WO2022052982A1 - USE OF PPARα (PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α) LIGAND IN PREPARATION OF MEDICINE - Google Patents

Abstract

Provided is use of a PPARα (peroxisome proliferator-activated receptor α) ligand in the preparation of a medicine. The medicine alone or in any combination has the following functions: (1) activating an activation signal in T cells; (2) activating T cells; (3) promoting the differentiation of T cells; (4) regulating the metabolic state of DCs; (5) restoring the regulatory function of DCs on T cells; (6) improving the antitumor effect of immune checkpoint inhibitors; and (7) improving the therapeutic effect of antitumor vaccines.

Description

Application of PPARα (peroxisome proliferator-activated receptor α) ligand in the preparation of medicine technical field

The invention belongs to the technical field of medicine and biology, and in particular relates to the application of PPARα (peroxisome proliferator-activated receptor α) ligands in the preparation of medicines.

Background technique

During tumor growth, tumor cells use various immune evasion strategies to evade T cell immune surveillance, such as: resisting T cell killing of tumor cells, high expression of death receptors to induce T cell death, immune checkpoint signaling or immunosuppression Sexual factors (TGF-β, PGE2, etc.) inhibit T cell activation and induce a large number of suppressive immune cell subsets (regulatory T cells or myeloid-derived suppressor cells). In short, the main problem of tumor immune escape is the inability of infiltrating T cells in tumor tissue to be fully activated and the inability to generate durable memory T cells. Therefore, enhancing or improving the activation and survival of T cells in the tumor microenvironment and generating memory T cells to maintain long-term anti-tumor activity will be the core task of developing tumor immunotherapy.

T cell activation initiates a series of intracellular signaling cascades, which ultimately lead to different biological events such as T cell proliferation, immune function, or death, depending on the strength and duration of the activation signal. The full activation of T cells requires two independent signals: the first signal is an antigen-specific signal, which is bound by the T cell receptor (TCR) on the surface of the T cell membrane and the antigen-MHC complex on the surface of the antigen presenting cell , causing activation of downstream tyrosine kinases; the second signal is mediated by cytokines or co-stimulatory molecules on the surface of antigen-presenting cells, such as B7.1 (CD80) and B7.2 (CD86). Receiving only the first signal would result in T cell inactivation, while activation of the second signal alone would not cause T cell activation. Therefore, the synergistic effect between the two signals is of great significance to the immune function of T cells. The sufficient activation of the first signal and the second signal will cause the phosphorylation of important kinases such as NF-κB and MAPK (JNK, p38 and ERK), and activate the downstream transcription factors to promote T cell activation. After activation of T cells, the above-mentioned kinases are further activated under the action of cytokines, such as IL-2, IL-7 and IL-15, to initiate T cell proliferation and differentiation into memory T cells ^1,2 . Studies have shown that TAK1 (TGF-β-activated kinase 1) can integrate signals that cause T cell development, activation, survival and other functions, and is at the core of T cell function regulation ³ .

There is an interaction between PPARα and TAK1. For example, after knocking out TAK1 in mouse hepatocytes, it was found that not only the mRNA level of PPARα was reduced, but also the expression of genes related to the control of lipid metabolism and fatty acid oxidation downstream of PPARα. The fatty acid oxidation capacity of hepatocytes leads to lipid deposition in hepatocytes ⁵ , indicating that activation of TAK1 can upregulate the intracellular expression of PPARα. As well as in PPARα knockout mice, it was found that the NF-κB and JNK pathways downstream of TAK1 in T cells were selectively activated, resulting in increased production of cytokines such as IFN-γ, TNF-α and IL-2 ⁶ . In addition, PPARα can inhibit the NF-κB cell pathway in the presence of activators, indicating that PPARα is an important negative regulator in T cells. However, based on the limitations of existing theories and inherent thinking, there is no report on whether PPARα inhibitors can have the same T cell regulation effect.

Although the original view is that activators of PPARα have anti-inflammatory effects, they can inhibit the activation of T cells by inhibiting the transcriptional activity of NF-κB. However, recent studies have shown that CD8+ T cells pretreated with the PPARα-selective activator Fenofibrate show superior antitumor effects in adoptive T cell therapy ⁸ . In addition, the combination strategy of pan-PPAR agonist Bezafibrate and immune checkpoint inhibitors can control the tumor well, and the survival time of tumor-bearing mice is significantly prolonged ⁹ . The above studies all support our theoretical inference, but their analysis suggests that the effect is due to the regulation of PPARα on T cell metabolic state rather than PPARα directly affecting T cell activation-related signaling pathways.

Through systematic research, we previously found that PPARα, as an intracellular activation signal checkpoint molecule of T cells, binds with PPARα ligand molecules to release the TAK1 kinase hidden by PPARα and increase the activation of downstream NF-κB and JNK kinases. , improve the sensitivity of T cells, strengthen T cell activation, and promote T cells to secrete more cytokines (such as IL-2 and IFN-γ) to promote their own proliferation and anti-tumor activity (Figure 13). And in combination with immunotherapy (including immune checkpoint inhibitors and anti-tumor therapeutic vaccines), it can enhance the control of tumor growth by immunotherapy, and can also promote the generation of memory T cells to prevent tumor recurrence.

In addition, the anti-tumor activity of T cells often requires the help of DCs, such as the presentation of tumor-associated antigens and the provision of costimulatory molecules and other signals. Therefore, the function of DCs is indispensable for a successful antitumor response. However, in the tumor microenvironment, immune cells are disadvantaged in the process of highly competing with tumor cells for nutrients such as glucose, fatty acids, and amino acids. Therefore, immune cells, such as DCs, are usually in a state of extreme nutritional starvation and cannot reach a fully activated or mature state. Therefore, the metabolic state of DC can reflect its activation state and function. Current studies have shown that events such as increased mitochondrial activity and enhanced fatty acid oxidation in DCs can cause immune dysfunction in DCs, preventing them from fully activating T cells. Activation capacity of T cells ^10,11 . As a member of the type II nuclear hormone receptor superfamily, PPARα can be activated by both endogenous and exogenous lipid molecular ligands ¹² . These lipid molecules include saturated fatty acids (palm, stearic acid), unsaturated fatty acids (oleic acid, linoleic acid, arachidonic acid), 2-oleoyl-1-palmitin glycerol-3-phosphocholine ( 16:0/18:0 GPC), hexadecylamide ethanol (PEA), etc. ¹³ . PPARα is sensitive to fatty acid sensing and can be rapidly activated to initiate transcription and regulate the expression of fatty acid β-oxidation and lipid metabolism-related genes ^14,15 . Regulation of the metabolic level of DC cells by PPARα ligands can improve the immunosuppression of DCs caused by the immune microenvironment, thereby improving the anti-tumor activity of T cells.

Our study shows that ligands of PPARα can reduce the level of fatty acid oxidation/oxidative phosphorylation in DC, increase the level of glycolysis, and restore the function of DC on CD8+ T cells, especially antigen-specific CD8+ T cells, while reducing CD4+ The generation of regulatory T cells greatly relieves the immunosuppression of the tumor microenvironment, thereby achieving better anti-tumor activity.

references:

1 Paul, S. & Schaefer, BCA new look at T cell receptor signaling to nuclear factor-κB. Trends in immunology 34, 269-281, doi: https://doi.org/10.1016/j.it.2013.02.002(2013) .

2 Berard, M., Brandt, K., Paus, SB&Tough, DFIL-15 Promotes the Survival of Naive and Memory Phenotype CD8⁺T Cells. The Journal of Immunology 170,5018-5026, doi: 10.4049/jimmunol.170.10.5018 (2003).

