell lung cancer, comprising administering a PD1 inhibitor and a second agent used in combination with the PD1 inhibitor to a patient diagnosed with non-small cell lung cancer, wherein the second agent is selected from Cordyceps sinensis and / or a fermentation product thereof.

[0013] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0014] The PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof;

[0015] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0016] Furthermore, the patient has at least one of the following protein changes: VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2, phosphorylated MEK1 / 2.

[0017] Furthermore, the patient has been diagnosed with non-small cell lung cancer and has at least one of the following protein alterations: VEGF, Ki67, RhoA, Raf-1, C-Fos.

[0018] Furthermore, the patient has been diagnosed with non-small cell lung cancer and has high expression of at least one of the following proteins: VEGF, Ki67, RhoA, Raf-1, c-Fos, preferably any one of VEGF, Ki67, RhoA, Raf-1, and c-fos is highly expressed, preferably VEGF and Ki67 are simultaneously highly expressed, more preferably RhoA, Raf-1, and c-fos are simultaneously highly expressed, preferably VEGF, Ki67, RhoA, Raf-1, and c-fos are simultaneously highly expressed.

[0019] Furthermore, the dosage of the second agent is about 91-910 mg / Kg / day, preferably 91-455 mg / Kg / day. Specifically, the dosage of the second agent refers to the patient dosage of the fermented bacterial powder. The dosage of the PD1 inhibitor is about 0.1-0.2 mg / Kg / day.

[0020] As used in this disclosure, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

[0021] In another aspect of the present disclosure, the present disclosure also provides a method for inhibiting protein expression in non-small cell lung cancer, comprising administering a therapeutically effective amount of a combination of Cordyceps sinensis and a PD1 inhibitor to a patient, wherein the protein expression includes at least one of the following: VEGF protein, Ki67 protein, RhoA protein, Raf-1 protein, c-fos protein, phosphorylated ERK1 / 2 protein and phosphorylated MEK1 / 2 protein.

[0022] or,

[0023] A method for inhibiting protein expression in non-small cell lung cancer, wherein the protein expression includes at least one of the following: VEGF protein, Ki67 protein, RhoA protein, Raf-1 protein, c-fos protein, phosphorylated ERK1 / 2 protein and phosphorylated MEK1 / 2 protein, and the method comprises: using Cordyceps sinensis and PD1 together as therapeutic agents.

[0024] Furthermore, the highly expressed protein is preferably non-small cell lung cancer in which any one of VEGF, Ki67, RhoA, Raf-1, and c-fos proteins is highly expressed, preferably VEGF and Ki67 proteins are highly expressed at the same time, more preferably RhoA, Raf-1, and c-fos proteins are highly expressed at the same time, and preferably VEGF, Ki67, RhoA, Raf-1, and c-fos proteins are highly expressed at the same time.

[0025] Furthermore, the cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0026] The PD1 inhibitor is selected from PD1 antibodies;

[0027] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0028] Furthermore, the therapeutically effective amount refers to a subject dosage of fermented bacteria powder of about 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d, and a subject dosage of the PD1 inhibitor of about 0.1-0.2 mg / Kg / d.

[0029] As used in this disclosure, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

[0030] In another aspect of the present disclosure, the present disclosure also provides a use of a non-small cell lung cancer therapeutic agent in the preparation of a drug, wherein the drug is used to treat non-small cell lung cancer; the non-small cell lung cancer therapeutic agent contains a PD1 inhibitor and a second agent used in combination with the PD1 inhibitor, wherein the second agent is selected from Cordyceps sinensis and / or its fermentation products.

[0031] Furthermore, the non-small cell lung cancer is a non-small cell lung cancer in which any one of VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein is highly expressed.

[0032] Furthermore, the cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder.

[0033] Furthermore, the PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof.

[0034] Furthermore, the PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0035] In another aspect, the present disclosure also provides a method for treating and / or preventing tumors or hyperproliferative diseases, particularly cancer, comprising administering the combination of Cordyceps sinensis and a PD1 inhibitor to a patient in need thereof.

[0036] Furthermore, the tumor or hyperproliferative disease includes non-small cell lung cancer, in particular non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, c-fos, phosphorylated ERK1 / 2 and phosphorylated MEK1 / 2, preferably non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1 and c-fos, preferably non-small cell lung cancer with high expression of VEGF and Ki67 proteins, more preferably non-small cell lung cancer with high expression of RhoA, Raf-1 and c-fos proteins, preferably non-small cell lung cancer with high expression of VEGF, Ki67, RhoA, Raf-1 and c-fos proteins.

[0037] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0038] Optionally, the PD1 inhibitor is selected from PD1 antibodies;

[0039] Optionally, the PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0040] In another aspect of the present disclosure, the present disclosure provides a use of Cordyceps sinensis for preparing a drug for treating non-small cell lung cancer as a basic treatment with a PD1 inhibitor.

[0041] In another aspect of the present disclosure, the present disclosure also provides a use of Cordyceps sinensis for preparing a drug for treating non-small cell lung cancer metastasis as a basic treatment with a PD1 inhibitor.

