Sequential treatment of cancer using 6-thio-dG, checkpoint inhibitors, and radiotherapy

Abstract

Disclosed herein are treatment methods for treating cancer with the telomerase-mediated telomere-targeting drug 6-thio-2'-deoxyguanosine (6-thio-dG), checkpoint inhibitors, and / or radiation therapy that result in innate and adaptive immune-dependent tumor regression in syngeneic and humanized mouse cancer models.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to U.S. Provisional Application No. 62 / 989,041, filed on 13 March 2020, the entire contents of which are incorporated herein by reference.

[0002] Statement of federal government grant support This invention was made with government support under grant number 2P50CA070907-21A1, awarded by the National Cancer Institute. The government has certain rights to this invention.

[0003] Areas of this disclosure This disclosure relates to the fields of medicine, pharmacology, molecular biology, and oncology. More specifically, this disclosure relates to methods and compositions for treating cancer using sequential therapy of 6-thio-dG, checkpoint inhibitors, and / or radiotherapy. [Background technology]

[0004] Background of this disclosure Immunotherapy has revolutionized the treatment of many cancers in the field of immuno-oncology (Brahmer et al., 2012; Hodi et al., 2010; Ribas and Wolchok, 2018; Topalian et al., 2012). The most commonly used immunotherapy is PD-L1 / PD-1 checkpoint blockade, which is FDA-approved for advanced cancers such as melanoma, non-small cell lung cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell carcinoma, gastric cancer, rectal cancer, and any solid tumor that cannot repair errors in its DNA that occur during replication (Garon et al., 2015; Ribas et al., 2016; Rizvi et al., 2015b; Socinski et al., 2018; National Cancer Institute). Despite the success of immunotherapy, many patients do not respond well to these therapies due to the immunosuppressive tumor microenvironment, tumor immunogenicity, and the emergence of primary and adaptive resistance (Chen and Han, 2015; Gide et al., 2018). Recent studies have shown that the abundance of tumor mutations and neoantigens partially determines the response of cancer patients to checkpoint blockade, but a significant number of patients with high levels of mutations and neoantigens still do not respond well (Le et al., 2017; Mandal et al., 2019; Rizvi et al., 2015a), suggesting that neoantigens are not sufficient to induce an antitumor immune response. Therefore, there is an urgent need to identify other factors for a better immune response and to develop novel approaches to improve overall patient survival.

[0005] The generation of an effective antitumor adaptive immune response requires the presentation of tumor antigens by antigen-presenting cells, and the activation of these antigen-presenting cells largely depends on sufficient innate sensing. Innate sensing is often provided by danger signals such as high-mobility group box-1 proteins, extracellular ATP, and tumor DNA released from stressed tumor cells (Kroemer et al., 2013; Pitt et al., 2017). Recent studies have highlighted the importance of cytoplasmic DNA sensing in radiotherapy and DNA damage therapy (Deng et al., 2014; Sen et al., 2019). For example, the presence of cytoplasmic DNA in the form of micronuclei (small DNA-containing organelles) that have lost their nuclear envelope membrane can trigger an immune response. Micronuclei are products of chromosomal damage resulting from genotoxic stress and chromosomal misseparation during cell division (Fenech et al., 2011). The cytoplasmic DNA sensor cGAS recognizes the micronucleus and converts GTP (guanosine triphosphate) and ATP (adenosine triphosphate) into the second messenger cGAMP (cyclic GMP-AMP) (Wu et al., 2013). Subsequently, the adapter protein IFN gene stimulator (STING) binds to cGAMP (Ablasser et al., 2013; Diner et al., 2013; Gao et al., 2013; Zhang et al., 2013). This complex process activates TANK-binding kinase 1 (TBK1) and IFN regulator 3 (IRF3) (Liu et al., 2015; Tanaka and Chen, 2012), further activating downstream transcription of type I IFN and other cytokines (reviewed in Li and Chen, 2018), ultimately increasing spontaneous sensing.

[0006] Eukaryotic linear chromosomes are capped by special structures called telomeres (TTAGGG), which are essential for maintaining chromosome stability (reviewed in Blackburn, 1991). Telomeres make up the last approximately 10kb of all human chromosomes and the last 12-80kb of all mouse chromosomes (Lansdorp et al., 1996; Zijlmans et al., 1997). In all somatic human cells, telomeres shorten with each cell division due to terminal replication problems and the lack of telomere maintenance mechanisms (reviewed in Greider, 1996). However, single-celled eukaryotes, germline cells, and immortal cancer cells almost always maintain their telomeres at a constant length by activating the enzyme telomerase (Greider and Blackburn, 1985; McEachern and Blackburn, 1996; Morin, 1989; Nakamura et al., 1997; Singer and Gottschling, 1994; Yu et al., 1990). Telomerase is a reverse transcriptase that elongates telomeres by adding TTAGGG repeats to the ends of chromosomes and is expressed in about 90% of human tumors but not in most normal cells (Shay and Bacchetti, 1997). Therefore, telomerase is an attractive target for developing anti-cancer therapies.

[0007] The nucleoside analog 6-thio-2'-deoxyguanosine (6-thio-dG) is a novel and effective therapeutic approach in the cancer field. Incorporation of 6-thio-dG by telomerase into de novo synthesized telomeres is known to induce damage to telomeric DNA (Mender et al., 2015a). This results in rapid tumor shrinkage or growth arrest in many tumor-derived xenograft models with minimal side effects (Mender et al., 2018; Sengupta et al., 2018; Zhang et al., 2018). The most important advantage of this telomere-targeting therapy over direct telomerase inhibitors is that 6-thio-dG does not have a long lag period for tumor killing effects. Furthermore, 6-thio-dG is not a direct telomerase inhibitor but is preferentially recognized by telomerase over other polymerases and incorporated into telomeres, resulting in immediate DNA strand termination. Importantly, the effect of 6-thio-dG is independent of the initial telomere length by hijacking tumor telomerase to create unstable telomeres (Mender et al., 2015b). Summary of the Invention

[0008] Summary of the Disclosure Thus, in one aspect of the disclosure, there is provided a method of treating cancer in a subject, comprising administering to the subject an effective amount of 6-thio-2'-deoxyguanosine (6-thio-dG) for each treatment cycle, followed by treatment with an immune checkpoint inhibitor. In some embodiments, the cancer is selected from one or more of pancreatic, lung, mesothelioma, gastric, esophageal, liver, biliary, bladder, head and neck, oral, nasopharyngeal, adult brain, colon, rectal, colorectal, prostate, ovarian, cervical, uterine, testicular, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma.

[0009] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor. In one embodiment, the immune checkpoint inhibitor is a combination of one or more CTLA-4 inhibitors, one or more PD-1 inhibitors, or one or more PD-L1 inhibitors.

[0010] In some embodiments, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, semipramab, JTx-4014, sasanalimab, budigalimab, BI754091, spartalizumab, camrelizumab, sintilimab, tislelizumab, zimberelimab, tripalimab, dostarlimab, INCMGA00012, AMP-224, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001 and AMP-515.

[0011] In some embodiments, the PD-L1 inhibitor is selected from one or more of atezolizumab, avelumab, cosibelimab, vintafusp alpha, durvalumab, MGD013, KNO35, KN046, AUNP12, CA-170 and BMS-9986189.

[0012] In some embodiments, the CTLA-4 inhibitor is selected from one or more of ipilimumab and tremelimumab.

[0013] In some embodiments of the methods disclosed herein, 6-thio-dG is administered for about 1 day to about 5 days per treatment cycle. In some embodiments, the checkpoint inhibitor is administered for about 1 day to about 3 days per treatment cycle.

[0014] As used herein, the term treatment cycle means from about 1 week to about 12 weeks between administrations of treatment.

[0015] In one embodiment of the method disclosed herein, 6-thio-dG and checkpoint inhibitors are administered in combination with chemotherapeutic agents, hormone therapy, toxin therapy, or surgery.

[0016] In another embodiment, a method for treating cancer in a subject requiring treatment is disclosed herein, comprising the step of administering 6-thio-dG to the subject, followed by treatment with cemiplimab (Libtayo®), wherein the cancer is selected from one or more of the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. In some embodiments of the method, 6-thio-dG is administered for approximately 1 to 5 days per treatment cycle. In some embodiments of the method, cemiplimab is administered for approximately 1 to 3 days per treatment cycle. In one embodiment of this method, 6-thio-dG and semiprimab are administered in combination with chemotherapy agents, hormone therapy, toxin therapy, or surgery.

[0017] In one embodiment, a method for treating cancer in a subject is disclosed herein, comprising the step of administering 6-thio-dG to the subject, followed by treatment with atezolizumab, wherein the cancer is selected from one or more of the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. In some embodiments of the method, 6-thio-dG is administered for about 1 to 5 days per treatment cycle. In some embodiments of the method, atezolizumab is administered for about 1 to 3 days per treatment cycle. In one embodiment of this method, 6-thio-dG and atezolizumab are administered in combination with chemotherapy agents, hormone therapy, toxin therapy, or surgery.

[0018] In another aspect of this disclosure, a method for treating cancer in a subject is disclosed herein, comprising the step of administering 6-thio-dG to the subject, followed by treatment with an immune checkpoint inhibitor administered in combination with radiotherapy. In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitor, a PD-1 inhibitor, or a CTAL-4 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from one or more of atezolizumab, avelumab, cosivelimab, vintrafusp alfa, durvalumab, MGD013, KNO35, KN046, AUNP12, CA-170, and BMS-9986189. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-1 inhibitor is selected from one or more of the following: pembrolizumab, nivolumab, cemiplimab, JTx-4014, sasanlimab, budigalimab, BI754091, spartalizumab, camrelizumab, cintilimab, tislerizumab, zinbererimab, tripalimab, dostallimab, INCMGA00012, AMP-224, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001, and AMP-515. In some embodiments, the PD-1 inhibitor is cemiplimab. In some embodiments, the CTLA-4 inhibitor is ipilimumab or tremelimumab. In some embodiments, the cancers treated are selected from one or more of the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. In some embodiments, the cancers treated are pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharynx cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervix cancer, testis cancer, lymphoma, leukemia, skin cancer, or breast cancer. In some embodiments, brain cancer is adult brain cancer. In some embodiments, radiotherapy is administered first, followed by the administration of one or more checkpoint inhibitors.In some embodiments, radiotherapy is administered after the administration of one or more checkpoint inhibitors.

[0019] In some aspects of the disclosed method, the cancers treated are those of the lung, colorectal, liver, melanoma, pancreas, ovary, or brain (adult).

[0020] In some aspects of the disclosed method, the cancers treated are pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, leukemia, skin cancer, or breast cancer.

[0021] In other embodiments of the disclosed method, the total dose of 6-thio-dG administered over a treatment period of approximately 1 to 5 days is approximately 10 mg to approximately 2000 mg, or approximately 15 mg to approximately 2000 mg, or approximately 20 mg to approximately 2000 mg, or approximately 10 mg to approximately 4800 mg per treatment cycle.

[0022] In one aspect of the disclosed method, the cancer being treated is metastatic.

[0023] In some aspects of the disclosed method, the cancer being treated is recurrent or will recur.

[0024] In some embodiments of the disclosed method, the cancer being treated is treatment-resistant. In one embodiment, the treatment-resistant cancer is resistant to checkpoint inhibitor treatment. In another embodiment, the treatment-resistant cancer is resistant to one or more PD-1, PD-L1, and / or CTLA-4 inhibitors. In some embodiments, the cancer is resistant to tyrosine kinase inhibitors, such as erlotinib, but is not limited to these.

[0025] In some embodiments of the methods disclosed herein, the subject to be treated has been previously treated with checkpoint inhibitor therapy. In one embodiment, the subject is PD-1 inhibitors PD-L1 inhibitors or CTLA-4 inhibitorsThe subject has been previously treated with one or more of the following. In another embodiment, the subject has been previously treated with tyrosine kinase inhibitor therapy.

[0026] In some embodiments of the methods disclosed herein, the administration of 6-thio-dG followed by treatment with a checkpoint inhibitor is repeated at least once.

[0027] In some embodiments of the methods disclosed herein, 6-thio-dG and the checkpoint inhibitor are administered systemically. In other embodiments, 6-thio-dG and the checkpoint inhibitor are administered topically or locally to the tumor site. In one embodiment, 6-thio-dG is administered topically or locally to the tumor site, and the checkpoint inhibitor is administered systemically.

[0028] In some embodiments of the methods disclosed herein, administration of 6-thio-dG and a checkpoint inhibitor results in inhibition of tumor growth.

[0029] In some aspects of the methods disclosed herein, administration of 6-thio-dG and a checkpoint inhibitor results in remission of the cancer being treated.

[0030] In some embodiments of the methods disclosed herein, administration of 6-thio-dG and one or more checkpoint inhibitors results in a reduction of tumor burden.

[0031] In some aspects of the methods disclosed herein, administration of 6-thio-dG and one or more checkpoint inhibitors results in inhibition of cancer cell metastasis.

[0032] In some embodiments of the methods disclosed herein, administration of 6-thio-dG and one or more checkpoint inhibitors results in tumor eradication.

[0033] In another aspect, a method for treating cancer in a subject is disclosed herein, comprising the step of administering a therapeutically effective dose of 6-thio-dG to the subject, followed by treatment with radiotherapy. In some embodiments, the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. In some embodiments, the cancer to be treated is pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharynx cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervix cancer, testis cancer, lymphoma, leukemia, skin cancer, or breast cancer. In some embodiments, brain cancer is adult brain cancer.

[0034] In another aspect, a method for treating cancer in a subject is disclosed herein, comprising the step of administering a therapeutically effective dose of 6-thio-dG to the subject, preceded by radiotherapy. In some embodiments, the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, brain (adult), colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. In some embodiments, the cancer to be treated is pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharynx cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, testis cancer, lymphoma, leukemia, skin cancer, or breast cancer. In some embodiments, the cancer is adult brain cancer.

[0035] In one embodiment of the method disclosed herein, the administration of 6-thio-dG and radiotherapy are repeated at least once.

[0036] Cancer may exhibit telomerase activity. 6-thio-dG and atezolizumab, avelumab, cosiverimab, vintrafusp alfa, durvalumab, MGD013, KNO35, KN046, AUNP12, CA-170, BMS-9986189, pembrolizumab, nivolumab, semiplimab, JTx-4014, sasanlimab, budigalimab, BI754091, spartalizumab, camrelizumab, cintilimab, tislerizumab, zinberelimab, PD-1, PD-L1, and CTLA-4 inhibitors such as tripalimab, dostallimab, INCMGA00012, AMP-224, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001AMP-515, ipilumumab, and tremelimumab may be administered in combination with chemotherapy, radiotherapy, hormone therapy, toxin therapy, or surgery. The daily dose of 6-thio-dG administered may range from approximately 0.15 mg / kg to approximately 70 mg / kg. The interval between administration of 6-thio-dG and administration of PD-L1, PD-1, and / or CTLA-4 inhibitors may be approximately 1 to 14 days, for example, approximately 1 to 4 days, or approximately 2 to 4 days, or approximately 2 to 5 days, or approximately 2 to 6 days, or approximately 2 to 7 days, or approximately 2 to 8 days, or approximately 2 to 9 days, or approximately 2 to 10 days, or approximately 2 to 11 days, or approximately 2 to 12 days, or approximately 2 to 13 days. The method may further include a step of evaluating telomerase activity in adult brain cancer cells from the subject. Administration of 6-thio-dG and PD-1, PD-L1, and / or CTLA-4 inhibitors may result in inhibition of tumor growth, remission of the cancer, reduction of tumor burden, inhibition of cancer cell metastasis, or eradication of the tumor.

