Use of PPAR-delta inhibitors in combination with immunotherapeutic agents to prepare an antitumor drug - Patent Application 20070229333

Abstract

The present invention relates to the pharmaceutical use of PPARδ inhibitors in combination with immunotherapeutic agents to prepare antitumor drugs, wherein the immunotherapeutic agents are immune agonists or immune checkpoint inhibitors, and the tumors are preferably melanoma, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, colon cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, bladder cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer.The pharmaceutical agents of the present invention show remarkable anticancer effect, strong targeting ability, and have few side effects.

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to the field of biomedical technology, in particular to the use of PPAR.delta. inhibitors in combination with immunotherapeutic agents for the preparation of antitumor drugs, and to antitumor drug compositions thereof.

[0002] [Description of Related Art] In recent years, tumors have become a major threat to human health. In addition to traditional surgery, radiation therapy, and chemotherapy, immunotherapy as an alternative treatment has attracted more and more attention because it can mobilize the immune system in vivo to specifically identify tumors, and has excellent and sustained therapeutic effects with fewer side effects compared with traditional treatments. Immunotherapy can be used alone or in combination with radiation therapy or chemotherapy. In addition, various types of immunotherapy can be used in combination to enhance the effect of immunotherapy on tumor treatment.

[0003] The goal of immunotherapy is to build new or promote existing antitumor immune responses by stimulating the immune system to specifically eliminate tumor cells and generate tumor-specific immune memory responses. There are also various ways in which tumors evade attacks from the immune system, the key being the use of co-inhibitory mechanisms that allow immunological evasion via immune-suppressive molecules such as CTLA-4 and PD-(L)1 (Beatty GL, Gladney WL. Clin Cancer Res. 2015; 21(4): 687-692.). The first generation of immune checkpoint inhibitors specific to these targets have made significant progress in tumor therapy. Among them, anti-PD-(L)1 monoclonal antibodies (mAbs) and anti-CTLA-4 mAbs have been approved for the treatment of dozens of cancers, but the overall efficacy rate is only about 20%. A prerequisite for these therapies to be effective is that the immune system generates sufficient immune responses against tumor antigens in advance, as in the case of hot tumors. Patients with these tumors have T cells that have an immune response to the tumor, but the function of these T cells is inhibited by PD-1 signaling and CTLA4. Disinhibition is effective in patients with this type of tumor. For cold tumors, or tumors in which the immune system does not respond adequately to tumor antigens, it is important to activate the immune system effectively so that it can work effectively.

[0004] Activating costimulatory signals is an important way to stimulate the immune system, and stimulating the pathway of CD40 costimulatory signals located on antigen-presenting cells (APCs) is an effective way to stimulate the immune system. CD40 molecules are primarily expressed on APC cells, including some monocytes, macrophages, and dendritic cells (DCs), and may alternatively be expressed on B cells, platelets, endothelial cells, and smooth muscle cells. CD40L is expressed on activated CD4 T cells, memory CD8 T cells, and activated NK cells. CD40 stimulators have been shown to promote the expression of DC cell costimulatory molecules CD80 and CD86, as well as major histocompatibility molecules, and the release of immunostimulatory cytokines, thereby enhancing antigen-presenting function in APCs and promoting T cell immunity in tumor models (French RR, Chan HT, Tutt AL, Glennie MJ. Nat Med. 1999; 5(5): 548-553.; Sotomayor EM, Borrello I, Tubb E, et al. Nat Med. 1999; 5(7)): 780-787.; Diehl L, den Boer AT, Schoenberger SP, et al. Nat Med. 1999; 5(7): 774-779.). Recombinant human CD40L has shown preliminary efficacy in treating advanced physical tumors and non-Hodgkin's lymphoma (Vonderheide RH, Dutcher JP, Anderson JE, et al. J Clin Oncol. 2001; 19(13): 3280-3287.), as well as in treating adenovirus containing CD40L DNA in bladder cancer (Malmstrom PU, Loskog AS, Lindqvist CA, et al, Clin Cancer Res. 2010; 16(12): 3279-3287.), as demonstrated in clinical trials. Recombinant human CD40L can effectively stimulate antitumor immune responses in CD8 T cells.As demonstrated in clinical trials where CD40 agonist antibodies were used to treat pancreatic cancer, CD40 signals can further activate macrophage immune surveillance, functionally transitioning tumor-infiltrating macrophages from pro-tumor to anti-tumor (Beatty GL, Chiorean EG, Fishman MP, et al, Science. 2011;331(6024):1612-1616.).

[0005] Although some anti-CD40 agonist antibodies have shown preliminary therapeutic effects clinically, their efficacy is limited when used alone, with the overall response rate being only about 14%. In view of this, for better immune effects, it has been considered to use anti-CD40 agonist antibodies in combination with other immune stimulants. The combined immune stimulants mainly include type I interferon, IL-2, and TLR receptor antagonists (US Patent No. US9095608B2; Bouchlaka MN, Schisel GD, Chen M, et al. The Journal of Experimental Medicine. 2013; 210(11): 2223-2237.; US Patent No. US7993659B2.). Another important factor limiting the clinical application of anti-CD40 agonist antibodies is their adverse side effects. The clinically effective dose of anti-CD40 agonist antibodies is close to its toxic dose, and the side effects mainly include dose-dependent cytokine release syndrome (Vonderheide RH, Flaherty KT, Khalil M, et al. J Clin Oncol. 2007; 25(7): 876-883.) and hepatotoxicity (e.g., increased transaminases, hepatocellular necrosis, etc.) (Medina-Echeverz J, Ma C, Duffy AG, et al. Cancer immunology Research. 2015; 3(5): 557-566.). Clinical trials have shown that when administered with IL-2, anti-CD40 agonist antibodies can cause fatal hepatotoxicity, which increases with age, as well as pulmonary and intestinal toxicity (Bouchlaka MN, Schisel GD, Chen M, et al. The Journal of Experimental Medicine. 2013; 210(11): 2223-2237.). As demonstrated in mouse models, the combination of anti-CD40 agonistic antibodies with chemotherapeutic drugs even causes lethal toxicity (Long KB, Gladney WL, Tooker GM, et al., Cancer Discov. 2016;6(4):400-413.).Therefore, it is clear that a relatively high dose of anti-CD40 agonist or the combination of anti-CD40 agonist and other immunostimulants causes serious side effects due to the activation of the whole immune system, which limits the clinical application of anti-CD40 agonist. Therefore, a method for reducing the toxicity caused by the administration of high doses of CD40 agonist, which does not affect the anti-tumor activity of the drug or even increases the anti-tumor activity of the drug, is an urgent issue that must be addressed before the clinical anti-tumor application of CD40 agonist can be further expanded.

[0006] Peroxisome proliferator-activated receptors (PPARs) are ligand-activated receptors in the family of nuclear hormone receptors. The three subtypes have been revealed in various species as regulators of many intracellular metabolic processes. The three subtypes of PPARs, namely α, γ, and δ, form a subfamily of nuclear receptors. This PPAR family is normally activated by endogenous fatty acids (HE Xu, MH Lambert, VG Montana et al. Molecular cell. 1999; 3(3): 397-403.) and by the transcription of activated target genes (RM Evans, GD Barish, YX Wang. Nature Medicine. 2004; 10(4): 355-361.), and can control the general metabolism for fatty acids. PPARγ or PPARδ is essential for the maturation of M2 macrophages. Blocking the pathways for PPARγ or PPARδ signaling can cause the polarization of macrophages to an M1 state (Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, et al. Nature. 2007; 447(7148): 1116-20.; Odegaard JI, Ricardo-Gonzalez RR, Eagle ARed, et al., Cell Metab. 2008; 7(6): 496-507.). In the tumor microenvironment, macrophages form the major group of infiltrating leukocytes. Macrophages have an M2-like phenotype (suppressed phenotype) and promote tumor progression and drug resistance in immunotherapy or chemotherapy. Polarizing and reversing the M1 state of macrophages is considered an important anti-cancer immunotherapy strategy (D Saha, RL Martuza, SD Rabkin. Cancer Cell. 2017;32(2):253-267.e255.; DG DeNardo, B Ruffell. Nat Rev Immunol. 2019;19(6):369-382.).