[0042] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0043] The PD1 inhibitor is selected from PD1 antibodies;

[0044] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0045] Furthermore, the non-small cell lung cancer refers to non-small cell lung cancer with high expression of any one of VEGF and Ki67 proteins, preferably non-small cell lung cancer with high expression of both VEGF and Ki67 proteins. The non-small cell lung cancer metastasis refers to non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, and c-fos proteins, preferably non-small cell lung cancer metastasis with high expression of both VEGF, Ki67, RhoA, Raf-1, and c-fos proteins.

[0046] In another aspect of the present disclosure, the present disclosure provides a use of a regulated immune cell population and at least two agents in the preparation of a drug for treating non-small cell lung cancer, comprising: contacting the immune cells with a sufficient amount of a composition comprising the at least two agents for a period of time sufficient to obtain a regulated immune cell population; wherein the regulated immune cell population comprises NK cells, and / or DC cells, CD4+T cells, CD8+T cells; and wherein the at least two agents comprise a PD1 inhibitor and Cordyceps sinensis.

[0047] Furthermore, the non-small cell lung cancer refers to non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, c-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein. Preferably, it is non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, and c-fos protein, preferably non-small cell lung cancer with high expression of both VEGF and Ki67 protein, more preferably non-small cell lung cancer with high expression of RhoA, Raf-1, and c-fos protein, preferably non-small cell lung cancer with high expression of VEGF, Ki67, RhoA, Raf-1, and c-fos protein.

[0048] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0049] The PD1 inhibitor is selected from PD1 antibodies;

[0050] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0051] Furthermore, the sufficient amount refers to a subject dosage of fermented bacteria powder of about 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d, and a subject dosage of the PD1 inhibitor of about 0.1-0.2 mg / Kg / d.

[0052] As used in this disclosure, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

[0053] In another aspect of the present disclosure, the present disclosure provides a use of a regulated serum factor and at least two agents in the preparation of a non-small cell lung cancer drug, comprising: contacting the serum factor with a sufficient amount of a composition comprising the at least two agents for a period of time sufficient to obtain the regulated serum factor; wherein the regulated serum factor group comprises any one of the following serum factors: TNF-α, IL-6, MDA, IL-10, SOD, GSH-Px, EPO and GM-CSF levels; and wherein the at least two agents comprise a PD1 inhibitor and Cordyceps sinensis.

[0054] Furthermore, the non-small cell lung cancer refers to non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, c-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein. Preferably, it is non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, and c-fos protein, preferably non-small cell lung cancer with high expression of both VEGF and Ki67 protein, more preferably non-small cell lung cancer with high expression of RhoA, Raf-1, and c-fos protein, preferably non-small cell lung cancer with high expression of VEGF, Ki67, RhoA, Raf-1, and c-fos protein.

[0055] The cordyceps sinensis is provided in the form of fermented cordyceps sinensis powder;

[0056] The PD1 inhibitor is selected from PD1 antibodies;

[0057] The PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab or toripalimab.

[0058] Furthermore, the sufficient amount refers to a subject dosage of fermented bacteria powder of about 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d, and a subject dosage of the PD1 inhibitor of about 0.1-0.2 mg / Kg / d.

[0059] As used in this disclosure, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that particular value.

[0060] The cordyceps sinensis and the PD1 inhibitor are administered simultaneously or sequentially.

[0061] The dosage or therapeutically effective amount or sufficient amount of the fermented bacteria powder is about 91-455 mg / Kg / d, 91-546 mg / Kg / d, 91-637 mg / Kg / d, 91-728 mg / Kg / d, 91-819 mg / Kg / d or 91-910 mg / Kg / d, and the dosage or therapeutically effective amount or sufficient amount of the PD1 inhibitor is about 0.1-0.2 mg / Kg / d.

[0062] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0064] Figure 1 shows the effect of Cordyceps sinensis on non-small cell lung cancer in mice. (A) Fluorescence images of mice 2 weeks after administration (n=3; ** P < 0.01 and *** P < 0.001; & P < 0.05, relative to PD1). (B) Body weight of mice in different treatment groups (n = 6). (C) Microscopic morphology of lung tumor tissue in each group (he, 20x). (a) Control group (Ctrl); (b) Model group (Model); (c) Cordyceps group (5 g / kg, BH); (d) 2 mg / kg PD1 inhibitor group (PD1); (e) 2 mg / kg PD1 inhibitor + 1 g / kg Cordyceps group (PD1+BL); (f) 2 mg / kg PD1 inhibitor + 5 g / kg Cordyceps group (PD1+BH).

[0065] Figure 2 shows the effects of Cordyceps sinensis on serum inflammatory cytokines and oxidative stress markers in mice with non-small cell lung cancer. (A-C) Serum TNF-α, IL-6, and IL-10 concentrations in different groups. (D-F) Serum oxidative stress markers MDA, GSH-Px, and SOD concentrations in mice. n = 6. Results are mean ± SD.### P < 0.001, vs. control; * P<0.05, ** P<0.01, *** P < 0.001, vs. model; & P<0.05, && P<0.01, &&& P<0.001, vs. PD1.

[0066] Figure 3 shows a schematic diagram of differentially expressed genes (GEGs) analysis of Cordyceps sinensis against non-small cell lung cancer. (A) Volcano plot of differentially expressed genes between mice with NSCLC and healthy mice. (B) Volcano plot of differentially expressed genes between mice with NSCLC and mice after BH treatment. (C) GO(BP) enrichment results for differentially expressed genes between the model / control group and the BH / model group. (E) GSEA enrichment results for differentially expressed genes between the model / control group and the BH / model group.