3 Wan, YY, Chi, H., Xie, M., Schneider, MD & Flavell, RA //www.nature.com/articles/ni1355#supplementary-information(2006).

4 Ajibade,AA,Wang,HY&Wang,R.-F.Cell type-specific function of TAK1 in innate immune signaling.Trends in immunology 34,307-316,doi:https://doi.org/10.1016/j.it. 2013.03.007 (2013).

5 Inokuchi-Shimizu, S. et al. TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis. The Journal of clinical investigation 124, 3566-3578, doi:10.1172/JCI74068(2014).

6 Dunn, SE et al. Peroxisome proliferator–activated receptor(PPAR)αexpression in T cells mediates gender differences in development of T cell–mediated autoimmunity. The Journal of experimental medicine 204,321-330,doi:10.1084/jem.20061839(2007) .

7 Bansal, T. et al. Arjunolic acid, a peroxisome proliferator-activated receptor alpha agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-beta signaling. The Journal of biological chemistry 292, 16440-16462, doi:10.1074/jbc. M117.788299 (2017).

8 Zhang, Y. et al. Enhancing CD8+T Cell Fatty Acid Catabolism within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy. Cancer cell 32,377-391.e379,doi:https://doi.org/10.1016/j .ccell.2017.08.004(2017).

9 Chamoto, K. et al. Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proceedings of the National Academy of Sciences 114, E761-E770, doi:10.1073/pnas.1620433114(2017) .

10 Zhao, F. et al. Paracrine Wnt5a-β-Catenin Signaling Triggers a Metabolic Program that Drives Dendritic Cell Tolerization. Immunity 48, 147-160.e147(2018).doi.org/10.1016/j.immuni.2017.12.004

11 Malinarich, F. et al. High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells. J Immunol 194, 5174-5186 (2015). DOI: https://doi.org/10.4049/jimmunol.1303316

12 Xu, HE et al. Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proceedings Of the National Academy Of Sciences Of the United States Of America 98, 13919-13924(2001).doi.org/10.1073/pnas .241410198

13 Forman, BM et al. Hypolipidemic drugs, polyunsaturatedfatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proceedings Of the National Academy Of Sciences Of the United States Of America 94,4312-4317(1997). doi.org/10.1073/pnas.94.9.4312

14 Yamazaki, K. et al. Microarray analysis of gene expression changes in mouse liver induced by peroxisome proliferator activated receptor alpha agonists. Biochemical And Biophysical Research Communications 290, 1114-1122 (2002).doi.org/10.1006/bbrc.2001.6319

15 Issemann, I. et al. Activation Of a Member Of the Steroid-Hormone Receptor Superfamily by Peroxisome Proliferators. Nature 347, 645-650 (1990). doi: 10.1038/347645a0.

SUMMARY OF THE INVENTION

The present invention first relates to the application of PPARα (peroxisome proliferator-activated receptor α) ligands in the preparation of medicines, and the functions of the medicines include any one or more of the following functions,

(1) Activating the activation signal in the T cell, the activation signal refers to an increase in the phosphorylation level of JNK, and the T cell is preferably a CD8+ T cell;

(2) Activating T cells, the activation of T cells refers to making T cells secrete more IFN-γ and IL-2 and at the same time increase the survival time of T cells, the T cells are preferably CD8+ T cells, more preferably , for tumor-infiltrating CD8+ T cells;

(3) promoting the differentiation of T cells, the differentiation refers to promoting the differentiation of T cells into memory T cells;

(4) Regulating the metabolic state of dendritic cells (Dendritic Cell, DC cells), the regulating metabolism refers to: down-regulating the fatty acid oxidation/oxidative phosphorylation level of DC cells, and up-regulating the level of glycolysis; the DC are bone marrow-derived DCs, more preferably, DCs infiltrating the tumor microenvironment;

(5) Restoring the regulatory function of DC on T cells, the said restoring the regulatory function of DC refers to: down-regulating the ratio of CD4+ T cells to regulatory T cells, increasing the ratio of tumor-killing CD8+ T cells, and activating tumor-killing The function of CD8+ T cells; the activation of tumor-killing CD8+ T cells refers to: making tumor-killing CD8+ T cells secrete more IFN-γ and IL-2 while increasing tumor-killing CD8+ T cells survival time;

(6) Improve the anti-tumor effect of an immune checkpoint inhibitor, the immune checkpoint inhibitor is preferably a PD-L1/PD-1 antibody, and preferably, the tumor is a tumor that is effectively treated by an immune checkpoint inhibitor ;

(7) To improve the therapeutic effect of anti-tumor vaccine, the anti-tumor vaccine is preferably a nano-vaccine loaded with polypeptide/protein tumor antigen through PEG2000-DSPE micelles.

The PPARα ligand is preferably an inhibitor of PPARα, more preferably, the inhibitor is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330, ZM0332, ZM0333.

The present invention also relates to a group of PPARα inhibitors, the inhibitors are ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330, ZM0332, ZM0333.

The present invention also relates to a method for treating tumors, which comprises administering a therapeutically effective amount of an immune checkpoint inhibitor and a PPARα ligand at the same time;

Preferably, the tumor is a tumor that is effectively treated by an immune checkpoint inhibitor, and the immune checkpoint inhibitor is a PD-L1/PD-1 antibody;

Preferably, the PPARα ligand is preferably an inhibitor of PPARα, more preferably, the inhibitor is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330 , ZM0332, ZM0333.

The present invention also relates to a method for treating tumors, which comprises administering a therapeutically effective amount of an anti-tumor vaccine and a PPARα ligand at the same time;

Preferably, the anti-tumor vaccine is preferably a nano-vaccine loaded with polypeptide/protein tumor antigens through PEG2000-DSPE micelles, and the preparation method of the micelles can refer to the records of CN201410570624;

Preferably, the PPARα ligand is preferably an inhibitor of PPARα, more preferably, the inhibitor is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330 , ZM0332, ZM0333.

The present invention also relates to an anti-tumor combined preparation comprising:

(1) A therapeutically effective amount of PPARα ligand;

(2) A therapeutically effective amount of an immune checkpoint inhibitor or a therapeutically effective amount of an antitumor vaccine;

(3) Necessary pharmaceutical excipients.

Preferably, the immune checkpoint inhibitor is PD-L1/PD-1 antibody;

Preferably, the anti-tumor vaccine is preferably a nano-vaccine loaded with polypeptide/protein tumor antigens through PEG2000-DSPE micelles;

Preferably, the PPARα ligand is preferably an inhibitor of PPARα, more preferably, the inhibitor is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330 , ZM0332, ZM0333.

Description of drawings

Figure 1. The PPARα inhibitor GW6471 in T cells enhances the phosphorylation of T cell activation-related kinases [in vitro experiments].

Figure 2. The PPARα inhibitor GW6471 enhances the survival and function of CD8+ T cells [in vitro experiment].

Figure 3. PPARα inhibitor GW6471 enhances the anti-tumor effect of PD-L1 antibody on MC38-OT I.

Figure 4. The PPARα inhibitor GW6471 prolongs the survival of PD-L1 antibody-treated MC38-OT I tumor-bearing mice.

Figure 5. PPARα inhibitor GW6471 enhances the anti-tumor effect of PD-1 antibody (PD-1 mAb) on MC38

Figure 6. Effect of PPARα inhibitor GW6471 on the function and activity of tumor-infiltrating T lymphocytes.

