CN110974964A

CN110974964A - Application of DPCPX inhibitor in preparation of PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicine

Info

Publication number: CN110974964A
Application number: CN201911359805.4A
Authority: CN
Inventors: 陈翔; 刘洪�; 匡欣薇
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2020-04-10
Anticipated expiration: 2039-12-25
Also published as: CN110974964B

Abstract

The invention provides an application of a DPCPX inhibitor in preparation of a PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament, in particular relates to an application of the DPCPX inhibitor in preparation of a PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament by targeted adenosine receptor inhibition ADORA1, and also provides an application of the DPCPX inhibitor and the PD-L1/PD-1 monoclonal antibody medicament in preparation of a tumor immune adjuvant therapy medicament by combination. Also provides application of ATF3 as a target site detection reagent combined with a DPCPX inhibitor in preparation of PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs. The tumor is a solid tumor, preferably melanoma or lung cancer, and more preferably melanoma or non-small cell lung cancer.

Description

Application of DPCPX inhibitor in preparation of PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicine

Technical Field

The invention relates to the field of biotechnology, in particular to the field of PD-L1/PD-1 monoclonal antibody tumor immunotherapy.

Background

In recent years, the relevant therapies for Immune Checkpoint Blockades (ICBs) have been extensively studied by global scientists and have provided enormous clinical benefit for the treatment of some malignancies, such as Malignant Melanoma (MM) and non-small cell lung cancer (NSCLC). The neutralizing antibody for blocking the interaction of programmed cell death protein 1(PD-1) and a ligand (PD-L1) thereof is clinically applied to multiple tumors, and brings breakthrough progress to tumor immunotherapy. However, as the research progresses, researchers find that the traditional Chinese medicine composition still has obvious defects, and only less than 40 percent of tumor patients respond to the PD-L1/PD-1 blocking therapy or have primary or secondary drug resistance in the treatment process. However, the mechanism by which these phenomena potentially occur is not clear. Recent studies have shown that tumor PD-L1 expression levels can be an important factor in assessing the clinical response of anti-PD-L1/PD-1 therapy, and are also common biomarkers. Therefore, the understanding of the molecular mechanism of tumor PD-L1 regulation is crucial, which is of great significance for improving the treatment effect and clinical prognosis of PD-L1/PD-1 antitumor.

Adenosine is a single nucleoside substance, can be accumulated in a tumor microenvironment, and plays a role in promoting the progress of tumors. Extracellular adenosine acts as a ligand to bind to adenosine receptors, thereby activating a downstream cascade of signaling reactions that affect tumor immune homeostasis. For example, the adenosine A2A receptor (ADORA2A) is believed to be a novel immune checkpoint that promotes tumor immune evasion by modulating the function of regulatory T cells. The adenosine A2B receptor (ADORA2B) promotes tumor growth by binding to bone marrow-derived suppressor immune cells. In addition, adenosine a1 receptor (adenosines a1 receptor, ADORA1) has been reported to have a tumor growth promoting effect in colorectal adenocarcinoma, human leukemia Jurkat cell, breast cancer, and renal cancer, but its role in tumor immunity is not clear.

cAMP-dependent transcription factor (ATF 3) is a member of the ATF/cAMP-responsive element-binding protein (CREB) family, and its expression is rapidly induced by a variety of cellular stresses, such as DNA damage, cellular damage, oxidative stress, and the like. Recent studies have shown that ATF3 inhibits tumor cell proliferation by inducing apoptosis of tumor cells. In innate immunity, ATF3 interacts as an immunomodulator in LPS-treated mice with NF-. kappa.B and the proinflammatory cytokines IL-6, IL-12B, toll-like receptor-4 (TLR-4), but its specific mechanism of action in the immune response is not clear.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new development direction of PD-L1/PD-1 monoclonal antibody tumor immunodetection or immunotherapy auxiliary drugs and develop a new drug combination mode.

