The present disclosure relates to the fields of molecular biology, medicine and oncology. More specifically, provided are a drug containing a transport protein inhibitor, a pharmaceutical composition containing a transport protein inhibitor, and use thereof.
Tumor remains one of the most fatal threats to human health. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain tumors, the overall 5-year survival rate for all tumors has improved only about 10% over the past 20 years. Tumors or malignant tumors grow and metastasize rapidly in an uncontrolled manner, which makes timely detection and treatment extremely difficult.
Tumor immunotherapy is another tumor treatment method that is developed after surgery, radiotherapy and chemotherapy and has definite efficacy on treating tumor. Tumor immunotherapy resists and kills tumor cells by activating the immune system of one's own body, and is one of the development directions of tumor treatment in the future.
Different patients treated by different immunotherapies will exhibit different information, thus requiring specific immune system evaluation for patients treated by tumor immunotherapy to monitor the therapeutic effects of tumor immunotherapy.
The programmed death receptor-1 (PD-1) is a protein that has been linked to the suppression of the response of immune system during chronic infections, pregnancy, tissue allografts, autoimmune diseases and tumors. PD-L1 modulates immune responses by binding to an inhibitory receptor, which is referred to as programmed death receptor-1 (PD-1) and is expressed on the surface of T cells, B cells and monocytes. The mechanism of PD-1 immunotherapy is designing a specific protein antibody against PD-1 or PD-L1, preventing the recognition process between PD-1 and PD-L1, and partially restoring the function of tumor-specific cytotoxic CD8+ lymphocytes (CTLs) with antigenic specificity, so as to enable CTLs to kill tumor cells.
However, despite the unprecedented success of the clinical application of programmed cell death receptor-1 (PD-1) antibodies against many types of tumors, the underlying mechanism of how PD-1 is regulated in tumor-specific cytotoxic CD8+ lymphocytes (CTLs) in tumor microenvironment remains poorly characterized. Meanwhile, considering the relationship between tumor relapse and the remaining tumorigenic cells, it is possible that CTLs, even after being relieved from the inhibitory effect of PD-1 signaling, may be incapable of destroying stem cell-like cancer cells that are able to repopulate tumors [tumor-repopulating cells (TRCs)]. These TRCs are a self-renewing and highly tumorigenic subpopulation of cancer cells that play crucial roles in the initiation, promotion and progression of tumorigenesis.
Furthermore, there still remains relapse in part of the treated patient population who initially respond and show extraordinary treatment outcomes. Therefore, medical personnel still need a novel regimen for treating tumor diseases.
In order to solve the above problems, the present disclosure provides a novel regimen for treating tumor diseases. Said regimen comprises providing a drug and a method for treating a disease accompanied with increased PD-1 expression, especially a tumor accompanied with increased PD-1 expression by using said drug.
The present disclosure provides use of a transport protein inhibitor in the preparation of a drug for preventing or treating a disease accompanied with increased PD-1 expression.
In the present disclosure, the disease is selected from diseases caused by tumor accompanied with increased PD-1 expression; preferably, the disease caused by tumor accompanied with increased PD-1 expression is selected from one or more of melanoma accompanied with increased PD-1 expression, liver cancer accompanied with increased PD-1 expression, breast cancer accompanied with increased PD-1 expression, lung cancer accompanied with increased PD-1 expression, colorectal cancer accompanied with increased PD-1 expression, ovarian cancer accompanied with increased PD-1 expression, or leukemia accompanied with increased PD-1 expression.
In the present disclosure, the transport protein is selected from one or a combination of a tryptophan transporter or a Kyn transporter; preferably, the tryptophan transporter is selected from SLC1A5, and the Kyn transporter is selected from SLC7A8 or PAT4.
In the present disclosure, the transport protein inhibitor is selected from one or more of GPNA, SAR, BCH, and pharmacologically acceptable salts thereof.
In the present disclosure, the drug further comprises other agent(s) for treating the disease accompanied with increased PD-1 expression; preferably, said other agent for treating the disease accompanied with increased PD-1 expression is selected from one or more of an immunomodulator, an IDO inhibitor, an AhR inhibitor, a radiotherapeutic agent or a chemotherapeutic agent; alternatively, the immunomodulator is selected from an immune checkpoint modulator or an agent that facilitates or mediates an antigen presentation that promotes a cell-mediated immune response.
The present disclosure also provides use of a transport protein inhibitor in the preparation of a drug for preventing or treating a disease accompanied with increased PD-1 expression in combination with other drug(s) capable of treating the disease accompanied with increased PD-1 expression.
In the present disclosure, the drug for treating the disease accompanied with increased PD-1 expression is selected from said other agents for treating the disease accompanied with increased PD-1 expression according to claim 5.
The present disclosure also provides a drug for preventing or treating a disease accompanied with increased PD-1 expression, wherein the drug comprises a transport protein inhibitor.
In the present disclosure, the transport protein is selected from one or a combination of a tryptophan transporter or a Kyn transporter; preferably, the tryptophan transporter is selected from SLC1A5, and the Kyn transporter is selected from SLC7A8 or PAT4; alternatively, the transport protein inhibitor is selected from one or more of GPNA, SAR, BCH, and pharmacologically acceptable salts thereof.
In the present disclosure, the drug further comprises said other agent(s) for treating the disease accompanied with increased PD-1 expression according to claim 5.
The present disclosure also provides a method for preventing or treating a disease accompanied with increased PD-1 expression, wherein the method comprises a step of administering a prophylactically or therapeutically effective amount of a drug.