Figure 7. PPARα inhibitor GW6471 increases the proportion of memory CD8+ T cells induced by PD-L1 antibody.

Figure 8. The antitumor effect of PPRAα inhibitor GW6471 mainly depends on CD8+ T cells.

Figure 9. The anti-tumor effect of PPARα inhibitor GW6471 combined with PD-L1 antibody mainly depends on DC cells.

Figure 10. Antitumor effect of PPARα-/-DC.

Figure 11. The PPARα inhibitor GW6471 enhances the activation of CD8+ T cells by tumor-infiltrating DCs.

Figure 12. Tumor-infiltrating DCs of PPARα-/- enhance the proportion and activity of tumor antigen-specific CD8+ T cells.

Figure 13. Regulation of DC metabolic state by PPARα inhibitor GW6471 [in vitro experiment].

Figure 14. The PPARα inhibitor GW6471 inhibits the induction of regulatory T cells by tumor exosomes [in vitro experiment].

Figure 15. The PPARα inhibitor GW6471 restores the ability of DC to activate CD8+ T cells [in vitro experiment].

Figure 16. PPARα inhibitor GW6471 enhances the anti-tumor effect of E7 vaccine on TC-1.

Figure 17. The PPARα inhibitor GW6471 prolongs the survival of E7 vaccine-treated TC-1 tumor-bearing mice.

Figure 18. Diagram of the mechanism of interaction between PPARα and TAK1 in T cells.

Figure 19. The effects of GW6471 and its structural analogs on the expression of the downstream target gene Cpt1a controlled by PPARα were detected by qRT-PCR.

detailed description

mouse model:

1. CD11c-DTR mouse model, which is characterized by the presence of a diphtheria toxin response element (DTR, DT: diphtheria toxin, R: response Original), in the case of intraperitoneal injection of this toxin, CD11c-positive DC cells derived from mouse bone marrow will be eliminated. This model was constructed because the method of using antibodies in the DC deletion experiment was very inefficient, so the method of transgenic mice was adopted.

2. The mouse CD11c-DTR/PPARα-/- chimeric bone marrow transfer model, that is, on the basis of the first model, the PPARα in the mouse genome is further knocked out, which is characterized by the simultaneous transfer of the same amount of CD11c-DTR bone marrow Cells and PPARα-/- myeloid cells, when mice were treated with diphtheria toxin, only PPARα-/- DC cells remained in the body, but no wild-type PPARα function DC cells. In this case, the role of the PPARα signaling pathway in DC cells on tumor growth and in anti-tumor immunity can be investigated without the need for antibody or small molecule drug treatment, and only by observing tumor growth.

Example 1: The PPARα inhibitor GW6471 enhances the phosphorylation of T cell activation-related kinases in T cells

Cell culture and processing methods:

(1) After the wild-type C57BL6 mice were sacrificed, spleen cells were taken, red blood cells were lysed, and CD8+ T cells were separated by CD8 magnetic beads (StemCell).

(2) After CD8+ T cells were treated with different concentrations of Fenofibrate (20μM) or GW6471 (10μM) for 20 minutes, 2.5ug/ml anti-CD3 antibody and 0.5ug/ml anti-CD28 antibody were used to activate T cells for 20 minutes. CD8+ T cells were collected, and the cells were lysed with Lysis Buffer on ice, and the intracellular proteins were extracted. Western Blot experiments were performed to investigate the changes of PPARα, JNK phosphorylation and I-kB degradation.

The results are shown in Figure 1. Compared with the anti-CD3 and anti-CD28 antibody activation groups alone, the T cells in the GW6471 (structure shown in Formula 1 below) and Fenofibrate pretreatment groups showed stronger JNK phosphorylation levels in the cells. , while the degradation of I-kB did not become more intense. At the same time, the content of PPARα in the cytoplasm was significantly reduced, indicating that after pretreatment with GW6471 and Fenofibrate, PPARα is likely to undergo a conformational change, break away from the physical association with TAK1, and instead form a transcriptional regulatory complex with co-stimulators/co-repressors and enter the nucleus. , and perform other functions in parallel. This result well confirms our speculation that ligand treatment of PPARα can release the physical binding of PPARα and TAK1, thereby releasing TAK1, activating its downstream kinase function, and promoting T cell activation, survival and even differentiation into memory T cells. .

Example 2: PPARα inhibitor GW6471 and agonist Fenofibrate enhance the survival and function of CD8+ T cells

Cell culture and processing methods:

(1) After the wild-type C57BL6 mice were sacrificed, spleen cells were taken, red blood cells were lysed, and then CD8+ T cells were separated with CD8a magnetic beads (StemCell).

(2) After CD8+ T cells were treated with different concentrations of Fenofibrate (20μM) or GW6471 (10μM) for 24, 48, and 72 hours, the cells were harvested, and 0.5ug/ml anti-CD3 antibody and 0.1ug/ml anti-CD3 antibody were used again. 72 hours after CD28 antibody activation of CD8+ T cells, the cell culture supernatant was collected, and the levels of IFN-γ and IL-2 in the supernatant were detected by ELISA.

(3) After treating CD8+ T cells with different concentrations of Fenofibrate, GW6471, and analogs for 24 hours, the cells were harvested, and mRNA was extracted by Trizol method. After reverse transcription, qRT-PCR was performed to detect the expression of Cpt1a.

The results in Figure 2 show that after pretreatment with GW6471, the state of CD8+ T cells was adjusted, and after restimulation, more IFN-γ and IL-2 could be secreted. In particular, the CD8+ T cells in the 24-hour pretreatment group could secrete 3 times the IFN-γ produced by the CD8+ T cells in the unpretreated and restimulated group, while the CD8+ T cells in the 72-hour pretreatment group could be stimulated by Activates and produces more IL-2 that maintains T cell survival. In contrast, CD8+ T cells pretreated with Fenofibrate for 24, 48 and 72 hours were able to produce more IFN-γ, but not IL-2.

Further, we repeated the above stimulation experiments on CD8+ T cells using a panel of structural analogs of the GW6471 compound (Table 1). The results showed that the treatment of GW6471 and some of its analogs could not only enhance the function of CD8+ T cells, but also promote their long-term survival, while Fenofibrate treatment could enhance the function of CD8+ T cells, but did not affect the proliferation of CD8+ T cells. and survival (Table 2). In addition, to verify whether the structural analogs of GW6471 (Table 1) have similar functions to GW6471, we used qRT-PCR to examine how these structural analogs (Table 1) affected the expression of the PPARα-controlled downstream target gene Cpt1a, Among them, Fenofibrate is a PPARα agonist control, and GW6471 is a PPARα inhibitor control. As shown in Figure 19, the structural analogs of GW6471 (Table 1) were all able to inhibit the expression of Cpt1a, indicating that they could inhibit the activity of PPARα and change the ability of PPARα to physically bind to TAK1 through a mechanism similar to that of GW6471.

Table 1. Schematic diagram of the structural analogs of GW6471

Table 2. Effects of PPARα ligands on CD8+ T cells

PPARα ligand name/number	IC50	EC 200
GW6471	34.61uM	0.0022uM
Fenofibrate	>40uM	0.0076uM
ZM0282	31.49uM	0.0058uM
ZM0283	0.3537uM	0.0087uM
ZM0284	7.495uM	0.0258uM
ZM0285	0.1303uM	0.7892uM
ZM0286	12.71uM	Unable to reach within lethal dose
ZM0325	30.9uM	Unable to reach within lethal dose
ZM0326	2.417uM	Unable to reach within lethal dose
ZM0329	40.96uM	Unable to reach within lethal dose
ZM0330	8.423uM	Unable to reach within lethal dose
ZM0332	>40uM	Unable to reach within lethal dose

ZM0333

>40uM

Unable to reach within lethal dose

_EC200 : Minimum dose to double IFN-γ production compared to no treatment group

IC ₅₀ : median lethal dose.