[0007] Furthermore, on the one hand, there are differences in technical understanding in the field, and on the other hand, the inventors have studied a large number of literatures and patents when making the present invention, but due to length limitations, all of the details and contents cannot be listed in detail, but this by no means means that the present invention does not have the features of the prior art, on the contrary, the present invention already has all the features of the prior art, and the applicant reserves the right to add relevant prior art into the background.

[0008] [Summary of the Invention] According to the above-mentioned prior art knowledge, the researcher who proposes the present invention hypothesized that PPARγ or PPARδ inhibitors can enhance anti-CD40 agonist antibodies in terms of therapeutic effect. The applicant has found in the study that the combination of anti-CD40 agonist antibodies and PPARδ inhibitors can provide better therapeutic effect on melanoma than the use of anti-CD40 agonist antibodies alone. The combination therapy showed 100% efficacy rate. Meanwhile, the combination therapy of anti-CD40 agonist antibodies and PPARδ inhibitors has a borderline effective dose range than the use of anti-CD40 agonist antibodies alone. In the combination of anti-CD40 agonist antibodies and PPARδ inhibitors, low and high doses of anti-CD40 agonist antibodies showed equally good therapeutic effect on tumors, and the combined low dose anti-CD40 agonist antibodies caused almost no hepatotoxicity. Surprisingly, in a melanoma model, only the combination of an anti-CD40 agonist antibody with a PPARδ inhibitor showed improved therapeutic efficacy, whereas the use of the same dose of anti-CD40 agonist antibody as the PPARγ inhibitor did not show a synergistic antitumor effect, and the use of the PPARδ inhibitor alone did not provide a therapeutic effect.

[0009] Based on the applicant's findings above, the applicant claims the following technical solutions: In a first aspect of the present invention there is provided the pharmaceutical use of a PPAR.delta. inhibitor in combination with an immunotherapeutic agent in an antitumor agent.

[0010] The tumor is preferably melanoma, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, esophageal cancer, colon cancer, colon cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, bladder cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer.

[0011] In the above-mentioned medical use, the immunotherapeutic agent is an immune agonist or an immune checkpoint inhibitor, Preferably, the immune agonist is an agonist specific for a costimulatory molecule, the costimulatory molecule comprising OX40, 4-1BB (CD137), CD27, GITR, CD28, and / or ICOS; Preferably, the immune agonist is a CD40 agonist; Preferably, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PDL1 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, a CD47 inhibitor; preferably, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, and an anti-CTLA-4 antibody; Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0012] In the above-mentioned medical use, the CD40 agonist is selected from an anti-CD40 agonist antibody, a CD40L protein, an expression vector of a CD40L protein, or a fragment, derivative, and / or polymer thereof; Preferably, the CD40L protein is a recombinant CD40L protein, and preferably, the CD40 agonist is an anti-CD40 agonist antibody.

[0013] In the above-mentioned medical use, the PPARδ inhibitor is a compound capable of inhibiting PPARδ in a targeted manner, or a nucleic acid molecule capable of inhibiting the effect of PPARδ mRNA, or a molecule capable of degrading PPARδ, Preferably, the nucleic acid molecule is an siRNA or shRNA, Preferably, the PPARδ inhibitor is GSK3787.

[0014] A second aspect of the present invention provides an anti-tumor pharmaceutical composition, comprising a PPARδ inhibitor and an immunotherapeutic agent that are prepared separately and then packaged together, or a formulation prepared by mixing a PPARδ inhibitor and an immunotherapeutic agent together, The tumor is preferably melanoma, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, colon cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, bladder cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer; The tumor is preferably a melanoma, bladder cancer, or non-small cell lung cancer (NSCLC).

[0015] Preferably, the PPARδ inhibitor is a compound capable of inhibiting PPARδ in a targeted manner, or a nucleic acid molecule capable of inhibiting the effect of PPARδ mRNA, or a molecule capable of degrading PPARδ, Preferably, the nucleic acid molecule is an siRNA or shRNA, and preferably, the PPAR.delta. inhibitor is GSK3787.

[0016] Preferably, the immunotherapeutic agent is an immune agonist or an immune checkpoint inhibitor; More preferably, the immune agonist is an agonist specific for a costimulatory molecule, the costimulatory molecule comprising OX40, 4-1BB (CD137), CD27, GITR, CD28, and / or ICOS; More preferably, the immune agonist is a CD40 agonist; More preferably, the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PDL1 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, and a CD47 inhibitor; More preferably, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, and an anti-CTLA-4 antibody.

[0017] The above-mentioned antitumor pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and is prepared into a pharma- ceutically acceptable formulation; Preferably, the formulation is an injectable formulation, a targeting formulation, or a nanoformulation.

[0018] A further aspect of the invention provides the use of a PPARδ inhibitor in combination with an immunotherapeutic agent for treating a tumor, comprising administering to a patient a low dose of the immunotherapeutic agent while administering the PPARδ inhibitor during the course of treatment to enhance the immunotherapeutic effect, to reduce side effects of the immunotherapeutic agent, or to prevent an increase in the dose of the immunotherapeutic agent, Immunotherapeutic agents are immune agonists or immune checkpoint inhibitors. A PPARδ inhibitor is a compound capable of inhibiting PPARδ in a targeted manner, or a nucleic acid molecule capable of inhibiting the effect of PPARδ mRNA, or a molecule capable of degrading PPARδ, Preferably, the nucleic acid molecule is an siRNA or shRNA, and preferably, the PPAR.delta. inhibitor is GSK3787.

[0019] the immune agonist is an agonist specific for a costimulatory molecule, the costimulatory molecule including OX40, 4-1BB (CD137), CD27, GITR, CD28, and / or ICOS; Preferably, the immune agonist is a CD40 agonist; The immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PDL1 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, and a CD47 inhibitor, preferably, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, and an anti-CTLA-4 antibody; Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0020] B cells have two roles in tumor growth. As demonstrated in many studies, B cells and plasma cells can promote tumor growth in the microenvironment, which has stimulated the treatment of physical tumors by eradicating B cells. However, some studies carried out on human tumors have revealed that B cells can form tertiary lymphoid structures (TLS) in human tumors, which is different from the pro-tumor effect of B cells obtained in mouse tumor models. In TLS, B cells may promote the response to immunotherapy by facilitating the presentation of tumor antigens to CD4 T cells, which correlates with a better prognosis for patients (F Petitprez, A Reynies, EZ Keung, et al. Nature. 2020; 577(7791): 556-560.; BA Helmink, SM Reddy, J Gao, et al. Nature. 2020; 577(7791): 549-555.; R Cabrita, M Lauss, A Sanna, et al. Nature. 2020; 577(7791): 561-565.). Clearly, therefore, eradication of B cells may cause adverse outcomes in most patients. Decisions regarding the use of B cell targeted therapies, and their combination with other therapies, will be based on a better understanding of the general nature of tumor-immune interactions mediated by various B cell subpopulations. The reason why B cells behave differently in human and mouse tumor models may be the fact that B cells represent an inherently heterogeneous population with various effects, and the mouse model experiments in the aforementioned studies were designed to investigate the role of B cells in tumor initiation and development by eradicating the entire B cell population. Therefore, targeting B cells with immunosuppressive effects is an attractive strategy.

