CN116999551A

CN116999551A - Binding/inhibitors of PLK3 and their applications

Info

Publication number: CN116999551A
Application number: CN202210463493.7A
Authority: CN
Inventors: 梁国新; 尚红
Original assignee: First Hospital of China Medical University
Current assignee: First Hospital of China Medical University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-11-07

Abstract

The invention relates to the field of biological medicine. In particular to the application of binding/inhibitor or gene editing aiming at Polo-like kinase 3 (PLK 3) in anti-tumor immunity. The present invention provides a novel strategy capable of promoting immune killing of tumor cells by cytotoxic T lymphocytes (Cytotoxic T lymphocyte, CTLs), particularly cd8+ T cells, by binding/inhibiting PLK3 protein in the cells to alter its kinase activity, or by applying a method of gene editing (elimination/mutation) PLK3 to alter its kinase activity. The invention also relates to the combined use of a PLK3 binding/inhibitor and a PD-1 inhibitor, such as an anti-PD-1 antibody, in anti-tumor immunization. Provides a brand new target and application strategy for anti-tumor treatment, and has important clinical significance.

Description

Binding/inhibitors of PLK3 and uses thereof

Technical Field

The invention relates to the field of biological medicine. In particular to the application of binding/inhibitor or gene editing aiming at Polo-like kinase 3 (PLK 3) in anti-tumor immunity. More specifically, the present invention provides a substance capable of binding to/inhibiting PLK3 protein in cytotoxic T lymphocytes (Cytotoxic T lymphocyte, CTLs), particularly cd8+ T cells, thereby altering its kinase activity, or applying gene editing (elimination/mutation) of PLK3 thereby altering its kinase activity, thereby providing a method capable of promoting killing of tumor cells by CTLs, particularly cd8+ T cells. The invention also relates to the use of a combination of a PLK3 binding/inhibitor and a PD-1 inhibitor in the treatment of various tumours.

Background

Worldwide, the incidence and mortality of malignant tumors have been on the rise, and have become the main cause of human death. Cancer has begun to become a major cause of death in china in 2010, severely affecting people's life expectancy and impeding socioeconomic development. Finding an effective way to treat malignant tumors, including cancers, thoroughly has been the goal of continuous efforts in the scientific community. At present, the traditional treatment methods of malignant tumors mainly comprise operation treatment, radiotherapy and chemotherapy, targeted treatment and the like, but the side effects of the traditional treatments are large, adverse reactions such as hematological toxicity, gastrointestinal tract reaction and the like are often caused to patients, and serious patients even endanger lives. Thereby bringing about a serious drop in quality of life and an increase in economic burden. Even more difficult, these therapies rely on early findings, which are not apparent to patients with intermediate and advanced cancer, and therefore, finding effective methods for thoroughly treating malignant tumors, including cancers, has been the goal of ongoing efforts in the scientific community. And the research and development of new anti-tumor therapeutic drugs has important significance for improving the survival time of cancer patients and improving the prognosis of patients.

In recent years, the latest and most promising antitumor therapy is immunotherapy, and has the remarkable advantages of still effectiveness, lasting curative effect, less adverse reaction and the like for patients with middle and advanced malignant tumors. Tumor immunotherapy mainly includes the following two major categories: the first class of immunotherapeutic drugs is to block the binding of immune checkpoints (such as PD-1) and their ligands by inhibiting the action of tumor immune negative regulatory receptors, i.e., directly acting on the autoimmune system, and to reactivate the immune response of T cells to tumors to achieve the anti-tumor effect. However, the efficacy of the current PD-1 monoclonal antibodies is not satisfactory, and only a small fraction of patients have efficacy in the treatment of PD-1 monoclonal antibodies, and most patients cannot benefit therefrom. The second type is immune cell therapy, namely, tumor surface specific antigen receptor is artificially installed on CD8+ T cells or Natural Killer (NK) cells and is amplified in vitro and returned to the body of a patient, so that the artificially modified CD8+ T cells can specifically identify, attack and kill tumor cells, namely CAR-T, CAR-NK therapy, but the therapy is still in clinical research stage at present, and the operation is complex and is not easy to be widely popularized. Therefore, development of a novel immunotherapeutic strategy for anti-tumor treatment is urgently needed, and the development of a wide range of tumor patients is benefited.

The Polo Like Kinase (PLK) family belongs to a highly conserved serine/threonine kinase, which family includes five members PLK1-5, which play an essential role in cell proliferation and growth as key mitotic kinases and cell cycle regulators. The PLK3 was found to be involved in cellular stress and repair of gene double strand breaks. PLK3 protein binds to the centrosome in a microtubule-dependent manner, is involved in mitosis and is localized to the mitotic apparatus. There is no report on the functional mechanism of PLK3 in the immune system, especially CTL killing.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a new intervention target, which can enhance the recognition and killing function of cytotoxic T lymphocytes (Cytotoxic T lymphocyte, CTL), especially cd8+ T cells, on tumor cells by using a substance capable of altering the kinase activity of PLK3 protein or by gene editing (elimination/mutation) PLK3 to alter the kinase activity thereof, and restore and enhance the antitumor immune function thereof, thereby playing an important role in the treatment of diseases such as malignant tumor. The invention also provides the effect of the combination of the PLK3 binding/inhibiting agent and other medicines such as PD-1 antibody medicines in enhancing the anti-tumor immunity.