Experimental example 3: The typical PPARα inhibitor GW6471 enhances the anti-tumor effect of PD-1/PD-L1 antibody on MC38-OT I, activates the function of tumor-infiltrating T lymphocytes, increases the proportion of memory T cells, and prolongs the survival of mice. Survival period

Cell culture and animal model modeling methods:

(1) Recovery and culture of MC38-OT I cells (with their own antigen ovalbumin257-264, which is easy to detect and recognize tumor antigen-specific T lymphocytes): use DMEM medium (containing 10% fetal bovine serum). The MC38-OT I tumor cell cryopreservation tube was taken out from the liquid nitrogen storage tank, immediately placed in a 37°C water bath to dissolve rapidly, and then the cell suspension was transferred into a centrifuge bucket containing 10ml of culture medium, centrifuged at 900rpm for 5 minutes, and then removed. After resuspending with fresh medium, transfer the cells to a cell culture flask, add 10-15ml medium to suspend the pelleted cells, adjust the cell concentration, and place it in a 37°C, volume fraction 5% CO ₂ saturated humidity incubator cultivated in. During the maintenance culture, the cell status was observed every day and fresh medium was replaced in time. When the cells adhered to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, trypsinize the cells that grow well and reach 90% confluence, neutralize the trypsin with fresh medium, centrifuge at 900 rpm, discard the supernatant, add sterile PBS to resuspend, count, and count the cells in the suspension. The density was adjusted to 5×10 ⁶ /ml for use.

(2) Inoculation of tumor cells: female wild-type C57 mice were subcutaneously inoculated with 0.1 ml per mouse (50×10 ⁴ MC38-OTI cells were inoculated per mouse).

Experimental protocol (1): PPARα inhibitor GW6471 enhances the anti-tumor effect of PD-L1 antibody (PD-L1 mAb) on MC38-OT I and prolongs the survival of mice

(1) The mouse-derived colon cancer cell line MC38-OT I (with its own antigen ovalbumin257-264, which is easy to detect and recognize tumor antigen-specific T lymphocytes) was used to subcutaneously inoculate C57 mice, which can be measured in about 5 days to tumor formation (average tumor volume was approximately 50 mm ³ ).

(2) Group design of tumor-bearing mice, the experiment was divided into 4 groups, respectively

a) tumor-bearing mice control group (CTR);

b) Antibody treatment group of tumor-bearing mice (PD-L1 mAb);

c) Tumor-bearing mice administration group (GW6471);

d) Tumor-bearing mice combined treatment group (GW6471+PD-L1 mAb);

A total of 4 groups of tumor-bearing mice were tested, with 6 mice in each group.

(3) Treatment scheme: As shown in Figure 3a, according to the day of inoculation, the mice were treated with PD-L1 antibody on the 7th, 10th and 17th days after inoculation, 200ug per mouse, intraperitoneal injection (PD-L1 antibody) L1 mAb was diluted in PBS at a concentration of 2 mg/ml, and each mouse was injected intraperitoneally with 0.1 ml). At the same time, the mice were treated with GW6471, 10 mg/kg, intraperitoneally, on the 11th day after inoculation, and GW6471 was given once every other day, for a total of 10 times (GW6471 stock solution was 25 mg/ml dissolved in DMSO, waiting for Before administration, it was diluted in pre-warmed PBS at 55°C until completely dissolved, the concentration was 2 mg/ml, and each mouse was injected intraperitoneally with 0.1 ml).

(4) Statistics of tumor growth and survival period: During the administration period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn, and the small size was recorded. Mouse death.

The results are shown in Figure 3b. Compared with the control group, the tumor growth trend of the mice in the GW6471 treatment group was slightly slowed down, but the effect was not significant. Although the tumor volume of the mice in the PD-L1 antibody group was also relieved, on the basis of this treatment, the combination of GW6471 could significantly slow down the growth of MC38-OT I tumors. At the same time, as shown in Figure 4, the combination of GW6471 and PD-L1 antibody also significantly improved the survival of mice.

Experimental protocol (2): PPARα inhibitor GW6471 enhances the anti-tumor effect of PD-1 antibody (PD-1 mAb) on MC38-OTI

(1) The mouse-derived colon cancer cell line MC38-OT I (with its own antigen ovalbumin257-264, which is easy to detect and recognize tumor antigen-specific T lymphocytes) was used to subcutaneously inoculate C57 mice, which can be measured in about 5 days to tumor formation (average tumor volume was approximately 50 mm ³ ).

(2) Group design of tumor-bearing mice, the experiment is divided into 3 groups, respectively

a) tumor-bearing mice control group (CTR);

b) Antibody treatment group of tumor-bearing mice (PD-1 mAb);

c) Tumor-bearing mice combined treatment group (GW6471+PD-1 mAb);

There were 3 groups of tumor-bearing mice in the experiment, with 7 mice in each group.

(3) Treatment plan: Calculated as day 0 of inoculation, mice were treated with PD-1 antibody on the 8th and 11th day after inoculation, 50ug per mouse, intraperitoneal injection (PD-1 mAb was diluted in PBS, the concentration of 0.5mg/ml, each mouse was intraperitoneally injected with 0.1ml). At the same time, the mice were treated with GW6471, 30 mg/kg, orally administered daily on the 5th day after inoculation. GW6471 was ground with a mortar, diluted and suspended in 0.3% CMC-Na solution to reach a concentration of 3 mg/ml and stored at 4°C. The drug was taken out before administration every day, and it was administered by gavage after equilibrating to room temperature.

(4) Statistics of tumor growth and survival period: During the administration period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn. On the 28th day after receiving the tumor, the change trend of the tumor volume was calculated, that is, the tumor volume on the 28th day of each mouse was compared with the tumor volume on the 8th day, and the change fold was calculated. If the change fold is 0, it means that the mouse tumor has been eliminated; if the change fold is greater than 0 and less than 1, it means that the mouse tumor is controlled and the volume has not further increased; if the change fold is greater than 1, the mouse tumor is not controlled and continues to progress.

The results are shown in Figure 5a. Compared with the control group, the tumor growth trend of the mice in the PD-1 antibody group and the PD-1 antibody combined with GW6471 group was significantly slowed down. The average tumor volume of the mice in the PD-1 antibody group showed a certain recurrence trend on the 28th day after receiving the tumor, while the combination group still controlled the tumor volume well, showing a significant curative effect advantage. A similar trend can be observed from Figure 5b, that the combination of PD-1 antibody with GW6471 significantly slowed the growth of MC38-OT I tumors.

Experimental protocol (3): PPARα inhibitor GW6471 enhances the function and activity of tumor-infiltrating T lymphocytes

(1) The MC38-OT I tumor-bearing mouse model was constructed. After the model was constructed, the mice were divided into 4 groups with 6 mice in each group, and were treated with PD-L1 antibody and GW6471 respectively. This method is completely the same as the experimental scheme (1).

(2) On the 21st day after inoculation, the tumor tissue of each group of mice was collected. The tissue was minced with surgical scissors and placed in 2 mg/ml collagenase IV for 1 h at 37°C and 220 rpm. The tissue was then ground and filtered on a filter screen, and the supernatant was discarded by centrifugation, and then resuspended in PBS to prepare a single-cell suspension.

