Application of tetrahydrobiopterin pathway as target spot in resisting QDPR deletion type tumor

Abstract

The invention belongs to the field of biological medicine, and relates to application of a tetrahydrobiopterin pathway as a target spot in resisting QDPR deletion type tumors. Particularly relates to application of a tetrahydrobiopterin bioactive agonist in preparation of an immunotherapy sensitizer or a drug resistance reversal agent for QDPR low-expression or deletion tumors. It is found for the first time that the biological activity of tetrahydrobiopterin is improved, the sensitivity of QDPR low-expression or deletion tumors to immunotherapy can be greatly improved, and the treatment effectiveness of an immune checkpoint blocking agent serving as an anti-tumor drug is improved. The invention has an extremely important meaning for treating tumors resisted by immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of tetrahydrobiopterin pathway as a target in the resistance to QDPR-deficient tumors. Background Art

[0002] Tumor immune checkpoint blockers (ICBs) are a treatment method that has emerged in recent years and have shown good efficacy in some tumors. With the application of ICBs in the treatment of various tumors, it has gradually been found that some tumors are insensitive / ineffective to ICB treatment, and the main reason is that these tumors are in an immunosuppressive state. Therefore, current research in this field is mainly focused on revealing the mechanisms behind these tumors that are insensitive to ICB treatment, and finding new targets based on the mechanisms, and using new targets to sensitize tumors to the ICB efficacy.

[0003] Among tumors that are insensitive to ICB treatment, pancreatic cancer is particularly prominent. The fundamental reason is that it has a highly suppressive tumor immune microenvironment. Currently reported clinical trials of ICB treatment for pancreatic cancer have basically ended in failure. Therefore, it is urgent to find new targets that can sensitize pancreatic cancer, which is ineffective for ICB treatment.

[0004] Tetrahydrobiopterin (referred to herein as BH4) is a naturally occurring biogenic amine of the pterin family that is a cofactor for a variety of different enzymes, including phenylalanine hydroxylase (PAH), tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase. BH4 has been approved as an agent for phenylketonuria and hyperphenylalaninemia. Summary of the invention

[0005] On the one hand, the present application provides the use of a tetrahydrobiopterin bioactive agonist in the preparation of an immunotherapy sensitizer or resistance reversal agent for QDPR low-expression or QDPR-deficient tumors.

[0006] Resistance reversal agents refer to the situation that when some immune checkpoint blockers are used as anti-tumor drugs to treat tumors, there are some tumors that are not very sensitive to immune checkpoint blockers, or these tumors are resistant to immune checkpoint blockers. At this time, immune checkpoint blockers can be used in combination with tetrahydrobiopterin bioactive agonists (as resistance reversal agents) to reverse the tumor's resistance to the immune checkpoint blockers; or, vice versa, the same is true.

[0007] In one aspect, the present invention provides the use of a tetrahydrobiopterin biological activity agonist in the preparation of an anti-tumor enhancer or drug resistance reversal agent of an immune checkpoint blocker.

[0008] In one aspect, the present invention provides a composition for treating QDPR low-expression or QDPR-deficient tumors, comprising:

[0009] A tetrahydrobiopterin activity agonist and / or a pharmaceutically acceptable salt thereof, and

[0010] Immune checkpoint blockers and / or pharmaceutically acceptable salts thereof.

[0011] In one aspect, the present invention provides the use of the composition in the preparation of a drug for treating a tumor, wherein the tumor has low QDRP expression or QDRP deficiency.

[0012] In some embodiments, the QDRP low-expressing tumor is an mRNA low-expressing tumor and / or a protein low-expressing tumor; in some embodiments, the QDRP-deficient tumor is an mRNA-deficient tumor and / or a protein-deficient tumor.

[0013] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. In some embodiments, the composition is in the form of a lyophilized powder, an injection, a tablet, a capsule or a patch.

[0014] In one aspect, the present invention provides an electronic device for treating QDPR-deficient tumors, comprising:

[0015] An acquisition detection module is configured to obtain the expression level of the subject's QDPR;

[0016] A judgment module is configured to judge whether the subject is a medication subject according to the obtained expression level of the subject's QDPR; when the expression level of the subject's QDPR is lower than a normal level, the subject is judged to be a medication subject;

[0017] The drug administration module is configured to provide a tetrahydrobiopterin bioactive agonist and an immune checkpoint blocker to a subject determined to be a medication target based on the result of the determination module.