In the present disclosure, the method further comprises administering a therapeutically effective amount of said other agent for treating the disease accompanied with increased PD-1 expression according to claim 5; alternatively, the method further comprises a step of surgical treatment.
In the present disclosure, the drug further comprises a pharmaceutical excipient.
The drug provided by the present disclosure is capable of effectively reducing the expression of PD-1 in specific CD8+ T cells and enhancing the killing effect of specific CD8+ T cells on tumor cells.
Alternatively, diseases accompanied with increased PD-1 expression may be effectively treated by the drug provided by the present disclosure and by administering said drug or the combination of said drug and other related drugs.
When used in combination with the term “comprise” in claims and/or specification, the wording “a” or “an” may refer to “one”, but may also refer to “one or more”, “at least one” and “one or more than one”.
As used in claims and specification, the wording “comprise”, “have”, “include” or “contain” means inclusive or open-ended, and does not exclude additional and unmentioned elements, method or steps.
Throughout the application document, the term “about” means that a value includes the standard deviation of the error of the device or method used to determine the value.
Although the definition of the term “or” as being an alternative only and as “and/or” are both supported by the disclosed content, the term “or” in claims means “and/or” unless it is explicitly indicated that the term “or” only means an alternative or the alternatives are mutually exclusive.
The term “transport protein” or “transporter” refers to a carrier protein on the cell membrane of a tissue. In the present disclosure, the above terms may be used interchangeably and have the same meaning.
The definition of the term “drug” is a substance capable of preventing or treating a disease. A drug may comprise one effective ingredient for treating disease, and may also comprise two or more effective ingredients for treating disease. Alternatively, said drug may be a vaccine. Said “drug” may also be added with ingredients as needed, for example, ingredients that facilitate extended storage period of drug, stability or better absorption by the patient. Illustratively, said ingredients may be selected from pharmaceutical excipients.
The term “immunomodulator” comprises an immune checkpoint modulator. For example, immune checkpoint protein receptors and their ligands mediate the suppression of T cell-mediated cytotoxicity and are often expressed by tumors or expressed on anergic T cells in the tumor microenvironment, thus permitting the tumor to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands may overcome the immunosuppressive tumor environment, so as to permit cytotoxic T cell attack on tumor. Examples of immune checkpoint proteins include but are not limited to PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT and CD103. Modulation (including inhibition) of the activity of such protein may be accomplished by an immune checkpoint modulator, which may include, for example, an antibody, an aptamer, a small molecule, a soluble form of a checkpoint receptor protein and the like that target a checkpoint protein. PD-1-targeting inhibitors include the approved drug agents pembrolizumab and nivolumab, while ipilimumab is an approved CTLA-4 inhibitor. Antibodies specific for PD-L1, PD-L2, LAG3, TIM3, TIGIT and CD103 are known and/or commercially available, and may also be produced by those skilled in the art.
In addition to immune checkpoint modulators, immunomodulators also include an agent that facilitates or mediates an antigen presentation that promotes a cell-mediated immune response. Such immunomodulators may include, for example, a tumor antigen vaccine. A tumor antigen vaccine may include a preparation comprising a particular tumor antigen or a set of known tumor antigens, with or without a subject's own dendritic cells or an adjuvant. Or, a tumor antigen vaccine may comprise a relatively crude preparation of a tumor cell antigen derived from a patient's tumor. When said tumor cell antigen is exposed ex vivo to the dendritic cells generated in vitro from a patient's cells, T cell-mediated attack on tumor is allowed when the dendritic cell vaccine is introduced into the patient. In one embodiment, immunomodulators include a tumor antigen vaccine. In another embodiment, the tumor antigen vaccine comprises a dendritic cell tumor antigen vaccine.
The term “IDO” (indoleamine 2,3-dioxygenase) refers to a protease involved in the metabolism of tryptophan. Indoleamine 2,3-dioxygenase 1 (IDO1) modulates immune cell function to a suppressive phenotype, and is therefore partially accountable for tumor escape from host immune surveillance. IDO1 degrades the essential amino acid tryptophan into kynurenine and other metabolites. These metabolites and the lack of tryptophan lead to the suppression of the function of effector T cells and enhanced differentiation of regulatory T cells. IDO inhibitors are known and/or commercially available, and may also be produced by those skilled in the art.
The term “kynurenine (Kyn)” refers to an intermediate metabolite of tryptophan generated in human body.
The term “AhR (aryl hydrocarbon receptor) inhibitor” or “AhR antagonist” refers to such an agent or compound that inhibits one or more AhR signaling and downstream effector pathways, including constitutive AhR signaling. Therefore, the term “AhR inhibitor” refers to such an agent that inhibits the expression of AhR polypeptide or the polynucleotide encoding AhR, or binds to partially or completely block the stimulating effect of said AhR polypeptide or the polynucleotide encoding AhR, reduces, prevents and delays the activating effect of said AhR polypeptide or the polynucleotide, and inactivates, desensitizes or downregulates the activity of said AhR polypeptide or the polynucleotide encoding AhR. Such AhR inhibitors are capable of, for example, inhibiting AhR expression (for example, AhR translation, the post-translational processing of AhR, the stabilization, degradation, nuclear localization or cytoplasmic localization of AhR polypeptide, or the transcription, post-transcriptional processing, stabilization or degradation of the polynucleotide encoding AhR), or binding to partially or completely block the stimulating effect, DNA binding, or the transcription factor activity of AhR. AhR inhibitors are capable of taking effect directly or indirectly. AhR inhibitors are known and/or commercially available, and may also be produced by those skilled in the art.
The term “radiotherapeutic agent” includes drugs that cause DNA damage. Radiotherapy has been widely used in the treatment of cancer and diseases, and includes those commonly referred to as γ-ray and X-ray and/or targeted delivery of radioisotopes to tumor cells.