(3) Cell staining: The above cell suspension was fluorescently stained, and the color scheme was APC-CD45, PE/Cy7-CD3, APC/Cy7-CD8, PE-Perforin, FITC-Granzyme B, Percp/Cy5.5-IFNγ , BV421-Ki67. Among them, CD45, CD3 and CD8 are antibodies against surface antigens, which were directly stained at 4°C for 30 min in the dark. The rest are antibodies against intracellular antigens, which need to be fixed overnight and stained after permeabilization. After staining, the cells were washed and resuspended for flow cytometry.

The results are shown in Figure 6. On the basis of PD-L1 antibody treatment, GW6471 can significantly improve the ability of tumor-infiltrating CD8+ T lymphocytes to secrete perforin, Granzyme B and IFNγ, that is, the function of killing tumor cells. Activity of T cells (Ki67) .

Experimental protocol (4): PPRAα inhibitor GW6471 increases the proportion of memory CD8+ T cells induced by PD-L1 antibody

(1) The MC38-OT I tumor-bearing mouse model was constructed. After the model was constructed, the mice were divided into four groups as above, with 6 mice in each group, and were treated with PD-L1 antibody and GW6471 respectively. This method is completely the same as the experimental scheme (1).

(2) On the 15th day after inoculation, the tumor tissue of each group of mice was collected. The tissue was minced with surgical scissors and placed in 2 mg/ml collagenase IV for 1 h at 37°C and 220 rpm. The tissue was then ground and filtered on a filter screen, and the supernatant was discarded by centrifugation, and then resuspended in PBS to prepare a single-cell suspension.

(3) Cell staining: The above cell suspension was fluorescently stained, and the color scheme was BV605-CD45, APC/Cy7-CD8, PE-CD62L, FITC-CD44, Percp/Cy5.5-CD3, APC-KLGR1. All the above antibodies were antibodies against surface antigens, and were directly stained at 4°C for 30 min in the dark. After staining, the cells were washed and resuspended for flow cytometry, and CD45+CD3+CD8+CD44+KLRG1-CD62L+ cells were regarded as a memory CD8+ T cell population.

The results are shown in Figure 7. Compared with the control group, the use of GW6471 alone cannot increase the proportion of memory CD8+ T cells in the tumor tissue, while the PD-L1 antibody and GW6471 combined with the PD-L1 antibody group can significantly increase the proportion. . More importantly, on the basis of PD-L1 antibody treatment, GW6471 can further significantly increase the proportion of memory CD8+ T cells in tumor tissue , which brings long-term benefits to tumor-bearing mice, and supports the results in Figure 4, that is, GW6471 Combination with PD-L1 antibody significantly prolonged the survival of tumor-bearing mice.

Experimental scheme (5): The anti-tumor effect of PPRAα inhibitor GW6471 mainly depends on CD8+ T cells

(1) The MC38-OT I tumor-bearing model was constructed as above.

(2) Group design of tumor-bearing mice, the experiment was divided into 4 groups, respectively

a) tumor-bearing mice control group (CTR);

b) tumor-bearing mice combined treatment group (GW6471+PD-L1 mAb);

c) CD4 deletion group in tumor-bearing mice (GW6471+PD-L1 mAb+α-CD4 Ab)

d) CD8 deletion group in tumor-bearing mice (GW6471+PD-L1 mAb+α-CD8 Ab)

A total of 4 groups of tumor-bearing mice were tested, with 6 mice in each group.

(3) Treatment plan: Calculated as day 0 of inoculation, mice were treated with PD-L1 antibody on days 7, 10 and 17 after inoculation, 200ug per mouse, intraperitoneal injection (PD-L1 mAb diluted in PBS) , the concentration is 2mg/ml, each mouse is intraperitoneally injected with 0.1ml). At the same time, the mice were treated with GW6471, 10 mg/kg, intraperitoneally, on the 11th day after inoculation, and GW6471 was given once every other day, for a total of 10 times (GW6471 stock solution was 25 mg/ml dissolved in DMSO, waiting for Before administration, it was diluted in pre-warmed PBS at 55°C until completely dissolved, the concentration was 2 mg/ml, and each mouse was injected intraperitoneally with 0.1 ml).

(4) Statistics of tumor growth: During the administration period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn.

The results are shown in Figure 8. The tumor growth after deletion of CD8+ T cells was the same as that of the control group, which significantly offset the combined treatment effect of PD-L1 antibody and GW6471, while the effect of deletion of CD4+ T cells was not so obvious. . The above shows that the combined therapeutic effect of GW6471 and PD-L1 antibody mainly depends on CD8+ T cells.

Example 4: The anti-tumor effect of typical PPARα inhibitor GW6471 combined with PD-L1 antibody mainly depends on DC cells, especially PPARα-/-DC

Animal model modeling and cell culture methods:

(1) Construction of CD11c-DTR and CD11c-DTR/PPARα-/- chimeric mouse models: First, wild-type C57 mice (female) were irradiated with 10 Gy whole body for 10 minutes to kill bone marrow cells. On the second day, the bone marrow cells of CD11c-DTR mice were taken and injected into the irradiated mice by tail vein according to the number of cells per mouse at 5×10 ⁶ . To construct CD11c-DTR/PPARα-/- chimeric mice, CD11c-DTR mice and PPARα-/- mouse bone marrow cells were mixed 1:1 and injected into the irradiated mice via tail vein. After two weeks, the antibiotic ampicillin was given to observe their living conditions, and the experiment could be carried out after two months.

(2) Recovery and culture of MC38-OT I cells: Before recovering the cells, the ultra-clean bench in the cell culture room was irradiated with ultraviolet light for 30 minutes, and the DMEM medium (containing 10% fetal bovine serum) was placed at room temperature for use. After the preparation is over, take the tumor cell cryopreservation tube out of the liquid nitrogen storage tank, immediately put it into a 37°C water bath to dissolve quickly, and then transfer the cell suspension into a centrifuge bucket containing 10 ml of culture medium, and centrifuge at 900 rpm for 5 minutes. Remove the supernatant, resuspend it with fresh medium and transfer it to a cell culture flask, add 10-15ml medium to suspend the precipitated cells, adjust the cell concentration, and place it in a 37°C, volume fraction 5% CO ₂ saturated humidity incubator for cultivation . During the maintenance culture, the cell status was observed every day and fresh medium was replaced in time. When the cells adhered to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, trypsinize the cells that grow well and reach 90% confluence, neutralize the trypsin with fresh medium, centrifuge at 900 rpm, discard the supernatant, add sterile PBS to resuspend, count, and count the cells in the suspension. The density was adjusted to 5×10 ⁶ /ml for use.

(3) Inoculation of tumor cells: Use a 1 ml sterile syringe to subcutaneously inoculate the model mice constructed above, 0.1 ml per mouse (ie, 50×10 ⁴ MC38-OT I cells per mouse). Take care to mix the cells well before each pipetting of the cell suspension.

Experimental scheme (1): The anti-tumor effect of PPARα inhibitor GW6471 combined with PD-L1 antibody mainly depends on DC cells

(1) The mouse-derived colon cancer cell line MC38-OT I (with its own antigen ovalbumin257-264, which is convenient for the detection of specific T lymphocytes) was used to subcutaneously inoculate the constructed CD11c-DTR mice. Tumor formation of about 50 ^mm3 was seen.