[0037] Cancer can be pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, leukemia, or skin cancer. Cancer can be metastatic and / or recurrent and / or resistant to treatment. Treatment-resistant cancer is PD-L1 inhibitors , PD-1 inhibitors and / or CTLA-4 inhibitors This could be due to resistance to checkpoint inhibitor treatment, such as PD-L1. Inhibitor therapy , PD-1 Inhibitor therapy and / or CTLA-4 inhibitors The tumor may have been previously treated with checkpoint inhibitor therapy, such as pharmacotherapy. Administration of 6-thio-dG and subsequent treatment with PD-1, PD-L1, and / or CTLA4 inhibitors should be repeated at least once. 6-thio-dG and PD-1, PD-L1, and / or CTLA4 inhibitors may be administered systemically or locally to the tumor site. 6-thio-dG may be administered via the same or a different route than the PD-1, PD-L1, and / or CTLA4 inhibitors.

[0038] [Invention 1001] A process comprising administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, followed by treatment with an immune checkpoint inhibitor. A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. [Invention 1002] The method of the present invention 1001, wherein the immune checkpoint inhibitor is a PD-1 inhibitor. [Invention 1003] The method of the present invention 1001, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor. [Invention 1004] The method of the present invention 1001, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor. [Invention 1005] The method of the present invention 1001, wherein the immune checkpoint inhibitor is a combination of one or more CTLA-4 inhibitors and one or more PD-1 inhibitors. [Invention 1006] The method of the present invention 1001, wherein the immune checkpoint inhibitor is a combination of one or more CTLA-4 inhibitors and one or more PD-L1 inhibitors. [Invention 1007] The method according to any of items 1001 to 1006 of the present invention, wherein the administration of 6-thio-dG is for approximately 1 to 5 days per treatment cycle. [Invention 1008] A method according to any one of the present invention 1001 to 1006, wherein a checkpoint inhibitor is administered for approximately 1 to 3 days per treatment cycle. [Invention 1009] A method according to any one of the present invention 1001 to 1008, wherein 6-thio-dG and a checkpoint inhibitor are administered in combination with a chemotherapeutic agent, hormone therapy, toxin therapy, or surgery. [Invention 1010] A process of administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, followed by treatment with semiprimab (Libtayo®). A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. [Invention 1011] The method of the present invention 1010, wherein 6-thio-dG is administered for approximately 1 to 5 days per treatment cycle. [Invention 1012] The method according to either 1010 or 1011 of the present invention, wherein semiprimab (Libtayo®) is administered for approximately 1 to 3 days per treatment cycle. [Invention 1013] A method according to any one of items 1010 to 1012 of the present invention, wherein 6-thio-dG and semiprimab (Libtayo®) are administered in combination with a chemotherapy agent, hormone therapy, toxin therapy, or surgery. [Invention 1014] A process comprising administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, followed by treatment with an immune checkpoint inhibitor administered in combination with radiotherapy. A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. [Invention 1015] A method according to any of items 1001 to 1014 of the present invention, wherein the total dose of 6-thio-dG administered over a treatment period of approximately 1 to 5 days is approximately 20 to 2000 mg. [Invention 1016] The method according to any one of items 1001 to 1014 of the present invention, wherein the cancer is of the lung, colorectal, liver, melanoma, or glioblastoma. [Invention 1017] The method according to any of the present invention 1001 to 1014, wherein the cancer is metastatic. [Invention 1018] The method of any of the present invention 1001 to 1014 wherein the cancer is recurrent. [Invention 1019] A method according to any of the present invention 1001 to 1014, wherein the cancer is resistant to treatment. [Invention 1020] The method of the present invention 1010, wherein the treatment-resistant cancer is resistant to checkpoint inhibitor treatment. [Invention 1021] The method of the present invention 1001, wherein the treatment-resistant cancer is resistant to one or more PD-1, PD-L1, and CTLA-4 inhibitors. [Invention 1022] Any method 1001 to 1009 of the present invention, wherein the subject has been previously treated with checkpoint inhibitor therapy. [Invention 1023] The method of the present invention 1022, wherein the subject has been previously treated with one or more of the PD-1, PD-L1, and CTLA-4 therapies. [Invention 1024] A method according to any one of items 1001 to 1023 of the present invention, wherein the administration of 6-thio-2-deoxyguanosine (6-thio-dG) and subsequent treatment with a checkpoint inhibitor are repeated at least once. [Invention 1025] A method according to any one of the present invention 1001 to 1023, wherein 6-thio-dG and a checkpoint inhibitor are administered systemically. [Invention 1026] A method according to any one of items 1001 to 1023 of the present invention, wherein 6-thio-dG and a checkpoint inhibitor are administered locally or regionally to the tumor site. [Invention 1027] A method according to any one of the present invention 1001 to 1023, wherein 6-thio-dG is administered locally or locally to the tumor site, and a checkpoint inhibitor is administered systemically. [Invention 1028] A method according to any one of the present invention 1001 to 1023, wherein administration of 6-thio-dG and a checkpoint inhibitor results in inhibition of tumor growth. [Invention 1029] A method according to any one of the present invention 1001 to 1023, wherein administration of 6-thio-dG and a checkpoint inhibitor results in remission of the cancer. [Invention 1030] Any method 1001 to 1023 of the present invention, wherein administration of 6-thio-dG and a checkpoint inhibitor results in a reduction of tumor burden. [Invention 1031] A method according to any one of the present invention 1001 to 1023, wherein administration of 6-thio-dG and a checkpoint inhibitor results in inhibition of cancer cell metastasis. [Invention 1032] Any method 1001 to 1023 of the present invention, wherein administration of 6-thio-dG and a checkpoint inhibitor results in tumor eradication. [Invention 1033] A process of administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, followed by treatment with radiotherapy. A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. [Invention 1034] A process of administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, wherein radiotherapy treatment is performed prior to the administration of 6-thio-2'-deoxyguanosine (6-thio-dG). A method for treating cancer in a subject, wherein the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. [Invention 1035] The method of the present invention 1029 or 1030, wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, brain cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, testicular cancer, lymphoma, leukemia, skin cancer, and breast cancer. [Invention 1036] A method according to any one of items 1029 to 1031 of the present invention, wherein the administration of 6-thio-2-deoxyguanosine (6-thio-dG) and radiotherapy are repeated at least once. [Invention 1037] A process comprising administering 6-thio-2'-deoxyguanosine (6-thio-dG) to a target, followed by treatment with an immune checkpoint inhibitor and radiotherapy. A method for treating cancer in a subject, including the following. In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitor, a PD-1 inhibitor, or a CTAL-4 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from one or more of atezolizumab, avelumab, cosivelimab, vintrafusp alfa, durvalumab, MGD013, KNO35, KN046, AUNP12, CA-170, and BMS-9986189. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-1 inhibitor is selected from one or more of the following: pembrolizumab, nivolumab, semiprimab, JTx-4014, sasanlimab, budigalimab, BI754091, spartalizumab, camrelizumab, cintilimab, tislerizumab, zinbererimab, tripalimab, dostallimab, INCMGA00012, AMP-224, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001, and AMP-515. In some embodiments, the PD-1 inhibitor is cemiprimab (Libtayo®) administered in combination with radiotherapy, and the cancer is selected from the group consisting of pancreatic, lung, mesothelioma, stomach, esophagus, liver, biliary tract, bladder, head and neck, oral cavity, nasopharynx, adult brain, colon, rectum, colorectal, prostate, ovary, cervix, uterus, testis, lymphoma, leukemia, skin, breast, kidney, neuroblastoma, Merkel cell carcinoma, myelodysplastic syndrome, myelofibrosis, and multiple myeloma. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating specific aspects of this disclosure, are given only as examples, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description.

[0039] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure may be better understood by referring to one or more of these drawings in conjunction with a detailed description of the particular aspects presented herein. [Brief explanation of the drawing]