[0018] In some embodiments, the tetrahydrobiopterin activity agonist is selected from: sepiapterin reductase activator, GTP cyclohydrolase 1 activator, protein tyrosine phosphatase activator, aldehyde keto reductase family member C3 activator, aldehyde keto reductase family member BIO activator, dihydrofolate reductase activator, pterin-4-methanolamine dehydratase activator, dihydropterin reductase activator or a combination thereof; or the following substances or pharmaceutically acceptable salts thereof: tetrahydrobiopterin, sapropterin, sepiapterin, 1',2'-diacetyl-5,6,7,8-tetrahydrobiopterin , 6-methyl-5,6,7,8-tetrahydrobiopterin, trihydrobiopterin, dihydrobiopterin, 6-pyruvoyltetrahydropterin, 6-lactoyltetrahydropterin, 6-hydroxypropyl-tetrahydropterin, (6R)-L-erythro-5,6,7,8-tetrahydropterin, (6R,S)-5,6,7,8-tetrahydropterin, 6-hydroxymethyl-5,6,7,8-tetrahydropterin, 6-phenyl-5,6,7,8-tetrahydropterin, 7,8-dihydroneopterin, 1'-hydroxy-2'-oxopropyltetrahydropterin, L-cympterin, 7,8-dihydrobiopterin, pyruvoyltetrahydropterin and lactoyltetrahydropterin.

[0019] In some embodiments, the tumor is a malignant tumor; in some embodiments, the malignant tumor is a solid cancer or a blood cancer; in some embodiments, the solid cancer is one or more cancers selected from the group consisting of malignant melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell carcinoma, clear cell renal cell carcinoma, breast cancer, ovarian cancer, serous ovarian cancer, ovarian clear cell carcinoma, nasopharyngeal cancer, uterine cancer, anal cancer, colorectal cancer, rectal cancer, colon cancer, hepatocellular carcinoma, esophageal cancer, esophageal adenocarcinoma, gastric cancer, esophagogastric junction cancer, small intestine cancer, pancreatic cancer, urothelial carcinoma, prostate cancer, fallopian tube cancer, primary peritoneal cancer, malignant pleural mesothelioma, gallbladder cancer, bile duct cancer, biliary tract cancer, skin cancer, testicular cancer, vaginal cancer, vulvar cancer, penile cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, spinal tumors, brain tumors, squamous cell carcinoma, bone / soft tissue sarcoma, and Kaposi's sarcoma.

[0020] In some embodiments, the immune checkpoint blocker is an anti-PD-1 antibody or an antibody functional fragment, an anti-PD-L1 antibody or an antibody functional fragment, a PD-1 antagonist, a PD-L1 / VISTA antagonist, a PD-L1 / TIM3 antagonist, an anti-PD-L2 antibody or an antibody functional fragment, a PD-L1 fusion protein, a PD-L2 fusion protein, an anti-CTLA-4 antibody or an antibody functional fragment, an anti-LAG-3 antibody or an antibody functional fragment, a LAG-3 fusion protein, an anti-Tim3 antibody or an antibody functional fragment, an anti-KIR antibody or an antibody functional fragment, an anti-BTLA antibody or an antibody functional fragment, an anti-TIGIT antibody or an antibody functional fragment, an anti-VISTA antibody or an antibody functional fragment, an anti-CSF-1R antibody or an antibody functional fragment, or a CSF-1R inhibitor.

[0021] In some embodiments, the anti-PD-1 antibody is Nivolumab, Cemiplima b, Pembrolizumab, Spartalizumab, Tislelizumab, AMP-514, Dostarlimab, Toripalimab, Camrelizumab, Genolizumab, Sintilimab, STI-A1110, ENUM388D4, ENUM244C8, GLS010, MGA01 2. AGEN2034, CS1003, HLX10, BAT-1306, AK105, AK103, BI754091, LZM009, CMAB819, Sym021, GB 226, SSI-361, JY034, HX008, ISU106, ABBV181, BCD-100, PF-06801591, CX-188 or JNJ-63723283.

[0022] In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, BMS-936559, STI-1014, KN035, LY3300054, HLX20, SHR-1316, CS1001, MSB2311, BGB-A333, KL-A167, CK-301, AK106, AK104, ZKAB001, FAZ053, CBT-502, JS003, or CX-072.