(2) Group design of tumor-bearing mice

The experiment was divided into 3 groups, which were

a) tumor-bearing mice control group (CTR);

b) Tumor-bearing mice injected with DT (CTR(DT));

c) Antibody treatment group of tumor-bearing mice (GW6471+PD-L1 mAb);

d) Tumor-bearing mice combined with DT group (GW6471+PD-L1 mAb(DT));

A total of 4 groups of tumor-bearing mice were tested, with 5 mice in each group.

(3) Treatment plan: Calculated according to the day of inoculation as the 0th day, on the 6th day after inoculation, each mouse in group b) and group d) was intraperitoneally injected with 500ng DT, once every other day to delete DC. Mice were treated with PD-L1 antibody on days 7, 10 and 17, 200ug per mouse, i.p. (PD-L1 mAb diluted in PBS at a concentration of 2mg/ml, 0.1ml per mouse i.p.) . At the same time, on the 8th day after inoculation, mice were treated with GW6471, 10 mg/kg, intraperitoneal injection, and GW6471 was administered every other day (GW6471 stock solution was 25 mg/ml dissolved in DMSO, before administration, Dilute in pre-warmed PBS at 55°C to complete dissolution, the concentration is 2mg/ml, and each mouse is injected intraperitoneally with 1ml).

(4) Statistics of tumor growth and survival period: During the administration period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn, and the small size was recorded. Mouse death.

The results are shown in Figure 9. Compared with the GW6471+PD-L1 mAb treatment group, the tumor volume of the mice lost control after DT (that is, the deletion of DC) , which was the same as the control group, indicating that the combined treatment regimen depends on DC cells. participation .

Experimental scheme (2): PPARα ^-/- DCs play an important role in the anti-tumor effect

(1) Wild-type C57 mice were inoculated with MC38-OT I cells. After the model was constructed, they were divided into 3 groups with 9 mice in each group, namely the tumor-bearing control group (CTR) and the wild-type DC treatment group (WT). DC) and PPARα knockout DC treatment group (PPARα ^-/- DC).

(2) Treatment plan: Calculated as day 0 of inoculation, DC injection was performed on the mice on the 6th, 13th and 20th days after inoculation, and 5×10 ⁶ DC cells were injected subcutaneously near the tumor site of each mouse. The DCs received in the wild-type DC treatment group and the PPARα knockout DC treatment group were obtained from wild-type C57 mice and PPARα knockout mice, respectively. After the extracted bone marrow cells were differentiated into mature DCs in vitro, they were stimulated with 2 mg/ml OVA antigen overnight, and then the DCs were collected, counted, and injected for treatment.

(3) Tumor growth statistics: During the tumor growth period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn.

The results are shown in Figure 10. Compared with the control group, the wild-type DC treatment group could effectively control the tumor growth. The treatment effect of PPARα knockout DC treatment group was significantly better than that of wild-type DC treatment group, which further reflected the important role of PPARα ^-/- DC in anti-tumor.

Experimental Protocol (3): Effects of GW6471 and PPARα Knockout on the Function of DC-Activated T Cells

(1) After wild-type mice were inoculated with MC38-OTI tumor cells, the mice were divided into two groups: tumor-bearing control group and GW6471 administration group. The GW6471 administration group received intraperitoneal injection of 10 mg/kg GW6471 from the 7th day after tumor receiving, once every two days, for a total of 6 times. On the 22nd day after receiving the tumor, the mice were sacrificed, and the tumor tissue was surgically removed, ground and digested to obtain tumor tissue cells.

(2) In vitro treatment: tumor-infiltrating DCs (CD45+MHCII+CD11c+F4/80-) were sorted by flow cytometry, incubated with 2 mg/ml OVA antigen for 48 hours, and then washed away with antigen. The OTCD8+ T cells (pre-stained with CFSE) obtained by magnetic bead sorting from the spleen of OTI transgenic mice were co-incubated at a ratio of 1:8. After 3 days, the proliferation status and secretion of IFN- gamma level.

(3) Detection method: Flow cytometry was used to detect the dilution ratio of CFSE to determine the division state of OTI CD8+ T cells. Generally speaking, cell proliferation is about obvious, and the higher the dilution of CFSE, the lower the average fluorescence intensity. The level of IFN-γ secreted by OTI CD8+ T cells can also be detected by ELISA kit to indicate the activation state of OTI CD8+ T cells.

The results showed that compared with the tumor receiving control group, tumor-infiltrating DCs in the GW6471-administered group could more effectively activate OTI CD8+ T cells—proliferated more strongly (Fig. 11a) and secreted more IFN-γ (Fig. 11a). Figure 11b)

Experimental protocol (4): The effect of PPARα knockout on the function of DC-activated T cells

(1) Wild-type and PPARα knockout mice were inoculated with MC38-OTI tumor cells, respectively. On the 20th day after receiving the tumor, the mice were sacrificed, and the tumor tissue was surgically removed, ground and digested to obtain tumor tissue cells.

In vitro treatment: pre-incubate anti-mouse IFN-γ antibody in an ELISPOT plate one day before the isolation of mouse intratumoral T cells at a concentration of 5 μg/ml, incubate overnight at 4°C, discard the antibody the next day, and add blocking solution ( RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and β-mercaptoethanol) was blocked at room temperature for 2 h. Then, the isolated mouse tumor T cells were added to the plate at 3 × 105 per well, and incubated with 15 μg/ml OVA257-264 antigen peptide for 48 h in a 37°C incubator. After 48 h, the culture medium was discarded, and deionized water was added to lyse the cells, 5 min/time, 2 times in total. Wash with PBST (1×PBS containing 0.05% Tween-20) solution for 3 times, then add biotin-labeled anti-mouse IFN-γ antibody, and incubate at room temperature for 2 h. After washing with PBST solution for 4 times, streptavidin-labeled horseradish peroxidase (streptavidin-HRP) was added and incubated for 1 h at room temperature. Finally, after washing with 1×PBS for 3 times, AEC substrate was added for 5-60 min, and deionized water was added to stop the reaction. After washing, the ELISPOT plate was dried.

Cell staining: Fluorescent staining of the above cell suspension, the color scheme is APC-CD45, PE/Cy7-CD3, APC/Cy7-CD8, PE-Tetramer (Tet), after mixing the antibody with the cells, stain at 4°C in the dark 30min. The staining was terminated by washing once with PBS containing 2% FBS.

(2) Detection method: Ellispot results were scanned and counted by luciferase spot analyzer CTL analyzer LLC. Another part of the tumor tissue single cell suspension was directly stained. Since the PE-Tetramer (Tet) antibody we used can directly recognize T cell receptors on the surface of T cells that can pair with tumor-associated antigens, this analysis method can directly detect The ratio of tumor antigen-specific Tet+CD8+ T cells was obtained.

The results showed that compared with wild-type mice, the tumor tissues of PPARα knockout mice contained a higher proportion of antigen-specific Tet+CD8+ T cells (Fig. 12a), and these cells were able to produce strong IFN-γ signal (Fig. 12b). These results suggest that tumor-infiltrating DCs from PPARα knockout mice have stronger ability to present antigen and activate CD8+ T cells.

Experimental example 5: PPARα inhibitor GW6471 regulates the metabolic state of DC, thereby regulating the function of DC

Cell culture and processing methods:

Acquisition of bone marrow-derived DC: Take 6-8 week old C57BL/6 mice, dissect the long bones and tibias, blow out the bone marrow, lyse the red blood cells with 2 ml of erythrocyte lysate for 1 min, neutralize them in serum-free medium, and then resuspend them to obtain small cells. The mouse bone marrow cell suspension was seeded in a 10cm petri dish at 3×10 ⁶ cells/dish. Complete medium (RPMI 1640 containing 10% FBS, 50 μM mercaptoethanol) was added with 20ng/ml mGM-CSF (recombinant mouse granulocyte-macrophage colony stimulating factor). Medium (containing 20ng/ml mGM-CSF). On day 6, unadherent cells were collected as immature BMDCs. The purity of CD11c+ DCs identified by flow cytometry was more than 90% and could be used in subsequent experiments.