[0067] Figure 4 shows a schematic diagram of the proteomic analysis of DEPs in the Cordyceps sinensis anti-NSCLC setting. (A) Proteomic analysis flow chart. (B) Volcano plot of DEPs in the model and control groups. (C) Volcano plot of DEPs in the BH and model groups. (D) Enrichment of GO(BP) DEPs in the model / control groups. (E) Enrichment in the BH / model groups.

[0068] Figure 5 shows the effect of Cordyceps sinensis on the expression level of MAPK proteins in mice with non-small cell lung cancer. (AB) Immunohistochemical staining using different types of antibodies (20×). (CD) Protein level analysis of key targets in tumor tissues by Western blotting, including RhoA, Raf-1, c-fos, ERK1 / 2, MEK1 / 2 and their phosphorylated forms in the MAPK signaling pathway. (E) Analysis of serum hematopoietic growth factor (EPO and GM-CSF) concentrations in all groups of mice. (F) CD4 + T cells, CD8 + The proportions of T cells, dendritic cells, and NK cells. All values ​​are expressed as mean ± SD with n = 6 per group. ### P < 0.001, vs. control; * P<0.05, ** P<0.01, *** P < 0.001, vs. model; & P<0.05, && P<0.01, &&& P<0.001, VS. PD1. DETAILED DESCRIPTION

[0069] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0070] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0071] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0072] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0073] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0074] According to an embodiment of the present disclosure, the second therapeutic agent is provided in the form of fermented Cordyceps sinensis powder.

[0075] In the text, the term "fermented cordyceps sinensis powder" refers to the dry powder of mycelium obtained by liquid fermentation culture of the asexual generation of the ergot fungus Cordyceps sinensis isolated from the fruiting body of Cordyceps sinensis. In some cases, it is equivalent to "fermented cordyceps sinensis" in combination with the context. For example, the fermented cordyceps sinensis powder can be the dry powder of mycelium obtained by liquid fermentation culture of the asexual generation of the ergot fungus Cordyceps sinensis - Acanthopanax sinensis isolated from fresh Cordyceps sinensis in Qinghai, or it can be the dry powder of mycelium obtained by liquid fermentation culture of the asexual generation of Acanthopanax sinensis mycelium isolated from the fruiting body of Cordyceps sinensis in Tibet. In some embodiments, the fermented cordyceps sinensis powder is derived from Bailing capsules, and its fingerprints are consistent with those of wild Cordyceps sinensis in terms of nucleosides, sugar alcohols, sterols, and amino acids. Therefore, in terms of medicinal value, the two also have similar effects. Because wild Cordyceps sinensis contains high levels of heavy metals, the use of an industrial fermentation process with precise control of reaction conditions ensures that the metal content in the fermented Cordyceps sinensis powder meets safety standards while also ensuring consistent and controllable quality across batches, alleviating consumer concerns. Furthermore, the price is significantly lower than that of wild Cordyceps sinensis.

[0076] According to an embodiment of the present disclosure, the PD1 inhibitor is selected from a PD1 antibody. Cordyceps sinensis and the PD1 antibody can further synergistically inhibit the proliferation of non-small cell lung cancer cells, thereby better achieving the therapeutic purpose.

[0077] As used herein, the term "PD1 antibody" refers to a substance that can directly or indirectly neutralize, block, inhibit, reduce, or impede the activity of PD1. PD1 antibodies can neutralize, block, inhibit, reduce, or impede the activity of PD1 by binding to one or more PD1s. PD1 antibodies include: anti-PD1 antibodies and antigen-binding fragments thereof, receptor molecules, and derivatives that specifically bind to PD1 to isolate it from binding to one or more receptors. According to an embodiment of the present disclosure, the PD1 antibody is selected from tislelizumab, nivolumab, panlevuzumab, atezolizumab, durvalumab, avelumab, or toripalimab.

[0078] As used herein, the term "treatment" refers to the process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug described herein to an individual in need.

[0079] According to an embodiment of the present disclosure, the second therapeutic agent and the PD1 inhibitor are administered simultaneously or sequentially.

[0080] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0081] Example 1

[0082] 1 Drugs and reagents

[0083] Cordyceps sinensis (Bailing Capsules) were provided by Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. (Hangzhou, China), and its main ingredient is fermented Cordyceps sinensis powder. Cordyceps sinensis solution was prepared by dispersing Cordyceps sinensis in 0.5% sodium carboxymethylcellulose.

[0084] Tislelizumab injection (20 mL / kg) was purchased from BeiGene Co., Ltd. (Beijing, China).

[0085] Physiological saline (0.9%) was provided by Shandong Kelun Pharmaceutical Co., Ltd. (B22082402A, Shandong, China). Matrigengel matrix was obtained from Shanghai Novartis Medical Technology Co., Ltd. (0827045; Shanghai, China).

[0086] 2 Experimental methods

[0087] 2.1 Construction of NSCLC mouse model

[0088] Lewis lung carcinoma (LLC) cells (obtained from Zhongqiao Xinzhou Biotechnology) were cultured and passaged in DMEM medium supplemented with 10% fetal bovine serum and 1% double-antibody at 37°C in a 5% CO2 incubator. When LLC cells reached 80%-90% growth, they were trypsinized, harvested, and washed with phosphate-buffered saline (PBS). The cells were then added to Matrigel at a 1:1 ratio.