The term “chemotherapeutic agent” refers to a chemical compound useful for treating cancer. Classes of chemotherapeutic agents include but are not limited to: an alkylating agent, an antimetabolite, a kinase inhibitor, a spindle poison plant alkaloid, a cytotoxic/antitumor antibiotic, a topoisomerase inhibitor, a photosensitizer, an anti-estrogen, a selective estrogen receptor modulator, an anti-progesterone, an estrogen receptor downregulator, an estrogen receptor antagonist, a luteinizing hormone-releasing hormone agronist, anti-androgens, an aromatase inhibitor, an EGFR inhibitor, a VEGF inhibitor, an antisense oligonucleotide that inhibits the expression of gene(s) involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents that may be used in the treatment method of the present disclosure include a cell growth inhibitor and/or a cytotoxic agent.
The term “pharmaceutical excipient” refers to a substance that is a basic material and an important constituent part of a pharmaceutical preparation. A pharmaceutical excipient not only endows a drug with a certain dosage form, but is also closely related to the improvement of drug efficacy and the reduction of adverse reaction. In addition to endowing with a dosage form, serving as a carrier and enhancing the stability, pharmaceutical excipients also have important functions such as solubilizing, aiding solubilization and extending or controlling the release, and are important ingredients that may influence the quality, safety and effectiveness of drugs. Said pharmaceutical excipients may be classified into various types, such as a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesive agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid, and a release retarder.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that, the detailed description and specific examples (although representing the specific embodiments of the present disclosure) are given for illustrative purpose only, since various changes and modifications made within the spirits and scope of the present disclosure will become apparent to those skilled in the art after the browsing of this detailed description.
The experimental animals, reagents and materials used in the present disclosure were as follows.
Female C57BL/6 mice (6- to 8-weeks old), OT-1 transgenic mice, Pmel-1 transgenic mice, AhR−/− mice and MMTV-PyMT mice were all purchased from commercial institutions. These animals were maintained in the corresponding facilities owned by the applicant under pathogen-free conditions. All studies involving mice were approved by the Animal Care and Use Committee.
Mouse tumor cell lines OVA-B16 (melanoma), H22 (hepatocellular carcinoma) and ID8 (ovarian cancer) as well as human tumor cell lines A375 (melanoma) and HepG2 (hepatocellular carcinoma) were purchased from China Center for Type Culture Collection (Beijing, China) and cultured in DMEM (Thermo Scientific) with 10% fetal bovine serum (FBS) (Gibco, USA), except that H22 cells were grown in RPMI 1640 culture medium (Gibco, USA) containing 10% FBS.
pGFP-C-shLenti-shIDO1, pGFP-C-shLenti-shSLC1A5, pGFP-V-RS-shPAT4, pGFP-V-RS-shSLC7A8 and pCMV6-Entry-DDK-SLC1A5 were purchased from Origene (MD, USA). Kynurenine, kynurenic acid, indoxyl sulfate, melatonin, 1-L-MT, 3′,4′-dimethoxyflavone (DMF), TCDD, sarcosine (SAR), 2-amino-2-norbornanecarboxylic acid (BCH), L-γ-Glutamyl-p-nitroanilide (GPNA) and anti-PAT4 antibody were from Sigma-Aldrich (ST, USA). Cyclosporine A was from SelleckChemicals. Anti-IFN-γ, TNF-α, PD-1 and PD-L1 neutralizing antibody as well as anti-PD-1, CD3, CD8, IFN-γ, TNF-α and CD107a antibodies were purchased from BioLegend. IFN-γ was purchased from R & D Systems. Salmon fibrinogen, thrombin and dispase were from Reagent Proteins (CA, USA). Puromycin was from Invitrogen (SD, USA).
Human peripheral blood and resected human breast or colon cancer tissues were obtained from patients at hospitals. Ethical permission was granted by the Clinical Trial Ethics Committee of the corresponding hospital. All patients provided written informed consent to participate in this study.
Experimental Methods
(1) Detection Methods of Biomarkers
In any of the aforementioned methods, the presence and/or expression level/amount of a biomarker was determined by measuring the protein expression level of the biomarker. In certain embodiments, said method comprised bringing a biological sample into contact with an antibody that specifically binds to the biomarker described herein (for example, an anti-PD-1 antibody) under a condition where the binding of the biomarker was allowed, and detecting whether a complex was formed by the antibody and the biomarker. Such method may be an in-vitro or in-vivo method. In some cases, an antibody such as a biomarker used for selecting individuals was used to select subjects eligible for adopting PD-1 axis binding antagonist therapy. Any method for measuring the protein expression level that was known in the art or provided herein could be used. For example, in some embodiments, the protein expression level of a biomarker (for example, PD-1) was determined by a method selected from the group consisting of the following methods: flow cytometry (for example, fluorescence-activated cell sorting (FACS™)), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunoassay, surface plasmon resonance, spectroscopy, mass spectrometry, and HPLC.
(2) Tumor Cells Cultured on 2D Rigid Dish or in 3D Fibrin Gels
For conventional 2D cell culture, tumor cells were maintained in a rigid dish with complete culture medium. Specifically, fibrinogen (Searun Holdings Company, Freeport, Me.) was diluted to 2 mg/ml with T7 buffer (pH 7.4, 50 mM Tris, 150 mM NaCl). Then, a 1:1 (volume) mixture of fibrinogen and cell solution was made. 250 μl of the cell/fibrinogen mixture was seeded into a 24-well plate and mixed well with 5 μl pre-added thrombin (0.1 U/μl, Searun Holdings Company). After 30 min of incubation at 37° C., these cells were supplemented with 1 ml of complete culture medium. After 5 days of incubation, dispase II was added to and digested the cultured gels for 10 min at 37° C. The spheroids were harvested and further digested with 0.25% trypsin for 3 min to obtain a single cell suspension for the following experiments.