In order to achieve the above purpose, the present invention provides the following technical solutions.

The invention provides an application of an agent or a method for inhibiting PLK3 activity in preparing a product for enhancing CTL (cytotoxic T lymphocyte) anti-tumor function, which is characterized in that the product enhances the CTL anti-tumor function by inhibiting PLK3 kinase activity in CTL.

Further, the agent that inhibits PLK3 activity includes at least one of the following I-V:

I. small molecule compounds of the PLK3 inhibitor class, such as: wortmannin, BI2536, BI6727 (Volasertib), centrinone (LCR-263), rigosertib sodium, GW843682X, GSK461364, R03280, poloxin, MLN0905, NMS-1286937, LFM-AB, SEB13 Hydrochloride, TC-S7005, HMN-214, poloxin, TAK-960 dihydrochloride and Cyllpolin9;

II. Specific antibodies to PLK3, or antigen-binding fragments thereof, including monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, receptors, and chimeras;

III, sgRNA of CRISPR-Cas 9 as a target PLK3 gene;

IV, siRNA or shRNA as PLK3 targeting inhibitors;

v, expression vector containing the targeted inhibitor siRNA or shRNA in III.

Preferably, the target sequence of the sgRNA, siRNA or shRNA is shown as SEQ ID NO. 1; the siRNA sequence is shown as SEQ ID NO. 2-7; the shRNA sequence is shown in SEQ ID NO. 8-10.

Further, the method for inhibiting PLK3 activity comprises mutating PLK3 gene sequence at DNA level or knocking out PLK3 sequence by adopting gene editing technology, so that PLK3 protein can not be expressed or normal PLK3 protein can not be expressed.

Further, the product comprises a reagent, a kit, an immunotherapy or a medicament.

Further, the CTLs include cd8+ T cells.

Further, the tumor includes any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

The invention also provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises any agent for inhibiting PLK3 activity as claimed in claim 2 and a pharmaceutically acceptable carrier; the carrier comprises any one or more of slow release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant.

Further, the application of the pharmaceutical composition in preparing antitumor drugs.

Preferably, the tumor comprises any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

Further, the pharmaceutical composition comprises any one of an external preparation, an oral preparation or an injection preparation.

Further, the external preparation is spray or aerosol; the oral preparation is any one of granules, capsules, tablets or vesicles; the injection preparation adopts intradermal, subcutaneous, intramuscular, local or intravenous injection as an administration mode.

The invention also provides a combination composition, which is characterized in that the combination composition comprises the pharmaceutical composition of claim 7 and a PD-1 antibody.

Further, the PD-1 antibody includes Nivolumab (Nivolumab), avelumab, durvalumab (devaluzumab), toripalimab (terlipressin Li Shan antibody), keytruda (Pembrolizumab), tisselizumab (tirelizumab), pembrolizumab (pamphleab), or Atezolizumab (alemtuzumab).

Further, the application of the combined medicine composition in preparing antitumor medicines.

The invention also provides a method for enhancing the anti-tumor function of CTL, which is characterized in that the method adopts the pharmaceutical composition or the combination pharmaceutical composition to remove or kill tumor cells through extracorporeal circulation of blood, including extracorporeal operation of dialysis. The combined application can kill tumor cells more efficiently, and has more excellent therapeutic effect.

Compared with the prior art, the invention has the beneficial effects.

The present invention provides a novel therapy capable of promoting immune killing of CTL, particularly cd8+ T cells, against tumor cells by applying a substance capable of binding/inhibiting PLK3 protein in CTL, particularly cd8+ T cells, thereby altering its kinase activity, or by subjecting PLK3 to gene editing (elimination/mutation) to alter its kinase activity. In addition, the invention also provides a combined scheme for combining PLK3 binding/inhibitor or gene knockout/mutation with other antitumor drugs such as PD-1 antibody drugs and treating malignant tumors, and the combined application of the two can kill tumor cells more efficiently, has a better treatment effect than the prior PD-1 monoclonal antibody drug immunotherapy, solves various bottleneck problems in the prior immunotherapy, and comprises the following steps: the invention has the advantages of low response rate, immune tolerance and the like of the immunosuppressive PD-1 antibody medicaments, provides a new target point and a brand new treatment strategy for anti-tumor immunotherapy, and has very important practical value.

Drawings

Fig. 1: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human lymphoblastic cell line CEM.T2; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of human lymphoblastic line cem.t2 by cd8+ T cells.

Fig. 2: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human osteosarcoma cell line 143B; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing effect of cd8+ T cells on human osteosarcoma cell line 143B.