[0021] Therefore, in the present invention, the PPARδ inhibitor inhibits CD19, which has an immunosuppressive effect. + CD24 hi IgD lo / - CD19 targets only B cells while stimulating the immune system + CD24 lo IgD hiIt does not affect B cells, which prevents the adverse effects caused by the complete eradication of B cells. Furthermore, the therapeutic strategy of combining PPARδ inhibitors with immunotherapy drugs not only enhances the efficacy of immunotherapy, but also provides improved safety by preventing patients from being exposed to ineffective immunotherapy and the serious toxic side effects brought about by immunotherapy.

[0022] According to the results of our study, in the combination therapy model of PPARδ inhibitors and immunotherapy drugs, PPARδ inhibitors may have provided therapeutic effects through mechanisms other than affecting the polarization of tumor-associated macrophages. During the evaluation of the combination therapy mechanism of anti-CD40 agonist antibodies and PPARδ inhibitors for melanoma, we revealed that the number and percentage of T cell-suppressing B cells in the draining lymph nodes were significantly increased. Using PPARδ inhibitors to eradicate B cells or suppress the immunosuppressive effect of B cells significantly enhanced the therapeutic effect of low-dose anti-CD40 agonist antibodies. In addition, eradicating all B cells or using PPARδ inhibitors also helped to enhance the antitumor effect of anti-PD-1 antibodies. Further studies showed that the tumor itself increased the CD19 expression in the draining lymph nodes. + CD24 hi IgD lo / - These results suggest that CD19 can induce an increase in B cells. + CD24 hi IgD lo / - B cells express more PPARδ and have stronger proliferation ability and immunosuppressive effect, whereas CD19 + CD24 lo IgD hi B cells have better immune stimulation. PPARδ inhibitors suppress CD19 + CD24 hi IgD lo / - It can reduce B cell proliferation and immunosuppressive effects, and is safe because it has very few side effects.

[0023] Another aspect of the present invention relates to a method for treating a tumor, comprising administering to a subject in need of said treatment an effective amount of (1) a PPARδ inhibitor, and (2) an immune stimulant or immune checkpoint inhibitor, wherein (1) and (2) may be administered simultaneously or separately. In some embodiments, the tumor is melanoma, bladder cancer, or non-small cell lung cancer (NSCLC).

[0024] In some embodiments, the PPARδ inhibitor comprises a small molecule inhibitor of PPARδ. In some embodiments, the PPARδ inhibitor comprises an siRNA or srRNA or shRNA. In some embodiments, the PPARδ inhibitor comprises GSK3787.

[0025] In some embodiments, the PPARδ inhibitor is administered daily for a period of 1-14 days, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days for a period of 2-30 days, or every 2, 3, or 4 weeks for a period of 2-24 weeks. The PPARδ inhibitor may be administered orally, intramuscularly, intravenously, or intraperitoneally.

[0026] In some embodiments, the PPARδ inhibitor is GSK3787 and is administered at a unit dose of 1-100 mM / kg body weight, 1-30 mM / kg body weight, 1-10 mM / kg body weight, 1-3 mM / kg body weight, and is administered at a unit dose of 1-100 mM / kg body weight, 1-30 mM / kg body weight, 1-10 mM / kg body weight, 1-3 mM / kg body weight, 3-100 mM / kg body weight, 3-30 mM / kg body weight, 3-10 mM / kg body weight, 10-100 mM / kg body weight, 10-30 mM / kg body weight, or 30-100 mM / kg body weight.

[0027] In some embodiments, the immune stimulatory agent or immune checkpoint inhibitor is a CD40 stimulatory agent. In some embodiments, the CD40 stimulatory agent is an anti-CD40 antibody.

[0028] In some embodiments, the immune stimulant or immune checkpoint inhibitor is administered daily for a period of 1-14 days, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days for a period of 2-30 days, or every 2, 3, or 4 weeks for a period of 2-24 weeks. The immune stimulant or immune checkpoint inhibitor may be administered orally, intramuscularly, intravenously, or intraperitoneally.

[0029] In some embodiments, the immune stimulator is an anti-CD40 antibody and is administered at a unit dose of 0.1-30 mg / kg body weight, 0.1-10 mg / kg body weight, 0.1-3 mg / kg body weight, 0.1-1 mg / kg body weight, 0.1-0.3 mg / kg body weight, 0.3-30 mg / kg body weight, 0.3-10 mg / kg body weight, 0.3-3 mg / kg body weight, 0.3-1 mg / kg body weight, 1-30 mg / kg body weight, 1-10 mg / kg body weight, 1-3 mg / kg body weight, 3-30 mg / kg body weight, 3-10 mg / kg body weight, or 10-30 mg / kg body weight.

[0030] The present invention has at least the following technical advantages: 1) PPARδ inhibitors inhibit tumor CD19 + CD24 hi IgD lo / - It can reduce the proliferation and suppressive effect of B cells, and can be combined with CD40 agonists for anti-tumor therapy. The present invention enhances the ability of CD40 agonists to stimulate immune responses while reducing the required amount of CD40 agonists, and provides a greatly improved tumor-curing effect in enhance CD40 agonists, thereby preventing the toxic side effects of CD40 agonists used at high doses. 2) The present invention further demonstrates that, when administered to tumor-bearing mice, the combination of a PPARδ inhibitor with a low dose of a CD40 agonist effectively stimulates the treated organism to generate antitumor T cells and induces a memory response in the antitumor T cells, thus significantly enhancing the antitumor effect of the antitumor T cells. 3) The present invention demonstrates that PPARδ inhibitors can be used as an alternative in combination with various immunotherapeutic agents (immune agonists or immune checkpoint inhibitors) for antitumor treatment. 4) CD19 + CD24 lo IgD hi Compared with B cells, CD19 + CD24 hi IgD lo / - B cells express higher PPARδ. In the present invention, in a combination therapy model of a PPARδ inhibitor and an immunotherapeutic drug, the PPARδ inhibitor suppresses the immunosuppressive CD19 + CD24 hi IgD lo / - It only reduces B cell proliferation and suppressive effects, and not immunostimulatory CD19 + CD24 lo IgD hi The effect on B cells is very small, which prevents the adverse outcomes in most patients that could otherwise be caused by B cell depletion, makes the combination therapy highly target specific, and makes it very safe with few side effects. 5) The combination therapy in this disclosure allows for the use of lower doses of anti-CD40 antibodies to reduce liver toxicity. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is a graph showing the dose-dependent antitumor effect of FGK45.5 obtained in the experiment of Example 1 of the present invention. [Diagram 2] FIG. 2 is a graph showing the therapeutic results of a combined use of a PPARδ inhibitor and FGK45.5 obtained in a mouse experiment in Example 2 of the present invention. [Diagram 3] FIG. 3 shows the immune effect of the combined therapy of FGK45.5 and a PPARδ inhibitor obtained in Example 3 of the present invention. [Figure 4] FIG. 4 graphically depicts the changes in lymphocyte subpopulations and phenotypes in the draining lymph nodes of tumor-bearing mice in different treatment groups obtained in Example 4 of the present invention. [Diagram 5]FIG. 5 is a graph showing the experimental results demonstrating that the PPARδ inhibitor obtained in Example 5 of the present invention reduced the immunosuppressive effect of B cells. [Figure 6] FIG. 6 is a graph showing the therapeutic effect of combination therapy of a PPARδ inhibitor and an anti-PD-1 antibody on B16 cancer-bearing mice obtained in Example 6 of the present invention. [Figure 7] FIG. 7 is a graph showing the experimental results of a PPARδ inhibitor that reduces the proliferation of CD19+CD24hiIgDlo / - B cells obtained in Example 7 of the present invention. [Figure 8] FIG. 8 is a graph showing the experimental results of a PPARδ inhibitor that reduces the suppressive effect of CD19+CD24hiIgDlo / - B cells obtained in Example 8 of the present invention. [Figure 9] FIG. 9 graphically depicts the PPARδ inhibitor enhancing effect of FGK45.5 in treating murine MB49 tumors. [Figure 10] FIG. 10 graphically depicts PPARδ inhibitors reducing the immunosuppressive effect of CD19+CD24hiIgDlo / - B cells from non-small cell lung cancer (NSCLC) patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] In the graph, rat IgG represents the control antibody group, FGK represents the anti-CD40 agonist antibody FGK45.5, T007 represents the PPARγ inhibitor T0070907, GSK represents the PPARδ inhibitor GSK3787, Naive B represents B cells from natural untreated mice, No B represents the group in which B cells were not added to the co-culture system, Naive represents the group of natural mice, and NS represents no statistical significance; * P < 0.05, ** P < 0.01, *** P<0.001, **** P<0.0001.