[0089] C57BL / 6J mice (n = 36, body weight 20 ± 3 g; Beijing Life River Laboratory Animal Technology Co., Ltd., Beijing, China) were acclimated to experimental conditions for 1 week. They had free access to food and water, and the temperature was 25 ± 2°C, the relative humidity was 50%-60%, and the lighting schedule was consistent with the circadian rhythm (12 h light, 12 h dark). The animal experiments were approved by the Center for the Care and Use of Laboratory Animals of the China Academy of Chinese Medical Sciences (No. 2022B139). After 7 days of acclimation, 36 male C57BL / 6J mice were randomly divided into 6 groups (6 mice per group). After the mice were anesthetized, the prepared Matrigel-cell mixture was vertically injected into the left lung at a depth of approximately 4 mm at a concentration of 1 million cells / mouse. The control group was injected with the same volume of saline.

[0090] 2.2 Grouping and Management

[0091] LLC cells used for modeling were stably transfected with luciferase, which emits fluorescence when stimulated by the substrate. Each mouse was intraperitoneally injected with 150 μL of D-luciferin and observed for 15 minutes. Fluorescence images and fluorescence intensity were then acquired using a small animal imaging system. Mice with successful modeling were further divided into the following groups (n = 6 per group):

[0092] 1) Model group;

[0093] 2) Cordyceps group (BL & BH): Oral administration of Cordyceps solution at a dose of 5g / kg mouse; the low dose of Cordyceps animal 1g / kg is equivalent to a human dose of 91mg / kg; the high dose of Cordyceps combination 5g / kg is equivalent to a human dose of 455mg / kg;

[0094] 3) Inhibitor (PD1): Tislelizumab injection was intraperitoneally injected at a dose of 2 mg / kg mouse;

[0095] 4) PD1 inhibitor + Cordyceps sinensis (PD1+BH): Oral administration of Cordyceps sinensis solution at a dose of 5 g / kg mouse, and intraperitoneal injection of Tislelizumab injection at a dose of 2 mg / kg mouse;

[0096] 5) PD1 inhibitor + Cordyceps sinensis (PD1+BL): Oral administration of Cordyceps sinensis solution at a dose of 1 g / kg mouse, and intraperitoneal injection of Tislelizumab injection at a dose of 2 mg / kg mouse.

[0097] Mice in the control and model groups were treated with distilled water. Two weeks later, blood and lung tumor tissue were collected for biochemical analysis, pathological evaluation, immunohistochemistry, transcriptomic sequencing, proteomics, and western blotting. Specific analysis and testing methods are as follows:

[0098] 2.2.1 Histological observation of lungs

[0099] Mouse lung tissue was fixed overnight in 4% paraformaldehyde. Ethanol dehydration was performed before paraffin embedding, followed by sectioning, dewaxing, and rehydration. For histopathological analysis, tissue sections were stained with hematoxylin and eosin (H&E).

[0100] 2.2.2 Serum biochemical analysis by ELISA

[0101] ELISA kits for interleukin (IL)-6, IL-10, tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), erythropoietin (EPO), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from Shanghai ELISA Biotechnology Co., Ltd. (ml002095, ml063159, ml037873, ml016824, ml037757, ml643059) in Shanghai, China. Serum biochemical analysis of the above factors was performed according to the manufacturer's instructions.

[0102] 2.2.3 Transcriptome sequencing and data analysis

[0103] RNA was extracted from lung tissue of mice (n = 3 per group), and Illumina TruSeq RNA libraries were constructed and sequenced using an Illumina NovaSeq 6000 (San Diego, CA, USA). A 2-fold cutoff was used to define the difference, and the statistical significance threshold for screening differentially expressed genes was set at P < 0.05. Finally, Gene Ontology (GO) functional enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed using the R programming language.

[0104] 2.2.4 Proteomic detection and mass spectrometry analysis

[0105] Protein was extracted from the lung tissue of mice in the control, model, and drug-treated groups. Briefly, 0.1 g of lung tissue was obtained from each sample, and the appropriate lysis solution (RIPA + 1X protease inhibitor cocktail) was added to quickly grind and extract protein. The supernatant was obtained after centrifugation (15,000 g, 4°C for 20 minutes). After quantification, 100 μg / 100 μL of protein from each group was incubated in 5 mM dithiothreitol at 37°C in the dark for 30 minutes to chemically reduce disulfide bonds. Next, the protein was alkylated by incubation with 20 mM iodoacetamide at 37°C in the dark for 30 minutes. The samples were washed three times by the following steps: 400 μL of methanol (pre-cooled at -80°C); 100 microliters of dichloromethane; and 200 microliters of ultrapure water. The supernatant was discarded, and 500 μL of pre-cooled methanol was added to the pellet obtained after centrifugation (15,000 g, 4°C for 3 minutes). Then, by adding 200 μ L 200mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer solution of pH8.5 in protein precipitation, dissolve protein, and further digest 17h (protein: enzyme=100 μ g: 1 μ g) at 37 ℃ with trypsin.Use commercial C18 column (Waters, Milford, MA, USA) that sample is desalted, is dissolved in 50 μ L 0.1% formic acid (FA), and centrifuges (15,000x g, 30 minutes).Analyze supernatant (10 μ L) by liquid chromatography and tandem mass spectrometry, be used for protein identification.