The technical scheme of the invention comprises the following steps:

the technical scheme of the invention is as follows:

the ADORA1 is used as biomarker before tumor immunotherapy of PD-L1/PD-1 monoclonal antibody.

Application of ADORA1 as a target site detection reagent in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicine.

The application of the combination of the inhibitor of ADORA1 and the PD-L1/PD-1 monoclonal antibody in preparing tumor immune adjuvant therapy medicaments.

The second technical scheme of the invention:

application of DPCPX inhibitor in preparing PD-L1/PD-1 monoclonal antibody tumor immunity adjuvant treatment medicine is provided.

The DPCPX inhibitor can be used for preparing a PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament by targeted inhibition of an adenosine receptor ADORA 1.

The application of the DPCPX inhibitor and the PD-L1/PD-1 monoclonal antibody drug in preparing tumor immune adjuvant therapy drugs.

The third technical scheme of the invention is as follows:

application of ATF3 as a target site detection reagent in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs.

The technical scheme of the invention is as follows:

application of ATF3 as a target site detection reagent combined with a DPCPX inhibitor in preparation of PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs.

The ATF3 is used as a target site detection reagent and is combined with a PD-L1/PD-1 monoclonal antibody medicament to prepare the tumor immunotherapy medicament.

The technical scheme of the invention is as follows:

application of ADORA1-ATF3 axis as a target site detection reagent in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs.

The ADORA1-ATF3 axis is used as a target site detection reagent and is combined with a PD-L1/PD-1 monoclonal antibody drug to prepare the tumor immunotherapy drug.

In the five previous technical schemes: the tumor is preferably a solid tumor. The tumor is further preferably melanoma or lung cancer. The tumor is further preferably melanoma or non-small cell lung cancer.