[0033] [Detailed Description of the Invention] The following detailed description is provided in conjunction with the accompanying drawings. The present invention will be further described below in conjunction with examples, but the present invention is not limited thereby.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples are available through commercial channels unless otherwise specified.

[0035] Primary sources of reagents: PPARγ inhibitor T0070907 was purchased from Selleck Chemicals, USA.

[0036] The PPARδ inhibitor GSK3787 was purchased from Selleck Chemicals, USA. FGK45.5 (mouse anti-CD40 agonist antibody) was purchased from BioXcell, USA (West Lebanon, NH).

[0037] Rat IgG: Clone No. 2A3 was purchased from BioXcell, USA (West Lebanon, NH). Anti-CD19 antibody: Clone No. 1d3 was purchased from BioXcell, USA (West Lebanon, NH).

[0038] Anti-PD-1 antibody: Clone No. RMP1-14 was purchased from BioXcell, USA (West Lebanon, NH). Anti-CD3ε antibody: clone No. 145-2C11 was purchased from Biolegend, USA (San Diego, Calif.).

[0039] Anti-CD28 antibody: clone No. 37.51 was purchased from Biolegend, USA (San Diego, Calif.). B16 tumor cells: ATCC were purchased from the US (American Type Culture Collection) cell bank.

[0040] Example 1: Anti-CD40 agonistic antibodies showed dose-dependent anti-tumor effects, but high doses of anti-CD40 agonistic antibodies could cause obvious liver toxicity.

[0041] Anti-CD40 agonist antibodies can promote the transition from cold tumors to hot tumors and have moderate antitumor effects on some tumors, but the effective dose range is narrow and they are toxic to the liver and some other tissues. As reported, the toxic effects can be reduced by intra-tissue injection and sustained release of anti-CD40 agonist antibodies in the tumor drainage area.

[0042] First, we tested whether peritumoral injection of anti-CD40 agonistic antibodies would result in significant toxicity. We used a murine B16 melanoma model (C57BL / 6B16 tumor-bearing mouse model), which has been widely used in research and has relatively high immunogenicity. C57BL / 6 mice (8–10 weeks old, n = 5 / group) were inoculated with 7 × 10 5 Mice were inoculated subcutaneously with B16 tumor cells (mouse melanoma cells) and administered an anti-CD40 agonist antibody when the tumor became palpable approximately 8 days after tumor cell inoculation.

[0043] Mice were monitored daily and tumors were measured every 4 days using calipers. Tumor length (a) and width (b), and tumor volume = ab 2 / 2 was measured. Dosage regimen: The tumor-bearing mice were treated with a low dose of 25 μg FGK45.5 (a mouse anti-CD40 agonist antibody) three times for a total of 75 μg (days 8, 11, and 14), or a high dose of 40 μg five times for a total of 200 μg (days 8, 11, 14, 17, and 20), or 40 μg isotype control rat IgG five times (days 8, 11, 14, 17, and 20). Subcutaneous injections were made between the tumor and the draining lymph nodes in the groin. 72 hours after the last injection in each group, samples of mouse serum were tested for alanine transaminase (ALT, a known indicator of liver tissue damage).

[0044] The results are shown in Figure 1a. Mice injected with FGK45.5 had lower tumor volumes than controls, but the high-dose FGK45.5 group showed a more obvious antitumor effect.

[0045] The results show that the antitumor effect of FGK45.5 was dose-dependent. ALT was analyzed to assess the toxicity of the treatment. Mice in the low dose FGK45.5 group had less liver damage, with serum ALT of about 100 units, slightly higher than the control's of about 40 units. It should also be noted that the high dose FGK45.5 group had increased toxicity. Mice treated with high dose FGK45.5 showed more severe liver damage, with a significant increase in ALT levels to about 500 units (Figure 1b).

[0046] These data show that high doses of agonistic CD40 antibody administered by local subcutaneous injection are helpful in enhancing antitumor effect in this therapy, but cause relatively severe liver toxicity.Therefore, the strategy of enhancing antitumor effect by increasing the dose of anti-CD40 agonistic antibody is not feasible.Therefore, a method of reducing the toxic side effects of agonistic CD40 antibody while ensuring the antitumor effect is crucial to expand its clinical application.

[0047] Example 2: PPARδ inhibitors enhanced the therapeutic effect of low-dose anti-CD40 agonist antibodies without incurring significant hepatotoxicity, whereas PPARγ inhibitors did not provide such an effect.

[0048] As reported, PPARγ or PPARδ is a sensor of fatty acids and is essential for the maturation of M2 (inhibited phenotype) macrophages. Blocking the PPARγ or PPARδ pathway leads to the polarization of macrophages to the M1 state. In the tumor microenvironment, macrophages form the major group of infiltrating lymphocytes, have an M2-like phenotype, and promote tumor progression and drug resistance in immunotherapy or chemotherapy. Polarizing and reversing these cells to the M1 state is considered an important anti-cancer immunotherapy strategy. Given their ability to convert tumor-associated macrophages (TAMs) to a predominantly M1 phenotype, we used the B16 tumor-bearing mouse model of Example 1 and a low-dose FGK45.5 dosing regimen to test whether a PPARγ inhibitor (T0070907, Selleck, USA) or a PPARδ inhibitor (GSK3787, Selleck, USA) and an anti-CD40 agonist antibody have a combined anti-tumor effect.

[0049] The establishment of B16 melanoma-bearing mouse model and the administration schedule of low-dose and high-dose FGK45.5 were according to Example 1. The low-dose FGK45.5 and T0070907 or GSK3787 combination group was administered T0070907 or GSK3787 by intraperitoneal injection once a day from day 7 to day 16 after injection of B16 cells at the doses marked in Figure 2a, 2b. The high-dose FGK45.5 and T0070907 or GSK3787 combination group was administered T0070907 or GSK3787 by intraperitoneal injection once a day from day 7 to day 22 after injection of B16 cells at the doses marked in Figure 2b. T0070907 or GSK3787 only groups received T0070907 or GSK3787 by intraperitoneal injection once a day from day 7 to day 16 after B16 cell injection at the doses marked in Figures 2a, 2b.