(3) Statistical Analysis Methods
All experiments were performed at least three times. Results were expressed as mean±SEM and analyzed by Student's t-test. P value <0.05 was considered statistically significant. The analysis was conducted using the Graphpad 6.0 software. Sample exclusion was never carried out.
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) Anti-CD3/CD28 bead-activated OVA-CTLs were cultured alone or together with OVA-B16 cells (differentiated OVA-B16 cells) from conventional culture flasks or OVA-B16 TRCs grown in 3D fibrin matrices for 24 hours. PD-1 expression was determined by flow cytometry. The determination results were as shown in
(2) The co-culture supernatants of CTLs with either differentiated OVA-B16 cells or OVA-B16 TRCs or unmodified culture medium (negative control) were concentrated by freeze-drying and incubated with anti-CD3/CD28 bead-activated OVA-CTLs for 24 hours.
PD-1 expression was determined by flow cytometry. The determination results were as shown in
(3) CD3/CD28 bead-activated OVA-CTLs were cultured for 24 hours and the supernatant was analyzed by ELISA. The determination results were as shown in
(4) Anti-CD3/CD28 bead-activated OVA-CTLs were co-cultured with OVA-B16 TRCs for 24 h. IFN-γ production in T cells and tumor cells was analyzed by flow cytometry. The determination results were as shown in
(5) Anti-CD3/CD28 bead-activated OVA-CTLs were treated with PBS (phosphate buffered saline) or the conditioned medium from CTLs or OVA-B16 TRCs stimulated by IFN-γ for 24 h. PD-1 expression of OVA-CTLs was subsequently determined by flow cytometry. The determination results were as shown in
(6) OVA-B16 cells from conventional culture flasks or OVA-B16 TRCs from 3D fibrin gels were co-cultured with anti-CD3/CD28 bead-activated OVA-CTLs at a ratio of 1:30 for 4 h. CD45− tumor cell apoptosis was determined by flow cytometry. The determination results were as shown in
(7) OVA-B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OVA-CTLs at a ratio of 1:30 in the presence of either a neutralizing anti-PD-1 antibody or an IgG isotype control. After 4 h, cell viability was analyzed by flow cytometry. The determination results were as shown in
As could be seen from the results of Example 1, by using B16 melanoma TRCs expressing the model tumor antigen ovalbumin (OVA) and OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice, the inventors found that TRCs strongly induced anti-CD3/CD28 bead-activated OT-1 T cells to upregulate PD-1 expression during co-incubation (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) Anti-CD3/CD28 bead-activated OVA-CTLs were co-cultured with OVA-B16 cells from conventional culture flasks or OVA-B16 TRCs grown in 3D fibrin matrices for 24 hours. Kyn in the supernatant was determined by ELISA. The determination results were as shown in
(2) CD3/CD28 bead-activated OVA-CTLs were co-cultured with OVA-B16 TRCs for 24 hours in the presence of a neutralizing anti-IFN-γ antibody or an isotype control. Kyn levels in the supernatants were determined by ELISA. The determination results were as shown in
(3) OVA-B16 cells grown in culture flask or OVA-B16 TRCs were treated with IFN-γ (10 ng/ml) for 24 h. Kyn levels in the supernatants were determined by ELISA. The determination results were as shown in
(4) The isolated splenic CD8+ T cells were incubated with anti-CD3/CD28 beads in the presence of different concentrations of Kyn. PD-1 expression was determined by flow cytometry. The determination results were as shown in
(5) The experimental steps were the same as those of step (4), except that CD8+ T cells were treated with 200 μM Kyn for different time periods as shown in
(6) Anti-CD3/CD28 bead-activated CTLs were treated with PBS, 200 μM Kyn or other metabolites (500 μM) from the tryptophan pathway (melatonin, kynurinic acid and indoxyle sulphate). After 24 h, PD-1 expression was determined by flow cytometry. The determination results were as shown in
(7) Scramble- or shIDO1-(Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 T cells. After 4 h or 24 h, TRC apoptosis (as shown in
(8) Tryptophan levels in cell lysates were determined by ELISA in scramble- and shSLC1A5-(Sh1 or Sh2)-B16 TRCs in the presence or absence of IFN-γ for 24 h. The determination results were as shown in
(9) Scramble- and shSLC1A5-(Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 T cells. After 4 h or 24 h, TRC apoptosis (as shown in
(10) B16 TRCs were transiently transfected with vector or Flag-SLC1A5 plasmid. The over-expression of SLC1A5 was confirmed by western blot (left). Tryptophan levels in the supernatants were determined by ELISA and the Trp consumption was calculated (right). The confirmation results were as shown in
(11) Vec- or SLC1A5-B16 cells cultured in rigid plate were treated with PBS or IFN-γ (10 ng/ml) for 24 h. Kyn levels in the supernatants were determined by ELISA. The determination results were as shown in
As could be seen from the results of Example 2, much higher levels of Kyn were found in the supernatants of B16 TRC-T cell co-culture compared to the supernatants of differentiated B16 cell-T cell co-culture (
Meanwhile, basal IDO1 expression was much higher in all types of TRCs relative to their differentiated counterparts, and it could be further upregulated by IFN-γ. Also, when co-cultured with activated OT-1 T cells, IDO1 expression was upregulated in OVA-B16 TRCs, but TDO was not detectable. On the other hand, blocking IDO1 activity by the inhibitor 1-MT or knocking down IDO1 in OVA-B16 TRCs resulted in more TRC apoptosis in the co-culture system (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) CD8+ T cells isolated from spleen were stimulated with 200 μM Kyn or Kyn+anti-CD3/CD28 beads for 48 h. CD8+ T cells were fixed, stained with an anti-AhR antibody and imaged by confocal microscopy. The imaging results were as shown in