[0023] In some embodiments, the anti-CTLA-4 antibody is Ipilimumab, AGEN1884, or Tremelimumab.

[0024] In some embodiments, the immune checkpoint blocker is an anti-ICB antibody or an antibody functional fragment. For example, an antibody, an antibody functional fragment, a peptide, or a peptidomimetic that binds to any functional domain of any part of the ICB. Wherein, the antibody may be a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., a bispecific antibody), a nanobody, and / or an antibody fragment connected to the ICB. The antibody may be a chimeric antibody, a humanized antibody, a CDR-transplanted antibody, or a human antibody. The antibody fragment may be, for example, Fab, Fab', F(ab')2, Fv, Fd, a single-chain Fv (scFv), a disulfide-bonded FV (sdFv), or a VL, VH domain. The antibody may be in a conjugated form, for example, in combination with a label, a detectable marker, or a cytotoxic agent. The antibody may be an isotype IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA, IgM, IgE, or IgD.

[0025] On the one hand, the present invention provides the use of a QDPR detection reagent in the preparation of a reagent for the treatment evaluation and prognosis of immunotherapy for anti-tumor.

[0026] In some embodiments, the immunotherapy is anti-tumor with immune checkpoint blockers; in some embodiments, the QDPR detection reagent is selected from one or more reagents for detecting the expression level of QDPR mRNA, or detecting the expression level of QDPR protein, or detecting the biological activity of QDPR protein; in some embodiments, the reagent for detecting the expression level of QDPR protein is selected from one or more of antibodies, antibody functional fragments or conjugated antibodies.

[0027] In a specific embodiment, through multi-omics combined analysis and verification, it was identified that the gene QDPR involved in the biopterin metabolism process was significantly lowly expressed in pancreatic cancer and was associated with poor prognosis. In vivo and in vitro experiments confirmed that QDPR deficiency can promote the malignant phenotype of pancreatic cancer in an immune system-dependent manner and lead to the formation of an immunosuppressive microenvironment, thereby making pancreatic cancer resistant to treatment with immune checkpoint blockers.

[0028] In a specific embodiment, mechanistic studies have shown that QDPR deficiency can lead to the accumulation of the metabolite BH2, thereby upregulating the BH2 / BH4 ratio, leading to increased intracellular ROS generation, promoting the expression of tumor cell-activated chemokine CXCL1, and then recruiting immunosuppressive cells MDSC to the tumor microenvironment to promote immunosuppression, leading to resistance to immune checkpoint blocker therapy.

[0029] In a specific embodiment, further analysis in multiple cancer types revealed that QDPR expression levels can be used as a biomarker for evaluating the efficacy of immunotherapy (immune checkpoint blockers) and treatment prognosis.

[0030] In a specific embodiment, the QDPR discovered based on the experiments of the present invention disrupts the BH2 / BH4 ratio, thereby causing ineffective ICB treatment of tumors. In order to reverse this treatment insensitivity, the metabolite BH4 (tetrahydrobiopterin) is supplemented. The results of preclinical animal model experiments confirm that after BH4 supplementation, the BH2 / BH4 ratio can be reversed and the tumors that are insensitive to ICB treatment can be re-sensitized. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The immunohistochemistry (IHC) was performed to detect the QDPR protein level in human pancreatic cancer tissue sample microarray (TMA, n=116) in Example 1. Representative immunohistochemistry images (A), the protein level of QDPR in cancer and adjacent tissues was significantly reduced (B). The QDPR IHC staining score was analyzed for survival curves in the pancreatic cancer cohort (C).

[0032] Figure 2 1×10 6 Qdpr - / - or control KPC-luc cells. Luminescence images and quantification of luminescence intensity (A, n = 5). Kaplan-Meier survival curves of each group (B, n = 10). C57BL / 6J mice were implanted with 1×10 6 QUR - / - (Second sgRNA, Qdpr - / - sg2) or control KPC-luc cells. Representative luminescence images and quantification of luminescence intensity (C, n = 5). Kaplan-Meier survival curves of each group (D, n = 10).

[0033] Figure 3 1×10 6 QUR - / - Or control KPC-luc cells. After 12 days, the proportion of CD8 and G-MDSCs in the tumor was detected by flow cytometry (A). 3×10 6 QUR - / -or control KPC-luc cells and received dual immune checkpoint blockade (ICB) (anti-PD1 mAb + anti-CTLA4 mAb) or IgG isotype control at the indicated time points. Schematic diagram of the treatment plan (B). Representative luminescence images (C) and quantification of luminescence intensity (D).