Fig. 3: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human breast cancer cell line (ER positive) MCF 7; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of cd8+ T cells by human breast cancer cell line (ER positive) MCF 7.

Fig. 4: the left panel shows that PLK3 kinase inhibitors, siPLK3, shPLK3 significantly enhance the killing effect of cd8+ T cells on human breast cancer cell line (ER negative) HCC 1954; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of human breast cancer cell line (ER negative) HCC1954 by cd8+ T cells.

Fig. 5: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human non-small cell lung cancer cell line A549; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on human non-small cell lung cancer cell line a 549.

Fig. 6: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human small cell lung cancer cell line H446; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on human small cell lung cancer cell line H446.

Fig. 7: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human gastric cancer cell line MKN-45; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on human gastric cancer cell line MKN-45.

Fig. 8: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human colon cancer cell line LoVo; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on the human colon cancer cell line LoVo.

Fig. 9: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human malignant melanoma cell line A375; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of cd8+ T cells against human malignant melanoma cell line a 375.

Fig. 10: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human liver cancer cell line HepG 2; the right panel shows that the PLK3 kinase inhibitor alone or in combination with PD-1 mab can significantly enhance the killing effect of CD8+ T cells on human hepatoma cell line HepG 2.

Fig. 11: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human cervical cancer cell line MEG-01; the right panel shows that the PLK3 kinase inhibitor alone or in combination with PD-1 mab can significantly enhance killing of CD8+ T cells against human cervical cancer cell line MEG-01.

Fig. 12: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human Hodgkin lymphoma cell line L1326; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of human hodgkin's lymphoma cell line L1326 by cd8+ T cells.

Fig. 13: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on T lymphocyte leukemia cell line Jurkat; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing effect of cd8+ T cells on the T lymphocyte leukemia cell line Jurkat.

Fig. 14: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human ovarian cancer cell line Caov-3; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance killing of human ovarian cancer cell line Caov-3 by cd8+ T cells.

Fig. 15: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human thyroid cancer cell line TPC-1; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of cd8+ T cells against human thyroid cancer cell line TPC-1.

Fig. 16: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human nasopharyngeal carcinoma cell line CNE-1; the right panel shows that the PLK3 kinase inhibitor alone or in combination with PD-1 mab can significantly enhance the killing effect of CD8+ T cells on human nasopharyngeal carcinoma cell line CNE-1.

Fig. 17: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human head and neck cancer cell line HN-5; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of cd8+ T cells against human cervical cancer cell line HN-5.

Fig. 18: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human esophageal cancer cell line EC 109; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing effect of cd8+ T cells on human esophageal cancer cell line EC 109.

Fig. 19: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human kidney cancer cell line A498; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing effect of cd8+ T cells on human renal carcinoma cell line a 498.

Fig. 20: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human cholangiocarcinoma cell line HUCCT 1; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on human cholangiocarcinoma cell line HUCCT 1.

Fig. 21: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human pancreatic cancer cell line PANC-1; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing effect of cd8+ T cells on human pancreatic cancer cell line PANC-1.

Fig. 22: the left graph shows that PLK3 kinase inhibitor, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on human brain glioma cell line BT 325; the right panel shows that PLK3 kinase inhibitors alone or in combination with PD-1 mab significantly enhance the killing effect of cd8+ T cells on human glioma cell line BT 325.

Fig. 23: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human bladder cancer cell line T24; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of human bladder cancer cell line T24 by cd8+ T cells.

Fig. 24: the left graph shows that PLK3 kinase inhibitors, siPLK3 and shPLK3 can obviously enhance the killing effect of CD8+ T cells on a human prostate cancer cell line PC-3; the right panel shows that PLK3 kinase inhibitors, alone or in combination with PD-1 mab, significantly enhance the killing of human prostate cancer cell line PC-3 by cd8+ T cells.

Fig. 25: it was shown that PLK3 kinase inhibitors alone significantly inhibited the expression of cd8+ T cell PD-1. Wherein the left graph is a typical flow graph and the right graph is a statistical graph.

Fig. 26: the PLK3 antibody alone or in combination with PD-1 monoclonal antibody was shown to significantly enhance killing of CD8+ T cells against human lymphoblastic line CEM.T2.

Detailed Description

The following detailed description of the invention in connection with specific embodiments is intended to provide the public with a better understanding of the teachings, rather than to limit the teachings, and in fact, modifications that come within the spirit of the invention are desired to be protected.

Example 1.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human lymphoblast CEM.T2 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human lymphoblastic cem.t2 as shown in the left panel of figure 1.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human lymphoblast CEM.T2 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human lymphoblastic CEM.T2, as shown in the right graph of figure 1.

Example 2.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human osteosarcoma cell line 143B cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: the PLK3 kinase inhibitor can obviously enhance the killing of CD8+ T cells on human osteosarcoma cell line 143BInjury effect, as shown in the left panel of fig. 2.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human osteosarcoma cell line 143B cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human osteosarcoma cell line 143B, as shown in the right graph of FIG. 2.