[0040] (Figure 1A) Figures 1A-1G. The therapeutic effect of 6-thio-dG depends on CD8+ T cells. (Figure 1A) Cell viability of 6-thio-dG in MC38 cells (IC 50Cells were treated with 6-thio-dG for 5 days. (Figures 1B and 1C) Colony formation assay of 6-thio-dG in MC38 cells at indicated doses for 13 days. Cells were treated with 6-thio-dG every 3 days, then fixed and stained with crystal violet. Representative images of three biological replicates are shown in Figure 1B, and quantified data are shown in Figure 1C. (Figures 1D and 1E) WT (Figure 1D) or Rag1- / - (Figure 1E) C57BL / 6 mice (n=5) were treated with 5 × 10⁶ cells. 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). (Figure 1F and Figure 1G) 5 × 10⁶ C57BL / 6 mice (n=5) were inoculated. 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). 200 μg of anti-CD4 (Figure 1F) or anti-CD8 (Figure 1G) was administered one day prior to treatment, followed by twice-weekly administration for three weeks. Tumor growth was measured every three days. Data are shown as mean ± SEM from 2-3 independent experiments. p-values ​​were determined by unpaired two-sided t-tests (Figure 1C) or two-way ANOVA (Figures 1D-G). See also Figures 9A-9D. (Figure 1B) See the explanation for Figure 1A. (Figure 1C) See the explanation for Figure 1A. (Figure 1D) See the explanation for Figure 1A. (Figure 1E) See the explanation for Figure 1A. (Figure 1F) See the explanation in Figure 1A. (Figure 1G) See the explanation for Figure 1A. (Figure 2A) Figures 2A to 2F. 6-thio-dG treatment increases tumor-specific T cell response. (Figures 2A and 2B) C57BL / 6 mice (n=4-5) were treated with 5 × 10⁻¹⁴⁻¹ 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Six days after the last treatment, tumor-infiltrating T cells were analyzed for the frequency of total T cells (Figure 2A) and Ki67+CD8+ T cells (Figure 2B). (Figure 2C) C57BL / 6 mice (n=5) with MC38-OVA tumors were treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Three days after the last treatment, H-2Kb -OVA 257~264 For OVA-specific CD8+ T cells containing tetramers, tumor infiltrating T cells were analyzed. (Figs. 2D and 2E) Using the same experimental scheme as in (A), splenocytes were collected and restimulated with irradiated MC38 tumor cells for 48 hours. IFN-γ-producing cells were determined by ELISPOT assay. Representative spots are shown in Fig. 2D, and quantitative data (n = 5) are shown in Fig. 2E. (Fig. 2F) 5 × 10 5 MC38 tumor cells were inoculated into IFN-γ reporter mice (n = 3) and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Eleven days after the last treatment, tumors were minced and digested for flow cytometric detection of YFP+ T cells. p-values were determined by unpaired two-sided t-test (Figs. 2A - C, 2E, and 2F). See also Figs. 10 A ~F. (Fig. 2B) See the description of Fig. 2A. (Fig. 2C) See the description of Fig. 2A. (Fig. 2D) See the description of Fig. 2A. (Fig. 2E) See the description of Fig. 2A. (Fig. 2F) See the description of Fig. 2A. (Fig. 3A) Figs. 3A - 3F. 6-thio-dG treatment enhances the cross-priming ability of dendritic cells. (Fig. 3A) 5 × 10 5 MC38 tumor cells were inoculated into C57BL / 6 mice (n = 5) and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). 200 μg of anti-CSF1R was administered 1 day before the start of treatment and then twice weekly for 3 weeks. (Fig. 3B) 5 × 10 5MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Tumor growth was measured every 3 days. (Figure 3C) Percentage of tumor-free mice in WT and Batf3- / - mice (n=5) after 6-thio-dG treatment. (Figure 3D) BMDCs were cultured with MC38 tumor cells pretreated overnight with 200 nM 6-thio-dG or vehicle, then the DCs were purified and co-cultured with naive OT-1 T cells. After 48 hours, the supernatant was collected and tested for IFN-γ production by cytometry bead array (CBA). (Figure 3E) BMDCs were cultured with MC38 tumor cells pretreated for 18 hours with 200 nM 6-thio-dG or vehicle, and the supernatant was collected for IFN-β ELISA. (F) 5 × 10⁶ cells were cultured in Ifnar1- / - mice (n=5). 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Tumor growth was measured every 3 days. Data are shown as mean ± SEM from 2-3 independent experiments. The p-value was determined by two-way ANOVA (Figures 3A, 3B, and 3F) or unpaired two-sided t-test (Figures 3C-E). (Figure 3B) See the explanation for Figure 3A. (Figure 3C) See the explanation for Figure 3A. (Figure 3D) See the explanation in Figure 3A. (Figure 3E) See the explanation for Figure 3A. (Figure 3F) See the explanation for Figure 3A. (Figure 4A) Figures 4A-4G. STING signaling in the host is required for innate sensing induced by 6-thio-dG. (Figures 4A and 4B) 5 × 10⁻¹⁵ mice (n=5) of Myd88- / - (Figure 4A) or Tmem173- / - (Figure 4B) were given 5 × 10⁻¹⁵ units. 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Tumor growth was measured every 3 days. (Figures 4C and 4D) 5 × 10⁶ C57BL / 6 mice (n=5) 5Individual Tmem173KO (Figure 4C) or Mb21d1KO (Figure 4D) MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Tumor growth was measured every 3 days. (Figures 4E and 4F) MC38 tumor cells were treated with 1 μM 6-thio-dG for 24 hours. A TIF (telomere dysfunction-induced lesion) assay was used to confirm the induction of TIF in MC38 cells by 6-thio-dG treatment. n=100 (control), n=100 (6-thio-dG). (Figure 4G) BMDCs were cultured with HCT116 human colon cancer cells pretreated with 500 nM 6-thio-dG or vehicle for 4 hours, then the DCs were purified and cytoplasmic DNA was extracted. The relative abundances of MT-CO1 and human 18S in the cytoplasm of the DCs were detected by qPCR. Data are presented as the mean ± SEM of 2-3 independent experiments. p-values ​​were determined by two-way ANOVA (A-D) or unpaired two-tailed t-tests (Figures 4F and 4G). See also Figures 11A-11H. (Figure 4B) See the explanation for Figure 4A. (Figure 4C) See the explanation for Figure 4A. (Figure 4D) See the explanation for Figure 4A. (Figure 4E) See the explanation for Figure 4A. (Figure 4F) See the explanation for Figure 4A. (Figure 4G) See the explanation for Figure 4A. (Figure 5A) Figures 5A-5F. 6-thio-dG overcomes PD-L1 blockade resistance in an advanced tumor model. (Figure 5A) C57BL / 6 mice (n=4-5) with MC38 tumors were treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Seven days after the first treatment, PD-1+CD8+ T cell frequency (left) and PD-1 MFI (right) were tested. (Figures 5B and 5C) C57BL / 6 mice (n=5) were subjected to 5 × 10⁶ treatment. 5MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 10 and 11). 50 μg of anti-PD-L1 antibody was administered on days 13 and 17. Tumor growth (Figure 5B) and viability (Figure 5C) were shown. (Figure 5D) C57BL / 6 mice (n=5) with MC38 tumors were treated with 6-thio-dG (3 mg / kg, days 10 and 11), anti-PD-L1 (2.5 kg / mg, day 10), or a combination of both. Seven days after the initial treatment, the afferent lymphoid system was harvested and stimulated with MC38 tumor cells or LLC tumor cells irradiated for IFN-γ ELISPOT. (Figures 5E and 5F) 1 × 10⁶ C57BL / 6 mice (n=5) were treated. 6 LLC mouse lung tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 4, 5, 6, and 10, 11). 200 μg of anti-PD-L1 antibody was administered on days 8 and 13. Tumor growth was measured every 3-4 days (Figure 5E). After 6 weeks, tumor-free mice (n=4) and control mice in the sequential treatment group were given 5 × 10⁶ cells. 6 Individual LLCs (right flank) and 5 × 10 6 Individual MC38 (left flank) tumor cells were reloaded. Tumor growth was measured every 3–4 days (Figure 5F). Data are shown as the mean ± SEM of two independent experiments. The p-value was determined by an unpaired two-sided t-test (Figures 5A, 5D), two-way ANOVA (Figures 5B, 5E, and 5F), or log-rank test (Figure 5C). See also Figure 12. (Figure 5B) See the explanation for Figure 5A. (Figure 5C) See the explanation for Figure 5A. (Figure 5D) See the explanation for Figure 5A. (Figure 5E) See the explanation for Figure 5A. (Figure 5F) See the explanation for Figure 5A. (Figure 6A) Figures 6A-6E. 6-thio-dG reduces human colon cancer burden in a humanized mouse model. (Figure 6A) Overall survival in patients with high and low TERT (telomerase reverse transcriptase, a catalytic subunit of telomerase) expression of colorectal adenocarcinoma, from the TCGA database. (Figure 6B) Cell viability of 6-thio-dG in HCT116 human colon cancer cells (IC 50). Cells were treated with 6-thio-dG for 5 days. (Figure 6C) Schematic diagram of a humanized mouse tumor model. (Figures 6D and 6E) 1 × 10⁻¹⁶ cells in NSG-SGM3 mice (n=5) (Figure 6D) or humanized NSG-SGM3 mice (n=4) (Figure 6E). 6 Individual HCT116 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 8, 9, and 10). Tumor growth was measured every 3 days. Data are shown as the mean ± SEM of two independent experiments. The p-value was determined by log-rank test (Figure 6A) or two-way ANOVA (Figures 6D and 6E). See also Figures 13A–13F. (Figure 6B) See the explanation for Figure 6A. (Figure 6C) See the explanation for Figure 6A. (Figure 6D) See the explanation for Figure 6A. (Figure 6E) See the explanation for Figure 6A. (Figure 7) Schematic diagram of 6-thio-dG induction by c-GAS / STING / IFN. (Figure 8) Figures 8A to 8B. 6-thio-dG and subsequent PD-L1 inhibitors This is evidence that it leads to complete tumor remission and immunogenic memory. (Figure 9A) Figures 9A to 9D (related to Figures 1A to 1G). (Figure 9A) Cell viability of 6-thio-dG in LLC mouse lung cancer cells (IC 50 Cells were treated with 6-thio-dG for 4 days. (Figure 9B) 1 × 10⁻¹⁶ C57BL / 6 mice (n=5) 6 LLC tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 4, 5, and 6). Tumor growth was measured every 3 days. (Figure 9C) IC6 of 6-thio-dG in CT26 mouse colon cancer cells 50 (Figure 9D) BALB / C mice (n=5) with 5 × 10 5 Individual CT26 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 5, 6, and 7). Tumor growth was measured every 3 days. Data are presented as the mean ± SEM of two independent experiments. The p-value was determined by two-way ANOVA. (Figure 9B) See the explanation for Figure 9A. (Figure 9C) See the explanation for Figure 9A. (Figure 9D) See the explanation for Figure 9A. (Figure 10A) Figures 10A to 10F (related to Figures 2A to 2G). (Figures 10A to 10D) C57BL / 6 mice (n=4 to 5), 5 × 10 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Seven days after the initial treatment, the tumor was analyzed for CD8+ T cells among CD45+ cells (Figure 10A) and CD8+ T cells among all tumor cells (Figure 10B), and CD4 + Foxp3 + Tumor-infiltrating T cells were analyzed for the frequency of Treg cells (Figure 10C) and NK cells (Figure 11D). (Figure 10E) 5 × 10⁶ C57BL / 6 mice (n=5) 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). 200 μg of anti-NK1.1 was administered one day prior to the start of treatment, followed by twice-weekly administration for 3 weeks. (Figure 10F) IFN-γ reporter mice (n=3) were given 5 × 10⁶ doses. 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7, 8, and 9). Eleven days after the last treatment, the tumors were finely chopped and digested for flow cytometry detection of YFP+ T cells. Representative flow cytometry gating was shown. Data are shown as the mean ± SEM of two independent experiments. The p-value was determined by an unpaired two-sided t-test (Figures 10A-D) or two-way ANOVA (Figure 10E). (Figure 10B) See the explanation for Figure 10A. (Figure 10C) See the explanation for Figure 10A. (Figure 10D) See the explanation for Figure 10A. (Figure 10E) See the explanation for Figure 10A. (Figure 10F) See the explanation for Figure 10A. (Figure 11A) Figures 11A to 11H (related to Figures 4A to 4G). (Figure 11A) BMDCs were cultured with MC38 tumor cells pretreated with 0.2 μM or 1 μM 6-thio-dG for 6 hours, then the DCs were purified with magnetic beads and subjected to Western blotting. (Figure 11B) BMDCs from wild-type (WT) or Tmem173KO mice were cultured with MC38 tumor cells pretreated overnight with 200 nM 6-thio-dG, then the DCs were purified with magnetic beads, and the relative abundance of IFN-β was tested by qPCR. (Figures 11C and 11D) 5 × 10⁶ C57BL / 6 mice (n=3) 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 10, 11, and 12). Three days after the last injection, the mice were sacrificed; tumors were collected and fixed for TIF (telomere dysfunction-induced lesion) staining. Images were obtained by fluorescein microscopy (100x). Red dots indicate DNA damage (γ-H2AX), green dots indicate telomeres, and yellow dots indicate TIF (DNA damage on telomeres). Scale bar, 10 μM. (Figures 11E and 11F) 6-thio-dG treatment induced micronuclei in MC38 cells. (Figure 11E) Representative images of two daughter cells in anaphase MC38 cells show telomere signals as well as coated and uncoated micronuclei. Green dots represent telomere signals, and red represents lamin A / C (nuclear envelope biomarkers). (Figure 11F) Quantification of micronuclei induced by 1 μM 6-thio-dG treatment after 48 hours. (Figures 11G and 11H) 100,000 MC38 cells were seeded in a 6-well plate and labeled with 25 μM EdU. After 2 days, the cells were washed and incubated overnight in fresh medium with 1 μM 6-thio-dG. The cells were then washed and co-cultured overnight with DCs. The next day, DCs were purified with magnetic beads. The purified DCs were then fixed and cytospinned for immunoFISH. Telomere probe: green, EdU: red, DAPI: blue. Images were captured at 63x magnification using an Axio Imager Z2 with an automated acquisition system and analyzed with ISIS software (camera: coolcube 1-metasystems). Representative imaging (Figure 11G) and quantification data (Figure 11H) are shown, n=100. Data are presented as the mean ± SEM of 2-3 independent experiments. p-values ​​were determined by unpaired two-sided t-tests (B, F, and H). (Figure 11B) See the explanation for Figure 11A. (Figure 11C) See the explanation for Figure 11A. (Figure 11D) See the explanation for Figure 11A. (Figure 11E) See the explanation for Figure 11A. (Figure 11F) See the explanation for Figure 11A. (Figure 11G) See the explanation for Figure 11A. (Figure 11H) See the explanation for Figure 11A. (Figure 12) Figure 12 (related to Figures 5A to 5G). 5 × 10⁻¹⁴ C57BL / 6 mice (n=5) 5 Individual MC38 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 10 and 11). 50 μg of anti-PD-L1 antibody was administered on days 13 and 17. Mice were weighed. Data are shown as mean ± SEM. (Figure 13A) Figures 13A-13F (related to Figures 6A-6E). (Figures 13A-13C) Human CD45+ cells and CD3+ T cells in mouse peripheral blood were examined by flow cytometry 12 weeks after humanized mouse rearrangement. Representative flow cytometry plots are shown in Figure 13A. The frequencies of CD45 and CD3 in the pre-treatment control group and the 6-thio-dG group are shown in Figures 13B and 13C, n=5. (Figure 13D) A375 Cell viability of 6-thio-dG in human melanoma cancer cells (IC 50 ). Cells were treated with 6-thio-dG for 4 days. (Figure 13E) 2 × 10⁻¹⁶ cells were given to NSG-SGM3 mice (n=5). 6 Individual A375 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 7 and 8), anti-PD-L1 + anti-CTLA-4 (200 μg intraperitoneally, days 10 and 13), or a combination of 6-thio-dG + anti-PD-L1 and anti-CTLA-4. Tumor growth was measured every 3 days. (Figure 13F) Humanized NSG-SGM3 mice (n=5~7) were given 2 × 10⁶ cells. 6Individual A375 tumor cells were inoculated and treated with 6-thio-dG (3 mg / kg, days 13 and 14), anti-PD-L1 + anti-CTLA-4 (200 μg intraperitoneally, days 16 and 19), or a combination of 6-thio-dG + anti-PD-L1 and anti-CTLA-4. Tumor growth was measured every 3 days. Data are shown as mean ± SEM. P-values ​​were determined by unpaired two-sided t-tests (Figures 13B and 13C, non-significant p>0.05) or two-way ANOVA (Figure 13F). (Figure 13B) See the explanation for Figure 13A. (Figure 13C) See the explanation for Figure 13A. (Figure 13D) See the explanation for Figure 13A. (Figure 13E) See the explanation for Figure 13A. (Figure 13F) See the explanation for Figure 13A. (Figure 14) Figure 14 shows the effect of the anti-PD-1 agent semiprimab (Libtayo®) and 6-thio-dG on tumor volume in mice with LLC cell-derived tumors (NSCLC). The administration was 3 mg / kg of 6-thio-dG (intraperitoneal) and 10 mg / kg of semiprimab (intraperitoneal). Different groups were administered as shown in the table below. Day 1 (December 31, 2020): 35 B6 mice were inoculated with 1000K LLC cells. Days 11-13: Experiment started. 3 mg / kg of 6-thio-dG and 10 mg / kg of Libtayo were used in this study. (Table A) Dosage schedule Figure 15 (TIFF0007829491000001.tif54167) shows the effect of the anti-PD-1 agent semiprimab (Libtayo®) and 6-thio-dG on tumor volume in mice with LLC cell-derived tumors (NSCLC). Administration was 3 mg / kg of 6-thio-dG (intraperitoneal) and 10 mg / kg of semiprimab (intraperitoneal). Different groups were administered as shown in the table above. Day 1 (December 31, 2020): 35 B6 mice were inoculated with 1000K LLC cells. Days 11-13: Experiment started. 3 mg / kg of 6-thio-dG and 10 mg / kg of Libtayo were used in this study. (Figure 16) Figure 16 shows the effect of 6-thio-dG in combination with the PD-1 agent pembrolizumab in a humanized mouse model of small cell lung cancer (SCLC). (Figure 17) Figure 17 shows 6-thio-dG in combination with PD-L1 inhibitors and radiation in an HCC mouse model. (Figure 18A) Figures 18A to 18D show PD-L1 in the HCC mouse model. inhibitors And 6-thio-dG in combination with radiation is shown. Figure 18A: Dosage schedule. (Figure 18B) Figures 18A to 18D show PD-L1 in the HCC mouse model. inhibitors Figure 18B shows 6-thio-dG in combination with radiation. HCC53N liver cancer cells (p53 and NRAS knockout) treated in vivo with an initial local IR, followed by three doses of 6-thio-dG, and then two treatments with anti-PD-L1 antibody resulted in complete tumor remission. (Figure 18C) Figures 18A to 18D show PD-L1 in the HCC mouse model. inhibitors Figure 18C shows 6-thio-dG in combination with radiation. Although the tumor was reloaded with 10 times more HCC53N cells, it did not regrow, suggesting immunological memory. (Figure 18D) Figures 18A to 18D show PD-L1 in the HCC mouse model. inhibitors And 6-thio-dG in combination with radiation is shown. Figure 18D When naive mice were tested, the tumors grew rapidly. [Modes for carrying out the invention]

[0041] Detailed explanation Telomerase is almost universally expressed in tumor cells. 6-thio-dG, a telomere-targeting drug mediated by telomerase, directly induces telomere damage in telomerase-positive cancer cells but not in normal cells where telomerase is silent, thereby reducing the delay between initial treatment and the response to therapy. In this study, we aimed to investigate whether 6-thio-dG, which induces telomere stress in telomerase-positive cancer cells, can initiate rapid DNA damage for spontaneous sensing. Using syngeneic wild-type and gene-deficient mice, we evaluated how 6-thio-dG triggers spontaneous sensing and how it contributes to host anti-tumor immunity. Importantly, we demonstrate that 6-thio-dG overcomes PD-L1 blockade resistance in advanced tumors. Unexpectedly, 6-thio-dG induced activation of DNA-mediated spontaneous sensing and immune responses in a host STING-dependent manner, resulting in an improved anti-tumor effect. Furthermore, 6-thio-dG, followed sequentially by anti-PD-L1 therapy, can completely eliminate advanced tumors. Therefore, 6-thio-dG is a tumor-targeted immunostimulant that can benefit patients with telomerase-positive and PD-L1-resistant cancers in clinical practice.

[0042] These and other aspects of this disclosure are described in detail below.

[0043] I. Telomeres, telomerase, and telomere dysfunction During mitosis, a cell makes copies of its genetic material. Half of the genetic material is transferred to each new daughter cell. To ensure that information is successfully transmitted from one generation to the next, each chromosome has a special protective cap called a telomere located at the end of its "arm." Telomeres are controlled by the presence of the enzyme telomerase.

[0044] Telomeres are repetitive DNA sequences (e.g., TTAGGG) at the ends of chromosomes in the body. Telomeres can reach a length of 15,000 base pairs. Telomeres function by preventing chromosomes from losing base pairs at their ends. They also prevent chromosomes from fusing together. However, with each cell division, some telomeres are lost (usually 25-200 base pairs per division). If telomeres become too short, the chromosomes reach a "critical length" and can no longer replicate. This means the cell ages and dies or deteriorates through a process called apoptosis. Telomere activity is controlled by two mechanisms: erosion and addition. Erosion, as mentioned above, occurs with each cell division because lagging strand DNA synthesis cannot be completed. Addition is determined by the activity of telomerase.

[0045] Telomerase, also known as telomere terminal transferase, is an enzyme composed of protein and RNA subunits that elongates chromosomes by adding a TTAGGG sequence to the ends of existing chromosomes. Telomerase is found in fetal tissue, adult germ cells, and tumor cells. Telomerase activity is regulated during development and is extremely low, almost undetectable, in somatic cells. These somatic cells age because they do not normally use telomerase. Cellular aging is the result of bodily aging. If telomerase is activated within a cell, the cell continues to grow and divide. This "immortal cell" theory is important in two research areas: aging and cancer.

[0046] Cellular aging, or senescence, is the process by which cells age, cease to grow, or die. Cellular aging is caused by the shortening of telomeres on chromosomes until the chromosomes reach a critical length. Cellular aging is like a wind-up clock. If the clock is kept wound, the cell becomes immortal and constantly produces new cells. If the clock is unwound, the cell stops producing new cells and experiences what is called replication senescence, or it dies. Cells are constantly aging. If we could extend the replication capacity of the cells in the body, it would certainly create some interesting possibilities, especially for diseases associated with the inheritance of short telomeres (called telomeropathy or telomere spectrum disorders). Therefore, telomerase research can yield important discoveries related to the aging process.