In the existing research, PD-1 blocking therapy shows better anticancer effect, but the overall response rate is still not satisfactory. Part of the reasons for this phenomenon are thought to be positively correlated with PD-L1 expression, and we are still not aware of the regulation mechanism of tumor PD-L1. From the research data we show, in example 1, as shown in fig. 1, we constructed a mouse model of subcutaneous transplantation tumor (melanoma) of gene knock-down adenosine receptor ADORA1, and by treating mouse tumor in combination with PD-1 monoclonal antibody, we found that targeted inhibition of adenosine receptor ADORA1 can significantly enhance the anti-mouse tumor proliferation of PD-1 monoclonal antibody, promote the survival of mouse, and has no significant toxic side effect. As shown in table 1 and fig. 2, we observed the histopathological staining results and clinical prognosis of non-small cell lung cancer patients who had been clinically treated with PD-1mab (nivolumab), and by comparing the expression of ADORA1 and PD-L1 in tumors and correlating the progression-free survival time and total survival time of patients, we found that patients with low expression of ADORA1 had higher expression of PD-L1, better response effect of patients receiving PD-1mab, longer progression-free survival time and total survival time of patients, and significant statistical differences. The results all indicate that the ADORA1 is a new target, and can provide a new immunoassay target for PD-L1/PD-1 monoclonal antibody tumor immunotherapy, develop a new research and development direction of detection reagents or immunotherapy auxiliary drugs and develop a new drug combination mode. In example 2, as shown in fig. 3 and 4, a subcutaneous transplantation tumor (melanoma and non-small cell lung cancer) immune mouse model is constructed, mouse tumors are treated by combining ADORA1 specific inhibitor DPCPX with PD-1 monoclonal antibody, and we find that the targeted inhibition of adenosine receptor ADORA1 can significantly enhance the mouse tumor proliferation resistance of PD-1 monoclonal antibody and promote the survival effect of mice without significant toxic side effects. As shown in fig. 5, from the mRNA sequencing result, we found that the expression of ATF3 can be significantly up-regulated by gene-knock-down ADORA1, and in vitro studies further confirm that the gene-knock-down ADORA1 or DPCPX inhibiting ADORA1 receptor can significantly activate the expression of downstream ATF 3; the tumor TCGA database is analyzed through bioinformatics, the fact that in various tumor types, the ATF3 and the expression of PD-L1 have obvious positive correlation is found, and in vitro research also finds that gene knock-down ATF3 can actually cause the expression of tumor PD-L1 to be down-regulated; similarly, as shown in fig. 6, we can promote the expression of PD-L1 by up-regulating ATF3, and further through luciferase reporter gene detection and ChIP detection, we find that ATF3 can be directly combined with PD-L1 promoter, thereby transcriptionally regulating the expression of tumor PD-L1, while there is no research report on the direct regulation of tumor PD-L1 by ATF3, which is a transcription factor, and we believe that the direct regulation of tumor PD-L1 can be achieved by targeted inhibition of ATF3, which is similar to PD-L1/PD-1 monoclonal antibody, thereby inhibiting the promotion of tumor immune escape by PD-L1/PD-1, which is a pair of immune inspection points. As shown in fig. 7 and 8, a gene knock-down transcription factor ATF3 subcutaneous transplantation tumor (melanoma and non-small cell lung cancer) immune mouse model is constructed, and tumors of mice are treated by combining with a specific inhibitor DPCPX of ADORA1, so that the combined intervention group has stronger tumor proliferation capacity against mice, longer survival time of the mice and no obvious toxic and side effects compared with a single drug group or a control group. The results all indicate that the development of the targeted ADORA1 inhibitor can provide a new development direction of immunotherapy adjuvant drugs for PD-L1/PD-1 monoclonal antibody tumor immunotherapy and develop a new drug combination mode. In example 3, as shown in table 2 and fig. 9, we observed the histopathological staining results and clinical prognosis of non-small cell lung cancer patients who had been clinically treated with PD-1mab (nivolumab), and by comparing the expression of ADORA1, ATF3 and PD-L1 in tumors and correlating the progression-free survival time and total survival time of patients, we found that patients with lower ADORA1 expression had higher ATF3 and PD-L1 expression, and that patients received PD-1mab had better response, longer progression-free survival time and total survival time, and both had significant statistical differences. The results all indicate that the ADORA1-ATF3 axis can provide a new immunoassay target for PD-L1/PD-1 monoclonal antibody tumor immunotherapy, develop a detection reagent or provide a new research and development direction of immunotherapy auxiliary drugs and develop a new drug combination mode.

Our research shows that the target adenosine receptor ADORA1 can directly regulate the transcription level of tumor PD-L1 by up-regulating downstream transcription factor ATF3, thereby enhancing the curative effect of blocking the tumor treatment by PD-1 monoclonal antibody. The mechanism directly discusses the existing problems of PD-1 blocking agent action, and develops a new drug combination mode for providing a new research and development direction of PD-L1/PD-1 monoclonal antibody tumor immunodetection or immunotherapy adjuvant drugs.

Name interpretation: the "/" in PD-L1/PD-1 means "and" or ".

The detailed structure of the present invention will be further described with reference to the accompanying drawings and the detailed description.