Acquisition of Tumor-derived Exosomes (TDE): Tumor cells were cultured in a medium containing 10% exosome-depleted fetal bovine serum (100,000 g, 2 h) for 48 h, and the supernatant was collected. First, cell debris in the supernatant was removed by centrifugation at 4000 rpm for 2 h. Then, the fractions larger than 100 kDa in the supernatant were collected by using a 100 kDa ultrafiltration tube at a speed of 4000 rpm, 30 min/time. The collected components were added to the exosome extraction reagent (EXOTC10A-1, SBI) at a ratio of 5:1 (v:v), mixed well and placed at 4°C for at least 12 hours. After 12 hours, the precipitate appeared, centrifuged at 3000 rpm for 30 min, and the precipitate was separated, which was exosomes. After resuspending in PBS according to a certain volume, the protein content was determined by BCA to standardize the content of exosomes, and the packaged and stored at -80°C for future use.

Processing of tumor exosomes can alter the metabolic state and function of bone marrow-derived DCs, mimicking the effects of the tumor microenvironment.

Experimental Protocol (1): Regulation of DC Metabolic Status by GW6471

(1) Investigate the oxidative phosphorylation level of DC cells:

Cell processing: Seahorse XF24 cell culture plate by Seahorse (Total Cell Metabolism Level Detection Kit, purchased from Agilent) uses Corning ^™ Cell-Tak Cell and Tissue Adhesive Reagent (to promote the adhesion of suspended cells to the bottom of the cell culture plate) 3.5 μg/cm ² After adherence pretreatment, bone marrow-derived DCs pretreated with exosomes or GW6471 for 48h were seeded in culture plates at 300,000 per well, with five replicate wells per group, 0.5ml system, and cells adhered for 4h.

Seahorse hippocampal bioenergetics assay (levels of overall cellular metabolism): Probe plates were hydrated overnight. Before assay, cells were replaced with pre-warmed 525 μl XF Base medium (Seahorse Bioscience) supplemented with 10 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate, and adjusted to pH 7.4 with NaOH Available after filtering. Oligomycin (oligomycin), FCCP (trifluoromethoxyphenylhydrazone carbonyl cyanide), Rotenone (rotenone) and Antimycin A (antimycin A) were diluted in XF basal medium to 1 mM, 1.5 mM, 100 nM and 1 mM, respectively , a volume of 75 μl was added to wells A, B, and C of the probe plate (Rotenone and antimycin-A were mixed together in well C). After the preparation is completed, the oxidative phosphorylation level of the cells can be detected on the machine.

(2) To investigate the fatty acid oxidation level of DC cells:

Cell treatment: Seahorse XF24 cell culture plates were pretreated with Corning ^TM Cell-Tak Cell and Tissue Adhesive reagent 3.5μg/cm ² for adherent pretreatment, and bone marrow-derived DCs pretreated with exosomes or GW6471 for 48h were prepared at a rate of 300,000 per well. Inoculated in culture plates, five replicate wells per group, 0.5 ml system, and cells were plated in substrate-limiting medium (DMEM containing only 0.5 mM glucose, 1.0 mM L-glutamine, 0.5 mM carnitine and 1% FBS). ) overnight.

Seahorse hippocampal bioenergetics assay (level of fatty acid oxidation): Probe plate hydrated overnight. 45 min before the test, the cells were replaced with pre-warmed 450 μl FAO assay medium (fatty acid oxidation assay medium, containing 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO ₄ , 1.2 mM Na ₂ HPO ₄ , 2.5 mM glucose, 0.5 mM meat Alkaline and 5 mM HEPES sulfonic acid buffered saline). 75 μl of 100 μM Etomoxir (abbreviated as ETO, an inhibitor of carnitine palmitoyl transporter 1) was prepared by using the medium, and added to well A of the probe plate. After the preparation is complete, you can go on the machine to detect the degree of fatty acid oxidation.

(3) Investigate the lactate secretion level of DC cells:

Cell treatment: DCs derived from bone marrow were seeded in 24-well cell culture plates at 300,000 per well, and the supernatant was collected after pretreatment with exosomes or GW6471 for 48 hours.

Detection method: Lactate Colorimetric Assay Kit (Biovision) was used to detect the lactic acid content in the supernatant, and the absorbance at 570 nm was read for quantitative analysis.

The results showed that tumor exosomes (TDE) were able to significantly increase DC cell oxygen consumption (OCR), a marker of cellular oxidative phosphorylation levels (Fig. 13a), and fatty acid oxidation levels (Fig. 13b), while reducing lactate levels. Secretion (Figure 13c) - Annotation of cellular glycolysis levels. Therefore, tumor exosomes can mimic the regulation of the metabolic state of DCs by the tumor microenvironment in vitro. On this basis, GW6471 could significantly reduce the level of oxidative phosphorylation and fatty acid oxidation of DC, and restore the level of glycolysis (Fig. 13), and the metabolic state change caused by GW6471 contributed to the recovery of DC function.

Experimental protocol (2): The effect of GW6471 on DC induction of regulatory T cells

Introduction to the mouse model:

Foxp3-GFP transgenic mice are characterized by gene editing to place the gene expressing green fluorescent protein (GFP) behind the promoter of the transcription factor Foxp3 responsible for the development of CD4+ regulatory T cells. Therefore, we can easily detect the transformation of CD4+ T cells into regulatory T cells in vivo or in vitro by the method of fluorescence detection (GFP positive means Foxp3 positive), rather than the complex intracellular protein staining method in the past. condition.

OTI (C57BL/6-Tg(TcraTcrb) 1100Mjb/J) transgenic mice are characterized by gene editing, so that the T cell receptors of the CD8+ T cells of the mice can specifically recognize the 257-264 of the OVA protein. The structure of the polypeptide is activated and proliferated. The transgenic mice are commonly used in studies investigating antigen-specific immune responses.

(1) Cell treatment: BMDCs were first pretreated with exosomes and 15uM GW6471 for 48h. In addition, splenocytes of Foxp3-GFP transgenic mice were taken, and CD4+ cells were sorted by magnetic beads, and counted after centrifugation. Then, the above pretreated DCs (2×10 ⁴ ) were combined with CFSE (carboxyfluorescein diacetate succinimidyl ester, commonly used to label and monitor the proliferation state of cells) labeled CD8+ from the spleen of OTI transgenic mice. T cells (2 x ¹⁰⁵ ) were co-cultured at a ratio of 1:10 in U-bottom 96-well plates. On the fourth day, 100U/well of recombinant mouse IL-2 cytokine was added to promote the survival of T cells.

(2) Detection method: After 7 days of co-culture, the proportion of CD4+Foxp3+ cells was analyzed by flow cytometry.

The results showed that DCs pretreated with tumor exosomes were able to induce more regulatory T cells (CD4+Foxp+) production (Fig. 14), while GW6471 could significantly reduce regulatory T cells and restore them to untreated controls group level (Figure 14).