[0050] Consistent with previous studies, the use of low doses of FGK45.5 alone (75 μg total) showed only very small antitumor effects, and the use of PPARγ inhibitors or PPARδ inhibitors alone for treatment provided little antitumor effect. However, when FGK45.5 was used in combination with a PPARδ inhibitor, it significantly reduced the rate of tumor growth. On the other hand, FGK45.5 and PPARγ inhibitors did not produce a synergistic antitumor effect (Figure 2a).

[0051] Next, we further investigated the effects of PPARδ inhibitors and combination therapy with different doses of FGK45.5. PPARδ inhibitors and low-dose FGK45.5 jointly provided therapeutic effects essentially equivalent to those provided by the same inhibitors together with high-dose FGK45.5, and matched the therapeutic effects of high-dose FGK45.5. All three treatment regimens showed obvious and nearly equivalent antitumor effects.

[0052] The antitumor effects in the high-dose FGK45.5 and PPARδ inhibitor combination group, the low-dose FGK45.5 and PPARδ inhibitor combination group, and the high-dose FGK45.5 alone group, measured as tumor incidence by tumor growth volume curve, were 62% (5 / 8), 77% (6 / 9), and 87% (7 / 8), respectively, with efficacy rates all being 100%, while the tumor incidence and efficacy rates in the low-dose FGK45.5 alone group were 100% (8 / 8) and 50% (4 / 8), respectively (Figure 2b).

[0053] More importantly, the combination group of low-dose FGK45.5 and PPARδ inhibitor showed a marked decrease in hepatotoxicity index (ALT level in serum) when compared with the combination group of high-dose FGK45.5 and PPARδ inhibitor. 24 hours after the last injection of PPARδ inhibitor (GSK3787), mouse tail vein blood was collected for each group. Serum was separated and ALT levels were examined. The combination group of low-dose FGK45.5 and PPARδ inhibitor had a serum ALT level of about 100 IU / L, which was equivalent to the serum ALT level of the group of mice administered low-dose FGK45.5 alone, while the combination group of high-dose FGK45.5 and PPARδ inhibitor had a serum ALT level of about 500 IU / L. The difference between them is statistically significant (Figure 2c).

[0054] Inducing immune system to generate antitumor immune memory response is considered to be the key for immune system to provide sustained antitumor effect and prevent tumor recurrence.To further verify whether FGK45.5 and PPARδ inhibitor in combination can induce antitumor immune memory response in organism, the following experiment was carried out.

[0055] In the above-mentioned B16 tumor-bearing mouse model and treatment plan, after completion of the treatment, both the group of mice administered low doses of FGK45.5 in combination with PPARδ inhibitors and the group of mice administered high doses of FGK45.5 alone had some tumor-free mice. These tumor-free mice were then used in the memory experiment described below. 90 days after the first treatment, 5×10 5 B16 tumor cells were injected into the tail vein of each tumor-free mouse to reactivate the immune system. The control group consisted of age-matched mice that had not been injected with B16 tumor cells.

[0056] It should be noted that 92% of mice in the two treatment groups survived for more than 40 days, while all mice in the control group died within 31 days. Furthermore, 80% of mice in the combination therapy group with low doses of FGK45.5 and PPARδ inhibitors survived for more than 100 days, while only 50% of mice in the group treated with high doses of FGK45.5 alone survived for more than 100 days. However, the difference between them was not statistically significant. These results indicate that the combination therapy with low doses of FGK45.5 and PPARδ inhibitors can similarly induce the immune system and generate antitumor memory responses comparable to those induced using high doses of FGK45.5 alone (Figure 2d).

[0057] The above results suggest that in the group of mice administered FGK45.5 and PPARδ inhibitor in combination, PPARδ inhibitor significantly enhances the therapeutic effect of low-dose anti-CD40 agonist antibody without increasing liver damage.The combination therapy of low-dose FGK45.5 and PPARδ inhibitor achieved both good safety and good efficacy.For safety, all subsequent experiments were carried out using low-dose FGK45.5, which applies to all dosing schedules, regardless of whether the components are used alone or in combination.

[0058] Example 3: Combination therapy of FGK45.5 and a PPARδ inhibitor effectively increased the infiltration depth of CD8 T cells in tumors. The number of immune-effecting cells in tumors from the group treated with FGK45.5 alone, the group treated with a combination of low-dose FGK45.5 and a PPARδ inhibitor, and the control group was quantified using flow cytometry, tissue sections, and immune cell staining.

[0059] The results are shown in Figure 3. The combination therapy significantly reduced CD45 expression in treated tumors compared to treatment with FGK45.5 alone. + CD8 in cells + There was only a small increase in the proportion of T cells, the difference not having statistical significance, and CD45 T cells were upregulated in tumors treated with the combination therapy when compared to the control group. +CD8 in cells + The percentage of cytotoxic T cells was significantly increased. In the treated group of mice that received FGK45.5 in combination with a PPARδ inhibitor, CD45 + CD8 in lymphocytes + The percentage of T cells increased to approximately 25%, indicating that CD45 + CD8 in lymphocytes + The proportion of T cells was approximately 15%, while the control group had CD45 + CD8 in lymphocytes + The proportion of T cells was only 10%. In comparison, CD45 + CD4 in cells + The percentages of T cells, B cells, and myeloid-derived suppressor cells (MDSCs) did not show significant differences between treatment groups (Figure 3a). Interestingly, CD8 + T cells were mainly present in the periphery, but in the FGK45.5 and PPARδ inhibitor combination therapy group, CD8 + T cells infiltrated deeper into the tumor (Figure 3b).

[0060] Example 4: FGK45.5 led to an increase in B lymphocytes in the draining lymph nodes. Antigen-induced T cell activation occurs in lymph nodes, and cellular variations in lymph nodes of tumor-bearing mice caused by different treatments should be more evident than in other areas. Subsequent experiments were performed to identify lymphocyte subpopulations and phenotypic changes in draining lymph nodes in tumor-bearing mice treated with different dosing regimens.

[0061] C57BL / 6B16 tumor-bearing mouse model (7×10 5Mice inoculated subcutaneously with B16 cells (8 days after tumor cell inoculation, tumors were palpable) were used. The experiment included a low-dose FGK45.5 treatment group, a low-dose FGK45.5 treatment group in combination with a PPARδ inhibitor, a PPARδ inhibitor treatment group alone, and an isotype control rat IgG treatment group. The treatment schedule of Example 2 shown in Figure 2a was followed. 24 hours after the last injection of GSK3787 (i.e., 72 hours after the last FGK45.5 injection), mice from all groups were sacrificed. Lymph node cells of mice from all groups were harvested for flow cytometry analysis and cell counting.