[0034] Figure 4 The concentrations of BH2 and BH4 in the lysates of KPC cells in the QDPR-deficient and control groups were detected by Elisa in Example 2. The ratio of BH2 to BH4 was calculated (A). The ROS levels of the two groups of KPC cells were reflected by fluorescence intensity, and the excitation / emission wavelengths on the fluorescence microplate reader were 650 / 675nm (B). ELISA detected the CXCL1 protein level in the supernatant of each group of KPC cells (C). Chemotaxis assay detected the chemotaxis of the conditioned medium of each group of KPC cells on MDSCs (D).

[0035] Figure 5 For Example 3, Panel A: Comparison of QDPR expression between non-responders (NR) and responders (R) in two published ICB-treated cancer cohorts (STAD and Melanoma). Panel B: Overall survival Kaplan-Meier curves of four independent ICB-treated cancer cohorts according to QDPR mRNA levels.

[0036] Figure 6 In Example 4, after BH4 supplementation, ELISA was used to detect BH2 and BH4 levels, and the BH2 / BH4 ratio was calculated (A). C57BL / 6J mice were orthotopically implanted with 3×10 6 Qdpr - / - KPC-luc cells and received dual ICB (anti-PD1 mAb + anti-CTLA4 mAb) or IgG isotype control. Study design (B). Representative luminescence images and quantification of luminescence intensity (C). DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0038] the term:

[0039] As used herein, an "anti-tumor" drug is capable of negatively affecting tumor / cancer cells in a subject, for example, by promoting the killing of tumor / cancer cells, inducing apoptosis of tumor / cancer cells, reducing the growth rate of tumor / cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumor / cancer cells, promoting an immune response against tumor / cancer cells, preventing or inhibiting the progression of tumor / cancer cells, or prolonging the life span of a subject suffering from tumor / cancer cells.

[0040] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. Therapeutic benefits include, but are not limited to, eradication, inhibition, reduction, or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit is achieved by eradication, inhibition, reduction, or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, but the patient may still suffer from the underlying disorder.

[0041] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a preventive benefit. To obtain a preventive benefit, a pharmaceutical composition may be administered to a patient at risk of developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0042] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in the therapeutic compositions of the present invention is contemplated. Supplementary active ingredients may also be added to the pharmaceutical compositions.

[0043] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic reaction, etc., within the scope of reasonable medical judgment, and consistent with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds described include salts derived from suitable inorganic and organic acids and bases.

[0044] As used in the present invention, "low expression or absence of QDPR" means: taking tumor tissue, detecting the expression level of QDPR in cancer and adjacent tissues by immunohistochemistry, quantifying the immunohistochemical results, and comparing the expression levels of QDPR in cancer and adjacent tissues. If the expression level of QDPR in cancer is higher than that in adjacent tissues, QDPR is considered to be low expressed in the sample; if the immunohistochemical staining of QDPR in cancer tissue is negative, it is considered to be absent.

[0045] Example 1 QDPR is significantly underexpressed in pancreatic cancer and is associated with poor prognosis. QDPR deficiency can promote the malignant phenotype of pancreatic cancer in an immune system-dependent manner and lead to the formation of an immunosuppressive microenvironment, thereby making pancreatic cancer resistant to immune checkpoint blocker therapy

[0046] Material

[0047] Pancreatic cancer and adjacent tissue microarrays (116 pairs), QDPR antibody (Abcam, #ab126150), immunohistochemistry kit (KeyGEN, #KGOS300), pancreatic cancer primary cells overexpressing luciferase (KPC-luc), C57BL / 6 mice, immune system-deficient NOG mice (purchased from Beijing Weitong Lihua), luciferase substrate (intraperitoneal injection, used to monitor pancreatic in situ tumor growth); small animal in vivo imaging system; isoflurane gas anesthetic. Flow cytometry antibodies: APC anti-mouse CD3ε(#100312,Biolegend), Alexa Fluor 700anti-mouse CD4(#100430,Biolegend), BrilliantViolet 510anti-mouse CD8a(#100752,Biolegend), APC / Cyanine7 anti-mouse CD45(#103116,Biolegend),PerCP / Cyanine5.5 anti-mouse / human CD11b(#101228,Biolegend),Brilliant Violet 785anti-mouse Ly-6G(#127645,Biolegend),FITC anti-mouse Ly-6G / Ly-6C(Gr-1)(#108406, Biolegend). Immunotherapy antibodies: anti-PD1 mAb (BioXcell, #BE0146), anti-CTLA4 mAb (BioXcell, #BP0032).