(2) The experimental steps were the same as those of step (1), except that the cytoplasmic and nuclear protein fractions were seperated prior to the western blot analysis of AhR. The intensity relative to that in the resting/Kyn group was calculated. The calculation results were as shown in
(3) Western blot analysis of AhR expression in both resting CD8+ T cells and anti-CD3/CD28 bead-activated CD8+ T cells treated with PBS or Kyn (200 μM) for 48 h was carried out. AhR ratio of Nuc/Cyt was calculated relative to that in the Kyn (−) group. The analysis results were as shown in
(4) CD8+ T cells activated by anti-CD3/CD28 beads were treated with PBS, Kyn or Kyn/DMF (10 or 20 μM) for 72 h. The expression of PD-1 was determined by flow cytometry. The determination results were as shown in
(5) Anti-CD3/CD28 bead-activated wild-type or AhR−/− OT-1 mice were co-cultured with OVA-B16 cells or OVA-B16 TRCs. Tumor cell apoptosis (as shown in
(6) HEK293T cells were transiently co-transfected with the luciferase reporter PGL3 coupled to the PD-1 promoter together with an AhR or an empty plasmid for 24 h and then treated with Kyn for another 48 h. The test results were as shown in
(7) Activated CD8+ T cells were treated with TCDD for 48 h and then PD-1 expression was analyzed by qPCR (left) and flow cytometry (right). The analysis results were as shown in
As could be seen from the results of Example 3, the inventors found that the addition of Kyn resulted in the translocation of cytoplasmic AhR to the nucleus of activated T cells, as demonstrated by fluorescent microscopy and western blot (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) Resting or anti-CD3/CD28 bead-activated CD8+ T cells (1×104 cells) were lysed and Kyn levels were analyzed by ELISA. The analysis results were as shown in
(2) Activated CD8+ T cells were treated with PBS or Kyn (200 μM) for 24 h. The expression of PAT4 and SLC7A8 was determined by real-time PCR (left) and immunostaining (right). The determination results were as shown in
(3) Scramble shRNA or shRNA against SLC7A8 and PAT4 were delivered via retrovirus to activated CD8+ T cells for 24 h, and then the transduced T cells were treated with Kyn (200 μM) for 48 h. Kyn levels in the supernatants were determined by ELISA and the Kyn consumption was calculated. The determination results were as shown in
(4) The experimental steps were the same as those of step (3), except that the expression of PD-1 was analyzed by flow cytometry. The analysis results were as shown in
(5) Activated OT-1 T cells were transfected with scramble shRNA, PAT4 shRNAs (Sh1 and Sh2) or SLC7A8 shRNAs (Sh1 and Sh2) for 72 h, and then co-cultured with OVA-B16 TRCs. TRC apoptosis was analyzed by flow cytometry. The analysis results were as shown in
(6) The mRNA expression of PAT4 and SLC7A8 was determined in activated CD8+ T cells treated with PBS, Kyn, Kyn/CH-223191 (10 μM) or Kyn/DMF (20 μM) for 48 h. The determination results were as shown in
(7) Resting or activated CD8+ T cells were treated with PBS or Kyn for 48 h. Chip-qPCR analysis was performed with an anti-AhR antibody and qPCR primers specific for the SLC7A8 or PAT4 promotor. The analysis results were as shown in
(8) HEK293T cells were transiently co-transfected with AhR vector and the luciferase reporter PGL3 coupled to the SLC7A8 or PAT4 promotor, and then treated with Kyn for another 48 h. The determination results were as shown in
As could be seen from the results of Example 4, the inventors went on to investigate how exogenous Kyn entered T cells. Endogenous Kyn was barely detectable in CD8+ T cells (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) CD8+ T cells were treated with Kyn (200 μM) for the time periods. The expression of PD-1 was determined by flow cytometry. The determination results were as shown in
(2) PD-1 expression was determined by flow cytometry in resting or anti-CD3/CD28 bead-activated CD8+ T cells isolated from wild type or AhR−/− mice. The determination results were as shown in
(3) PD-1 expression in resting and CD3/CD28 bead-activated CD8+ T cells was determined by flow cytometry at the time points as shown in
(4) CD8+ T cells were pretreated with anti-CD3/CD28 beads for 4 h, and then treated with PBS or Kyn for 24 h. PD-1 expression was determined by flow cytometry. The determination results were as shown in
(5) The mRNA level of SLC7A8 or PAT4 in resting or anti-CD3/CD28 bead-activated CD8+ T cells was determined. The determination results were as shown in
(6) Anti-CD3/CD28 bead-activated CD8+ T cells were treated with CsA (1 μM) for 48 h. The mRNA expression of PAT4 and SLC7A8 was determined by real-time PCR. The determination results were as shown in
(7) Activated CD8+ T cells were treated with Kyn (200 μM) in the presence or absence of CsA (1 μM) for 48 h. The cells were lysed and the Kyn level was determined by ELISA assay. The determination results were as shown in
(8) Resting or activated CD8+ T cells were treated with PBS, Kyn (200 CsA (1 μM), Kyn/CsA for 48 h. The PD-1 level was determined by flow cytometry. The determination results were as shown in
(9) Chip-qPCR analysis was performed in resting or activated CD8+ T cells with an anti-NFATc1 antibody and qPCR primers specific for the PAT4 or SLC7A8 promotor. The analysis results were as shown in
(10) Splenic APCs pulsed with OVA257-264 (2 μg/ml) were co-cultured with OT-1 CD8+ T cells in the presence of IgG or anti-PD-1 neutralizing antibody (10 μg/ml) for 72 h. IFN-γ expression was measured by flow cytometry (left). Or, CD8+ T cells from OT-1 mice were treated with Kyn (200 μM) for 72 h, then co-cultured with OVA-peptide loaded APCs and treated with or without anti-PD-1 neutralizing antibody for 72 h (right). The determination results were as shown in Fig. J. **p<0.01. The data represented mean±SEM.