[0047] Cancer cells become malignant by evading the normal short-telomere aging process. Malignant cells grow uncontrollably, proliferating to form tumors that spread to distant tissues throughout the body. Telomerase is detected in almost all human cancer cells. This confers the advantage of selective growth to many types of tumors. If telomerase activity is inhibited, telomeres in cancer cells will gradually shorten, similar to telomeres in normal body cells. This prevents cancer cells from dividing uncontrollably in the early stages of development. If a tumor is already fully developed, it can be removed, and anti-telomerase therapy can be administered to prevent recurrence. Essentially, preventing telomerase from performing its function will transform cancer cells from immortal to lethal. However, direct telomerase inhibitors require a delay period from the start of treatment until tumor reduction occurs, and their clinical development has not progressed much due to increased toxicity. Therefore, the present invention provides a method that shortens the delay period, requires effective telomerase activity, and potentially reduces side effects.

[0048] II. Treating cancer A. Therapeutic agents for sequential treatment 1. In some embodiments, the PD-L1 inhibitor is selected from one or more of atezolizumab, avelumab, cosivelimab, vintrafusp alfa, durvalumab, MGD013, KNO35, KN046, AUNP12, CA-170, and BMS-9986189. In some embodiments, the PD-L1 inhibitor is atezolizumab.

[0049] Atezolizumab (brand name Tecentriq®) is a fully humanized, engineered monoclonal antibody of the IgG1 isotype against the protein, programmed cell death ligand 1 (PD-L1). In 2015, atezolizumab was in clinical trials as an immunotherapy for several types of solid tumors. In May 2016, atezolizumab was approved by the FDA for the treatment of bladder cancer, but in May 2017, it failed a Phase III trial for secondary bladder cancer. In October 2016, the FDA approved atezolizumab for the treatment of urothelial carcinoma and metastatic non-small cell lung cancer (NSCLC) patients whose disease has progressed during or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor abnormalities should have had disease progression at the time of FDA-approved treatment for these abnormalities before receiving atezolizumab. In September 2018, research findings presented at the 19th World Conference on Lung Cancer (WCLC) in Toronto, Canada, showed that atezolizumab extends survival in patients with advanced small cell lung cancer. In October 2018, a clinical trial of atezolizumab in combination with nab-paclitaxel in patients with advanced triple-negative breast cancer was completed. In March 2019, as determined by the FDA-approved trial, atezolizumab was approved in the United States in combination with protein-conjugated paclitaxel for adult patients with unresectable locally advanced or metastatic triple-negative breast cancer (TNBC) whose tumor expresses PD-L1 (PD-L1 stained tumor-infiltrating immune cells of any intensity cover 1% or more of the tumor area). In March 2019, atezolizumab was approved in the United States as a first-line treatment for adult patients with advanced small cell lung cancer (ES-SCLC) in combination with carboplatin and etoposide. The most common adverse effects in the study were fatigue, loss of appetite, nausea, and infection. Urinary tract infection was the most common severe adverse effect.

[0050] Atezolizumab blocks the interaction of PD-L1 with programmed cell death protein 1 (PD-1) and the CD80 receptor (B7-1R). PD-L1 can be highly expressed on certain tumors, and its absence is thought to lead to reduced activation of immune cells (particularly cytotoxic T cells) that can recognize and attack cancer. Inhibition of PD-L1 by atezolizumab can eliminate this inhibitory effect, thereby inducing an antitumor response. This is one of several methods to block inhibitory signals associated with T cell activation, a more common strategy known as immune checkpoint inhibition. For some cancers (particularly bladder), the probability of benefiting is related to PD-L1 expression, but most cancers with PD-L1 expression still do not respond, while some cancers without PD-L1 expression (about 15%) do respond.

[0051] Avelumab (Bavencio®) is a fully human IgG1 antibody developed by Merck Serono and Pfizer. Avelumab is approved by the FDA for the treatment of metastatic Merkel cell carcinoma. Avelumab failed in a Phase III clinical trial for gastric cancer.

[0052] Durvalumab (Imfinzi®) is a fully human IgG1 antibody developed by AstraZeneca. Durvalumab is approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer following concurrent chemoradiotherapy.

[0053] KN035 is the only PD-L1 antibody currently available in a subcutaneous formulation undergoing clinical evaluation in the United States, China, and Japan.

[0054] AUNP12 is the first cancer treatment developed by Aurigene and Laboratoires Pierre Fabre to be evaluated in clinical trials for treating cancer. peptide This is a 29-mer peptide used as a PD-1 / PD-L1 inhibitor.

[0055] CA-170, discovered by Aurigene / Curis as a PD-L1 and VISTA antagonist, is currently in Phase I clinical trials for the treatment of mesothelioma.

[0056] 2. PD-1 inhibitors such as semiprimab, pembrolizumab, nivolumab, JTx-4014, sasanlimab, budigalimab, BI754091, spartalizumab, camrelizumab, cintilimab, tislerizumab, zinbererimab, tripalimab, dostallimab, INCMGA00012, AMP-224, REGN2810, BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100, JS001, and AMP-515. In some embodiments, the PD-1 inhibitor is semiprimab or pembrolizumab.

[0057] Semiprimab, marketed under the trademark Libtayo®, is a monoclonal antibody drug for the treatment of squamous cell carcinoma, basal cell carcinoma, and non-small cell lung cancer. Semiprimab belongs to a class of drugs that bind to programmed death receptor-1 (PD-1) and block the PD-1 / PD-L1 pathway. In September 2018, semiprimab was approved by the U.S. Food and Drug Administration (FDA) for the treatment of people with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for therapeutic surgery or therapeutic radiation. Semiprimab is being studied for the treatment of melanoma, cervical cancer, brain cancer, head and neck cancer, renal cell carcinoma, and Hodgkin lymphoma.

[0058] Pembrolizumab (formerly lambrolizumab, marketed under the trademark Keytruda®) is a humanized antibody used in cancer immunotherapy. Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved pembrolizumab for any unresectable or metastatic solid tumors with certain genetic abnormalities (mismatch repair failure or microsatellite instability). Approved indications for Keytruda® currently include, among others, metastatic melanoma, NSCLC, head and neck cancer, Hodgkin lymphoma, and metastatic esophageal squamous cell carcinoma. Pembrolizumab is administered by slow intravenous injection.

[0059] 3. Thiopurines, such as 6-thioguanine and 6-mercaptopurine, are currently used in clinical practice as anti-inflammatory, anti-leukemic, and immunosuppressant agents. In the activation reaction, 6-thioguanine is converted to 6-thioguanosine monophosphate by the hypoxanthine guanine phosphoribosyltransferase (HPRT) enzyme. 6-thioguanosine monophosphate is then further metabolized by kinases and RNA reductases to 6-thio-2'-deoxyguanosine 5'-triphosphate, which can ultimately be incorporated into the DNA strand during DNA replication. Once incorporated into DNA, 6-thioguanine can also generate reactive oxygen species, which can cause further damage to DNA, proteins, and other cellular macromolecules, thus inhibiting cell replication. While thiopurines are clinically used for the treatment of several types of leukemia, their usefulness for treating solid tumors is limited, partly due to increased toxicity and the development of other therapies.

[0060] One specific thiopurine is 6-thio-dG. This compound is a nucleoside analog and has been found to be a telomere disruption compound mediated by telomerase. Therefore, cancer cells are highly sensitive to 6-thio-dG, and observed ICs 50The values ​​range from 0.7 to 2.9 μM, depending on the cell type, including treatment-resistant cancers (Mender et al., 2018). The structure is shown below. TIFF0007829491000002.tif51128

[0061] B. Treatment regimen This disclosure relates to 6-thio-dG treatment and subsequent PD-L1 Inhibitor therapy , PD-1 Inhibitor therapy and / or CTLA-4 inhibitors We provide sequential treatment for cancer using therapeutic agents. The duration of each treatment may vary, and short intervals between treatments are considered advantageous. For example, 6-thio-dG treatment may last as little as 2 days, but it may also last 3 days, 4 days, or more, including 2-4 days. PD-L1 inhibitors , PD-1 inhibitors and / or CTLA-4 inhibitors The interval before treatment should be at least one day, and may be up to 14 days, for example, 2-4 days. Due to the potentially harmful effects of 6-thio-dG on activated effector T cells, 6-thio-dG and PD-L1 inhibitors , PD-1 inhibitors and / or CTLA-4 inhibitors Overlap between them should be avoided.

[0062] The daily dose of 6-thio-dG is 0.5 mg / kg to 10 mg / kg, preferably intravenously or orally. PD-L1 inhibitors , PD-1 inhibitors and / or CTLA-4 inhibitors The dosage falls within and is consistent with the approved current dosing schedule.

[0063] C. Telomerase-positive cancer Telomerase-positive cancers are far more susceptible to the effects of the methods disclosed herein than telomerase-negative cancers. Therefore, testing a biopsy to determine whether a cancer is telomerase-positive is not essential but highly useful.

[0064] The most common method for detecting telomerase activity is the telomere repeat amplification protocol (TRAP), which allows for semi-quantitative and quantitative analysis using several modifications of TRAP (referred to as ddTRAP, as opposed to droplet digital TRAP). These modifications include scintillation proximity assays, hybridization protection assays, transcription amplification assays, and magnetic bead-based extraction assays.

[0065] The telomere repeat amplification protocol can be subdivided into three main stages: primer extension, amplification of DNA synthesized by telomerase, and finally, detection. In the extension stage, telomere repeat sequences are added to telomere-mimicking oligonucleotides by telomerase present in the cell extract. PCR amplification of the DNA synthesized by telomerase is performed using telomere-mimicking and reverse primers. Different labels can be incorporated into the DNA synthesized by telomerase. Detection then follows this stage (e.g., electrophoretic separation and imaging of the PCR product).

[0066] Further methods include quantitative isolation of telomerase and subsequent measurement of the total telomerase activity from a given cell mass, where the total telomerase activity can be compared to a suitable standard. Once telomerase is isolated and tested in vitro, a wide variety of labeling and detection methods can be used.

[0067] D. Drug-resistant cancer Antineoplasmic resistance, often used interchangeably with chemotherapy resistance, refers to the resistance of neoplasms (cancerous cells), i.e., the ability of cancer cells to survive and grow despite anti-cancer treatment. In some cases, cancer can develop resistance to multiple drugs, known as multidrug resistance.

[0068] Based on the concept of heterogeneity in cancer cells, there are two common causes of failure in antineoplastic therapy: intrinsic genetic features that confer resistance to cancer cells and acquired resistance after drug exposure. Characteristics of resistant cells include altered membrane transport, enhanced DNA repair, defects in apoptotic pathways, and changes in target molecules, proteins, and pathway mechanisms, such as enzymatic inactivation. Since cancer is a genetic disease, two genomic events—genomic alterations (e.g., gene amplification and deletion) and epigenetic modifications—underlie acquired drug resistance. Cancer cells constantly utilize a variety of tools, including genes, proteins, and altered pathways, to ensure their survival against antineoplastic drugs.

[0069] Antineoplasmic resistance, synonymous with chemotherapy resistance, is the ability of cancer cells to survive and grow despite different anti-cancer treatments, i.e., multidrug resistance of cancer cells. There are two common causes of failure in antineoplasmic treatment: (i) innate resistance, such as genetic characteristics that confer resistance to cancer cells from the outset, rooted in the concept of heterogeneity of cancer cells; and (ii) acquired resistance after drug exposure.

[0070] Since cancer is a genetic disease, two genomic events—genomic changes (e.g., gene amplification and deletion) and epigenetic modifications—underlie these mechanisms of acquired drug resistance.

[0071] Chromosomal rearrangements due to genomic instability can lead to gene amplification and deletion. Gene amplification is an increase in the copy number of a region of a chromosome, which frequently occurs in solid tumors and can contribute to tumor progression through altered gene expression.

[0072] A 1993 hamster cell study showed that amplification in the DHFR gene, which is involved in DNA synthesis, begins with chromosomal breaks beneath the gene, and that the subsequent cross-linking-breaking-fusion cycle leads to large intrachromosomal repeats. Overamplification of oncogenes can occur in response to chemotherapy and is thought to be a mechanism underlying several classes of resistance. For example, DHFR amplification occurs in response to methotrexate, TYMS (involved in DNA synthesis) amplification occurs in response to 5-fluorouracil, and BCR-ABL amplification occurs in response to imatinib mesylate. Determining the regions of gene amplification in cells derived from cancer patients has extremely great clinical significance. Gene deletion is the opposite of gene amplification, and drug resistance arises from the loss of chromosomal regions and tumor suppressor genes such as TP53.

[0073] Genomic instability can occur when replication forks are obstructed or stalled during their movement. This can happen with replication fork barriers, proteins such as PTIP, CHD4, and PARP1, which are usually removed by BRCA1 and BRCA2, which are cellular DNA damage sensors, investigators, and responders.

[0074] Epigenetic modifications in antineoplastic drug resistance play a major role in cancer development and drug resistance because they contribute to the regulation of gene expression. The two main types of epigenetic regulation are DNA methylation and histone methylation / acetylation. DNA methylation is the process of adding methyl groups to DNA, usually in the upstream promoter region, which stops DNA transcription in that region and effectively suppresses the expression of individual genes. Histone modifications, such as deacetylation, alter chromatin formation and repress the expression of large chromosomal regions. In cancer cells where the normal regulation of gene expression is disrupted, oncogenes are activated via hypomethylation, and tumor suppressor factors are repressed via hypermethylation. Similarly, in the development of drug resistance, epigenetic modifications have been suggested to lead to the activation and overexpression of pro-drug resistance genes.

[0075] Studies on cancer cell lines have shown that hypomethylation (loss of methylation) of the MDR1 gene promoter leads to overexpression and multidrug resistance.

[0076] In methotrexate-resistant breast cancer cell lines lacking drug uptake and folate carrier expression, administration of the DNA methylation inhibitor DAC improved both drug uptake and folate carrier expression.

[0077] Acquired resistance to the alkylating agent fotemustine in melanoma cells was associated with high MGMT activity due to hypermethylation of the MGMT gene exon.

[0078] In imatinib (Gleevec®)-resistant cell lines, it has been shown that methylation-mediated arrest of SOCS-3 gene expression leads to STAT3 protein activation, which in turn causes uncontrolled proliferation.

[0079] Cancer cells can become resistant to multiple drugs through altered membrane transport, enhanced DNA repair, defects in apoptotic pathways, and changes in target molecules, proteins, and pathway mechanisms, such as enzymatic inactivation.

[0080] Many classes of antineoplastic drugs act on intracellular components and pathways such as DNA and nuclear components, meaning that many classes of antineoplastic drugs need to enter cancer cells. P-glycoprotein (P-gp), or multidrug resistance protein, is a phosphorylated and glycosylated membrane transporter that can move drugs out of cells, thereby reducing or eliminating drug efficacy. This transporter protein is encoded by the MDR1 gene and is also called the ATP-binding cassette (ABC) protein. MDR1 has irregular substrate specificity, allowing it to transport many structurally diverse compounds, mainly hydrophobic compounds, across the cell membrane. Studies have found that the MDR1 gene can be activated and overexpressed in response to drugs, and thus may form the basis for resistance to many drugs. Overexpression of the MDR1 gene in cancer cells is used to maintain intracellular levels of antineoplastic drugs below cytotoxic levels.

[0081] For example, the antibiotic rifampicin has been found to induce MDR1 expression. Experiments with different drug-resistant cell lines and patient DNA have revealed gene rearrangements that initiate MDR1 activation or overexpression. The C3435T polymorphism in exon 226 of MDR1 has also been shown to be strongly correlated with p-glycoprotein activity.