Example 3.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human breast cancer cell line (ER positive) MCF7 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 uM, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 excitation The enzyme inhibitor can significantly enhance the killing effect of CD8+ T cells on human breast cancer cell line (ER positive) MCF7, as shown in the left graph of FIG. 3.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human breast cancer cell line (ER positive) MCF7 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human breast cancer cell line (ER positive) MCF7, as shown in the right graph of figure 3.

Example 4.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human breast cancer cell line (ER negative) HCC1954 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 uM, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was individually aligned with target cells at a 4:1 ratioProportional mixing, co-culturing for 2 days, and flow cytometry to detect killing of target cells by CD8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells on the human breast cancer cell line (ER negative) HCC1954, as shown in the left panel of fig. 4.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human breast cancer cell line (ER negative) HCC1954 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the combination of PLK3 kinase inhibitor with PD-1 mab resulted in greater killing of human breast cancer cell line (ER negative) HCC1954 by cd8+ T cells, as shown in the right panel of fig. 4.

Example 5.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The non-small cell lung cancer cell line A549 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 uM, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: shRNA lentivirus (available from Dhmarcon) infection with PLK3Killer cd8+ T cells were stained and screened for puromycin to give stably transduced cells. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on non-small cell lung cancer cell line a549, as shown in the left panel of fig. 5.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The non-small cell lung cancer cell line A549 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on a non-small cell lung cancer cell line A549, and the killing effect is shown in the right graph of FIG. 5.

Example 6.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human small cell lung cancer cell line H446 cell lysate, tumor cell antigen, was added to stimulate for 16H and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: transfection of siPLK3 (purchased from Sigma) with Lipo3000 chemistry Injury cd8+ T cells 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human small cell lung cancer cell line H446, as shown in the left panel of fig. 6.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human small cell lung cancer cell line H446 cell lysate, tumor cell antigen, was added to stimulate for 16H and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human small cell lung cancer cell line H446, as shown in the right graph of FIG. 6. Killing by human small cell lung carcinoma cell line H446 is shown in the right panel of FIG. 6.

Example 7.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human gastric cancer cell line MKN-45, namely tumor cell antigen, is added to stimulate for 16h and TNF-alpha is added to induce the tumor cell antigen into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D:shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: the PLK3 kinase inhibitor can significantly enhance the killing effect of CD8+ T cells on a human gastric cancer cell line MKN-45, as shown in the left graph of FIG. 7.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human gastric cancer cell line MKN-45, namely tumor cell antigen, is added to stimulate for 16h and TNF-alpha is added to induce the tumor cell antigen into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human gastric cancer cell line MKN-45, as shown in the right graph of FIG. 7.

Example 8.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human colon cancer cell line LoVo cell lysate, i.e., tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitionFormulation group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on the human colon cancer cell line LoVo, as shown in the left panel of fig. 8.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human colon cancer cell line LoVo cell lysate, i.e., tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human colon cancer cell line LoVo, as shown in the right graph of FIG. 8.

Example 9.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human malignant melanoma cell line A375 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ Co-cultivation per mLAnd (5) culturing for 7 days. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human malignant melanoma cell line a375, as shown in the left panel of fig. 9.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human malignant melanoma cell line A375 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human malignant melanoma cell line A375, as shown in the right graph of FIG. 9.

Example 10.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human liver cancer cell line HepG2 cell lysate, namely tumor cell antigen, is added to stimulate for 16 hours and TNF-alpha is added to induce the tumor cell antigen into mature dendritic cells. Will be Autologous CD8+ T cells and antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: the PLK3 kinase inhibitor can significantly enhance the killing effect of CD8+ T cells on a human liver cancer cell line HepG2, as shown in the left graph of figure 10.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human liver cancer cell line HepG2 cell lysate, namely tumor cell antigen, is added to stimulate for 16 hours and TNF-alpha is added to induce the tumor cell antigen into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on a human liver cancer cell line HepG2, as shown in the right graph of figure 10.

Example 11.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Adding human cervical cancer cell line MEG-01 cell lysate, i.e. tumor cell antigen, to stimulate for 16h andTNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: the PLK3 kinase inhibitor can significantly enhance the killing effect of CD8+ T cells on human cervical cancer cell line MEG-01, as shown in the left graph of FIG. 11.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human cervical cancer cell line MEG-01, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human cervical cancer cell line MEG-01, as shown in the right graph of FIG. 11.

Example 12.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Adding human jojoba The cell lysate of the odd-gold lymphoma cell line L1326, tumor cell antigen, was stimulated for 16h and induced into mature dendritic cells by addition of TNF- α. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human hodgkin's lymphoma cell line L1326, as shown in the left panel of fig. 12.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human hodgkin lymphoma cell line L1326 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human Hodgkin lymphoma cell line L1326, as shown in the right graph of FIG. 12.