As could be seen from the results of Example 5, the inventors found that the Kyn-AhR pathway and TCR signaling could induce PD-1 expression independent of each other. Despite the aforementioned PD-1 upregulation by Kyn in the presence of T cell activation signals (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) WT C57BL/6 mice were i.p. injected with PBS or Kyn (50 μg, once per day) for 5 days, and CD8+ T cells were sorted by FACS from the spleen and mesenteric lymph nodes. PD-1 expression was determined by flow cytometry (
(2) The experimental steps were the same as those of step (1), except that AhR−/− mice were used. The experimental results were as shown in
(3) PD-1 expression in resting or anti-CD3/CD28 bead-activated splenic CD8+ T cells was determined, wherein said cells were isolated from AhR−/− C57BL/6 mice injected with PBS or Kyn for 5 days. The determination results of the expression were as shown in
(4) WT or AhR−/− C57BL/6 mice were injected with or without anti-CD3 antibody (5 μg, i.p.) for different time periods and PD-1 expression was determined by flow cytometry. The determination results were as shown in
(5) C57BL/6 mice were injected with anti-CD3 antibody for 72 h, and then these mice were given Kyn (50 μg). The PD-1 expression in splenic CD8+ T cells was determined by flow cytometry after 48 h. The determination results were as shown in
(6) C57BL/6 mice with 5×5 mm OVA-B16 melanoma were adoptively transferred with CFSE-labeled OT-1 CD8+ T cells (4×106 cells/mouse, once every three days) on three occasions. At the same time, some mice were i.p. injected with Kyn (50 μg/mouse, once every two days) for three times. On Day 7 after transferring, CFSE+CD8+ T cells were isolated from tumor for flow cytometric analysis of PD-1 expression. The analysis results were as shown in
(7) 4×106 CD45.2 OT-1 T cells transfected with scramble-, shPAT4- or shSLC7A8-retrovirus were adoptively transferred into CD45.1 mice with 5×5 mm OVA-B16 melanoma for three times (once every three days). The tumor growth was measured (left) and the long-term survival rate was analyzed (right). The measurement results were as shown in
(8) The experimental steps were the same as those of step (7), except that C57BL/6 mice were treated with Kyn, DMF (5 μg/mouse, o.t., twice every day) or Kyn+DMF for 6 days. CFSE+CD8+ T cells from tumor were isolated for flow cytometric analysis of PD-1 expression. The analysis results were as shown in
(9) C57BL/6 mice with 5×5 mm B16 melanoma were treated with PBS, Kyn (50 μg/mouse, i.p., once every two days), DMF (5 μg/mouse, o.t., once every day) or Kyn+DMF for 6 days. PD-1 expression in CD8+ T cells isolated from tumors was determined by flow cytometry (n=5). The determination results were as shown in
(10) C57BL/6 mice were intraperitoneally injected with 1×106 ID8 ovarian cancer cells. After 10 days, mice were intraperitoneally administered with PBS, Kyn, DMF or Kyn+DMF for 6 days. CD8+ T cells from ascites were isolated and PD-1 expression was determined by flow cytometry (n=5). The determination results were as shown in
(11) CD45−ALDH+ and CD45−ALDH− tumor cells were sorted from spontaneously formed breast tumor in MMTV-PyMT mice. The cells were seeded into 90 Pa soft 3D fibrin gels, respectively. After 4 days, the number of colonies was counted. The counting results were as shown in
(12) CD45+, CD45−ALDH+ and CD45−ALDH− cells were lysed and the Kyn levels were determined by ELISA. The determination results were as shown in
(13) C57BL/6 mice with 5×5 mm OVA-B16 melanoma were adoptively transferred with or without OT-1 CD8+ T cells (4×106 cells/mouse) once per three days for three times. At the same time, these mice were treated with PBS, DMF (5 μg/mouse, o.t., twice every day) or anti-PD-1 neutralizing antibody (250 μg/day, once every two days, twice) for 20 days. The tumor growth was measured (left) and the long-term survival rate was analyzed (right). The measurement results were as shown in
(14) The experimental steps were the same as those of step (9), except that the tumors were weighed. The weighing results were as shown in
As could be seen from the results of Example 6, the analysis of spleen and lymph nodes showed that CD8+ T cells did not undergo proliferation but upregulated PD-1 expression on Day 5 (
Next, the inventors adoptively transferred scramble, shPAT4 or shSLC7A8 OT-1 T cells into CD45.1 mice bearing 3×3 mm OVA-B16 melanoma. The inventors found that PAT4 or SLC7A8 knockdown remarkably decreased PD-1 expression but increased the expression of IFN-γ and TNF-α in OT-1 T cells, concomitant with retarded tumor growth as well as prolonged survival of the mice (
In spontaneously formed breast cancer MMTV-PyMT mice, CD45−ALDH+ tumor cells had been identified as tumorigenic cells. The inventors further identified them as TRCs, since only CD45−ALDH+ but not CD45−ALDH− tumor cells grew into colonies in 90 Pa 3D soft fibrin gels (
The inventors co-cultured OVA-B16 TRCs, B16 TRCs, OVA-B16 cells or B16 cells with OVA-specific CD45.2 OT-1 T cells and OVA-nonspecific CD45.1 CD8+ T cells for 24 hours. The inventors found that PD-1 upregulation in non-specific CD45.1 CD8+ T cells was not induced by the above tumor cell types; PD-1 upregulation in antigen-specific OT-1 T cells was only slightly induced by OVA-B16 cells; however, PD-1 upregulation in OT-1 T cells was strikingly induced by OVA-B16 TRCs, suggesting that PD-1 expression was not only antigen-restricted but also controlled by local Kyn concentration. The adoptive transfer of OVA-specific CD8+ T cells generated the therapeutic effect against OVA-B16 melanoma in mice, which was enhanced by the administration of PD-1 antibody (
The steps of the technical solution adopted in this example and the corresponding results were as follows.