Drawings

FIG. 1 is a graph of experimental data of a mouse model according to example 1, in which FIG. 1A is a schematic view of a mouse experimental flow, FIG. 1B is a graph of a change in volume of a mouse subcutaneous graft tumor measured in real time, the volume of the mouse tumor is measured every three days from the start of intervention, a volume curve is drawn, the mouse is taken out of the body for in vitro measurement and a histogram is drawn on the day of sacrifice, the data is represented by mean. + -. standard error, the statistical analysis of the results is performed by one-way analysis of variance for overall group-to-group comparison and further performed by Dunnett-t test for two-group comparison, NS, P > 0.05; p < 0.01; p < 0.001. FIG. 1C is a statistical plot of the volume of subcutaneously transplanted tumor bodies measured scatter plots for mice on the day of sacrifice, data are presented as mean. + -. standard error, statistical analysis of results is performed using one-way anova for overall group-to-group comparisons and further performed using the Dunnett-t test for two group comparisons, NS, P > 0.05; p < 0.01; p < 0.001. FIG. 1D is a graph of the change in mouse body weight measured in real time, from the start of intervention, every three days the general state of the mouse was observed and the mouse body weight was measured and a body weight curve was plotted, with the data expressed as mean. + -. standard error, statistical analysis of results using one-way analysis of variance for overall group comparisons and further using the Dunnett-t test for two group comparisons. FIG. 1E is a Kaplan-Meier survival graph of mice, with comparisons of differences between groups using the Gehan-Breslow-Wilcoxon test. NS, P > 0.05; p < 0.05; p < 0.01.

FIG. 2 is a graph showing the expression levels of ADORA1 and PD-L1 in tumor tissue samples of NSCLC patients treated with PD-1 monoclonal antibody in example 1, which are labeled by immunofluorescence staining, the non-progression survival time (A, B) and the total survival time (C, D) of the patients are respectively compared according to the level of gene expression, a Kaplan-Meier survival curve graph is drawn, and the difference between groups is compared by using Gehan-Breslow-Wilcoxon test.

FIG. 3 is a graph of experimental data for a mouse model of melanoma according to example 2, wherein FIG. 3A is a schematic flow chart of a mouse experiment;

FIG. 3B is a graph showing the volume change of the subcutaneous transplanted tumor body of a mouse measured in real time; FIG. 3C is a statistical plot of the volume of subcutaneous transplanted tumor bodies measured in vitro for mice on the day of sacrifice; FIG. 3D is a graph of the change in body weight of mice measured in real time; FIG. 3E is a Kaplan-Meier survival graph for mice.

FIG. 4 is a graph of experimental data for a mouse model of lung cancer according to example 2, wherein FIG. 4A is a schematic flow chart of a mouse experiment; FIG. 4B is a graph showing the volume change of the subcutaneous transplanted tumor body of a mouse measured in real time; FIG. 4C is a scattergram of in vitro measurements of the volume of subcutaneous transplanted tumor bodies in mice on the day of sacrifice; fig. 4D is a graph of the change in body weight of mice measured in real time.

FIG. 5 is a graph of data from in vitro studies conducted on the ADORA1-ATF3-PD-L1 axis of examples 2 and 3, wherein FIG. 5A is a volcano plot of the melanoma cell line Sk-mel-28 gene knock-down ADORA1 versus the transcriptome sequencing analysis of the unloaded group, and a corrected p-value of 0.05 or less indicates a statistical difference; FIG. 5B is a graph showing the correlation between the expression of ATF3 and PD-L1 in the bioinformatics analysis of the inter-tumor TCGA database, wherein the statistical analysis is performed by using Spearman's rho correlation analysis, the red dots indicate the statistical difference, and p is less than or equal to 0.05; FIGS. 5C and 5E represent the results of western blot assays for ADORA1, ATF3 and PD-L1 expression, respectively, following in vitro gene knock-down of ADORA1 or ATF3 in melanoma cell lines; FIGS. 5D and 5F are corresponding statistical results of gray scale analysis, data expressed as mean ± standard error, results were compared between whole groups using one-way analysis of variance, and two groups were further compared using Dunnett-t test,. P < 0.01; p < 0.001.