Example 13.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ With 10% FB/mLS RPMI1640 medium. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The T lymphocyte leukemia cell line Jurkat cell lysate, tumor cell antigen, was added for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on the T lymphocyte leukemia cell line Jurkat, as shown in the left panel of fig. 13.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The T lymphocyte leukemia cell line Jurkat cell lysate, tumor cell antigen, was added for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on T lymphocyte leukemia cell line Jurkat, as shown in the right graph of FIG. 13.

Example 14.

Extracting 50-100m healthy personL peripheral blood, ficoll method for isolation of monocytes (PBMC), purification of CD8+ T cells and purification of CD8+ T cells according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human ovarian cancer cell line Caov-3 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on the human ovarian cancer cell line Caov-3, as shown in the left panel of fig. 14.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human ovarian cancer cell line Caov-3 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human ovarian cancer cell line Caov-3, as shown in the right graph of FIG. 14.

Example 15.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human thyroid cancer cell line TPC-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against the human thyroid cancer cell line TPC-1, as shown in the left panel of fig. 15.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human thyroid cancer cell line TPC-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger CD8+T cell against human thyroid cancer cell line TPC-1Killing as shown in the right panel of fig. 15.

Example 16.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human nasopharyngeal high differentiation squamous cell carcinoma cell line CNE-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human nasopharyngeal highly differentiated squamous cell carcinoma cell line CNE-1, as shown in the left panel of fig. 16.

50-100ml peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human nasopharyngeal high differentiation squamous cell carcinoma cell line CNE-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the mixture is mixed with target cells according to the ratio of 4:1 for co-culture for 2 days, and the target cells are subjected to detection of CD8+ T cells by flow cytometryIs a killing condition of (a). The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human nasopharyngeal high differentiation squamous cell carcinoma cell line CNE-1, as shown in the right graph of FIG. 16.

Example 17.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human head and neck cancer cell line HN-5 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human cervical cancer cell line HN-5, as shown in the left panel of fig. 17.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human head and neck cancer cell line HN-5 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Treating killer CD8+ T cells with solvent control, PD-1 mab (10 μg/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, available from MCE company), PLK3 kinase inhibitor+PD-1 mab for 24 hr, and then respectively mixing with target cellsMixing according to the ratio of 4:1, co-culturing for 2 days, and detecting the killing condition of CD8+T cells on target cells by using a flow cytometry method. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on HN-5 of a human head and neck cancer cell line, as shown in the right graph of FIG. 17.

Example 18.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human esophageal cancer cell line EC109 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human esophageal cancer cell line EC109, as shown in the left panel of fig. 18.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human esophageal cancer cell line EC109 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Respectively adopting solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor+PD-1 monoclonal antibodyAfter 24 hours of killing CD8+ T cells, the cells are respectively mixed with target cells according to the ratio of 4:1, and then co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human esophagus cancer cell line EC109, as shown in the right graph of FIG. 18.

Example 19.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human kidney cancer cell line A498 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human renal carcinoma cell line a498, as shown in the left panel of fig. 19.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human kidney cancer cell line A498 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 mab (10. Mu.g/mL, tislelizumab), PLK3 kinase inhibitor (5 uM,purchased from MCE company), PLK3 kinase inhibitor+pd-1 mab treated killer cd8+ T cells for 24 hours, and then mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined the killing of cd8+ T cells to target cells. The results show that: the combination of PLK3 kinase inhibitor and PD-1 mab resulted in stronger killing of human kidney cancer cell line a498 by cd8+ T cells, as shown in the right panel of fig. 19.

Example 20.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human bile duct cancer cell line HUCCT1 cell lysate, i.e. tumor cell antigen, is added to stimulate for 16h and TNF-alpha is added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human cholangiocarcinoma cell line HUCCT1, as shown in the left panel of fig. 20.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human bile duct cancer cell line HUCCT1 cell lysate, i.e. tumor cell antigen, is added to stimulate for 16h and TNF-alpha is added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control and PD-1 monoclonal antibody (10 mug/mL,tislelizumab (Tislelizumab)), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor+PD-1 monoclonal antibody treat killer CD8+ T cells for 24 hours, then mix with target cells according to the ratio of 4:1 respectively, co-culture for 2 days, and flow cytometry detects the killing condition of CD8+ T cells on target cells. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human cholangiocarcinoma cell line HUCCT1, as shown in the right graph of FIG. 20.

Example 21.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human pancreatic cancer cell line PANC-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human pancreatic cancer cell line PANC-1, as shown in the left panel of fig. 21.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. The human pancreatic cancer cell line PANC-1 cell lysate, i.e. tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 ug/ml, tislelizumab), PLK3 kinase inhibitor (5 mu M, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killer CD8+ T cells for 24 hours, and then the killer CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human pancreatic cancer cell line PANC-1, as shown in the right graph of figure 21.