[0082] MDR1 is activated via NF-κB, a protein complex that acts as a transcription factor. In rats, the NF-κB binding site is adjacent to the mdr1b gene, and since the mutated NF-κB gene or the inhibitory IκB gene in tumor cells is mutated under chemotherapy, NF-κB may be active in tumor cells. In colorectal cancer cells, inhibition of NF-κB or MDR1 induced increased apoptosis in response to chemotherapeutic agents.

[0083] Enhanced DNA repair plays a crucial role in cancer cells' ability to overcome drug-induced DNA damage.

[0084] Platinum-based chemotherapy, such as cisplatin, targets tumor cells by crosslinking their DNA strands, causing mutations and damage. Such damage can trigger programmed cell death (e.g., apoptosis) in cancer cells. Cisplatin resistance occurs when cancer cells develop an enhanced ability to reverse such damage by removing cisplatin from their DNA and repairing the resulting damage. Cisplatin-resistant cells upregulate the expression of excision-repair cross-complementation (ERCC1) genes and proteins.

[0085] Some chemotherapy drugs are alkylating agents, meaning they attach alkyl groups to DNA, preventing it from being read. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme that removes alkyl groups from DNA. MGMT expression is upregulated in many cancer cells, protecting them from alkylating agents. Increased MGMT expression has been found in colorectal cancer, lung cancer, non-Hodgkin lymphoma, breast cancer, glioma, myeloma, and pancreatic cancer.

[0086] TP53 is a tumor suppressor gene that codes for the p53 protein and responds to DNA damage through either DNA repair, cell cycle arrest, or apoptosis. Loss of TP53 due to gene deletion may allow cells to replicate continuously despite DNA damage. Tolerance to DNA damage can confer a method of resistance to drugs that normally induce apoptosis via DNA damage to cancer cells.

[0087] Other genes involved in drug resistance related to apoptotic pathways include h-ras and bcl-2 / bax. Oncogenic h-ras has been found to increase ERCC1 expression, resulting in enhanced DNA repair (see above). Inhibition of h-ras has been found to increase cisplatin sensitivity in glioblastoma cells. Since Bcl-2 is a pro-survivability oncogene, upregulated expression of Bcl-2 in leukemia cells (non-Hodgkin lymphoma) resulted in reduced levels of apoptosis in response to chemotherapy agents.

[0088] During targeted therapy, the target often modifies itself, reducing its expression to the point where the treatment is no longer effective. One example of this is the loss of estrogen receptors (ER) and progesterone receptors (PR) during anti-estrogen treatment of breast cancer. Tumors with loss of ER and PR no longer respond to tamoxifen or other anti-estrogen treatments, and while cancer cells may retain some responsiveness to estrogen synthesis inhibitors, they eventually become unresponsive to endocrine manipulation and no longer depend on estrogen for growth.

[0089] Another lineage of therapeutic agents used to treat breast cancer involves targeting kinases from the EGFR family, such as human epidermal growth factor receptor 2 (HER2). Mutations often occur in the HER2 gene upon treatment with inhibitors, and it has been found that approximately 50% of lung cancer patients have the EGFR-T790M gatekeeper mutation.

[0090] Treatment for chronic myeloid leukemia (CML) includes a tyrosine kinase inhibitor called imatinib, which targets the BCR / ABL fusion gene. In some individuals who are resistant to imatinib, the BCR / ABL gene is either reactivated or amplified, or has a single point mutation. These point mutations promote autophosphorylation of the BCR-ABL protein, leading to stabilization of the ATP binding site into an active form that cannot be bound by imatinib for proper drug activation.

[0091] Due to its crucial role as an enzyme in DNA replication, topoisomerase is a valuable target for cancer treatment, and many topoisomerase inhibitors have been developed. Resistance can occur when topoisomerase levels are reduced or when different topoisomerase isoforms are distributed differently within cells. Mutant enzymes have been reported in patient leukemia cells, and mutations in other cancers that confer resistance to topoisomerase inhibitors have also been reported.

[0092] One mechanism of antineoplasmic resistance is the overexpression of drug-metabolizing enzymes or carrier molecules. By increasing the expression of metabolic enzymes, drugs are more rapidly converted into drug conjugates or inactive forms that can then be excreted. For example, the electrophilic properties of glutathione allow it to react with cytotoxic agents to inactivate them, so increased expression of glutathione promotes drug resistance. In some cases, reduced or lost expression of drug-metabolizing enzymes confers resistance because enzymes are required to process drugs from their inactive to active forms. Arabinosides, commonly used chemotherapy drugs for leukemia and lymphoma, are converted to cytosine arabinoside triphosphate by deoxycytidine kinase. Mutations or loss of expression of deoxycytidine kinase result in resistance to arabinosides. This is a form of enzymatic inactivation.

[0093] Growth factor expression levels can also promote resistance to antineoplastic therapy. In breast cancer, drug-resistant cells were found to express high levels of IL-6, while susceptible cells did not express significant levels of growth factors. IL-6 activates the CCAAT enhancer-binding protein transcription factor, which activates MDR1 gene expression.

[0094] Another type of antineoplasm resistance is resistance to checkpoint inhibitors. Primary resistance to immune checkpoint blockade occurs in approximately 40%–65% of melanoma patients treated with anti-PD-1 based therapy. This clinical problem arises when an effective antitumor immune response cannot be induced at any of the three stages of the cancer immune cycle. To date, factors associated with primary resistance include elevated baseline serum LDH levels, increased baseline tumor volume, lack of PD-L1 expression in baseline melanoma tissue samples, lack of T cell infiltration, absence of PD-1 T cells and PD-L1 macrophages in melanoma biopsies taken early during treatment, insufficient neoantigens and low mutation counts, presence of innate anti-PD-1 resistance signature (IPRES) transcriptional signatures, or absence of interferon signatures.

[0095] Acquired resistance to immunotherapy can occur when there is a selection of subpopulations of tumor cells that possess genetic and epigenetic traits that allow tumor cells to evade the immune system. One example is the loss of B2M expression reported in patient-derived melanoma cell lines treated with immunotherapy and cytokine gene therapy. This results in a loss of MHC class I expression, and therefore CD8 +This resulted in a subsequent decrease in recognition by T cells. JAK1 / 2 mutations have also recently been identified as genetic markers of acquired resistance to immunotherapy in melanoma. These mutations in tumor cells result in reduced sensitivity to IFN-γ, ultimately suppressing IFN-γ-induced cell proliferation arrest. Loss-of-function mutations in the genes encoding JAK1 or JAK2 were found in recurrent tumors after whole exome sequencing of baseline and advanced biopsies; all patients had objective findings to pembrolizumab treatment and subsequently progressed. Furthermore, acquired resistance can also occur at the individual cell level, with tumor cells altering gene expression in response to immune molecules within the tumor microenvironment. For example, PD-L1 can be upregulated by tumor cells in response to immune cytokines such as IFN-γ released by T cells, thus limiting T cell function and potentially leading to both primary and acquired resistance.

[0096] III. Pharmaceutical preparations and routes of administration When a pharmaceutical composition is intended for clinical use, it is prepared in a form suitable for the intended purpose. Generally, this involves preparing a composition that is essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0097] Generally, it is desirable to use appropriate salts and buffers to stabilize the drug and enable uptake by target cells. The aqueous compositions of this disclosure contain an effective amount of drug dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically acceptable" means molecular entities and compositions that do not produce harmful, allergic, or other undesirable reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient of this disclosure, its use in therapeutic compositions is intended. Co-active ingredients can also be incorporated into compositions, provided that they do not inactivate the active ingredient of the composition.

[0098] The active compositions of this disclosure may include classic pharmaceutically acceptable preparations. Administration of these compositions according to this disclosure may be via any common route, insofar as the target tissue is available through that route, but generally includes systemic administration. This includes oral, nasal, or buccal administration. Alternatively, administration may be intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection, or intratumoral or local to the tumor, for example, by tumor vascular structures. Such compositions would typically be administered as pharmaceutically acceptable compositions, as described above.

[0099] The active compound may also be administered parenterally or intraperitoneally. For example, a solution of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations generally contain preservatives to prevent microbial growth.

[0100] Pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that they are easily injectable. The preparations should be stable under manufacturing and storage conditions and should be protected against microbial contamination such as bacteria and fungi. Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Inhibition of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0101] Sterile injectable solutions can be prepared by incorporating appropriate amounts of the active compound into a solvent along with any other components as desired (e.g., as listed above), and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other desired components, as listed above, for example. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques to obtain a powder of the active ingredient and any further desired components from a pre-sterilized filtered solution of the active ingredient and any further desired components.

[0102] The compositions of this disclosure can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.) (formed together with the free amino groups of the protein). Salts formed together with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0103] When formulated, the solution is administered in a therapeutically effective amount, preferably in a manner compatible with the administered formulation. The formulation can be readily administered in various dosage forms, such as injectable solutions and drug-release capsules. In the case of parenteral administration in aqueous solutions, for example, the solution is generally adequately buffered, and the liquid diluent is first made isotonic using, for example, sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, as is well known to those skilled in the art, and especially in light of this disclosure, a sterile aqueous medium is used. As an example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion solution, or injected into the proposed injection site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Depending on the condition of the subject being treated, some variation in the dosage will inevitably occur. The person responsible for administration will, in any case, determine the appropriate dose for each individual. Furthermore, for administration to humans, the preparation must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics standards.

[0104] IV. Combination Therapy In the context of this disclosure, it is also intended that anti-PD-L1 agents such as 6-thio-dG / atezolizumab, or anti-PD-1 agents such as 6-thio-dG / Libtayo®, or anti-CTAL-4 agents may be used in combination with chemotherapy or radiotherapy interventions or other treatments. In particular, it may be found that combining 6-thio-dG, anti-PD-L1, anti-PD-1, or anti-CTLA-4 agents with other therapies targeting different aspects of cancer cell function may be effective.

[0105] The methods and compositions of this disclosure are used to generally contact “target” cells with 6-thio-dG and at least one other active ingredient in order to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells. These compositions are provided in a range of amounts or combined amounts effective in killing cells or inhibiting cell proliferation. This process may involve contacting cells simultaneously with 6-thio-dG / anti-PD-L1, anti-PD-1, or anti-CTLA-4 and other active ingredients or factors. This may be achieved by contacting cells with a single composition or pharmacological formulation containing both active ingredients, or by contacting cells simultaneously with two different compositions or formulations, one of which comprises the interferon prodrug according to this disclosure and the other comprises the other active ingredient.

[0106] Alternatively, 6-thio-dG / anti-PD-L1, anti-PD-1, or anti-CTLA-4 therapy may precede or follow other activator treatments at intervals ranging from minutes to weeks. In embodiments where other activators and interferon prodrugs are applied to cells separately, efforts will generally be made to avoid significant time gaps between each delivery so that the activators and expression constructs can still exert a favorable synergistic effect on the cells. In such examples, it is assumed that cells will be brought into contact with both modalities within approximately 12 to 24 hours of each other, more preferably within approximately 6 to 12 hours of each other, with a delay of only about 12 hours being most preferable. In some situations, it may be desirable to significantly extend the time between treatments, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) may pass between each administration.

[0107] Two or more doses of either an interferon prodrug or other active agent may be desired. Various combinations may be used, as illustrated below, where 6-thio-dG / anti-PD-L1, anti-PD-1, or anti-CTLA-4 therapy is "A" and other therapies are "B". TIFF0007829491000003.tif18128

[0108] Other combinations are also conceivable. Similarly, to achieve cell death, both agents are delivered to the cells in a combined dose effective for cell death.

[0109] Suitable agents or factors for cancer treatment include any chemical compound or treatment method that induces DNA damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, and electron emission. Various chemical compounds, also referred to as "chemotherapeutic agents" or "genotoxic agents," may be used. This can be achieved by irradiating a localized tumor site; or by bringing tumor cells into contact with the agent by administering a therapeutically effective amount of the pharmaceutical composition to the target.

[0110] Various classes of chemotherapeutic agents are intended for use with the present disclosure. Imetelstat is described below. Other chemotherapeutic agents include selective estrogen receptor antagonists ("SERMs") such as tamoxifen, 4-hydroxytamoxifen (afimoxifen), faslodex, raloxifene, bazedoxifen, clomiphene, femarel, rasofoxifen, olmeroxifen, and toremifene. The active agents camptothecin, actinomycin D, and mitomycin C are commonly used chemotherapeutic agents. The present disclosure also includes the use of combinations of one or more DNA damaging agents, whether radioactive or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. The active agents may be prepared and used as combination therapeutic compositions.

[0111] Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. HSP90 inhibitors have been shown to be useful in the treatment of cancer. Such inhibitors include geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin, PU-H71, and rifabutin.

[0112] Therapeutic agents that directly crosslink DNA or form adducts are also conceivable. These agents, such as cisplatin and other DNA alkylating agents, may be used. Cisplatin is widely used to treat cancer, administered at 20 mg / m² for 5 days every 3 weeks, for a total of 3 therapeutic units. 2 The effective dose of cisplatin is used in clinical applications. Cisplatin is not absorbed orally and therefore must be delivered by injection intravenously, subcutaneously, intratumorally, or intraperitoneally.

[0113] Substances that damage DNA include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include doxorubicin (also known as adriamycin), etoposide, verapamil, and podophyllotoxin. These compounds, widely used in clinical practice for treating neoplasms, are administered at 25-75 mg / m² every 21 days for doxorubicin. 2 For etoposide, the dose is administered intravenously via bolus injection in the following range: 35-50 mg / m². 2 It is administered intravenously in doses within the specified range or orally at twice the intravenous dose. Microtubule inhibitors such as taxanes are also considered. These molecules are diterpenes produced by plants of the genus Taxus and include paclitaxel and docetaxel.

[0114] Epidermal growth factor receptor inhibitors such as Iressa, mTOR, and mammalian rapamycin target (also known as FK506-binding protein 12 rapamycin-related protein 1 (FRAP1)) are serine / threonine protein kinases that regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Therefore, rapamycin and its analogues ("rapalogs") are intended for use in cancer treatment according to this disclosure. Another EGFR inhibitor particularly useful here is gefitinib.

[0115] Another possible treatment is TNF-α (tumor necrosis factor-α), a cytokine involved in systemic inflammation and a member of the cytokine group that stimulates acute phase responses. The main role of TNF is in regulating immune cells. TNF can also induce apoptosis (programmed cell death), induce inflammation, and inhibit tumorigenesis and viral replication.

[0116] Active agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also cause DNA damage. Therefore, numerous nucleic acid precursors have been developed. Active agents that have undergone extensive testing and are readily available are particularly useful. Thus, active agents such as 5-fluorouracil (5-FU) are preferred by neoplastic tissues, making this agent particularly useful for targeting neoplastic cells. Although highly toxic, 5-FU is applicable to a wide range of carriers, including topical applications; however, intravenous administration at doses ranging from 3 mg / kg / day to 15 mg / kg / day is commonly used.

[0117] Other factors that cause DNA damage and have been widely used include those commonly known as gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damage factors, such as microwave and UV irradiation, are also considered. All of these factors are most likely to affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from 50–200 roentgens per day over a long period (3–4 weeks) to single doses of 2000–6000 roentgens. The dose range for radioisotopes varies considerably and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0118] Furthermore, it is also conceivable that separate immunotherapies, hormone therapies, toxin therapies, and / or surgeries may be used.

[0119] Those skilled in the art should refer to Chapter 33, particularly pages 624-652, of the 15th edition of Remington's Pharmaceutical Sciences. Some variation in dosage will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will, in any case, determine the appropriate dose for each individual subject. Furthermore, for administration to humans, preparations must meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics standards. [Examples]

[0120] V. Examples The following Examples section provides further details regarding examples of various embodiments. Those skilled in the art should understand that the techniques disclosed in the following Examples represent techniques and / or compositions that have been found to function well by the inventors. However, those skilled in the art should understand that many modifications can be made in light of the disclosed particular embodiments to still obtain similar or similar results without departing from the spirit and scope of the disclosure. These Examples are illustrative of the methods and systems described herein and are not intended to limit the scope of the disclosure. Non-limiting examples of such include, but are not limited to, those presented below.