FIG. 6 is the experimental data of the direct regulation mechanism study of ATF3 on PD-L1 in examples 2 and 3, wherein FIG. 6A represents the results of western blot detection of ATF3 and PD-L1 expression after gene overexpression of ATF3 in melanoma cell lines in vitro; FIG. 6B shows the corresponding statistical results of gray scale analysis, where the data are expressed as mean. + -. standard error, and the results are compared between two sets by unpaired t-test, where P is less than 0.01; p < 0.001; fig. 6C, 6D constructed a vector containing ATF3 mutant PD-L1 promoter sequence and wild-type PD-L1 promoter sequence as controls, transfected ATF3 multi-expression vector to melanoma cell line or treated with ADORA1 inhibitor DPCPX (50 μ M) to induce ATF3 up-regulation, respectively, and set empty load and vehicle as controls, and detected changes in PD-L1 luciferase activity by luciferase reporter gene detection method. Data are expressed as mean ± standard error, results were compared between whole groups using one-way analysis of variance, and further compared between two groups using Dunnett-t test, nsp >0.05,. P < 0.01,. P < 0.001; panel E is a predicted mutation map of the ATF3 binding site of the PD-L1 promoter region. (F) In melanoma cell lines, ATF3 was shown to bind significantly to PD-L1 by ChIP detection using anti-flag antibody and ATF3 primer for SYBR RT-PCR.

FIG. 7 is a graph of experimental data for example 3, wherein FIG. 7A is a schematic flow chart of a mouse experiment; FIG. 7B is a graph showing the volume change of the subcutaneous transplanted tumor body of a mouse measured in real time; FIG. 7C is a scattergram of in vitro measurements of the volume of subcutaneous transplanted tumor bodies in mice on the day of sacrifice; fig. 7D is a graph of the change in body weight of mice measured in real time. FIG. 7E is a Kaplan-Meier survival graph of mice, with comparison of differences between groups using the Gehan-Breslow-Wilcoxon test. NS, P > 0.05; p < 0.05; p < 0.01.

FIG. 8 is a graph of another set of experimental data from example 3, wherein FIG. 8A is a schematic flow chart of a mouse experiment; FIG. 8B is a graph showing the volume change of the subcutaneous transplanted tumor body of a mouse measured in real time; FIG. 8C is a scattergram of in vitro measurements of the volume of subcutaneous transplanted tumor bodies in mice on the day of sacrifice; fig. 8D is a graph of the change in body weight of mice measured in real time. FIG. 8E is a Kaplan-Meier survival graph of mice, with comparison of differences between groups using the Gehan-Breslow-Wilcoxon test. NS, P > 0.05; p < 0.05; p < 0.01.

FIG. 9 is a graph of tumor ATF3 and PD-L1 expression levels marked by immunofluorescence staining paraffin sections of tumor tissue samples of NSCLC patients treated with PD-1 monoclonal antibody in example 3, and the non-progression survival time (A, B) and the total survival time (C, D) of the patients are respectively compared according to the level of gene expression, so as to draw a Kaplan-Meier survival curve graph, and the difference between groups is compared by adopting Gehan-Breslow-Wilcoxon test.

Detailed Description

Example 1

1. In vivo studies of gene knock-down adenosine receptor ADORA1 in combination with PD-1mAb against proliferation of mouse transplantable tumors (melanoma).

1.1 group settings:

scramble + IgG2a (control)

shAdora1+ IgG2a (Adora1 knockdown group)

Scamble + PD-1mAb (PD-1 mAb treatment group)

ShAdora1+ PD-1mAb (combination intervention group)

1.2 Experimental scheme, see FIG. 1A:

about 6 days before the experiment: 6-8 weeks of C57BL/6 mice were injected subcutaneously with B16F10 melanoma shAdora1 knockdown cell line 5 x 10 at the right dorsal flank, respectively⁵20 mice each with and without the same number of Scramble cell lines (control).

Day 0 of the experiment: mouse body weight and tumor volume were recorded when tumor volume grew to about 100mm³Initially, the shAdora1 knockdown cell line and the Scramble cell line were inoculated into mice for PD-1mAb 100 ug/mouse/3 days, i.p., and isotype IgG control groups were set, 10 mice per group.

Experiment days

1, 4, 7, 10: the body weight and tumor volume of the mice were weighed every three days, and the volume thereof was pi/6 × length × width²And (4) formula calculation.