Example 22.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human brain glioma cell line BT325 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing effect of cd8+ T cells on human brain glioma cell line BT325, as shown in the left panel of fig. 22.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human brain glioma cell line BT325 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF-alpha was added to induce it to mature dendritic cells. Will beAutologous CD8+ T cells and antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 mug, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human brain glioma cell line BT325, as shown in the right graph of FIG. 22.

Example 23.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. The human bladder cancer cell line T24 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human bladder cancer cell line T24, as shown in the left panel of fig. 23.

50-100ml peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Adding human bladder cancer cell line T24 cell lysate, i.e. tumor cell antigen to stimulate16h and TNF- α was added to induce it into mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human bladder cancer cell line T24, as shown in the right graph of FIG. 23.

Example 24.

50-100mL peripheral blood was collected from healthy persons, mononuclear cells (PBMC) were isolated by Ficoll method, CD8+ T cells were purified and purified according to 2X 10 ⁶ The culture was performed in RPMI1640 medium containing 10% FBS. The remaining cells were induced into dendritic cells with GM-CSF and IL-4. Human prostate cancer cell line PC-3 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. They were then divided into 5 groups, a: solvent control, B: PLK3 kinase inhibitor group (5 μm, available from MCE company), C: siPLK3 group, D: shPLK3 group, E: a negative control, wherein: A. b, E group add reagent to treat cells for 24h, group c: siPLK3 (purchased from Sigma) was transfected with killer cd8+ T cells using Lipo3000 chemistry for 24h, group d: the killer cd8+ T cells were infected with shRNA lentivirus of PLK3 (available from Dhmarcon corporation) and screened for stable transduced cells by puromycin. Each group was mixed with target cells in a ratio of 4:1, co-cultured for 2 days, and flow cytometry examined killing of target cells by cd8+ T cells. The results show that: PLK3 kinase inhibitors significantly enhanced the killing of cd8+ T cells against human prostate cancer cell line PC-3, as shown in the left panel of fig. 24.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Adding inHuman prostate cancer cell line PC-3 cell lysate, tumor cell antigen, was stimulated for 16h and induced to mature dendritic cells by addition of TNF- α. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Solvent control, PD-1 monoclonal antibody (10 mug/mL, tislelizumab), PLK3 kinase inhibitor (5 uM, purchased from MCE company), PLK3 kinase inhibitor and PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the killing CD8+ T cells and the target cells are mixed according to the ratio of 4:1 respectively, and co-culture is carried out for 2 days, so that the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the PLK3 kinase inhibitor combined with PD-1 monoclonal antibody can obtain stronger killing effect of CD8+T cells on human prostatic cancer cell line PC-3, as shown in the right graph of FIG. 24.

Example 25.

50mL of peripheral blood of healthy people is extracted, mononuclear cells (PBMC) are separated and purified by a Ficoll method, CD8+ T cells are separated and purified, after CD3/28 magnetic beads are activated for 3 days, CD8+ T cells are treated by solvent control, PLK3 kinase inhibitor, siPLK3, shPLK3 and negative control respectively, and the expression level of PD-1 on the surface of the CD8+ T cells is detected by flow cytometry. The results show that: PLK3 kinase inhibitors were able to significantly inhibit the expression of cd8+ T cell PD-1 as shown in figure 25.

Example 26.

50-100mL of peripheral blood of healthy people is extracted, PBMC is extracted by a Ficoll method, CD8+ T cells are separated and purified, and the remaining cells are induced into dendritic cells by GM-CSF and IL-4. Human lymphoblast CEM.T2 cell lysate, tumor cell antigen, was added to stimulate for 16h and TNF- α was added to induce it to mature dendritic cells. Autologous CD8+ T cells were combined with antigen-loaded dendritic cells according to 2X 10 ⁶ The culture was carried out for 7 days in a total of/mL. Respectively using anti-IgG (10 ug/mL, R&D) PD-1 monoclonal antibody (10 ug/ml, tislelizumab), anti-PLK3 (10 ug/ml, abcam), anti-PLK3+PD-1 monoclonal antibody are used for treating killing CD8+ T cells for 24 hours, and then the two are respectively mixed with target cells according to the ratio of 4:1, and are co-cultured for 2 days, and the killing condition of the CD8+ T cells on the target cells is detected by flow cytometry. The results show that: the anti-PLK3 antibody combined with PD-1 monoclonal antibody can obtain stronger CD8+T cell to human lymphoblastic antigenKilling by cell cem.t2 as shown in figure 26.