[0062] The results are shown in Figure 4. In the draining lymph nodes, the group of mice that received a combination of low dose FGK45.5 and a PPARδ inhibitor and the group of mice that received FGK45.5 alone had essentially similar CD45 + The absolute number of cells was 100% in the PPARδ inhibitor group and the rat IgG control group, and both were CD45 + The absolute number of CD45 cells in the draining lymph nodes was significantly higher than that in the control lymph nodes. + The absolute number of cells was not changed (Figure 4a). + Cell counts were compared between the rat IgG control and the CD19 + The CD19 cell count was significantly higher in the group of mice treated with a low dose of FGK45.5 in combination with a PPARδ inhibitor. + The cell count was CD19 in the FGK45.5 group. + However, the CD45 cell counts in the group of mice treated with a combination of low-dose FGK45.5 and PPARδ inhibitors were significantly higher than those in the control group. + The percentage of CD19 cells in the FGK45.5 group was significantly higher than that of CD45 cells in the FGK45.5 group. + The percentage of CD45 cells in the FGK45.5 alone-treated group, the PPARδ inhibitor group, and the rat IgG control group was significantly lower than that of CD19 cells. + CD4 in cells + T cells and CD8 +Although the number and percentage of T cells were matched, the CD45 + CD4 in cells + T cells and CD8 + Both the number of T cells and their percentage were significantly higher than those in the group of mice administered FGK45.5 alone. + The absolute number and percentage of cells were essentially equivalent, and both groups had significantly higher values ​​than the PPARδ inhibitor group and the rat IgG control group. + The absolute number and percentage of cells are essentially the same (Fig. 4b). The percentages of CD4 / CD19 cells in the FGK45.5 group, the PPARδ inhibitor group, and the rat IgG group are essentially equivalent, but the values ​​increased significantly in the group of mice administered FGK45.5 and the PPARδ inhibitor in combination (Fig. 4c). The values ​​of CD8 / CD19 in the four experimental groups are similar to those of CD4 / CD19 cells in the four experimental groups (Fig. 4d).

[0063] Considering that M2 macrophages show important suppressive factors in the tumor microenvironment, and that PPARγ or PPARδ inhibitors have been reported to favor the polarization of macrophages from M2 to M1, we first hypothesized that PPARγ or PPARδ inhibitors would enhance the therapeutic effect of anti-CD40 agonist antibodies by promoting the change of TAMs from M2 to M1 after said polarization. However, the results revealed that the combination therapy of FGK45.5 with PPARδ inhibitors significantly reduced the tumor growth rate, and the combination therapy of FGK45.5 with PPARγ inhibitors showed no antitumor effect. These results indicate that PPARδ inhibitors may enhance the therapeutic effect of anti-CD40 agonist antibodies by a different mechanism, rather than changing TAMs from M2 to M1 via polarization. As can be seen from the above results, PPARδ inhibitors, when used alone, suppress the activation of CD4T, CD8T, CD19, and CD4T cells.+ B, and CD11c + FGK45.5 alone did not significantly affect the number of CD19 cells in lymph nodes. + B and CD11c + PPARδ inhibitors increased CD19 cells in the draining lymph nodes induced by FGK45.5. + It suppressed the increase in the number and percentage of B cells, but did not affect the increase in the number and percentage of CD11c cells induced by FGK45.5.

[0064] The above results indicate that the ability of PPARδ inhibitors to enhance the antitumor effect of low-dose FGK45.5 may be related to their effect on B cells. Example 5: PPARδ inhibitors reduced the immunosuppressive effect of B cells, thereby enhancing the immunotherapeutic effect of FGK45.5.

[0065] The next step was to test whether PPARδ inhibitors could enhance immunotherapy by affecting B cells. First, we tested in vitro the ability of B cells in the draining lymph nodes of differently treated tumor-bearing mice to inhibit the proliferation of activated T cells. Purified CD4 + T cells were labeled in vitro with 2.5 μM CFSE. CFSE-labeled cells were then cultured at 1 × 10 6 The cells were resuspended at 0.05 mg / ml and transferred to a 96-well plate coated with 10 μg / ml anti-CD3ε antibody and placed at 4°C overnight. B cells purified from draining lymph nodes and the above T cells (1:1) were co-cultured in the above 96-well plate containing 1 μg / ml anti-CD28 mAb for 72 h and analyzed by flow cytometry. The cell proliferation rate of CFSE-labeled T cells was calculated using the formula: (1-MFI value of CFSE of T cells in the experimental group / MFI value of CFSE of T cells without B cells in the control group)×100%. MFI refers to the mean fluorescence intensity.

[0066] The results reveal that B cells derived from draining lymph nodes in untreated tumor-bearing mice suppressed CD4 T cells, and B cells derived from draining lymph nodes in tumor-bearing mice treated with a PPARδ inhibitor reduced the inhibition. Treatment with FGK45.5 did not reduce the inhibition in B cells (Figure 5a). These results indicate that PPARδ inhibitors may enhance the immunotherapeutic effect of FGK45.5 by attenuating the immunosuppressive effect in B cells.

[0067] To test our hypothesis, we used antibodies to eradicate B cells in B16 tumor-bearing mice and further evaluated the role of PPARδ inhibitors in a combination regimen of FGK45.5 and PPARδ inhibitors.

[0068] A part of B16 tumor-bearing mice was first killed by anti-CD19 antibody, and then administered rat IgG, or FGK45.5, or FGK45.5 in combination with PPARδ inhibitor (GSK3787) for treatment. Tumor growth was observed. Treatment with rat IgG, FGK45.5, and FGK45.5 in combination with PPARδ inhibitor was adopted as control group. In these groups, the dose and administration schedule of rat IgG, FGK45.5, and PPARδ inhibitor (GSK3787) all followed the dose and administration schedule of Example 2, as shown in Figure 2a.

[0069] CD19 +B cell eradication experiment: 250μg of anti-CD19 antibody (clone 1d3) was administered by intraperitoneal injection to tumor-bearing mice on the 6th, 7th and 15th days after inoculation of B16 cells, and the eradication rate of B cells should be more than 90% as measured using FACS. The experimental results reveal that the tumors in the B cell eradicated group and the IgG-treated control group have similar growth status, which means that killing B cells alone does not provide a therapeutic effect on B16 tumors, and killing B cells significantly enhances the therapeutic effect of FGK45 on B16 tumors. More importantly, the combination with FGK45.5 and the eradication of B cells in the FGK45.5-PPARδ group had essentially the same therapeutic effect on B16 tumors. It is more noteworthy that, due to the eradication of B cells, FGK45.5 alone and the FGK45.5-PPARδ group had essentially equivalent therapeutic effects against B16 tumors, and the PPARδ inhibitor lost the function of enhancing the antitumor effect of FGK45.5 (Figure 5b).

[0070] These results indicate that B cells act as immunosuppressants in FGK45.5 treatment in a B16 tumor model and that a PPARδ inhibitor enhanced the antitumor effect of low-dose FGK45.5 by suppressing the immunosuppressive effect of B cells.

[0071] Example 6: Therapeutic effect of PPARδ inhibitor combined with anti-PD-1 antibody on B16 tumor-bearing mice Based on the above results, we predicted that eradicating B cells or using PPARδ inhibitors could enhance the efficacy of other immunotherapeutic agents in the B16 tumor-bearing mouse model.

[0072] To test this possibility, we treated B16 tumor-bearing mice with a PPARδ inhibitor or B cell eradication together with an anti-PD-1 antibody and evaluated the efficacy of each treatment.

[0073] Tumor-bearing mice in the same condition were treated with anti-PD-1 antibody, B cell eradication + anti-PD-1 antibody, or anti-PD-1 antibody + PPARδ inhibitor, respectively (GSK3787 (300 nmol) at the doses shown in Figure 2a, intraperitoneal injection, once a day, 7 to 16 days after injection of B16 cells). B cell eradication (e.g., "CD19" in Example 5) + A total of five parallel groups (n=8 / group) were used, including the "B cell eradication experiment" and the IgG treatment group (isotype control of rat IgG injection using the dosing regimen of Example 1) as a control group.