[0048] method

[0049] a) Immunohistochemistry: IHC analysis of QDPR protein expression in human pancreatic cancer tissues (116 pairs of human PDAC tumors and adjacent normal tissues) was performed using anti-QDPR antibody (#ab232983, IHC: dilution ratio 1:1000, Abcam). IHC staining was performed using an IHC detection kit (Solarbio, Beijing, China). Tumor tissues were removed and fixed in 10% formalin overnight and then embedded in paraffin. Next, the embedded tissues were cut into 4 μm thick sections and endogenous peroxidase was eliminated with 3% hydrogen peroxide. The sections were boiled in buffer (PH8.0) for 5 minutes in an electric pressure cooker for antigen retrieval, and then nonspecific sites were blocked using 3% BSA for 1 hour. Then, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1 hour, followed by the addition of DAB solution.

[0050] b) Cell culture: KPC cells were derived from a primary mouse pancreatic cancer cell line (named KPC) derived from spontaneous pancreatic tumors in mice (C57BL / 6J with LSL-KrasG12D / +; LSL-Trp53R172h / +; pdx-1-Cre). All cell lines were cultured in DMEM medium supplemented with 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin, and cultured in a humidified environment containing 5% CO2.

[0051] c) Tumor inoculation and detection: Female C57BL / 6J and NOG mice aged 6-8 weeks were used to establish pancreatic orthotopic or subcutaneous PDAC mouse models. KPC cells were suspended in 1× PBS and injected into the subcutaneous flank of mice (200 μl, 1×10 6 cells) or pancreas (50 μl, 1-3×10 6 The tumor growth of the orthotopic model was determined by bioluminescence imaging, and the tumor volume of the subcutaneous model was determined using the formula volume = length × width 2 The luminescence intensity of the orthotopic tumor was obtained by in vivo imaging software (Perkin Elmer).

[0052] d) Mouse survival: After the above mice were re-inoculated with tumors, the mortality of the mice was tracked and recorded daily, and the data were finally summarized to draw a survival curve of the mice.

[0053] e) Flow cytometry: Fresh pancreatic orthotopic tumors were finely minced and digested in RPMI 1640 medium containing 0.1 mg / mL collagenase IV (#17104019, Gibco), 0.02 mg / ml DNase I (#10104159001, sigma), 0.04 mg / ml hyaluronidase (#H3506, Sigma) and 1 mg / ml soybean trypsin inhibitor (#17075-029, Gibco) and 5% FBS, digested at 37°C for 1 hour, and then filtered through a 70 μM cell sieve (Corning) to prepare a single cell suspension. Mouse spleens were ground in PBS and transferred to a 70 μM cell sieve.

[0054] f) (Corning), single cell suspensions were prepared. Spleen cell suspensions and EDTA anticoagulated peripheral blood (PB) were treated with RBC lysis buffer (#420301, Biolegend). Cell surface markers were incubated in the dark at 4°C for 45 minutes using fluorescein-conjugated antibodies in CellStaining Buffer (#420201, Biolegend). For intracellular marker staining, cell suspensions were pre-incubated with PMA / ionomycin (Sigma) and Brefeldin A (Sigma) at 37°C for 4 hours, and then incubated with fluorescein-conjugated antibodies in Intracellular Staining Perm Wash Buffer (#421002, Biolegend) for 1 hour at 4°C. Flow cytometric analysis was performed on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software (Treestar).

[0055] result

[0056] like Figure 1 As shown in Figure 2, QDPR is significantly underexpressed in human pancreatic cancer tumor tissue samples (compared to adjacent tissues). Figure 1 AB), and this low expression is significantly associated with poor prognosis in pancreatic cancer ( Figure 1 C) Figure 2 As shown, QDPR deletion had no effect on tumor growth and mouse survival in immunodeficient NOG mice ( Figure 2 AB); significantly promoted tumor growth in immune-complete mice C57B / L6, resulting in worse survival of mice ( Figure 2 CD), suggesting that QDPR deficiency can promote the malignant phenotype of pancreatic cancer in an immune-dependent manner. Figure 3As shown, QDPR deficiency can lead to a significant decrease in anti-tumor immune cells CD8 T cells and a significant increase in immunosuppressive cells MDSC ( Figure 3 A). This suggests that QDPR deficiency can lead to the formation of an immunosuppressive microenvironment in pancreatic cancer tumor tissue. At the same time, QDPR deficiency leads to the ineffectiveness of ICB treatment in pancreatic cancer mouse models ( Figure 3 BD).