(1) CD8+ T cells were isolated from the peripheral blood of healthy volunteers (n=50) or patients with breast cancer (n=84), colon cancer (n=28) or lung cancer (n=15). Or, CD8+ T cells were isolated from breast tumor tissues (n=9) or colon tumor tissues (n=17). PD-1 expression was analyzed by flow cytometry. The analysis results were as shown in
(2) Kyn levels in the serum of healthy volunteers (n=31) and patients with breast cancer (n=73), lung cancer (n=10) or colon cancer (n=26) were measured. Or, lysates from fresh human breast tumor tissues (n=11) or colon tumor tissues (n=8) and the adjacent normal tissues were prepared and Kyn levels were determined. The determination results were as shown in
(3) The correlation between PD-1 expression and Kyn level in healthy people (left, n=37) and patients with breast cancer (right, n=62) was analyzed. The analysis results were as shown in
(4) CD8+ T cells isolated from PBMCs of patients with breast cancer or lung cancer were activated by anti-CD3/CD28 beads and treated with PBS, Kyn (200 μM) or Kyn/DMF (20 μM) for 48 h. The expression of PD-1 was determined by flow cytometry. The determination results were as shown in
(5) CD8+ T cells isolated from patients with breast cancer or lung cancer were treated with DMF (20 μM) for 48 h. PD-1 expression was determined by flow cytometry (n=6). The determination results were as shown in
(6) PD-1high CD8+ or PD-1dim CD8+ T cells were sorted from PBMCs of healthy donors or patients with breast cancer or lung cancer. The mRNA expression of AhR was analyzed by real-time PCR (n=8,
(7) Chip-qPCR analysis of AhR binding to the PD-1 promotor was performed. The analysis results were as shown in
(8) CD8+ T cells from breast cancer patients were treated with PBS, Kyn (200 Kyn/DMF (20 μM) or Kyn/CH (50 μM) for 48 h. The expression of PAT4 and SLC7A8 was determined by real-time PCR. The determination results were as shown in
(9) The mRNA expression of PAT4 or SLC7A8 in PD-1high CD8+ T cells or PD-1dim CD8+ T cells from breast cancer patients and lung cancer patients was determined. The results of expression were as shown in
(10) Chip-qPCR analysis was performed in PD-1high CD8+ T cells or PD-1dim CD8+ T cells isolated from the PBMCs of breast cancer patients with an anti-AhR antibody and qPCR primers specific for the promotor of SLC7A8 or PAT4. The analysis results were as shown in
(11) How PD-1 was upregulated in CD8+ T cells was shown in the schematic diagram. The results were as shown in
As could be seen from the results of Example 7, flow cytometric analysis showed that peripheral blood CD8+ T cells from breast, colon and lung cancer patients had higher PD-1 expression, compared to healthy donor controls (
The inventors used Kyn to treat the above CD8+ T cells from human samples in vitro with or without the stimulation of anti-CD3/CD28 microbeads. Kyn upregulated PD-1 expression in both resting and activated CD8+ T cells from healthy donors as well as breast and lung cancer patients (
The inventors compared the AhR activity between PD-1high and PD-1dim CD8+ T cells of cancer patients. The inventors found that PD-1high CD8+ T cells, sorted from the lung and breast cancer patients but not from healthy controls, expressed much higher levels of AhR mRNA than PD-1dimCD8+ T cells from the corresponding settings (
SLC7A8 and PAT4 were upregulated in human CD8+ T cells upon the addition of Kyn (
1. Experiment to Determine the Effect of GPNA on the Uptake of Tryptophan by Tumor Cells
1) Experimental Steps
Mouse OVA-B16 cells were cultured in RPMI-1640 culture medium in vitro and were group and treated as follows.
Control group 1 (PBS): culture medium+PBS;
Control group 2 (GPNA): culture medium+10 μM GPNA;
Experimental group 3 (IFN-γ): culture medium+50 ng/mL IFN-γ;
Experimental group 4 (IFN-γ+GPNA): culture medium+50 ng/mL IFN-γ+10 μM GPNA;
The cells in each group were collected after 24 h and ultrasonically broken, the concentrations of tryptophan in the lysates were determined by ELISA, and BCA protein assay was carried out at the same time.
2) Experimental Results
As could be seen from
2. Experiment to Determine the Effect of GPNA on PD-1 Expression in Specific CD8+ T Cells
1) Experimental Steps
3D-cultured OVA-B16 cells were grouped and treated with drug as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (GPNA): culture medium+10 μM GPNA;
After 12 h of treatment, the cells were co-cultured with OT-1 CD8+ T cells that had been activated for 48 h at a ratio of 1:10, and PD-1 expression in CD8+ T cells was determined by flow cytometry after 4 h.