[0106] 2.2.5 Database search and proteome analysis

[0107] An Xcalibur analysis system (Thermo Fisher, Waltham, MA, USA) was used to collect MS data. Protein identification was performed using Proteome Discoverer v2.4 using the Sequencing HT algorithm and alignment with the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) and UniProt database (http: / / www.uniprot.org / ). Differentially expressed proteins were screened based on the following criteria: up-regulated, FC > 1.5, and P < 0.05, down-regulated, FC < 0.67, P < 0.05. These differentially expressed proteins were then analyzed using GSEA.

[0108] 2.2.6 Immunohistochemical staining

[0109] The immunohistochemical staining protocol consisted of the following steps: First, mouse lung tissue was obtained from each group and fixed in 4% paraformaldehyde for 3 hours, then embedded in paraffin, deparaffinized in xylene, and dehydrated in graded ethanol. Deparaffinized sections were then immersed in water, then in citrate buffer (C1032 Solarbio, Beijing, China), and heated in a microwave oven to allow antigen retrieval. Each section was rinsed three times with PBS and blocked with 3% hydrogen peroxide for 10 minutes and 5% bovine serum albumin for 1 hour. Next, rabbit anti-mouse Ki-67 (9440s; CST, Danvers, MA, USA) and rabbit anti-mouse VEGFA polyclonal antibody (19003-1-AP; Proteintech, Wuhan, China) were added to the samples and incubated overnight in a refrigerator at 4°C. The next day, the samples were washed three times with PBS. HRP-conjugated goat anti-rabbit IgG (PR 30009; Proteintech; 100 drops) was then added to each slide and incubated at 25°C for 1 hour.

[0110] 2.2.7 Western blot analysis

[0111] Lung tissue protein was extracted from each group, measured using a BCA assay kit (Beyotime, Beijing, China), and adjusted to a final concentration of 1 μg / μL. Next, 5x loading buffer was added to the protein at a 1:4 ratio and heat-denatured at 96°C for 10 minutes. 30 μg of protein from each group was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% bovine serum albumin for 2 hours at room temperature, and the membrane was incubated with primary antibodies overnight at 4°C. Primary antibodies included anti-RhoA (1:1000; Bioss, Beijing, China), anti-raf1 (1:1000; Bioss), and anti-c-fos (1:1000; Bioss). After 24 hours, the membrane was washed three times with TBST (10 minutes each) and incubated with HRP-linked goat anti-rabbit / mouse IgG for 1 hour. The membrane was then washed three more times in TBST (10 minutes each). Finally, ECL luminescent liquid was added for visualization. The above experiment was repeated three times and the data were calculated using Image J software.

[0112] 2.2.8 Routine blood tests

[0113] Blood samples were collected from anesthetized mice, mixed in anticoagulant tubes containing EDTA-Na, and stored at 4°C. A SYSMEX XN-1000V instrument (Kobe, Japan) was used to measure platelets (PLTs), white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HGB).

[0114] 2.2.9 Flow cytometric analysis of immune cells

[0115] Flow cytometry was performed using a Beckman Coulter counter (CytoFLEX, Brea, CA, USA) according to the manufacturer's instructions. Blood samples were collected in tubes with sodium heparin anticoagulant. Red blood cell lysis buffer was then added and lysed on ice for 15 minutes (protected from light). Samples were then centrifuged (4°C, 400 x g, 5 minutes) and incubated with antibodies (4°C, 30 minutes) to isolate DCs, NK cells, and CD4+ and CD8+ T cells.

[0116] 3. Results and Analysis

[0117] 3.1 Alleviation of non-small cell lung cancer in mice

[0118] After 2 weeks of continuous oral administration, lung tissue images were collected from different groups of mice (n=3) using small animal in vivo imaging. Figure 1A shows that the fluorescence of the model group mice was 9.29×10 9 The fluorescence of mice in the drug-treated groups (alone and combination drug groups) was significantly reduced, among which the data of PD1 and the combination of PD1 and Bailing Capsule were statistically different from those in the model group, and the high-dose combination of PD1 and Bailing Capsule was statistically different from that of PD1 alone, indicating that the high-dose Bailing Capsule and PD1 group showed a synergistic effect.

[0119] Note: The more “+” there are, the higher the fluorescence intensity of lung cancer tissue is.

[0120] The high-dose Cordyceps group, combined with PD1, showed a statistically significant improvement compared to the model group. Compared to the PD1 inhibitor, PD1 was more effective in reversing these changes when combined with a high-dose Cordyceps (P<0.05). This finding suggests that Cordyceps inhibits tumor growth in mice in an orthotopic lung cancer model.

[0121] In addition, neither Cordyceps sinensis nor PD1 alone or in combination significantly changed the body weight of mice (Figure 1B), indicating that the drugs have good safety.

[0122] HE staining results showed that the bronchi and alveoli of healthy mice were intact, while the alveoli of NSCLC mice were severely damaged and structurally disordered, showing alveolar atresia, fissures, and collapse.

[0123] Note: The more “+” there are, the higher the degree of bronchial and alveolar damage is.