[0121] Example 1 - Materials and Method mouse Female C57BL / 6J, BALB / c, Myd88- / -, Tmem173- / -, Batf3- / - and OT-1 CD8+ T cell receptor transgenic mice, as well as NSG-SMG3 mice, were purchased from The Jackson Laboratory. Rag1- / - mice and IFN reporter mice (Ifng) were also purchased from the Jackson Laboratory. tm3.1Lky Ifnαr1- / - mice were purchased from the UT Southwestern Mouse Breeding Core. Ifnαr1- / - mice were provided by Dr. Anita Chong of the University of Chicago. All mice were maintained under specific pathogen-free conditions. Animal care and experiments were conducted in accordance with institutional and National Institutes of Health protocols and guidelines. This study was approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center.

[0122] Cell lines and reagents MC38, CT26, LLC A375, and HCT116 cells were purchased from ATCC. MC38-OVA cells were generated by lentiviral transduction of the OVA gene. All cell lines were routinely tested using a mycoplasma contamination kit (R&D) and cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% thermoactivated fetal bovine serum, 100 U / ml penicillin, and 100 U / ml streptomycin.

[0123] Anti-CD4 (GK1.5), anti-NK1.1 (PK136), anti-CD8 (53-5.8), and anti-CSF1R (AFS98) mAbs were purchased from BioXCell. Anti-PD-L1 (atezolizumab) and anti-CTLA-4 (ipilimumab) were obtained from UT Southwestern Simmons Cancer Center Pharmacy. 6-thio-dG was purchased from Metkinen Oy. For in vitro studies, 6-thio-dG was dissolved in DMSO / water (1:1) to prepare a 10 mM stock solution. For in vivo studies, 3 mg / kg of 6-thio-dG was prepared in 5% DMSO (1×PBS) for intraperitoneal injection. The drugs were kept frozen at -20°C until use.

[0124] Cell viability assay IC using a cell proliferation assay 50 To determine the optimal solution, mouse and human cancer cell lines were screened with 6-thio-dG using a 2-fold dilution series at eight different points in a 96-well plate. Cells were seeded 24 hours before drug addition, incubated for 4–5 days, and assayed using the Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions. Cell counts per well ranged from 1,000 to 10,000 cells per well, inversely proportional to the doubling time. Dose-response curves were constructed and ICs were performed using Graphpad Prism. 50The standard deviation was calculated. All samples were analyzed in triplets, and the standard deviation is from 2-3 independent experiments.

[0125] Colony formation assay MC38 cells were seeded at three different concentrations on 6-well plates (1000-4000 cells / well) and treated with various drug concentrations every 3-4 days. After 13 days of treatment, the cells were fixed and stained with 6% glutaraldehyde (Fisher Scientific) + 0.5% crystal violet (Sigma) solution. After washing with tap water, the cells were air-dried and images were captured using a G-BOX (Syngene, model: G-BOX F3).

[0126] Telomere Dysfunction-Induced Focus (TIF) and Micronucleus Assay The TIF assay is based on the co-localization detection of DNA damage using antibodies against DNA damage response factors such as γ-H2AX and 53BP1, and antibodies against telomere proteins or telomeres, with the use of telomere sequence-specific peptide nucleic acid (PNA) probes (Mender and Shay, 2015). Briefly, cells were seeded in 4-well chamber slides. The following day, cells were treated with 1 μM 6-thio-dG for 24 hours (for the TIF assay) or 1–3 μM 6-thio-dG for 48 hours (for the micronucleus assay). The slides were then rinsed twice with PBS and fixed with 4% formaldehyde in PBS (Thermo Fisher) for 10 minutes. The cells were then washed twice with PBS and permeabilized in 0.5% TritonX-100 in PBS for 10 minutes. After permeabilization, the cells were washed three times with PBS. The cells were blocked for 1 hour with 10% goat serum in 0.1% PBST (TritonX-100). γ-H2AX (TIF assay, mouse, 1:1000) (Millipore) or lamin A / C (micronucleus assay, mouse, 1:500) (Santa Cruz) were diluted in blocking solution and incubated on cells for 2 hours. The cells were washed three times with PBST (1×PBS in 0.1% Triton), then three times with PBS, and finally Alexafl. uoCells were incubated with r568-conjugated goat anti-mouse (1:500) (Invitrogen) for 40 minutes, then washed five times with 0.1% PBST. Cells were fixed in 4% formaldehyde in PBS at room temperature for 20 minutes. Slides were sequentially dehydrated with 70%, 90%, and 100% ethanol, and then denatured on a heat block at 80°C for 7 minutes in a hybridization buffer containing FAM-conjugated telomere sequence (C-rich) specific PNA probe, 70% formamide, 30% 2×SSC, 10% (w / v) MgCl2·6H2O (Fisher Sci), and 0.25% (w / v) blocking reagent (Roche) for nucleic acid hybridization and detection, followed by incubation overnight at room temperature. Slides were sequentially washed with 70% formamide (Ambion) / 0.6×SSC (Invitrogen) (2×1 hour), 2×SSC (1×15 minutes), and PBS (1×5 minutes), then sequentially dehydrated with 70%, 90%, and 100% ethanol, and subsequently mounted in Vectashield mounting medium containing DAPI (Vector Laboratories). Images were captured using a fluorescein microscope with a 100x objective lens. TIF was quantified using Image J.

[0127] Detection of DNA in bone marrow-derived dendritic cells Cells were labeled with EdU as described above (Min et al., 2019). Briefly, 100,000 MC38 cells were seeded in a 6-well plate and labeled with 25 μM EdU. After 2 days, the cells were washed and treated with 1 μM 6-thio-dG for 24 hours. The cells were washed again and co-cultured overnight with BMDCs. The following day, the DCs were sorted with magnetic beads, washed, fixed, and cytospinned. The slides were then stained with 6-carboxytetramethylrhodamine fluorescent azide (Invitrogen) in fresh, in-house prepared EdU staining solution (PBS containing 1 mM CuSO4 and 2 mM ascorbic acid) for 30 minutes. The slides were then vigorously washed with PBS for at least 1 hour, followed by a telomere FISH step using a FAM-TelG probe as described in the "Telomere Dysfunction-Induced Focus (TIF) and Micronucleus Assay" section. Images were captured at 63x magnification using an Axio Imager Z2 (Coolcube1 camera) equipped with an automated metaphase capture system, and analyzed with ISIS software (Metasystems).

[0128] Immunology FISH In short, 5 μM tissue sections were deparaffinized with xylene (2 × 5 min), 100% ethanol (2 × 2 min), 95% ethanol (1 × 2 min), 75% ethanol (1 × 2 min), and 50% ethanol (1 × 2 min), and then washed with tap water (2 × 3 min). The deparaffinized tissue sections were incubated in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6.0) under microwave for 20 minutes to activate the antigen. After cooling the tissue sections, they were rinsed with 1 × PBS for 5 minutes and then dehydrated in 95% ethanol for 3 minutes. Denaturation was performed on a heat block at 80°C for 7 minutes using a hybridization buffer containing a FITC-conjugated telomere sequence (TTAGGG)3-specific PNA probe (70% formamide, 30% 2×SSC, 10% (w / v) MgCl2·6H2O (Fisher Sci), 0.25% (w / v) blocking reagent (Roche)). Slides were sequentially washed with 70% formamide / 0.6×SSC (3×15 min), 2×SSC (1×15 min), PBS (1×5 min), and PBST (PBS + 0.1% Tween20; 1×5 min), and incubated with blocking buffer (4% BSA in PBST) for 30 minutes. Sections were incubated at room temperature for 1 hour with phosphohistone H2AX antibody (1:500) (Cell Signaling) in blocking buffer. After washing with PBST for 2×5 minutes, Alexafl in blocking buffer was used. uo Tissue sections were incubated with goat-anti-rabbit conjugates of r 568 at room temperature for 1 hour. Sections were sequentially washed with PBST (3 × 5 min) and PBS (1 × 5 min). Slides were mounted in Vectashield mounting medium containing DAPI. Images were captured using a fluorescein microscope with a 100x objective lens. TIF was quantified using ImageJ.

[0129] Tumor growth and treatment In 100 μL of phosphate-buffered saline (PBS), a total of 5 × 10¹⁴ ions were collected subcutaneously in the right dorsal flank of mice. 5 MC38, 5×10 5 1 CT26 or 1 × 106 Individual LLC cells were inoculated. The tumor was approximately 100 mm. 3 Once the tumors had proliferated, tumor-bearing mice were randomly divided into treatment groups. For a single 6-thio-dG treatment, 3 mg / kg of 6-thio-dG was administered intraperitoneally on days 7, 8, and 9 for MC38 and LLC tumors, and on days 5, 6, and 7 for CT26 tumors. For CSF1R, NK1.1, CD4+, and CD8+ T cell depletion, 200 μg of antibody was injected intraperitoneally one day before the start of treatment, and then intraperitoneally twice a week for two weeks. For PD-L1 blockade combination therapy in the MC38 model, 6-thio-dG was administered on days 10 and 11, and 50 μg of [unclear text] was administered on days 13 and 17. anti PD-L1 antibody The drug was administered by intraperitoneal injection. For PD-L1 blockade combination therapy in the LLC model, 6-thio-dG was administered on days 4, 5, 6, 10, and 11, and 200 μg of PD-L1 was administered by intraperitoneal injection on days 8 and 13. Tumor volume was measured by length (a), width (b), and height (h), and calculated as tumor volume = abh / 2.

[0130] Humanized mouse tumor model Humanized mouse rearrangements have been previously described (Qiao et al., 2019). Briefly, 4-week-old NSG-SGM3 female mice were irradiated with 100 cGy (X-ray irradiation using an X-RAD 320 device) one day before human CD34+ cell transduction. Umbilical cord blood was obtained from UT Southwestern Parkland Hospital. Human CD34+ cells were purified from the umbilical cord blood by density gradient centrifugation (Ficoll® Paque Plus, GE Healthcare), followed by positive immunomagnetic selection using anti-human CD34 microbeads (Stemcell). 1 × 10⁻⁶ 5 Individual CD34+ cells were intravenously injected into each recipient mouse. Twelve weeks after engraftment, 1 × 10⁶ humanized mice with more than 50% human CD45+ cell rearrangement and age- and sex-matched non-humanized mice were injected. 6Individual HCT116 tumor cells were inoculated subcutaneously into the right flank. 3 mg / kg of 6-thio-dG was administered intraperitoneally on days 7, 8, and 9. Tumor volume was measured twice weekly. The experiment was conducted in accordance with the UTSW Human Investigation Committee protocol and the UTSW Institutional Animal Care and Use Committee guidelines.

[0131] Tmem173 and Mb21d1 knockout MC38 cell lines The Tmem173 and Mb21d1 genes were knocked out in MC38 cells using CRISPR / Cas9 technology. A guide sequence for Tmem173 was also used. Guide array for TIFF0007829491000004.tif4128 and Mb21d1 TIFF0007829491000005.tif4128 was cloned into the px458 plasmid (a non-integrated plasmid with a GFP selection marker) and then transiently transfused into tumor cells using lipofectamine 2000 (Thermo Fisher). After 24 hours, GFP-positive cells were sorted and cultured for a further week. The sorted cells were then seeded into 96-well plates. After another week, GFP-negative clones were placed in 12-well plates and Western blotting was performed to identify the knockout clones. Finally, all knockout clones were pooled together for experimentation.

[0132] IFN-γ enzyme-coupled immunosorbent spot assay (ELISPOT) The MC38 tumor was subcutaneously injected into the right flank of C57BL / 6. For a single 6-thio-dG treatment, 3 mg / kg of 6-thio-dG was administered intraperitoneally on days 7, 8, and 9. For PD-L1 blockade combination therapy in the MC38 model, 3 mg / kg of 6-thio-dG was administered on days 10 and 11, along with 50 μg of [unclear]. anti PD-L1 antibodyThe drug was administered intraperitoneally 11.7 days after the last treatment, and the tumor inflow area lymphoid system and spleen were harvested from tumor-bearing mice to prepare single-cell suspensions. Tumor-specific T cells were restimulated using irradiated MC38 tumor cells and control LLC tumor cells. 1.5 × 10⁶ 5 Individual inflow area lymph node cells or spleen cells and 7.5 × 10 4 Irradiated tumor cells were co-cultured for 48 hours, and an ELISPOT assay was performed using the IFN-γ ELISPOT kit (BD Bioscience) according to the manufacturer's instructions. IFN-γ spots were counted using a CTL-ImmunoSpot® S6 Analyzer (Cellular Technology Limited).

[0133] In vitro co-culture of bone marrow dendritic cells (BMDCs) and T cells Single-cell suspensions of bone marrow (BM) cells were collected from the tibia and femur of C57BL / 6 mice. BM cells were placed in 10 cm dishes and cultured in complete RPMI1640 medium containing 20 ng / mL recombinant mouse GM-CSF (BioLegend). Fresh medium was added to the culture on days 3 and 6. BMDCs were collected on day 7. CD8+ T cells were isolated from the lymph nodes and spleen of OT-1 transgenic mice using a negative CD8+ T cell isolation kit (Stemcell). MC38-OVA cells were pre-treated with 200 nM 6-thio-dG for 4 hours. The drug was then washed off, and the tumor cells were cultured for 72 hours and collected on the same day as BMDC collection. MC38-OVA cells were then co-cultured overnight with BMDCs. The supernatant was collected for IFN-β ELISA (PBL). BMDCs were selected using a CD11c+ positive selection kit (Stemcell) and co-cultured with OT-1 CD8+ T cells for 48 hours. The supernatant was collected, and IFN-γ was measured by cytometry bead array assay (BD Biosciences).

[0134] Cytoplasmic DNA extraction and quantitative real-time PCR HCT116 cells were pretreated with 500 nM 6-thio-dG for 4 hours. The drug was then washed off, and the tumor cells were cultured for 72 hours and collected on the same day as BMDC collection. Subsequently, 1 × 10⁶ HCT116 cells were mixed in a 1:1 ratio. 6 The BMDCs were mixed with the BMDCs for 4 hours. The BMDCs were purified and divided into two equal, constant volumes. Using the Purelink Genomic DNA Kit (Invitrogen), one constant volume was extracted for whole genomic DNA and served as a standardization control. The other constant volume was resuspended in 100 μL of cytoplasmic extraction buffer containing 150 mM NaCl, 50 mM HEPES, and 25 mg / mL digitonin (Sigma), and incubated at room temperature for 10 minutes for plasma membrane permeabilization (West et al., 2015). The cells were then centrifuged to pellet intact cells. The cytoplasmic supernatant was collected and centrifuged at 12000 g for 10 minutes to pellet the remaining cell debris. Cytoplasmic DNA was then extracted using the Purelink Genomic DNA Kit (Invitrogen). Quantitative PCR was performed on both the whole cell extract and the cytoplasmic fraction using human DNA primers and mouse DNA primers (Xu et al., 2017).

[0135] Tumor digestion Tumor tissue was excised and digested with 1 mg / mL collagenase I (Sigma) and 0.5 mg / mL DNase I (Roche) at 37°C for 30 minutes. The tumor was then passed through a 70 μm cell filter to remove large fragments of undigested tumor. Tumor-infiltrating cells were washed twice with PBS containing 2 mM EDTA.