[0057] Example 2. QDPR deficiency can lead to the accumulation of the metabolite BH2, thereby upregulating the BH2 / BH4 ratio, leading to increased intracellular ROS generation, promoting the expression of tumor cell-activated chemokine CXCL1, and then recruiting immunosuppressive cells MDSC to the tumor microenvironment to promote immunosuppression, leading to resistance to immune checkpoint blocker therapy.

[0058] Material

[0059] KPC cells with QDPR knockout and blank control, MDSCs isolated in vitro, ELISA kit for detecting CXCL1, kit for detecting ROS, ELISA kit for detecting BH2 and BH4, MDSCs isolation kit, Transwell chamber for cell chemotaxis experiment.

[0060] method

[0061] a) ELISA experiment: To determine the concentration of BH2 and BH4 in KPC cells, KPC cells (2×10 5 Each well) was cultured in a six-well plate for 48 hours, and then the cells were collected and repeatedly frozen and thawed to obtain cell lysates, which were filtered through a 0.45 μm filter to exclude cell debris. The amount of BH2 and BH4 in the cell lysates was measured using a BH2 (tetrahydrobiopterin) ELISA kit (#E-EL-0111c, Elabscience) and a BH4 (tetrahydrobiopterin) ELISA kit (#E-EL-0110c, Elabscience). To detect the secretion of CXCL1, cells (2×10 5 Each well) was cultured in a six-well plate for 48 hours, and then the supernatant was collected and filtered through a 0.45 μm filter. The concentration of each target was detected according to the kit instructions.

[0062] b) ROS detection: In order to measure the level of reactive oxygen species (ROS) in KPC cells, KPC cells (4×10 4Each well) was seeded into a 96-well plate and incubated overnight, and then ROS measurement was performed using a cell ROS assay kit (#ab186029, Abcam). Briefly, cells were washed, replaced with fresh medium, incubated with ROSDeep Red dye for 60 minutes in a 37°C / 5% CO2 incubator, and then the fluorescence intensity was measured at Ex / Em=650 / 675nm using a fluorescence microplate reader.

[0063] c) Chemotaxis assay: MDSCs (myeloid suppressor cells) were isolated from the spleen of mice with PDAC orthotopic tumors (KPC cells) using a mouse MDSC isolation kit (#130-094-538, Miltenyi) and cultured in RPMI1640 supplemented with 10% FBS. MDSCs (1×10 5 Cells were seeded in the upper chamber of Transwell (manufactured by Corning) per well. Conditioned medium (CM) from specially treated KPC cell lines was collected and added to the lower layer of the Transwell chamber. After incubation at 37°C for 4 hours, MDSCs cells that migrated to the lower chamber were counted.

[0064] result

[0065] like Figure 4 As shown, QDPR deletion (QDPR - / - ) can significantly promote the increase of BH2 in pancreatic cancer tumor cells KPC cells, but has no effect on BH4 levels, ultimately leading to an increase in the BH2 / BH4 ratio (A). ROS detection results showed that QDPR deficiency led to a significant increase in ROS levels in KPC cells (B), and at the same time led to a significant increase in CXCL1 secretion (C). Chemotaxis assay results showed that the culture medium of KPC cells with QDPR deficiency can significantly increase the recruitment of MDSCs (D).

[0066] Example 3. QDPR expression level can be used as a biomarker for evaluating the efficacy of immunotherapy (immune checkpoint blockers) and treatment prognosis.

[0067] Material

[0068] Collect sequencing and survival information of the tumor ICB treatment cohort.

[0069] method

[0070] To estimate the differential expression of QDPRs between non-responders and responders in ICB-treated cancer patients, RNA-Seq datasets of two publicly available cancer cohorts (STAD and Melanoma) (https: / / doi.org / 10.1038 / s41591-018-0101-z.https: / / doi.org / 10.1016 / j.ccell.2019.01.003.) were downloaded and Wilcoxon test was performed. To investigate the prognostic significance of QDPR levels in ICB-treated cancer patients, we downloaded RNA-Seq datasets from four publicly available cancer cohorts (RCC, GBM, and Melanoma) (https: / / doi.org / 10.1038 / s41591-020-0839-y.https: / / doi.org / 10.1038 / s41591-019-0349-y.https: / / doi.org / 10.1016 / j.cell.2016.02.065.