Experimental protocol (3): GW6471 restores the activation effect of DC on CD8+ T cells

(1) Cell treatment: BMDCs were first incubated with exosomes, 2mg/ml OVA antigen, and 15uM GW6471 for 48h. In addition, spleen cells of OT I transgenic mice were taken, and CD8+ cells were sorted by magnetic beads and counted after centrifugation. Then, the above pretreated DCs (5×10 ⁴ ) were co-cultured with OTI CD8+ T cells (4×10 ⁵ ) at a ratio of 1:8 in a U-bottom 96-well plate.

(2) Detection method: After 3 days of co-culture, the supernatant was collected, and the IFN-γ secreted by OTI CD8+ T cells was detected by ELISA.

The results showed that DCs pretreated with tumor exosomes could inhibit the activation of CD8+ T cells, while GW6471 significantly increased the level of IFN-γ secreted by OTI CD8+ T cells (Fig. 15), which fully demonstrated that GW6471 restored the effect of DC on CD8+ T cells. activation of T.

Experimental Example 6: PPARα inhibitor GW6471 enhances the anti-tumor effect of E7 vaccine on TC-1 and prolongs the survival of mice

(1) Cell culture and animal model modeling methods:

(1) Recovery and culture of TC-1 cells: Before recovering the cells, the ultra-clean bench in the cell culture room was irradiated with UV light for 30 minutes, and the RPMI1640 medium (containing 10% fetal bovine serum) was placed at room temperature for use. After the preparations are over, take the tumor cell cryopreservation tube out of the liquid nitrogen storage tank, immediately put it into a 37°C water bath to dissolve quickly, and then transfer the cell suspension into a centrifuge bucket containing 10 ml of medium, and centrifuge at 900 rpm for 5 minutes. Remove the supernatant, resuspend it with fresh medium, transfer it into a cell culture flask, add 10-15ml medium to suspend the pelleted cells, adjust the cell concentration, and place it in a 37°C, volume fraction 5% CO ₂ saturated humidity incubator cultivated in. During the maintenance culture, the cell status was observed every day and fresh medium was replaced in time. When the cells adhered to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, trypsinize the cells that grow well and reach 90% confluence, neutralize the trypsin with fresh medium, centrifuge at 900 rpm, discard the supernatant, add sterile PBS to resuspend, count, and count the cells in the suspension. The density was adjusted to 5×10 ⁵ /ml until use.

(2) Inoculation of tumor cells: Use a 1 ml sterile syringe to subcutaneously inoculate female wild-type C57 mice with 0.1 ml per mouse (ie, inoculate 5×10 ⁴ TC-1 cells per mouse). Take care to mix the cells well before each pipetting of the cell suspension. Tumor formation of about 50 ^mm3 was seen in about 7 days.

(2) Treatment plan:

(3) Group design of tumor-bearing mice

The experiment was divided into 4 groups, which were

d) tumor-bearing mice control group (CTR);

e) tumor-bearing mice vaccine treatment group (E7 vaccine) (for the preparation method, please refer to CN201410570624);

f) Tumor-bearing mice administration group (GW6471);

g) tumor-bearing mice combined treatment group (GW6471+E7 vaccine);

There were 4 groups of tumor-bearing mice, 10 mice in each group.

(3) Treatment plan: Calculated as day 0 of inoculation, the mice were treated with E7 vaccine on the 7th, 14th and 21st days after inoculation (for the preparation of vaccine, please refer to the previous work of our laboratory: CN201410570624.7), E7 (20 )/MPLA/PEG-PE(20/10/1000w/w/w)/each mouse, subcutaneous injection. At the same time, the mice were treated with GW6471, 10 mg/kg, intraperitoneally, on the 8th day after inoculation, and GW6471 was given once every other day for a total of 10 times (GW6471 stock solution was 25 mg/ml dissolved in DMSO, waiting for Before administration, it was diluted in pre-warmed PBS at 55°C until completely dissolved, the concentration was 2 mg/ml, and each mouse was injected intraperitoneally with 1 ml).

(4) Statistics of tumor growth and survival period: During the administration period, the tumor growth of mice in each group was measured twice a week, the tumor volume was calculated according to length×width×width/2, and the tumor growth curve was drawn, and the small size was recorded. Mouse death.

The results are shown in Figure 16. Compared with the E7 vaccine treatment group, the combination of GW6471 can further significantly slow down the tumor growth of TC-1. At the same time, as shown in Figure 17, the combination of GW6471 and E7 vaccine also significantly improved the survival of mice.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art to understand the essence of the present invention, and do not limit the protection scope.

Claims (7)

1. The application of PPARα ligand in the preparation of medicine, the function of the medicine includes any one or more of the following functions:

   (1) an activation signal for activating T cells, the activation signal is to increase the phosphorylation level of JNK, and the T cells are preferably CD8+ T cells;

   (2) activate T cells,

   The activation of T cells refers to making T cells secrete more IFN-γ and IL-2 and at the same time increase the survival time of T cells,

   The T cells are preferably CD8+ T cells, more preferably, tumor-infiltrating CD8+ T cells;

   (3) promoting the differentiation of T cells, the differentiation refers to promoting the differentiation of T cells into memory T cells;

   (4) Regulate the metabolic state of dendritic cells (DC cells),

   The regulating metabolism refers to: down-regulating the fatty acid oxidation/oxidative phosphorylation level of DC cells, and up-regulating the level of glycolysis;

   The DCs are bone marrow-derived DCs, more preferably, DCs infiltrated in the tumor microenvironment;

   (5) Restore the regulatory function of DC on T cells,

   The restoration of the regulatory function of DC refers to: down-regulating the ratio of CD4+ T cells transformed into regulatory T cells, increasing the ratio of tumor-killing CD8+ T cells, and activating the function of tumor-killing CD8+ T cells;

   The activation of tumor-killing CD8+ T cells refers to: making tumor-killing CD8+ T cells secrete more IFN- and IL-2 while increasing the survival time of tumor-killing CD8+ T cells;

   (6) Improve the anti-tumor effect of an immune checkpoint inhibitor, the immune checkpoint inhibitor is preferably a PD-L1/PD-1 antibody, and preferably, the tumor is a tumor that is effectively treated by an immune checkpoint inhibitor ;

   (7) Improve the therapeutic effect of the anti-tumor vaccine, the anti-tumor vaccine is preferably a nano-vaccine with PEG2000-DSPE micelles loaded with polypeptide/protein tumor antigens.
2. The application according to claim 1, wherein the PPARα ligand is a PPARα inhibitor, preferably, the PPARα inhibitor is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284, ZM0285 , ZM0286, ZM0325, ZM0326, ZM0329, ZM0330, ZM0332, ZM0333.
3. A group of PPARα inhibitors, the inhibitors are ZM0282, ZM0283, ZM0284, ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330, ZM0332, ZM0333.
4. An anti-tumor combined preparation comprising:

   (1) A therapeutically effective amount of PPARα ligand;

   (2) A therapeutically effective amount of an immune checkpoint inhibitor or a therapeutically effective amount of an antitumor vaccine;

   (3) Necessary pharmaceutical excipients.
5. The combined preparation according to claim 4, wherein the PPARα ligand is an inhibitor of PPARα, preferably, the inhibitor of PPARα is GW6471 molecule and its structural analogs ZM0282, ZM0283, ZM0284 , ZM0285, ZM0286, ZM0325, ZM0326, ZM0329, ZM0330, ZM0332, ZM0333.
6. The combined preparation according to claim 4 or 5, wherein the immune checkpoint inhibitor is a PD-L1/PD-1 antibody.
7. The combined preparation according to claim 4 or 5, wherein the anti-tumor vaccine is a vaccine loaded with polypeptide/protein tumor antigens in PEG2000-DSPE micelles.

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