[0124] Compared with the model group, the lung damage in mice in the BH and PD1 groups was significantly improved. Similarly, the above phenomenon was also improved and alleviated by the combination treatment (PD1+BL and PD1+BH), as shown in Figure 1C.

[0125] 3.2 Significantly improved serum inflammatory factor levels and oxidative stress in mice with non-small cell lung cancer

[0126] Lung cancer cells secrete various mediators to recruit monocytes, neutrophils and other white blood cells to produce a series of inflammatory factors. Reactive oxygen species (ROS) are oxidative products of cellular metabolism that can destroy the functions of lipids, nucleic acids and proteins, disrupt the oxidative balance, induce oxidative stress, promote inflammation, and thus lead to various diseases. As shown in Figure 2, compared with the control mice, the serum TNF-α, IL-6 and MDA levels of NSCLC model mice were significantly increased, while the IL-10, SOD and GSH-Px levels were significantly decreased. The use of Cordyceps sinensis alone and the combination of Cordyceps sinensis and PD1 can significantly reverse the changes in the corresponding factors in the model mice, which is statistically different from the model group. In addition, when PD1 is used in combination with Cordyceps sinensis, the reversal effect is more significant.

[0127] 3.3 Regulatory effects on inflammation and immune-related genes in mice with non-small cell lung cancer

[0128] To identify key genes associated with the anti-NSCLC pneumonitis effect of Cordyceps sinensis, RNA sequencing (RNA-Seq) was performed on mouse lung cancer tissue samples from the control, model, and BH groups. As shown in Figures 3A-B (volcano plots), based on transcriptome data, different types of differentially expressed genes were found in the control, model, and BH groups. Comparison with the control group showed that 6515 differentially expressed genes were significantly altered in the model group, of which 2902 DEGs were upregulated and 3613 were downregulated. Comparison with the model group showed that 49 differentially expressed genes were significantly altered in the BH group, including 24 upregulated and 25 downregulated genes. Compared with the control group, upregulated differentially expressed genes in the model group were primarily enriched in pathways involved in biological processes such as leukocyte migration, oxidative stress response, and B cell proliferation (Figure 3C). Downregulated differentially expressed genes after oral administration of Cordyceps sinensis were primarily enriched in pathways involved in biological processes such as monocyte chemoattractant protein-1 production and negative regulation of inflammatory responses (Figure 3D). In addition, GSEA enrichment analysis of differentially expressed genes among different groups revealed that signaling pathways related to T cell differentiation and activation had higher enrichment scores (Figure 3E-H).

[0129] 3.4 Effectively regulate inflammation and immune-related proteins in lung tissue of mice with non-small cell lung cancer

[0130] Various quantitative proteomics data can accurately identify differentially expressed proteins (DEPs) between normal and tumor tissues. Protein profiling data were used to detect and analyze differentially expressed proteins in lung tissues of mice from different groups (Figure 4A). As shown in Figures 4B-C, 3414 DEPs (including 2951 upregulated and 463 downregulated DEPs) were identified between mice with NSCLC and healthy mice. Compared with the model group, there were 783 differentially expressed proteins in the BH group (including 368 upregulated and 415 downregulated DEPs).

[0131] GO enrichment analysis of the biological functions, metabolic pathways, and signal transduction effects of DEPs revealed that compared with the control group, the upregulated DEPs in the model group were mainly enriched in biological processes such as B cell differentiation, B cell activation, and regulation of MAP kinase activity in immune responses (Figure 4D). The downregulated DEPs in anti-NSCLC pneumonia after BH administration were mainly enriched in biological processes such as upregulation of mitogen-activated protein (MAP) kinase activity and downregulation of MAP kinase (MAPK) cascades (Figure 4E). B cells have an antigen-presenting function in the immune system, whereby antigens are presented to T cells in an MHC-restricted manner through the B cell receptor (BCR) to activate T cell immune responses and ultimately kill cancer cells. At the same time, the p38 MAPK pathway, a target downstream of the BCR, is involved in controlling cell proliferation.

[0132] 3.5 Inhibition of VEGF and Ki67 in mice with non-small cell lung cancer

[0133] VEGF has the effect of inducing the formation of microvessels in NSCLC, which further mediates the occurrence and metastasis of NSCLC. In addition, the Ki67 labeling index, a typical marker for measuring tumor proliferation, is commonly used to detect various cancers. VEGF and Ki67 protein expression levels in tumor tissues from different treatment groups were analyzed by immunohistochemistry to verify the results of transcriptome and proteome analysis in the study (Figure 5A-B). Note: The more “+” there are, the higher the protein expression level is.

[0134] Compared to the VEGF and Ki67 protein levels in the lung tissues of healthy mice, the VEGF and Ki67 protein levels detected in the lung tissues of the NSCLC mice of the present disclosure were significantly upregulated. Cordyceps sinensis alone and the combination of Cordyceps sinensis and PD1 significantly reduced VEGF and Ki67 protein levels compared to the model group (P < 0.001). The simultaneous use of the two drugs significantly reduced the protein levels of both drugs compared to either alone (P < 0.001).