[0136] Flow cytometry analysis Single-cell suspensions of cells were incubated with anti-FcγIII / II receptor (clone 2.4G2) for 15 minutes to block nonspecific binding, then stained with the conjugated antibody, and subsequently incubated with the indicated antibody in the dark at 4°C for 30 minutes. To eliminate dead cells, Fixable viability dye eFl was used. uo r 506 or eFl uor780 (eBioscience) was used. Foxp3 and Ki67 were stained intracellularly using the True-Nuclear Transcription Factor Buffer Set (BioLegend) according to the manufacturer's instructions. Data were collected using a CytoFLEX flow cytometer (Beckman Coulter, Inc.) and analyzed using FlowJo (Tree Star Inc., Ashland, OR) software.

[0137] Quantitative real-time PCR Different primer sets ( Using TIFF0007829491000006.tif56167), real-time PCR was performed using SsoAdvanced® Universal SYBR® Green Supermix (Bio-Rad) according to the manufacturer's instructions. Mouse GAPDH was used as an internal control. 2 -ΔΔCt The relative expression changes were calculated using the method.

[0138] Immunoblotting BMDC and MC38 treatment was the same as in the "In vitro co-culture of bone marrow dendritic cells". Six hours after co-culture, DCs were isolated using a CD11c+ positive selection kit (Stemcell). Protein sample preparation and immunoblotting procedures were performed as previously described (Liu et al., 2019). Proteins were detected using rabbit monoclonal antibodies against pSTING (Cell signaling, 72971), STING (Cell signaling, 50494), pTBK1 (Cell signaling, 5483), and TBK1 (Cell signaling, 3504). Protein loading was determined using an antibody against cyclophyllin A) (Cell signaling, 2175). Anti-rabbit antibody (1:2000 in 5% BSA) was used as the secondary antibody (Cell signaling, 7074). The membrane was developed using X-ray film (GeneMate, F-9024-8X10). For chemiluminescent Western blotting, Clarity Max Western ECL Substrate (Biorad, 1705062) or Supersignal West PicoPlus Chemiluminescent Substrate (Thermoscientific, 34577) was used.

[0139] Quantitative and statistical analysis All data analyses were performed using GraphPad Prism statistical software, and results are presented as mean ± SEM. P-values ​​were determined by two-way ANOVA for tumor growth, log-rank tests for survival, or unpaired two-sided t-tests for other analyses. A p-value < 0.05 was considered statistically significant.

[0140] Example 2 - Results The therapeutic effect of 6-thio-dG depends on CD8+ T cells. Previous studies using xenograft models have all shown that intensive daily treatment with 6-thio-dG for 10 days can partially control tumor growth in many tumor models (Mender et al., 2015a; Mender et al., 2018; Zhang et al., 2018). However, the potential role of this drug in the interaction between tumors and the adaptive immune system remains unclear. To investigate whether 6-thio-dG induces telomere-based DNA sensing for T cell responses, we first demonstrated the inhibition of cell viability by 6-thio-dG against telomerase-positive mouse colon cancer cells (MC38) in an immunocompetent host. MC38 tumor cells showed an IC50 of 370 nM. 50 Sensitivity to 6-thio-dG at various concentrations (Figure 1A). The inventors also confirmed 6-thio-dG sensitivity in MC38 cells by an independent colony-forming assay. In MC38 cells treated with 6-thio-dG every 3 days for 13 days, treatment with 0.5 μM 6-thio-dG resulted in less than 50% of cells forming colonies (Figures 1B and 1C). To evaluate whether 6-thio-dG reduces tumor burden in a syngeneic mouse model in vivo, the inventors subcutaneously inoculated immunoqualified wild-type (WT) C57BL / 6 mice with MC38 cells. Seven days after tumor inoculation (tumor volume approximately 100 mm³) 3 When 3 mg / kg of 6-thio-dG was administered daily for only 3 days (when the condition was met), tumor growth was significantly reduced compared to the control tumor (Figure 1D). We also observed inhibition of cell viability in vitro and significant delay of tumor growth in vivo in telomerase-positive LLC (Lewis lung mouse cancer derived from C57BL / 6 mice) and CT26 (colon mouse cancer derived from BALB / C mice) tumor models with only 3 days of treatment, so this was not a response specific to the MC38 tumor model (Figures 9A-9D).

[0141] The inventors hypothesized that 6-thio-dG may have an immunostimulatory role in vivo, as they achieved a better antitumor effect in a syngeneic mouse model by administering such a short-term treatment with 6-thio-dG compared to an intensive administration strategy (5 mg / kg daily for 2 weeks) in a xenograft model. Therefore, the inventors inoculated Rag1 knockout mice, which are unable to produce mature T cells and mature B cells, with tumors. Indeed, the therapeutic effect of 6-thio-dG was completely attenuated (Figure 1E), indicating that adaptive immune cells are primarily required for tumor control in vivo. To clarify which subset of T cells contributes to the 6-thio-dG-mediated antitumor effect, the inventors depleted CD4+ or CD8+ T cells while administering 6-thio-dG and observed the minor effect of CD4+ T cell depletion (Figure 1F). However, CD8+ T cell depletion completely abolished the therapeutic effect of 6-thio-dG (Figure 1G). In summary, this data can be interpreted as suggesting an essential role of CD8+ T cells in 6-thio-dG treatment.

[0142] 6-thio-dG treatment increases tumor-specific T cell responses. Since the therapeutic effect of 6-thio-dG is T cell dependent, the inventors hypothesized that 6-thio-dG treatment could alter the expansion and proliferation of immune cells in the tumor microenvironment. To test this, the inventors analyzed the number of tumor-infiltrating lymphocytes (TILs) six days after three daily doses of 6-thio-dG treatment. The inventors found an increase in the frequency of CD3+ T cells and CD8+ T cells in TILs after 6-thio-dG treatment (Figures 2A, 10A, and 10B). The inventors also observed a significant upregulation of CD8+ T cell proliferation, indicated by elevated Ki67 expression (Figure 2B), but no significant changes in Treg cells (Figure 10C). Tumor-infiltrating NK cells also increased, but the inventors did not see any effect of NK cell depletion on the therapeutic effect of 6-thio-dG (Figures 10D and 10E). Combined with CD8 depletion experiments, this suggests that while NK cells are not essential for the 6-thio-dG-mediated antitumor effect, a CD8+ T cell response is required.

[0143] The inventors further tested the antigen-specific T cell response after 6-thio-dG treatment by using the MC38-OVA tumor model, which allows tracking of antigen-specific T cells in tumor tissue. Indeed, the inventors observed increased tumor-specific CD8+ T cells in the tumor 6 days after 6-thio-dG treatment (Figure 2C). The inventors also observed an enhanced tumor-specific cytotoxic T cell response in the MC38 tumor model by measuring IFN-γ-producing T cells after 6-thio-dG treatment (Figures 2D and 2E). To directly evaluate the ability of T cells to produce IFN-γ in vivo, the inventors utilized an IFN-γYFP reporter mouse that allows tracking of IFN-γ-producing T cells with YFP expression (Reinhardt et al., 2009). 6-thio-dG treatment significantly increased YFP+ T cells in the tumor, suggesting an enhanced IFN-γ-producing capacity of T cells (Figures 2F and 10F). A prominent feature of adaptive immune responses is the formation of memory, which initiates a rapid recall response when the same antigen appears. To determine whether 6-thio-dG treatment induces a memory response, mice with tumors that had completely regressed after 6-thio-dG treatment were rested for 5 weeks and reloaded with the same MC38 tumors, but 10 times more numerous, on the contralateral flank (left flank), while LLC tumor cells were inoculated on the right flank as a control. When the same number of MC38 cells were injected into naive mice (never exposed to MC38 cells or 6-thio-dG), the tumors proliferated rapidly. Notably, all mice treated with 6-thio-dG spontaneously rejected the reloaded MC38 tumors.

[0144] Treatment with 6-thio-dG enhances the cross-priming ability of dendritic cells. Antigen cross-presentation by antigen-presenting cells (APCs), such as DCs or macrophages, is responsible for tumor-specific CD8+ T cell activation. To investigate which APC subsets contribute to 6-thio-dG-induced T cell activation, we first depleted macrophages using an anti-CSF1R antibody. We found that 6-thio-dG functioned more effectively in the macrophage-depleted group (Figure 3A), which can be explained by the additive effect of removing immunosuppressive tumor-associated macrophages. BATF3 (basic leucine zipper ATF-like transcription factor 3)-dependent DCs are critically important for priming antigen-specific CD8+ T cells (Broz et al., 2014; Edelson et al., 2010). 6-thio-dG treatment in Batf3-deficient mice partially delayed tumor growth but was significantly less effective compared to WT mice (Figure 3B). Notably, while 60% of the WT mice were completely tumor-free, none of the Batf3- / - mice were tumor-free (Figure 3C), suggesting a crucial role for BATF3-dependent DCs in the therapeutic effect of 6-thio-dG.

[0145] To directly demonstrate that 6-thio-dG treatment enhances the cross-priming ability of DCs, the inventors co-cultured MC38-OVA tumor cells pre-treated with 6-thio-dG overnight with bone marrow-derived DCs (BMDCs). The DCs were then purified and OVA 257~264Naive OT-1 transgenic CD8+ T cells expressing a TCR with specificity for epitope recognition were co-cultured with DCs. The inventors observed a significant increase in IFN-γ production by CD8+ T cells in the 6-thio-dG treated group (Figure 3D), which indicates increased cross-priming ability of DCs after 6-thio-dG treatment. Since IFN-I signaling promotes the cross-priming ability of DCs (Diamond et al., 2011; Le Bon et al., 2003; Sanchez-Paulete et al., 2017), the inventors co-cultured DCs with 6-thio-dG treated tumor cells and then tested IFN-β production by DCs. Indeed, IFN-β production significantly increased in the 6-thio-dG treated group, indicating increased spontaneous sensing by DCs (Figure 3E). The inventors further investigated whether the IFN-I pathway is essential for the antitumor effect mediated by 6-thio-dG. Using Ifnar1- / - mice, the inventors showed that loss of IFN-I signaling in the host abolished the antitumor effect of 6-thio-dG (Figure 3F), indicating the essential role of IFN-I signaling in 6-thio-dG treatment.

[0146] STING signaling in the host is necessary for spontaneous sensing induced by 6-thio-dG. Tumor cells under stress may release a risk-associated molecular pattern (DAMP) to initiate IFN-I signaling by engaging in the TLR / Myd88 pathway in APCs. Tumor-derived DNA can also trigger the cytoplasmic DNA-sensing cGAS / STING pathway, activating the IFN-I pathway (Deng et al., 2014; Li et al., 2019). To further illustrate which upstream pathways are essential in 6-thio-dG-triggered IFN-I signaling activation in host cells, we inoculated Myd88- / - and Tmem173- / - (Tmem173 encodes STING) mice with MC38 tumors. 6-thio-dG treatment adequately controlled tumor growth in Myd88- / - mice, but completely lost its effectiveness in Tmem173- / - mice (Figures 4A and 4B), suggesting an essential role in host STING signaling in spontaneous sensing triggered by 6-thio-dG. We further investigated whether 6-thio-dG treatment activates the host STING / IFN-I pathway. We observed increased TBK1 phosphorylation in DCs after co-culture with tumor cells pre-treated with 6-thio-dG, and phosphorylation was completely attenuated in Tmem173 DCs (Figure 11A). 6-thio-dG treatment induced IFN-β production in DCs in a STING-dependent manner (Figure 11B). Previous studies have reported that tumor-endogenous STING signaling is critically important in spontaneously sensing cancer treatment (Sen et al., 2019; Vanpouille-Box et al., 2017). Therefore, we investigated whether tumor-endogenous STING signaling also contributes to the efficacy of 6-thio-dG treatment. We used CRISPR / Cas9 to knock out Tmem173 and Mb21d1 (Mb21d1 encodes cGAS) in MC38 tumor cells. In contrast to other studies, 6-thio-dG treatment still controlled tumor growth in mice with Tmem173KO and Mb21d1KO tumor cells, thus indicating that tumor-endogenous STING signaling played a non-essential role (Figure 4C and Figure 4D).

[0147] Next, the inventors attempted to clarify how tumor cells treated with 6-thio-dG trigger spontaneous sensing in DCs. Since 6-thio-dG is a telomere-targeting drug, telomere stress induced by 6-thio-dG may contribute to spontaneous sensing in DCs by releasing DNA. Therefore, the inventors first analyzed telomere stress using a TIF (telomere dysfunction-induced lesion) assay and showed that 6-thio-dG induces telomere damage in MC38 cells (Figures 4E and 4F). Since telomeres represent only a small portion (approximately 1 / 6000) of genomic DNA, the co-localization of DNA damage and telomeres is significant. The inventors also observed a similar increase in TIF in 6-thio-dG-treated tumor tissue from MC38 tumor-bearing mice (Figures 11C and 11D). 6-thio-dG also induces an interphase bridge between two daughter cells during telophase, many of which contain telomere sequences, which may explain why many micronuclei contain telomere signals when the cell re-enters interphase after mitosis (Figure 11E). These cytoplasmic fragments form micronuclei with fragile nuclear envelopes (Figures 11E and 11F), which can eventually be recognized as danger signals. These DNA fragments are released from the cell and can be taken up by DCs.

[0148] To demonstrate this hypothesis, the inventors treated the human colon cancer cell line HCT116 with 6-thio-dG, co-cultured it with mouse BMDCs for 4 hours, then isolated the DCs and extracted cytoplasmic DNA. Short-term co-culture of human tumor cell lines with mouse BMDCs allowed the inventors to distinguish DNA from different origins. The inventors found an increase in human DNA (MT-CO1 and human 18S) in the cytoplasm of mouse DCs after 6-thio-dG treatment, suggesting that tumor-derived DNA enters host DCs (Figure 4G). To determine whether 6-thio-dG treatment increases the uptake of specific telomere DNA by DCs, the inventors labeled tumor cells with EdU and then washed the cells. Next, the inventors treated the cells with 6-thio-dG and washed them again. Finally, the inventors co-cultured the tumor cells with DCs and then isolated the DCs for analysis. DCs with tumor DNA uptake in the cytoplasm (EdU + In DC, the inventors observed increased telomere co-localization with EdU after 6-thio-dG treatment, suggesting significant uptake of tumor-derived telomere DNA (Figures 11G and 11H). Collectively, the inventors demonstrated that 6-thio-dG triggers spontaneous sensing through activation of the host cytoplasmic DNA-sensing STING / IFN-I pathway.

[0149] 6-thio-dG overcomes PD-L1 blockade resistance in advanced tumors. Treatment with 6-thio-dG activated CD8+ T cells and upregulated PD-1 expression at both the frequency of all CD8+ T cells and per cell (Figure 5A). PD-1 is a co-inhibitory molecule that limits T cell activation. Elevated PD-1 expression may ultimately inhibit cytotoxic CD8+ T cell function after 6-thio-dG treatment. Therefore, we hypothesized that the combination of 6-thio-dG and PD-1 / PD-L1 blockade may enhance the overall antitumor immune response, particularly in advanced tumor situations with a more immunosuppressive microenvironment containing multiple resistance mechanisms that limit the effectiveness of monotherapy. 6-thio-dG monotherapy was effective for approximately 100 mm 3Since it was only effective for relatively small tumor sizes, for the treatment of advanced tumors, the inventors have found that it is effective for tumor sizes of 150-200 mm. 3 The tumors were treated with 6-thio-dG and / or anti-PD-L1 therapy. In such advanced cancers, tumor volume was difficult to control with twice-daily treatment with 6-thio-dG or twice-daily treatment with anti-PD-L1 (Figure 5B). However, sequential administration of 6-thio-dG and anti-PD-L1 completely inhibited tumor growth (Figure 5B). Notably, only mice in the combination treatment group achieved 100% survival (Figure 5C), demonstrating a synergistic effect of 6-thio-dG treatment with PD-L1 blockade. Furthermore, the inventors observed no weight loss in mice in the combination treatment group (Figure 12). The inventors further analyzed tumor-specific T cell responses in inflow area lymph nodes (dLNs) and found that anti-PD-L1 therapy had little effect on T cell activation in advanced tumors. In contrast, the combination therapy significantly increased IFN-γ production compared to the other groups. Since there were virtually no IFN-γ spots in the control LLC tumor-stimulated group, the immune response was specific to the MC38 tumor (Figure 5D).