Day 12 of the experiment: half of the mice were sacrificed, tumor photographs were taken and tissue samples were saved for subsequent data analysis.

And (3) survival time observation: the remaining mice were observed for length of survival and recorded for time to death.

1.3 Experimental results:

as shown in fig. 1B and 1C, compared to the control group, the tumor volumes of the Adora1 knockdown group were not significantly different, the tumor volume of the PD-1 monoclonal antibody group was significantly reduced, and the combined intervention group was significantly smaller than that of the intervention group alone, which was statistically different.

As shown in fig. 1D, there was no significant statistical difference in mouse body weight between the four groups. Indicating that the drug has no significant toxic side effect on the general condition of the mice.

As shown in fig. 1E, compared to the control group, the survival time of the Adora1 knockdown group and the PD-1mab group was not significantly different, while the combined intervention group was significantly longer than the control group and the intervention group alone, and there was a statistical difference.

The results indicate that the gene-knock-down adenosine receptor ADORA1 can promote the curative effect of PD-1 monoclonal antibody anti-tumor (melanoma) immunotherapy.

2. Retrospective clinical observations analysis of the correlation of PD-L1 and ADORA1 expression levels in patients with NSCLC treated with PD-1mAb (nivolumab)

The experimental results are shown in Table 1, the expression levels of tumor PD-L1 and ADORA1 in NSCLC patients treated by the PD-1 monoclonal antibody are obviously and negatively correlated, and the statistical difference exists, and P is less than 0.05.

The attached Table 1 illustrates: table 1 shows that paraffin sections of tumor tissue samples of patients treated with NSCLC by PD-1 monoclonal antibody are subjected to immunofluorescence staining to mark the expression levels of tumor PD-L1 and ADORA1, the number of patients in each category is summarized in Table 1, and the Fisher's exact test (bilateral test) is used to evaluate the significance of the correlation between the two data.

3. Retrospective clinical observations of the relevance of PD-1mAb (nivolumab) treatment of tumor PD-L1 and ADORA1 expression in NSCLC patients to patient prognosis (progression-free and Total survival)

The experimental results are shown in fig. 2, compared with the low expression of tumor PD-L1 and the high expression of ADORA1, the high expression of PD-L1 and the low expression of ADORA1 show better prognosis in patients with NSCLC treated by PD-1 monoclonal antibody, and the statistical difference exists, P is less than 0.05; p < 0.01; p < 0.001.

Example 2

1. Specific inhibitor DPCPX targeted inhibition of adenosine receptor ADORA1 in combination with PD-1 monoclonal antibody against in vivo studies of proliferation of mouse transplantable tumors (melanoma, non-small cell lung cancer).

1.1 group settings:

vehicle + IgG2a (control group)

DPCPX + IgG2a (Adora1 inhibition group)

Vehicle + PD-1mAb (PD-1 mAb treatment group)

DPCPX + PD-1mAb (Combined intervention group)

1.2 Experimental scheme, see FIGS. 3A, 4A:

about 6 days before the experiment: 6-8 weeks C57BL/6 mice were injected subcutaneously with B16F10 melanoma 5 x 10 on the right dorsal flank, respectively⁵Single or LLC non-small cell lung cancer 1 x 10⁶40 mice each, a wild-type cell line.

Day 0 of the experiment: mouse body weight and tumor volume were recorded when tumor volume grew to about 100mm³At the beginning, the vaccinated mice were divided into four groups, and were injected intraperitoneally or vehicle control, PD-1mAb 100 ug/mouse/3 days intraperitoneally, with isotype IgG control, 10 mice per group, respectively.

Experiment days

1.3 Experimental results:

referring to fig. 3B, 3C, 4B, and 4C, compared to the control group, Adora1 showed no significant difference in tumor volume in the inhibition group, and the tumor volume in the PD-1mab group was significantly reduced, while the combined intervention group was significantly smaller than the intervention group alone, which was statistically different.