[0135] The Rho family mediates cell migration as a regulatory factor, among which RhoA is a key member that regulates tumor cell invasion and metastasis by affecting the cell cytoskeleton. This paper studied the effect of Cordyceps sinensis on the expression levels of key proteins in the MAPK signaling pathway by Western blot. As shown in Figures 5C-D, Cordyceps sinensis alone and in combination with PD1 significantly downregulated the expression of RhoA, Raf-1, and c-fos, which are related to tumor cell migration and invasion, and the combination therapy was superior to Cordyceps sinensis or PD1 alone (P < 0.01).

[0136] In addition, the total protein amount of ERK1 / 2 and MEK1 / 2 did not change significantly, but their phosphorylated forms were significantly downregulated when Cordyceps sinensis was used alone or in combination. To further evaluate the effect of Cordyceps sinensis on the immune function of mice with non-small cell lung cancer, the levels of hematopoietic growth factors (EPO and GM-CSF) were measured. As shown in Figure 5E, 5 days after modeling, the levels of hematopoietic factors (EPO and GM-CSF) in the model group decreased significantly (P<0.01). On the 14th day, the levels of EPO and GM-CSF in each drug-treated group increased significantly (P<0.01). After modeling, the number of CD4 + and CD8 + The percentage of lymphocytes was significantly lower than that in the control group (P<0.001); however, BH and PD1 could increase the number of CD4 + and CD8 + T cell levels (P>0.05).

[0137] Combination therapy can significantly increase CD4 + and CD8 + T cell levels, and the effect is better than that of single medication (Figure 5F). NK cells can recognize and eliminate stress cells during cancer, while DC cells can play an anti-tumor role by regulating the initiation, development and maintenance of immune responses during the anti-tumor process. As shown in Figure 5F, the levels of NK cells and DC cells in the model group were significantly lower than those in the control group (P<0.001). Consistent with the above transcriptomics and proteomics results, the drug-administered group reversed the above results and further proved that Cordyceps sinensis can activate immune cells and produce significant effects on mice with NSCLC. Compared with single treatment with Cordyceps sinensis or PD1, the combination of the two has better anti-tumor effects.

[0138] Taken together, these results indicate that Cordyceps sinensis can inhibit the expression of RhoA gene, recruit immune cells, and enhance immune function.

[0139] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A therapeutic agent for non-small cell lung cancer, which contains a PD1 inhibitor and a second agent used in combination with the PD1 inhibitor, and the second agent is selected from Cordyceps sinensis and / or its fermentation products.

2. The therapeutic agent according to claim 1, characterized in that, The second agent is provided in the form of fermented Cordyceps sinensis powder.

3. The therapeutic agent according to claim 1 or 2, characterized in that, The PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof.

4. The therapeutic agent according to any one of claims 1-3, characterized in that, The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

5. The therapeutic agent according to any one of claims 1-4, characterized in that, The non-small cell lung cancer is non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein.

6. The therapeutic agent according to any one of claims 1-5, characterized in that, The non-small cell lung cancer is non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, and C-Fos.

7. A method for treating non-small cell lung cancer, characterized in that, The method includes administering a PD1 inhibitor and a second agent used in combination with the PD1 inhibitor to a patient diagnosed with non-small cell lung cancer, and the second agent is selected from Cordyceps sinensis and / or its fermentation products.

8. The method according to claim 7, wherein The dose of the PD1 inhibitor is about 0.1 - 0.2 mg / Kg / d; The dose of the second agent is about 91 - 455 mg / Kg / d, 91 - 546 mg / Kg / d, 91 - 637 mg / Kg / d, 91 - 728 mg / Kg / d, 91 - 819 mg / Kg / d or 91 - 910 mg / Kg / d.

9. The method according to claim 7 or 8, characterized in that, The patient has at least one of the following protein alterations: VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2, phosphorylated MEK1 / 2 protein alterations.

10. The method according to any one of claims 7-9, characterized in that, The patient has been diagnosed with non-small cell lung cancer and has at least one of the following protein alterations: VEGF, Ki67, RhoA, Raf-1, C-Fos.

11. The method according to any one of claims 7-10, characterized in that, The patient has been diagnosed with non-small cell lung cancer and has high expression of at least one of the following: VEGF, Ki67, RhoA, Raf-1, C-Fos.

12. The method according to any one of claims 7-11, characterized in that, The second agent is provided in the form of fermented Cordyceps sinensis powder.

13. The method according to any one of claims 7-12, characterized in that, The PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof.

14. The method according to any one of claims 7-13, characterized in that The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

15. Use of a non-small cell lung cancer therapeutic agent in the preparation of a drug, characterized in that, The drug is used for the treatment of non-small cell lung cancer; The therapeutic agent for non-small cell lung cancer contains a PD1 inhibitor and a second agent used in combination with the PD1 inhibitor, and the second agent is selected from Cordyceps sinensis and / or its fermentation products.

16. The application according to claim 15, wherein, The non-small cell lung cancer is non-small cell lung cancer with high expression of any one of VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein.

17. The application according to claim 15 or 16, characterized in that, The Cordyceps sinensis is provided in the form of fermented Cordyceps sinensis powder.

18. The application according to any one of claims 15-17, characterized in that, The PD1 inhibitor is selected from a PD1 antibody or an antigen-binding fragment thereof.

19. The application according to any one of claims 15-18, characterized in that The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

20. A method for inhibiting protein expression in non-small cell lung cancer, characterized in that, Including: Administering to a patient a therapeutically effective amount of a combination of Cordyceps sinensis and a PD1 inhibitor, and the protein expression includes at least one of the following: VEGF protein, Ki67 protein, RhoA protein, Raf-1 protein, c-fos protein, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein.