2) Experimental Results
As could be seen from
1. Experiment to Determine the Effect of Kyn on the Expression of PAT4 and SLC7A8 in CD8+ T Cells
1) Experimental Steps
Splenic CD8+ T cells of OT-1 mice were obtained by magnetic bead sorting, and seeded into a 24-well plate (1 million cells/well) while anti-CD3/CD28 magnetic beads were added to activate T cells. The culture medium was RPMI-1640 added with 10 ng/mL mouse IL-2 and 50 nM β-mercaptoethanol. Cells were grouped and treated with drugs as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (Kyn): culture medium+200 μM Kyn;
RNA was collected from cells in each group after 48 h, and the expression of PAT4 and SLC7A8 was determined by fluorescence quantitative PCR.
2) Experimental Results
As could be seen from
SLC7A8 in mouse CD8+ T cells, indicating that the transporters PAT4 and SLC7A8 played an important role in the transport process of Kyn.
2. Experiment to Determine the Effect of SAR (Sarcosine) and BCH on PD-1 Expression in CD8+ T Cells
1) Experimental Steps
Mouse splenic CD8+ T cells were obtained by magnetic bead sorting, and seeded into a U-shaped bottom 96-well plate (100000 cells/well) while anti-CD3/CD28 magnetic beads were added to activate T cells. The culture medium was RPMI-1640 added with 10 ng/mL mouse IL-2 and 50 nM β-mercaptoethanol. Cells were grouped and treated with drugs as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (Kyn): culture medium+200 μM Kyn;
Experimental group 3 (Kyn+SAR): culture medium+200 μM Kyn+10 mM SAR;
Experimental group 4 (Kyn+BCH): culture medium+200 μM Kyn+100 μM BCH;
The expression level of PD-1 in CD8+ T cells of each group was determined by flow cytometry after 48 h.
2) Experimental Results
As could be seen from
3. Experiment to Determine the Effects of SAR and BCH on the Function of Specific CD8+ T Cells
1) Experimental Steps
Splenic CD8+ T cells of OT-1 mouse were obtained by magnetic bead sorting and seeded into a U-shaped bottom 96-well plate (100000 cells/well). Cells were grouped and treated with drugs as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (SAR): culture medium+10 mM SAR;
Experimental group 3 (BCH): culture medium+100 μM BCH;
After T cells were pretreated for 24 hours, 3D-cultured OVA-B16 cells were co-cultured with OT-1 CD8+ T cells at a ratio of 1:10, and the apoptosis of OVA-B16 cells was determined by flow cytometry after 4 hours.
2) Experimental Results
As could be seen from
1. Experiment to Determine the Effect of Kyn on the Expression of PAT4 and SLC7A8 in Human Peripheral Blood CD8+ T Cells
1) Experimental Steps
Mononuclear cells were isolated and obtained from the peripheral whole blood of breast cancer patients by using Ficoll. CD8+ T cells were sorted by magnetic beads, and seeded into a U-shaped bottom 96-well plate (100000 cells/well) while anti-CD3/CD28 magnetic beads were added to activate T cells. The culture medium was RPMI-1640 added with 10 ng/mL human IL-2. Cells were grouped and treated with drugs as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (Kyn): culture medium+200 μM Kyn;
RNA was collected from cells in each group after 48 h, and the expression of PAT4 and SLC7A8 was determined by fluorescence quantitative PCR.
2) Experimental Results
As could be seen from
2. Experiment to Determine the Effects of SAR and BCH on PD-1 Expression in Human Peripheral Blood CD8+ T Cells
1) Experimental Steps
Mononuclear cells were isolated and obtained from the peripheral whole blood of breast cancer patients by using Ficoll. CD8+ T cells were sorted by magnetic beads, and seeded into a U-shaped bottom 96-well plate (100000 cells/well) while anti-CD3/CD28 magnetic beads were added to activate T cells. The culture medium was RPMI-1640 added with 10 ng/mL human IL-2. Cells were grouped and treated with drugs as follows.
Control group 1 (PBS): culture medium+PBS;
Experimental group 2 (Kyn): culture medium+200 μM Kyn;
Experimental group 3 (Kyn+SAR): culture medium+200 μM Kyn+10 mM SAR; Experimental group 4 (Kyn+BCH): culture medium+200 μM Kyn+100 μM BCH;
The expression level of PD-1 in CD8+ T cells of each group was determined by flow cytometry after 48 h.
2) Experimental Results
As could be seen from
According to the results of Examples 8 to 10 of the present disclosure, both using the inhibitor GPNA to inhibit the tryptophan transporter in tumor cells and using the inhibitors SAR and BCH to respectively inhibit the Kyn transporters PAT4 and SLC7A8 in CD8+ T cells could effectively reduce PD-1 expression in specific CD8+ T cells and enhance the killing effect of specific CD8+ T cells on tumor cells.
The above-mentioned Examples of the present disclosure are merely exemplified to clearly illustrate the present disclosure rather than limitations to the embodiments of the present disclosure. For those of ordinary skill in the art, other changes or modifications in different forms may also be made based on the foregoing description. It is not necessary and impossible to enumerate all the embodiments. Any modification, equivalent replacement and improvement made within the spirits and principles of this disclosure shall be encompassed in the protection scope of the claims of the present disclosure.
Number | Date | Country | Kind |
---|---|---|---|
201810176164.8 | Mar 2018 | CN | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/CN2019/074548 | 2/2/2019 | WO | 00 |