[0150] MC38 is known to be an immunogenic tumor model. To test whether combination therapy can overcome PD-L1 blockade resistance even in less immunogenic tumor models, we used a mouse LLC tumor model that has been reported to be resistant to PD-L1 blockade (Bullock et al., 2019; Li et al., 2017). Consistent with previous reports, treatment with anti-PD-L1 alone had no therapeutic effect (Figure 5E). Notably, the combination of 6-thio-dG and anti-PD-L1 significantly reduced mouse tumor burden, and 40% of the mice eventually completely rejected the tumor (Figure 5E). To investigate the memory response, we reloaded tumor-free mice 6 weeks after tumor regression. All combination-treated mice spontaneously rejected the LLC tumor but not the MC38 tumor, suggesting long-lasting tumor-specific immunomemory (Figure 5F). Based on these results, 6-thio-dG treatment overcomes PD-L1 blockade resistance in advanced tumors. This has the potential to benefit patients with PD-1 / PD-L1 blockade resistance in clinical practice.

[0151] 6-thio-dG reduces the burden of human colon cancer in a humanized mouse model. Previous studies have shown that patients with high TERT (telomerase catalytic subunit) expression have poor clinical outcomes in various cancers, including non-small cell lung cancer and B-cell chronic lymphocytic leukemia (Terrin et al., 2007; Wang et al., 2002). Therefore, we analyzed colorectal adenocarcinoma patients from the TCGA database and found that patients with abnormally high TERT expression had significantly worse overall survival compared to colorectal cancer patients with low TERT expression (Figure 6A). To directly demonstrate whether 6-thio-dG-induced telomere stress can benefit cancer patients in a more clinically relevant model, we developed a humanized mouse model using NSG-SGM3 mice with transgenic expression of human SCF-1, GM-CSF, and IL-3, which support better development of human bone marrow cells. We reconstituted the human immune system in NSG-SGM3 mice using human CD34+ hematopoietic stem cells (HSCs). Twelve weeks after HSC transfer, the humanized mice possessed an average of over 60% human CD45+ cells and over 20% human T cells among the circulating human CD45+ cells (Figures 13A-13C). Subsequently, the inventors administered 0.73 μM IC50 to the mice. 50The human colon cancer cell line HCT116 (Figure 6B), which is sensitive to 6-thio-dG treatment, was inoculated into NSG-SGM3 control mice and humanized NSG-SGM3 mice. The humanized mouse control group had a similar human immune cell composition to the 6-thio-dG treated group before treatment was initiated (Figures 13B and 13C). After three doses of 6-thio-dG treatment, immunodeficient mice did not show a significant difference compared to the control group (Figure 6D). Notably, the humanized mice showed a significant delay in tumor growth after 6-thio-dG treatment (Figures 6C and 6E). Next, the inventors tested the human melanoma cell line A375, which is sensitive to 6-thio-dG in vitro (Figure 13D). The inventors administered 6-thio-dG treatment for a relatively short period, and therefore observed no effect in immunocompromised NSG-SGM3 mice (Figure 13E). Notably, the inventors found that treatment with two doses of 6-thio-dG partially delayed tumor growth in humanized mice. Furthermore, combination with checkpoint blockade further reduced tumor volume, suggesting that 6-thio-dG pretreatment sensitizes human tumors to checkpoint blockade (Figure 13F). Given that humanized mice only partially restore human immunity due to the deletion of several immune cells and a limited number of human T cells, it is not surprising that the inventors did not observe complete tumor regression.

[0152] The inventors demonstrated that treatment with 6-thio-dG for 3 days, followed by treatment with anti-PD-L1 2 days later, resulted in complete tumor remission in Lewis lung cancer (Figures 8A-8B). This is an extremely invasive tumor type, as illustrated in Figure 8A. The tumor, injected subcutaneously, was reduced to 1000m within 20 days. m 3The results were astonishing. The inventors observed the same rapid tumor growth with treatment with anti-PD-L1 alone. However, just three treatments with 6-thio-dG (THIO) resulted in significant tumor control for 20 days. Surprisingly, THIO and subsequent treatment with anti-PD-L1 (atezolizumab) resulted in complete tumor regression. The inventors maintained the cured mice for a further 5 weeks and reloaded the same mice with LLC tumors five more times, but no tumor growth was observed. However, when the inventors injected LLC-cured mice with MC38, the tumors grew, and anti-PD-L1 had no effect (Figure 8B). In control mice that had never been treated with THIO, the control LLC tumors grew similarly to those treated with MC38. This suggests tumor-specific immunological memory.

[0153] Overall, these data can be interpreted to support the idea that 6-thio-dG induces telomerase-dependent DNA damage and increases tumor DNA uptake by dendritic cells (DCs). The increased cytoplasmic DNA triggers the endogenous STING / IFN-I pathway in DCs, leading to enhanced cross-priming ability of DCs and subsequent tumor-specific T cell activation. Furthermore, 6-thio-dG overcomes PD-L1 blockade resistance in advanced tumors. This study identifies 6-thio-dG as a novel immunostimulant with potential benefits for a broad population of cancer patients in clinical practice.

[0154] Example 3 - Discussion High telomerase expression in tumor cells is recognized as a poor prognostic factor for cancer development (Zhang et al., 2018). Here, we report the previously uncertain role of telomerase-dependent telomere targeting therapy (6-thio-dG) in inducing antitumor immune responses in syngeneic colon and lung mouse models as well as in humanized mouse cancer models. This effect is mediated through the triggering of the cytoplasmic DNA-sensing STING / IFN-I pathway in dendritic cells (DCs), ultimately enhancing the cross-priming ability of DCs and subsequent tumor-specific T cell activation. This is a noteworthy finding, as telomerase is a universal tumor marker and may be applicable to many other telomerase-positive cancers. Furthermore, sequential administration of 6-thio-dG and anti-PD-L1 overcomes PD-L1 resistance in PD-L1 blockade-resistant tumors, suggesting that combination therapy may benefit PD-L1-resistant patients in clinical practice.

[0155] The current consensus is that 6-thio-dG treatment kills tumor cells primarily by damaging telomeres and inducing DNA damage. This study also demonstrates that this drug controls tumors primarily in a manner dependent on DNA sensing and T cell responses. Most previous studies have used xenograft models lacking a complete immune system. These models can only study tumor-endogenous actions or parts of the innate immune response. T cells are essential for long-term tumor control, even if they may be important factors. Furthermore, most previous studies tend to use high-dose or intensive drug strategies that more efficiently kill tumor cells directly, but in reality, they weaken the immune response due to either toxicity to immune cells or non-immunogenic death of tumor cells (Galluzzi et al., 2017; Kroemer et al., 2013). These intensive drug strategies also often lead to the emergence of tumor resistance mechanisms. In this study, the inventors utilized a fully immune-bodied syngeneic mouse model and a clinically more appropriate humanized mouse model to fully evaluate the effects of lower-dose and shorter treatment regimens with 6-thio-dG on the host immune response in tumor-bearing mice. This finding that 6-thio-dG is an immunostimulant may enable the design of better combination treatments, including immunotherapy, to amplify initial immunity.

[0156] Accumulating research indicates that spontaneous sensing mediated by tumor DNA is crucial for inducing antitumor immune responses, and that the STING / IFNI pathway is primarily involved in initiating these responses, but whether host or tumor-autonomous STING is more essential depends on the different treatment regimens (Deng et al., 2014; Li et al., 2019; Qiao et al., 2017; Sen et al., 2019; Vanpouille-Box et al., 2017; Woo et al., 2014). This discrepancy may be explained by the relative STING activation intensity of the host compared to tumor cells, for example, tumor cells may have suppressed or low-activity STING pathways (Xia et al., 2016). The inventors demonstrated that the spontaneous sensing of 6-thio-dG treatment is dependent on host STING signaling, as 6-thio-dG completely lost its effectiveness in Tmem173-deficient mice but not in Tmem173-deficient tumors. Since STING signaling is active in MC38 tumors, one explanation is that tumor-endogenous STING was activated after 6-thio-dG treatment, but most tumor cells died, so type I IFN could not be produced in large quantities. Another possibility is that there may be an endogenous mechanism that limits STING activation in tumor cells, but this remains largely undetermined. Recent reports have shown that STING signaling can also be involved in autophagy activation (Gui et al., 2019; Nassour et al., 2019), but the inventors did not observe autophagy activation in tumor cells after 6-thio-dG treatment (data not shown), making it less likely that 6-thio-dG contributes to the therapeutic effect. Furthermore, 6-thio-dG lost its effectiveness in Ifnar1-deficient mice, suggesting involvement of IFNI signaling. However, STING activation in autophagy is independent of IFNI signaling.

[0157] Compared to common DNA damage induction approaches, such as radiotherapy or chemotherapy which non-selectively induce DNA damage in all proliferating cells, one unique feature of 6-thio-dG is that it specifically induces telomere-associated DNA damage in telomerase-expressing cells, primarily tumor cells, but does not affect immune cells or other somatic cells where telomerase is silent. Importantly, 6-thio-dG can be preferentially incorporated into de novo-synthesized telomeres, leading to rapid tumor reduction. However, direct telomerase inhibitors work by inhibiting telomerase activity and rely on gradual telomere shortening. In contrast, 6-thio-dG exerts its effects rapidly regardless of initial telomere length. This is crucial in reducing toxicity compared to direct telomerase inhibitors (Gryaznov et al., 2007; Mender et al., 2015b). The inventors have shown that 6-thio-dG-induced DNA damage is significantly localized with telomeres, indicating the formation of telomere-induced lesions (TIFs). Since telomeres make up only about 1 / 6000th of genomic DNA, each TIF is highly significant. Furthermore, some TIFs are taken up by DCs, further triggering STING-dependent IFNI signaling.

[0158] Despite the overwhelming success of checkpoint blockade, particularly PD-1 / PD-L1 blockade, clinical practice shows that only a small number of patients respond well. Both primary and adaptive resistance limit the clinical benefit of PD-1 / PD-L1 therapy (Chen and Han, 2015; Gide et al., 2018; Zaretsky et al., 2016; Zou et al., 2016). We hypothesize that a lack of appropriate innate sensing may limit T cell activation within the tumor microenvironment, and therefore, combination therapies targeting both innate and adaptive immune cells are urgently needed. PD-L1 blockade reactivates the adaptive immune response by "releasing the brakes," while 6-thio-dG induces innate sensing by "adding fuel." We hypothesized that the combination of 6-thio-dG and PD-L1 blockade should enhance the overall anti-tumor immune response. In fact, this study demonstrated that sequential administration of 6-thio-dG and anti-PD-L1 has a synergistic effect in advanced tumors and PD-L1 blockade-resistant tumors. Further research should be conducted to determine the optimal combination regimen.

[0159] Overall, these results reveal a previously undefined role of 6-thio-dG, a telomerase-dependent telomere-targeting small molecule drug, in enhancing the anti-tumor immune response. Mechanistically, 6-thio-dG induces telomere dysfunction and increases cytoplasmic DNA release. Importantly, these telomere DNA fragments are taken up by dendritic cells (DCs), activating the DC-endogenous STING / IFN pathway, resulting in enhanced cross-priming ability of DCs and subsequent tumor-specific T cell activation. Furthermore, this study, demonstrating the remarkable efficacy of sequential administration of 6-thio-dG and anti-PD-L1 in advanced tumors and PD-L1-blockade-resistant tumors, provides strong scientific evidence to advance combination therapy into clinical trials. The inventors anticipate that these findings will be clinically applied in the near future, benefiting more patients in treatment.

[0160] All methods disclosed and claimed herein can be prepared and performed without undue experimentation in light of this disclosure. While the compositions and methods of this disclosure have been described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and the steps or sequences of the methods described herein without departing from the concepts, spirit, and scope of this disclosure. More specifically, the active agents described herein can be substituted with certain chemically and physiologically related active agents, and it will be apparent that the same or similar results can be achieved. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to fall within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0161] VI. References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement the details described herein. TIFF0007829491000007.tif194166TIFF0007829491000008.tif221166TIFF0007829491000009.tif203166TIFF0007829491000010.tif211166 TIFF0007829491000011.tif206166TIFF0007829491000012.tif216166TIFF0007829491000013.tif218166TIFF0007829491000014.tif191166

Claims

1. A pharmaceutical composition comprising 6-thio-2'-deoxyguanosine (6-thio-dG) for use in a method of treating cancer in a subject that is resistant to checkpoint inhibitors alone or to checkpoint inhibitors in combination with chemotherapy, The method includes the step of administering 6-thio-dG to the subject, followed by treatment with a checkpoint inhibitor selected from the group consisting of atezolizumab and semiprimab. A pharmaceutical composition wherein the cancer is selected from the group consisting of lung cancer, liver cancer, and colorectal cancer.

2. 6-thio-dG is administered for approximately 1 to 5 days per treatment cycle, and / or Checkpoint inhibitors are administered for approximately 1 to 3 days per treatment cycle. The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 1, wherein the checkpoint inhibitor is atezolizumab.

4. The pharmaceutical composition according to claim 1, wherein the checkpoint inhibitor is semiprimab.

5. The pharmaceutical composition according to claim 1, wherein the total dose of 6-thio-dG administered over a treatment period of approximately 1 to 5 days is approximately 20 to 2000 mg.

6. The pharmaceutical composition according to claim 1, wherein the cancer is resistant to chemotherapy.

7. The pharmaceutical composition according to claim 1, wherein the subject has been previously treated with checkpoint inhibitor therapy.

8. The pharmaceutical composition according to claim 7, wherein the subject has been previously treated with one or more of the following: PD-1 checkpoint inhibitor therapy or PD-L1 checkpoint inhibitor therapy.

9. The pharmaceutical composition according to claim 1, wherein the 6-thio-dG and the checkpoint inhibitor are administered systemically.

10. The pharmaceutical composition according to claim 1, wherein the 6-thio-dG and the checkpoint inhibitor are administered locally or regionally to the tumor site.

11. The pharmaceutical composition according to claim 1, wherein the 6-thio-dG is administered locally or locally to the tumor site, and the checkpoint inhibitor is administered systemically.

12. The pharmaceutical composition according to claim 1, wherein administration of 6-thio-dG and a checkpoint inhibitor results in inhibition of tumor growth.

13. The pharmaceutical composition according to claim 1, wherein administration of 6-thio-dG and a checkpoint inhibitor results in remission of the cancer.

14. The pharmaceutical composition according to claim 1, wherein administration of 6-thio-dG and a checkpoint inhibitor results in a reduction in tumor burden.

15. The pharmaceutical composition according to claim 1, wherein administration of 6-thio-dG and a checkpoint inhibitor results in inhibition of cancer cell metastasis.

16. The pharmaceutical composition according to claim 1, wherein administration of 6-thio-dG and a checkpoint inhibitor results in tumor eradication.

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