21. The method according to claim 20, characterized in that, The Cordyceps sinensis is provided in the form of fermented Cordyceps sinensis mycelia powder; The PD1 inhibitor is selected from PD1 antibodies; The dose of the PD1 inhibitor is about 0.1 - 0.2 mg / Kg / d; The dose of the Cordyceps sinensis is about 91 - 455 mg / Kg / d, 91 - 546 mg / Kg / d, 91 - 637 mg / Kg / d, 91 - 728 mg / Kg / d, 91 - 819 mg / Kg / d, or 91 - 910 mg / Kg / d.

22. The method according to claim 21, wherein The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

23. The method according to any one of claims 20 - 22, characterized in that, The patient has at least one of the following protein alterations: VEGF, Ki67, RhoA, Raf-1, C-Fos, phosphorylated ERK1 / 2, phosphorylated MEK1 / 2 protein alterations.

24. The method according to any one of claims 20-23, characterized in that, The patient has been diagnosed with non-small cell lung cancer and has at least one of the following protein alterations: VEGF, Ki67, RhoA, Raf-1, C-Fos.

25. The method according to any one of claims 20-24, characterized in that, The patient has been diagnosed with non-small cell lung cancer and at least one of the following proteins is highly expressed: VEGF, Ki67, RhoA, Raf-1, C-Fos.

26. A method for inhibiting the protein expression of non-small cell lung cancer, wherein the protein expression includes at least one of the following: VEGF protein, Ki67 protein, RhoA protein, Raf-1 protein, c-fos protein, phosphorylated ERK1 / 2 protein, and phosphorylated MEK1 / 2 protein, and the method includes: The PD1 inhibitor and Cordyceps sinensis are jointly used as therapeutic agents.

27. The method according to claim 26, wherein The Cordyceps sinensis is provided in the form of fermented Cordyceps sinensis mycelia powder; The PD1 inhibitor is selected from PD1 antibodies.

28. The method according to claim 27, wherein The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

29. A method for treating and / or preventing tumors or hyperproliferative diseases, the method comprising administering to a patient in need thereof a combination of a PD1 inhibitor and Cordyceps sinensis.

30. The method according to claim 29, wherein The Cordyceps sinensis is provided in the form of fermented Cordyceps sinensis mycelia powder; The PD1 inhibitor is selected from PD1 antibodies; The dose of the PD1 inhibitor is about 0.1 - 0.2 mg / Kg / d; The dose of the Cordyceps sinensis is about 91 - 455 mg / Kg / d, 91 - 546 mg / Kg / d, 91 - 637 mg / Kg / d, 91 - 728 mg / Kg / d, 91 - 819 mg / Kg / d, or 91 - 910 mg / Kg / d.

31. The method according to claim 30, characterized in that, The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab or toripalimab.

32. The method according to any one of claims 29-31, characterized in that, The tumor or hyperproliferative disease includes non-small cell lung cancer.

33. The method according to claim 32, wherein The non-small cell lung cancer described is non-small cell lung cancer with high expression of any one of the proteins VEGF, Ki67, RhoA, Raf-1, and C-Fos.

34. Use of Cordyceps sinensis in the preparation of a non-small cell lung cancer drug with a PD1 inhibitor as the basic treatment.

35. Use of Cordyceps sinensis in the preparation of a non-small cell lung cancer metastatic carcinoma drug with a PD1 inhibitor as the basic treatment.

36. Use of a conditioned immune cell population and at least two agents in the preparation of a medicament for treating non-small cell lung cancer, comprising: The time for the immune cells to contact a composition containing the at least two agents in an amount sufficient to obtain a population of modulated immune cells; Wherein the population of modulated immune cells comprises NK cells, and / or DC cells, CD4+ T cells, CD8+ T cells; and wherein the at least two agents comprise a PD1 inhibitor and Cordyceps sinensis. Use of a conditioned serum factor and at least two medicaments in the preparation of a drug for non-small cell lung cancer, comprising: The time for the serum factors to contact a composition containing the at least two agents in an amount sufficient to obtain modulated serum factors; Wherein the population of modulated serum factors comprises serum factors of any one of the following: TNF-α, IL-6, MDA, IL-10, SOD, GSH-Px, EPO, and GM-CSF content; and wherein the at least two agents comprise a PD1 inhibitor and Cordyceps sinensis.

38. The use according to any one of claims 34 to 37, characterized in that, The Cordyceps sinensis is provided in the form of fermented Cordyceps sinensis powder; The PD1 inhibitor is selected from PD1 antibodies; The dose of the PD1 inhibitor is about 0.1 - 0.2 mg / Kg / d; The dose of the Cordyceps sinensis is about 91 - 455 mg / Kg / d, 91 - 546 mg / Kg / d, 91 - 637 mg / Kg / d, 91 - 728 mg / Kg / d, 91 - 819 mg / Kg / d, or 91 - 910 mg / Kg / d; The non-small cell lung cancer described is non-small cell lung cancer with high expression of any one of the proteins VEGF, Ki67, RhoA, Raf-1, and C-Fos.

39. The use according to claim 38, characterized in that, The PD1 antibody is selected from tislelizumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, or toripalimab.

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