See fig. 3D, 4D, with no significant statistical differences in mouse body weight between the four groups. Indicating that the drug has no significant toxic side effect on the general condition of the mice.

Referring to fig. 3E, compared to the control group, the survival time of the Adora 1-inhibited group was not significantly different, the survival time of the PD-1 monoclonal antibody group was longer than that of the control group, while the combined intervention group was significantly longer than that of the control group and the intervention group alone, and there was a statistical difference.

The results indicate that the adenosine receptor ADORA1 specific inhibitor DPCPX can obviously enhance the curative effect of the anti-tumor (melanoma and non-small cell lung cancer) immunotherapy by combining with the PD-1 monoclonal antibody.

In vitro studies of ADORA1-ATF3 axis modulation of tumor PD-L1 expression levels.

2.1 Experimental methods: transcriptome sequencing, TCGA database information generation analysis, gene editing, cell gene intervention, DPCPX medicine intervention, Western blot, luciferase reporter gene, ChIP detection

2.2 Experimental results:

referring to fig. 5A, by transcriptome sequencing analysis, we found that gene knockdown ADORA1 was significantly upregulated in melanoma cell line Sk-mel-28 compared to the unloaded group, with statistical differences in ATF3 gene expression;

as shown in FIG. 5B, we found that the expression of ATF3 gene was significantly and positively correlated with PD-L1 in various tumor types by TCGA database bioassay analysis, and that the difference was statistically shown in red dots;

see 5C, 5D Western bolt detection gene knockdown tumor ADORA1, compared with the control group, the expression levels of ATF3 and PD-L1 can be obviously up-regulated, and statistical difference exists;

see 5E, 5F Western bolt detection gene knockdown tumor ATF3, compared with the control group, the expression level of PD-L1 can be obviously inhibited, and statistical difference exists;

see 6A, 6B Western bolt detection gene overexpression tumor ATF3, compared with a control group, the expression level of PD-L1 can be obviously promoted, and statistical difference exists;

see 6C and 6D luciferase reporter gene for detecting whether ATF3 is combined with PD-L1 promoter, compared with a control group, overexpression of ATF3 or DPCPX treatment can result in significant enhancement of the luciferase activity of tumor PD-L1 promoter, but when the ATF3 binding site of PD-L1 promoter is mutated, the effect disappears, and the ATF3 and PD-L1 are proved

The promoter is directly combined, and the result has statistical difference; the predicted mutation pattern of the ATF3 binding site of the PD-L1 promoter region is shown in detail in FIG. 6E.

See fig. 6F ChIP assay suggesting that ATF3 was significantly, statistically different from PD-L1 compared to the control.

The results indicate that the gene knock-down and specific inhibitor DPCPX can target and inhibit adenosine receptor ADORA1, and can significantly up-regulate ATF3, ATF3 as a transcription factor can directly regulate the transcription of PD-L1 from the mRNA level, so that the axis of the targeted adenosine receptor ADORA1-ATF3 can directly regulate the expression level of tumor PD-L1.

Example 3

1. In vivo studies of gene knock-down transcription factor ATF3 in combination with an adenosine receptor ADORA1 specific inhibitor DPCPX proliferation against mouse transplantable tumors (melanoma, non-small cell lung cancer).

1.1 group settings:

scramble + vehicle (control group)

Scramble + DPCPX (Adora1 inhibition group)

ShAtf3+ vehicle (Atf3 knockdown group)

Shatf3+ DPCPX (Combined intervention group)

1.2 Experimental scheme, see FIGS. 5A, 6A:

about 6-8 days before the experiment: 6-8 weeks of C57BL/6 mice were injected subcutaneously with B16F10 melanoma shAtf3 knockdown cell line 5 x 10 at the right dorsal flank, respectively⁵Single or LLC non-small cell lung carcinoma shATF3 knockdown cell line 1 x 10⁶One of the cells and the same number of Scramble cell lines (control group), 20 mice each.