Sequence listing

<110> affiliated first Hospital of university of medical science in China

<120> PLK3 binding/inhibitors and uses thereof

<141> 2022-04-24

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Ala Ala Ala Pro Ala Pro Pro Ala Gly Pro Gly Pro Pro Pro Ser Ala

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Leu Arg Gly Pro Glu Leu Glu Met Leu Ala Gly Leu Pro Thr Ser Asp

35 40 45

Pro Gly Arg Leu Ile Thr Asp Pro Arg Ser Gly Arg Thr Tyr Leu Lys

50 55 60

Gly Arg Leu Leu Gly Lys Gly Gly Phe Ala Arg Cys Tyr Glu Ala Thr

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Asp Thr Glu Thr Gly Ser Ala Tyr Ala Val Lys Val Ile Pro Gln Ser

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Arg Val Ala Lys Pro His Gln Arg Glu Lys Ile Leu Asn Glu Ile Glu

100 105 110

Leu His Arg Asp Leu Gln His Arg His Ile Val Arg Phe Ser His His

115 120 125

Phe Glu Asp Ala Asp Asn Ile Tyr Ile Phe Leu Glu Leu Cys Ser Arg

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Lys Ser Leu Ala His Ile Trp Lys Ala Arg His Thr Leu Leu Glu Pro

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Glu Val Arg Tyr Tyr Leu Arg Gln Ile Leu Ser Gly Leu Lys Tyr Leu

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His Gln Arg Gly Ile Leu His Arg Asp Leu Lys Leu Gly Asn Phe Phe

180 185 190

Ile Thr Glu Asn Met Glu Leu Lys Val Gly Asp Phe Gly Leu Ala Ala

195 200 205

Arg Leu Glu Pro Pro Glu Gln Arg Lys Lys Thr Ile Cys Gly Thr Pro

210 215 220

Asn Tyr Val Ala Pro Glu Val Leu Leu Arg Gln Gly His Gly Pro Glu

225 230 235 240

Ala Asp Val Trp Ser Leu Gly Cys Val Met Tyr Thr Leu Leu Cys Gly

245 250 255

Ser Pro Pro Phe Glu Thr Ala Asp Leu Lys Glu Thr Tyr Arg Cys Ile

260 265 270

Lys Gln Val His Tyr Thr Leu Pro Ala Ser Leu Ser Leu Pro Ala Arg

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Gln Leu Leu Ala Ala Ile Leu Arg Ala Ser Pro Arg Asp Arg Pro Ser

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Ile Asp Gln Ile Leu Arg His Asp Phe Phe Thr Lys Gly Tyr Thr Pro

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Asp Arg Leu Pro Ile Ser Ser Cys Val Thr Val Pro Asp Leu Thr Pro

325 330 335

Pro Asn Pro Ala Arg Ser Leu Phe Ala Lys Val Thr Lys Ser Leu Phe

340 345 350

Gly Arg Lys Lys Lys Ser Lys Asn His Ala Gln Glu Arg Asp Glu Val

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Ser Gly Leu Val Ser Gly Leu Met Arg Thr Ser Val Gly His Gln Asp

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Ala Arg Pro Glu Ala Pro Ala Ala Ser Gly Pro Ala Pro Val Ser Leu

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Val Glu Thr Ala Pro Glu Asp Ser Ser Pro Arg Gly Thr Leu Ala Ser

405 410 415

Ser Gly Asp Gly Phe Glu Glu Gly Leu Thr Val Ala Thr Val Val Glu

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Ser Ala Leu Cys Ala Leu Arg Asn Cys Ile Ala Phe Met Pro Pro Ala

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Glu Gln Asn Pro Ala Pro Leu Ala Gln Pro Glu Pro Leu Val Trp Val

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Ser Lys Trp Val Asp Tyr Ser Asn Lys Phe Gly Phe Gly Tyr Gln Leu

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Ser Ser Arg Arg Val Ala Val Leu Phe Asn Asp Gly Thr His Met Ala

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Leu Ser Ala Asn Arg Lys Thr Val His Tyr Asn Pro Thr Ser Thr Lys

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His Phe Ser Phe Ser Val Gly Ala Val Pro Arg Ala Leu Gln Pro Gln

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Leu Gly Ile Leu Arg Tyr Phe Ala Ser Tyr Met Glu Gln His Leu Met

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Lys Gly Gly Asp Leu Pro Ser Val Glu Glu Val Glu Val Pro Ala Pro

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Pro Leu Leu Leu Gln Trp Val Lys Thr Asp Gln Ala Leu Leu Met Leu

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Phe Ser Asp Gly Thr Val Gln Val Asn Phe Tyr Gly Asp His Thr Lys

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Leu Ile Leu Ser Gly Trp Glu Pro Leu Leu Val Thr Phe Val Ala Arg

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Asn Arg Ser Ala Cys Thr Tyr Leu Ala Ser His Leu Arg Gln Leu Gly

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Cys Ser Pro Asp Leu Arg Gln Arg Leu Arg Tyr Ala Leu Arg Leu Leu