[0074] Anti-PD-1 antibody was used in the following manner: From day 8 after tumor cell inoculation, anti-PD-1 antibody (clone RMP1-14) was administered to tumor-bearing mice by intraperitoneal injection at 200 μg each time, once every 4 days, for a total of 4 injections.

[0075] We found that when used alone, anti-PD-1 antibodies had a modest therapeutic effect against B16 tumors (with a response rate of 4 / 8), but eradicating B cells or using a PPARδ inhibitor further enhanced the therapeutic effect of anti-PD-1 antibody treatment against B16 tumors (with a response rate of 7 / 8) (Figure 6).

[0076] Example 7: PPARδ inhibitors inhibit CD19 + CD24 hi IgD lo / - It reduced B cell proliferation. Based on the above results, we hypothesized that tumor development or FGK45.5 treatment induces changes in B cells in the draining lymph nodes and in tumors, rendering these B cells suppressive to immunotherapy.

[0077] To verify this hypothesis, the experimental scheme of Example 4 was used. All groups of mice were sacrificed 24 hours after the last injection of GSK3787 (i.e., 72 hours after the last injection of FGK45.5), and various surface marker analyses were performed on B cells isolated from lymph nodes of tumor-bearing mice to identify surface markers of B cell subpopulations with immunosuppressive effects, and the effects of various treatments on B cell subpopulations with immunosuppressive effects.

[0078] According to known surface markers of regulatory B cells, and major development phenotypes, such as CD24, CD38, CD138 or IgD, CD19 in the different treatment groups. + CD24 hi IgD lo / - The following changes in B cell populations were revealed:

[0079] Twenty-four hours after the last GSK3787 injection, tumor-draining lymph nodes of differently treated mice were harvested. Lymph nodes from the same body parts of naive, tumor-free mice were also harvested as controls. Lymph nodes were mashed into single cell suspensions. Cells from different lymph nodes were stained using different fluorescently labeled antibodies for flow cytometry analysis.

[0080] Results showed that in the B cell population, CD19 + CD24 hi IgD lo / - B cells were the least abundant in naive mice. In tumor-bearing mice, CD19 + CD24 hi IgD lo / - The percentage of the B cell subpopulation was significantly increased. In the FGK-treated group, the percentage of this subpopulation was further increased. In the group of mice administered the PPARδ inhibitor alone, the percentage of this subpopulation of B cells was significantly decreased. In the group in which FGK was used together with the PPARδ inhibitor, the percentage of this subpopulation of B cells was much less than that of this subpopulation in the FGK-treated group (Fig. 7a,b). Furthermore, CD19 + CD24lo IgD hi Compared with B cell populations, CD19 + CD24 hi IgD lo / - B cells expressed higher PPARδ (Fig. 7c). + CD24 hi IgD lo / - Although FGK45.5 promoted PPARδ expression in B cells, stimulation caused by FGK45.5 was not associated with CD19 + CD24 hi IgD lo / - It did not result in any significant change in PPARδ expression in B cells (Fig. 7d).

[0081] Furthermore, as revealed by flow cytometry analysis, both tumor factors and FGKs express CD19 + CD24 hi IgD lo / - Promoting Ki67 expression in B cells, PPARδ inhibitors suppressed the CD19 expression caused by these two factors. + CD24 hi IgD lo / - It reduced the increase in Ki67 expression in B cells (Fig. 7e, f). However, CD19 + CD24 lo IgD hi The expansion of the B cell population was not affected by the various treatment factors (FIG. 7g).

[0082] Example 8: PPARδ inhibitors inhibit CD19 + CD24 hi IgD lo / - It reduced the inhibitory effect on B cells. CD19 + CD24 hi IgD lo / - To evaluate the functional modulation effect on B cells, the experimental scheme of Example 4 was used. 24 hours after the last GSK3787 injection (i.e., 72 hours after the last FGK45.5 injection), mice from all groups were sacrificed. CD19 + CD24 hi IgD lo / - B cell populations and CD19+ CD24 lo IgD hi B cell populations were isolated from the draining lymph nodes of differently treated tumor-bearing mice using flow cytometry. + T cells were labeled in vitro with 2.5 μM CFSE. CFSE-labeled cells were cultured at 1 × 10 in complete RPMI 1640 medium. 6 The cells were resuspended at 10 μg / ml and transferred to a 96-well plate coated with 10 μg / ml anti-CD3ε antibody and left overnight at 4° C. Purified B cells from draining lymph nodes and the above T cells (1:1) were co-cultured for 72 hours in the above 96-well plate containing 1 μg / ml anti-CD28 mAb for use in flow cytometry analysis. Proliferation of CFSE-labeled T cells was calculated using the formula: (1-MFI value of CFSE of T cells in experimental group / MFI value of CFSE of T cells without B cells in control group)×100%.

[0083] CD4 + T cells are differentiated into + CD24 hi IgD lo / - When cocultured with B cells, the tumor factor suppressed the proliferation of activated T cells. + CD24 hi IgD lo / - FGK increased the suppressive effect of B cells in the draining lymph nodes of tumor-bearing mice. + CD24 hi IgD lo / - GSK did not significantly affect the suppressive effect of B cells, and it did not upregulate CD19 in the draining lymph nodes of tumor-bearing mice. + CD24 hi IgD lo / - The suppressive effect of B cells was reduced (Figure 8a). + CD24 lo IgD hi In co-culture with B cell populations, CD19 + CD24 lo IgD hiThe B cell population promoted but did not inhibit the proliferation of activated T cells (Figure 8b).

[0084] Based on the results listed above, we have + CD24 hi IgD lo / - We found that B cells express PPARδ more highly and therefore have an immunosuppressive effect. PPARδ inhibitors mainly suppress the CD19 expression induced by tumors and FGK45.5. + CD24 hi IgD lo / - Reduces B cell proliferation and tumor-induced CD19 + CD24 hi IgD lo / - Reduce the increased suppressive effect of B cells, thereby inhibiting CD19 at the animal level. + CD24 hi IgD lo / - It reduces the immunosuppressive effects of B cells and promotes the immunotherapeutic effects of immune stimulatory and / or immune checkpoint antibodies against tumors.

[0085] Example 9: Therapeutic effect of PPARδ inhibitors in combination with FGK45.5 on mouse MB49 bladder cancer Based on the above results, we predict that PPARδ inhibitors may also enhance the therapeutic effect of immunotherapy drugs against tumors in other tumor models. To test this possibility, we investigated the therapeutic effect of PPARδ inhibitors in combination with FGK45.5 in MB49 bladder cancer cell-bearing mice. C57BL / 6 mice (8-10 weeks, n=5 / group, 4 groups) were treated with 7×10 5MB49 cells (mouse bladder cancer cells) were subcutaneously inoculated, and when the tumor became palpable about 8 days after tumor cell inoculation, rat IgG, or FGK45.5, or PPARδ inhibitor (GSK3787), or FGK45.5 in combination with PPARδ inhibitor (GSK3787) were administered. The tumor growth status was observed. The dose and administration schedule of rat IgG, FGK45.5, and PPARδ inhibitor (GSK3787) in all groups followed the dose and administration schedule of Example 2, as shown in Figure 2a.

[0086] We revealed that FGK45.5 (a murine anti-CD40 agonist antibody) used alone had a moderate therapeutic effect against MB49 tumors, while GSK3787 (a PPARδ inhibitor) used alone had no therapeutic effect against MB49 tumors, but the use of GSK3787 could further improve the effect of FGK45.5 in treating MB49 tumors (Figure 9).