Day 0 of the experiment: mouse body weight and tumor volume were recorded when tumor volume grew to about 100mm³At the beginning, the shAtf3 knockdown cell line and the Scramble cell line were inoculated into mice, and DPCPX 1 mg/Kg/mouse was injected into the abdominal cavity, and a vehicle control group was provided, 10 mice per group.

Experiment days

1.3 Experimental results:

the results, shown in fig. 7B, 7C, 8B, and 8C, show that the tumor volume of mice in the Adora 1-inhibited group is not significantly different, the tumor volume of the Atf 3-knockdown group is significantly reduced, and the combined intervention group is significantly smaller than that of the intervention group alone, and the statistical difference exists.

See fig. 7D, 8D, with no significant statistical differences in mouse body weight between the four groups. Indicating that the drug has no significant toxic side effect on the general condition of the mice.

Referring to fig. 7E and 8E, compared to the control group, the survival time of the Adora 1-inhibited group and the Atf 3-knockdown group was not significantly different, while the combined intervention group was significantly longer than the control group and the intervention group alone, and there was a statistical difference.

The animal experiment results show that the gene-knockdown transcription factor ATF3 can remarkably reduce the expression of PD-L1, can play a role in treating tumors similar to PD-1 monoclonal antibodies, and can remarkably inhibit tumor proliferation and enhance immune microenvironment antitumor (melanoma and non-small cell lung cancer) effects by combining with an adenosine receptor ADORA1 specific inhibitor DPCPX.

2. Retrospective clinical observations analysis of the correlation of tumor PD-L1 with ATF3 expression levels in patients with NSCLC treated with PD-1mab (nivolumab).

The results are shown in Table 2, the expression levels of tumor PD-L1 and ATF3 in NSCLC patients treated by PD-1 monoclonal antibody are in obvious positive correlation, the statistical difference exists, and P is less than 0.05

The attached Table 2 illustrates: table 2 shows that paraffin sections of tumor tissue samples of patients with NSCLC treated with PD-1 monoclonal antibody are subjected to immunofluorescence staining to mark the expression levels of tumor PD-L1 and ATF3, the number of patients in each category is summarized in Table 2, and the Fisher's exact test (bilateral test) is used to evaluate the significance of the correlation between the two data.

3. Retrospective clinical observations correlation of tumor PD-L1 and ATF3 expression levels in patients with NSCLC treated with PD-1 monoclonal antibody (nivolumab) with patient prognosis (progression-free and Total survival)

The experimental results are shown in fig. 9, in the NSCLC patients treated by the PD-1 monoclonal antibody, compared with the low expression of tumors PD-L1 and ATF3, the high expression of the gene indicates a better prognosis, and there is a statistical difference, P is less than 0.05; p < 0.01; p < 0.001.

The above description is for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the technical scope of the present invention, and the technical solution and the concept of the present invention are also intended to be covered by the scope of the claims of the present invention.

Claims

The application of the DPCPX inhibitor in preparing PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs.
2. The use of claim 1, wherein the DPCPX inhibitor can be used for preparing a PD-L1/PD-1 monoclonal antibody tumor immunotherapy medicament by targeted inhibition of adenosine receptor ADORA 1.
The application of the DPCPX inhibitor and the PD-L1/PD-1 monoclonal antibody drug in preparing tumor immune adjuvant therapy drugs.
Application of ATF3 as a target site detection reagent combined with a DPCPX inhibitor in preparation of PD-L1/PD-1 monoclonal antibody tumor immunotherapy drugs.
5. The use according to any one of claims 1 to 4, wherein the tumour is a solid tumour.
6. The use of claim 5, wherein the tumor is melanoma or lung cancer.
7. The use of claim 6, wherein the tumor is melanoma or non-small cell lung cancer.