[0071] https: / / doi.org / 10.1126 / science.aad0095.), and Kaplan-Meier analysis was performed.

[0072] result

[0073] like Figure 5 As shown, in the tumor ICB treatment cohort, the higher the QDPR expression level, the better the treatment response (A) and the longer the prognosis survival of ICB-treated tumor patients (B).

[0074] Example 4. Supplementation of BH4 can reverse the BH2 / BH4 ratio and resensitize tumors that are insensitive to ICB treatment.

[0075] Material

[0076] Metabolite BH4 (MCE, #HY-A0124A), pancreatic cancer primary cells overexpressing luciferase (KPC-luc), C57BL / 6 mice, luciferase substrate (ip injection, for monitoring pancreatic orthotopic tumor growth); small animal in vivo imaging system; isoflurane gas anesthetic.

[0077] method

[0078] a) Cell culture: QDPR knockout and control KPC cells were cultured in DMEM medium supplemented with 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin in a humidified environment containing 5% CO2. During the logarithmic growth phase of the cells, BH4 (10 μM) or control vehicle (DMSO) was added, and the levels of BH2 and BH4 in the cells were detected after 24 hours.

[0079] b) C57BL / 6J mice were first orthotopically induced with QDPR-knockout KPC cells to form pancreatic tumors. Six days later, BH4 (100 mg / kg per day for 11 consecutive days) and anti-CD8 mAb or IgG isotype control (200 μg per mouse, four injections at two-day intervals) were injected intraperitoneally. Tumor growth was detected by bioluminescence imaging on day 11 after treatment (n=7 per group).

[0080] result

[0081] like Figure 6 As shown, BH4 supplementation can reverse the imbalance of BH2 / BH4 ratio in KPC cells caused by QDPR knockout (A). We can see that in the QDPR knockout group, ICB treatment is ineffective, while BH4 supplementation can reverse the resistance of QDPR-deficient pancreatic cancer to ICB treatment and restore the therapeutic effect of ICB (BC).

Claims

1. Application of tetrahydrobiopterin bioactive agonists in the preparation of immunotherapy sensitizers or resistance reversal agents for QDPR low expression or deletion tumors.

2. Application of tetrahydrobiopterin bioactive agonists in the preparation of anti-tumor enhancers or resistance reversal agents of immune checkpoint blockers.

3. A composition for treating QDPR low-expression or QDPR-deficient tumors, characterized in that: Include: a tetrahydrobiopterin activity agonist and / or a pharmaceutically acceptable salt thereof, and Immune checkpoint blockers and / or pharmaceutically acceptable salts thereof.

4. Use of the composition according to claim 3 in the preparation of a drug for treating tumors, wherein the tumor has low QDRP expression or QDRP deficiency; Preferably, the QDRP low-expressing tumor is a mRNA low-expressing tumor and / or a protein low-expressing tumor; Preferably, the QDRP-deficient tumor is an mRNA-deficient tumor and / or a protein-deficient tumor.

5. An electronic device for treating QDPR-deficient tumors, characterized in that: Include: An acquisition detection module is configured to obtain the expression level of the subject's QDPR; A judgment module is configured to judge whether the subject is a medication subject according to the obtained expression level of the subject's QDPR; when the expression level of the subject's QDPR is lower than a normal level, the subject is judged to be a medication subject; The drug administration module is configured to provide a tetrahydrobiopterin bioactive agonist and an immune checkpoint blocker to a subject who is determined to be a medication target based on the results of the judgment template.