[0087] Example 10: PPARδ inhibitors inhibit CD19 expression in non-small cell lung cancer (NSCLC) patients (adenocarcinoma and squamous cell carcinoma) + CD24 hi IgD lo / - Immunosuppression in B cell subpopulations could be reduced.

[0088] To further demonstrate the clinical relevance of our study, we collected peripheral blood from non-small cell lung cancer (NSCLC) patients (including those with adenocarcinoma and squamous cell carcinoma) and demonstrated the following by flow cytometry in peripheral blood. Similar to the findings in the mouse model, CD19 expression in the peripheral blood of cancer patients was significantly higher than that of healthy controls. + CD24 hi IgD lo / - The proportion of B cells was significantly increased and showed human CD19 + CD24 hi IgD lo / - B cells express human CD19 + CD24 lo IgD hi They expressed higher levels of PPARδ than B cells (Fig. 10b).

[0089] B cells were isolated and collected from peripheral blood of multiple normal blood donors or patients and were identified as CD19 + CD24 hi IgD lo / - B cells were isolated from the collected B cells and cultured for 24 h in complete RPMI 1640 medium with or without the PPARδ inhibitor GSK3787. These B cells were then washed twice to remove the PPARδ inhibitor and re-cultured in complete RPMI 1640 medium at 1 × 10 6 CD4 + CD25 - T cells were isolated from the blood of normal blood donors. + CD24 hi IgD lo / - B cells and these T cells (1:1) were co-cultured for 48 hours, and Dynabeads were coated with anti-human CD3 mAb and anti-human CD28 mAb and plated in 96-well U-shaped culture plates, Golgi プラグ(Plug) (BD Biosciences), and PMA and ionomycin were added to the medium together and treated for 6 hours. + T cells were surface stained, membrane perforated, and stained intracellularly using fluorescently labeled anti-human IFNγ, and then analyzed using flow cytometry. + CD24 hi IgD lo / - B cells were cloned using CD19 immunoreactivity from healthy controls. + CD24 hi IgD lo / - We found that the IFNγ secretion of T cells activated by anti-human CD3 mAb and anti-human CD28 mAb was more potent than that of B cells (Fig. 10c). + CD24 hi IgD lo / - The suppressive activity of B cells was significantly reduced by GSK3787 treatment. These results are consistent with those observed in mice, where human CD19 + CD24 hi IgD lo / -B cells are also an important cancer-associated B cell subset with suppressive functions, and human CD19 + CD24 hi IgD lo / - The immunosuppressive function of B cells is highly dependent on PPARδ, and PPARδ inhibitors suppress the expression of CD19 from cancer patients. + CD24 hi IgD lo / - This shows that the immunosuppressive function of B cells could be reduced.

[0090] It should be noted that the above specific embodiments are illustrative, and those skilled in the art may be stimulated by the disclosure of the present invention to propose various solutions, which also belong to the scope of the disclosure of the present invention and are included in the scope of the present invention. It should be understood by those skilled in the art that the specification of the present invention and the accompanying drawings are illustrative, not limiting the scope of the claims. The protection scope of the present invention is defined by the claims and the equivalents of the claims.

Claims

1. The pharmaceutical use of a PPAR.delta. inhibitor in combination with an immunotherapeutic agent for the preparation of an antitumor medicament.

2. The pharmaceutical use of claim 1, wherein the tumor is melanoma, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, colon cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, bladder cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer.

3. 2. The pharmaceutical use of claim 1, wherein the immunotherapeutic agent is an immune agonist or an immune checkpoint inhibitor.

4. The pharmaceutical use of claim 3, wherein the immune agonist is an agonist specific to a costimulatory molecule, and the costimulatory molecule includes OX40, 4-1BB (CD137), CD27, GITR, CD28, and / or ICOS.

5. The pharmaceutical use of claim 3, wherein the immune agonist is a CD40 agonist.

6. The pharmaceutical use of claim 3, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PDL1 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, and a CD47 inhibitor.

7. The pharmaceutical use of claim 3, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, and an anti-CTLA-4 antibody.

8. The pharmaceutical use of claim 7, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

9. The pharmaceutical use according to claim 5, wherein the CD40 agonist is selected from an anti-CD40 agonist antibody, a CD40L protein, an expression vector of a CD40L protein, or fragments, derivatives and / or polymers thereof.

10. The pharmaceutical use of claim 9, wherein the CD40L protein is a recombinant CD40L protein.

11. The pharmaceutical use of claim 9, wherein the CD40 agonist is an anti-CD40 agonist antibody.

12. The pharmaceutical use according to any one of claims 1 to 11, wherein the PPARδ inhibitor is a compound capable of inhibiting PPARδ in a targeted manner, or a nucleic acid molecule capable of inhibiting the effect of PPARδ mRNA, or a molecule capable of degrading PPARδ.

13. The pharmaceutical use described in claim 12, wherein the nucleic acid molecule is siRNA or shRNA.

14. The pharmaceutical use of any one of claims 1 to 11, wherein the PPARδ inhibitor is GSK3787.

15. An anti-tumor pharmaceutical composition comprising a PPARδ inhibitor and an immunotherapeutic agent that are prepared separately and then packaged together, or comprising a formulation prepared by mixing the PPARδ inhibitor and the immunotherapeutic agent together.

16. The antitumor drug composition of claim 15, wherein the tumor is melanoma, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, esophageal cancer, colorectal cancer, colon cancer, lymphoma, brain tumor, sarcoma, cervical cancer, prostate cancer, bladder cancer, osteosarcoma, head and neck cancer, renal cell carcinoma, or gastric cancer.

17. The antitumor drug composition of claim 15, wherein the tumor is melanoma, bladder cancer, or non-small cell lung cancer (NSCLC).

18. The antitumor pharmaceutical composition of claim 15, wherein the PPARδ inhibitor is a compound capable of inhibiting PPARδ in a targeted manner, or a nucleic acid molecule capable of inhibiting the effect of PPARδ mRNA, or a molecule capable of degrading PPARδ.

19. The antitumor drug composition described in Claim 18, wherein the nucleic acid molecule is siRNA or shRNA.

20. The antitumor drug composition of claim 15, wherein the PPARδ inhibitor is GSK3787.

21. The antitumor pharmaceutical composition according to any one of claims 15 to 20, wherein the immunotherapeutic drug is an immune agonist or an immune checkpoint inhibitor.

22. The antitumor drug composition described in claim 21, wherein the immune agonist is an agonist specific to a costimulatory molecule, and the costimulatory molecule includes OX40, 4-1BB (CD137), CD27, GITR, CD28, and / or ICOS.

23. The antitumor pharmaceutical composition described in claim 21, wherein the immune agonist is a CD40 agonist.

24. The antitumor drug composition according to claim 21, wherein the immune checkpoint inhibitor is selected from a PD-1 inhibitor, a PDL1 inhibitor, a TIM3 inhibitor, a LAG3 inhibitor, and a CD47 inhibitor. thing.

25. The antitumor drug composition of claim 21, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, and an anti-CTLA-4 antibody.

26. An antitumor pharmaceutical composition according to any one of claims 15 to 25, further comprising a pharma- ceutically acceptable carrier and prepared into a pharma- ceutically acceptable formulation.

27. ​​The antitumor pharmaceutical composition of claim 26, wherein the formulation is an injectable formulation, a targeting formulation, or a nanoformulation.

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