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Arg Asp Arg Ser Pro Ala

645

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<213> artificial sequence

<400> 5

aaucaaucuc cacugcuugc cagug 25

<210> 6

<211> 25

<212> RNA

<213> artificial sequence

<400> 6

ggcccgaaau cguagugcuu guacu 25

<210> 7

<211> 25

<212> RNA

<213> artificial sequence

<400> 7

aguacaagca cuacgauuuc gggcc 25

<210> 8

<211> 57

<212> DNA/RNA

<213> artificial sequence

<400> 8

ccgggacgct gacaacatct acattctcga gaatgtagat gttgtcagcg tcttttt 57

<210> 9

<211> 57

<212> DNA/RNA

<213> artificial sequence

<400> 9

ccggagtgtg gaagaggtag aggtactcga gtacctctac ctcttccaca ctttttt 57

<210> 10

<211> 57

<212> DNA/RNA

<213> artificial sequence

<400> 10

ccgggaagag taagaatcat gcccactcga gtgggcatga ttcttactct tcttttt 57

Claims

1. Use of an agent or method for inhibiting PLK3 activity in the manufacture of a product for enhancing Cytotoxic T Lymphocyte (CTL) anti-tumor function, wherein said product enhances CTL anti-tumor function by inhibiting PLK3 kinase activity in CTL.

2. The use according to claim 1, wherein the agent that inhibits PLK3 activity comprises at least one of the following I-IV agents:

I. Small molecule compound inhibitors of PLK3, such as: wortmannin, BI2536, BI6727 (Volasertib), centrinone (LCR-263), rigosertib sodium, GW843682X, TAK960, GSK461364, R03280, poloxin, MLN0905, NMS-1286937, LFM-AB, SEB13 Hydrochloride, TC-S7005, HMN-214, poloxin, TAK-960 dihydrochloride, TAK-960 hydrochloride and Cyllpolin9;

II. Specific antibodies or antigen-binding fragments thereof for PLK3, including monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, receptors, chimeras, and the like;

III, sgRNA of CRISPR-Cas 9 as a target PLK3 gene;

IV, siRNA or shRNA as PLK3 targeting inhibitors;

v, expression vector containing the targeted inhibitor siRNA or shRNA in III.

3. The use according to claim 2, wherein the target sequence of the siRNA, shRNA or sgRNA is shown in SEQ ID No. 1; the siRNA sequence is shown as SEQ ID NO. 2-7; the shRNA sequence is shown in SEQ ID NO. 8-10.

4. The use according to claim 1, wherein the method of inhibiting PLK3 activity comprises mutating the PLK3 gene sequence at the DNA level or knocking out the PLK3 sequence using gene editing techniques such that it is not expressed or does not express normal PLK3 protein.

5. The use according to claim 1, wherein the product comprises a reagent, a kit, an immunotherapy or a medicament.

6. The use according to claim 1, wherein the CTLs comprise cd8+ T cells.

7. The use according to claim 1, wherein the tumour comprises any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

8. A pharmaceutical composition comprising any of the agents that inhibit PLK3 activity of claim 2 and a pharmaceutically acceptable carrier; the carrier comprises any one or more of slow release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant.

9. The use of a pharmaceutical composition according to claim 8 for the preparation of an antitumor drug.

10. Use of a pharmaceutical composition according to claim 9 for the preparation of an anti-tumour medicament, wherein the tumour comprises any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

11. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises any one of an external preparation, an oral preparation or an injectable preparation.

12. The pharmaceutical composition according to claim 11, wherein the external preparation is a spray or aerosol; the oral preparation is any one of granules, capsules, tablets or vesicles; the injection preparation adopts intradermal, subcutaneous, intramuscular, local or intravenous injection as an administration mode.

13. A combination composition comprising the pharmaceutical composition of claim 7 and a PD-1 antibody.

14. The combination composition of claim 13, wherein the PD-1 antibody comprises Nivolumab, avelumab, durvalumab (devaluzumab), toripalimab (terlipressin Li Shan antibody), keytruda (Pembrolizumab), tisleauzumab (tirelizumab), pembrolizumab (pamuzumab), or Atezolizumab (alemtuzumab).

15. Use of a combination composition according to claim 13 for the preparation of an antitumor drug.

16. The use of a combination according to claim 15 for the preparation of an anti-tumour medicament, wherein the tumour comprises any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral sphingoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdomyoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.

17. A method for enhancing CTL antitumor function, which comprises removing or killing tumor cells by extracorporeal circulation of blood, including dialysis-like extracorporeal operation, using the pharmaceutical composition of claim 9 or the combination composition of claim 13.

18. The method of claim 17, wherein the tumor comprises any of the following types: acute myeloid leukemia, adrenal tumor, AIDS-related cancer, soft tissue acinar sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, esophageal cancer, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, chromocytorenal cell carcinoma, clear cell carcinoma, colon cancer, rectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, ewing's tumor, extraosseous myxoid chondrosarcoma, incomplete bone fibrogenesis, fibrodysplasia of the bone, gall bladder cancer or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, glioblastoma, islet cell tumor, kaposi's sarcoma, renal cancer, leukemia, lipoma/benign lipoma liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, malignant mesothelioma, multiple endocrine tumor, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, non-small cell lung cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disease, renal cell carcinoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid metastatic cancer, ovarian cancer, and uterine cancer.