6. The use, composition or electronic device according to any one of claims 1 to 5, characterized in that: The tetrahydrobiopterin activity agonist is selected from: sepiapterin reductase activator, GTP cyclohydrolase 1 activator, protein tyrosine phosphatase activator, aldehyde-keto reductase family member C3 activator, aldehyde-keto reductase family member BIO activator, dihydrofolate reductase activator, pterin-4-carbinolamine dehydratase activator, dihydropterin reductase activator or a combination thereof; or The following substances or their pharmaceutically acceptable salts: tetrahydrobiopterin, sapropterin, sepiapterin, 1',2'-diacetyl-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydrobiopterin, trihydrobiopterin, dihydrobiopterin, 6-pyruvoyltetrahydropterin, 6-lactoyltetrahydropterin, 6-hydroxypropyl-tetrahydropterin, (6R)- L-erythro-5,6,7,8-tetrahydropterin, (6R,S)-5,6,7,8-tetrahydropterin, 6-hydroxymethyl-5,6,7,8-tetrahydropterin, 6-phenyl-5,6,7,8-tetrahydropterin, 7,8-dihydroneopterin, 1'-hydroxy-2'-oxopropyltetrahydropterin, L-cympterin, 7,8-dihydrobiopterin, pyruvoyltetrahydropterin and lactoylatetrahydropterin.

7. The use, composition or electronic device according to any one of claims 1 to 5, characterized in that: The tumor is a malignant tumor; Preferably, the malignant tumor is a solid cancer or a blood cancer; Preferably, the solid cancer is one or more cancers selected from the group consisting of malignant melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell carcinoma, clear cell renal cell carcinoma, breast cancer, ovarian cancer, serous ovarian cancer, ovarian clear cell carcinoma, nasopharyngeal cancer, uterine cancer, anal cancer, colorectal cancer, rectal cancer, colon cancer, hepatocellular carcinoma, esophageal cancer, esophageal adenocarcinoma, gastric cancer, esophagogastric junction cancer, small intestine cancer, pancreatic cancer, urothelial carcinoma, prostate cancer, fallopian tube cancer, primary peritoneal cancer, malignant pleural mesothelioma, gallbladder cancer, bile duct cancer, biliary tract cancer, skin cancer, testicular cancer, vaginal cancer, vulvar cancer, penile cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, spinal tumors, brain tumors, squamous cell carcinoma, bone / soft tissue sarcoma and Kaposi's sarcoma.

8. The use, composition or electronic device according to any one of claims 1 to 5, characterized in that: The immune checkpoint blocker is an anti-PD-1 antibody or antibody functional fragment, an anti-PD-L1 antibody or antibody functional fragment, a PD-1 antagonist, a PD-L1 / VISTA antagonist, a PD-L1 / TIM3 antagonist, an anti-PD-L2 antibody or antibody functional fragment, a PD-L1 fusion protein, a PD-L2 fusion protein, an anti-CTLA-4 antibody or antibody functional fragment, an anti-LAG-3 antibody or antibody functional fragment, a LAG-3 fusion protein, an anti-Tim3 antibody or antibody functional fragment, an anti-KIR antibody or antibody functional fragment, an anti-BTLA antibody or antibody functional fragment, an anti-TIGIT antibody or antibody functional fragment, an anti-VISTA antibody or antibody functional fragment, an anti-CSF-1R antibody or antibody functional fragment or a CSF-1R inhibitor; Preferably, the anti-PD-1 antibody is Nivolumab, Cemiplima b, Pembrolizumab, Spartalizumab, Tislelizumab, AMP-514, Dostarlimab, Toripalimab, Camrelizumab, Genolizumab, Sintilimab, STI-A1110, ENUM388D4, ENUM244C8, GLS010, MGA01 2. AGEN2034, CS1003, HLX10, BAT-1306, AK105, AK103, BI754091, LZM009, CMAB819, Sym021, GB 226, SSI-361, JY034, HX008, ISU106, ABBV181, BCD-100, PF-06801591, CX-188 or JNJ-63723283; Preferably, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, BMS-936559, STI-1014, KN035, LY3300054, HLX20, SHR-1316, CS1001, MSB2311, BGB-A333, KL-A167, CK-301, AK106, AK104, ZKAB001, FAZ053, CBT-502, JS003 or CX-072; Preferably, the anti-CTLA-4 antibody is Ipilimumab, AGEN1884 or Tremelimumab.

9. Application of QDPR detection reagents in the preparation of reagents for treatment evaluation and prognosis of immunotherapy against tumors.

10. The use according to claim 9, characterized in that The immunotherapy is anti-tumor with immune checkpoint blockers; Preferably, the QDPR detection reagent is selected from one or more reagents for detecting the expression level of QDPR mRNA, or detecting the expression level of QDPR protein, or detecting the biological activity of QDPR protein; Preferably, the reagent for detecting the expression amount of the QDPR protein is selected from one or more of antibodies, antibody functional fragments or conjugated antibodies.

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