Methods of treating uveal melanoma liver metastasis with therapeutically effective combination of one or more checkpoint inhibitors and toll-like receptor 9 agonist

Abstract

The present invention relates to a method for treating uveal melanoma liver metastasis in a human subject. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist having the following structure: 5 '-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1). The one or more checkpoint inhibitors are systemically administered to the subject and the TLR9 agonist is locally administered to the liver of the subject by means of hepatic arterial infusion at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor.

Description

[0001] Priority Statement This application claims priority to U.S. Provisional Application Serial No. 63 / 470,653, filed June 2, 2023, and U.S. Provisional Application Serial No. 63 / 546,424, filed October 30, 2023, the entire contents of which are hereby incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to methods for treating cancer, specifically liver cancer, and methods for delivering toll-like receptor (TLR) agonists to solid tumors in the liver via the vascular system using localized regional therapy. Background Technology

[0003] Cancer is a devastating disease involving the uncontrolled growth of cells that can lead to the growth of solid tumors in various organs such as the skin, liver, and pancreas. Tumors may originate in any number of organs or may be the result of metastasis or spread from other sites.

[0004] Melanoma is a clinically and molecularly diverse disease encompassing a wide range of subtypes and presentation characteristics. Melanoma has a number of rare subtypes and presentations, including melanoma with brain metastases and leptomeningeal disease, acral melanoma, pediatric melanoma, and melanoma of unknown primary origin, as well as uncommon presentation sites such as extradermal melanoma. Therefore, melanoma research must be at the forefront of development, including special populations that would otherwise be excluded from most therapeutic clinical trials. Uveal melanoma is a biologically unique disease, exhibiting significant cellular, molecular, and clinical differences from cutaneous melanoma (CM). Furthermore, UM and CM are distinct malignancies requiring different approaches.

[0005] Liver metastases (LM) are a leading cause of morbidity and mortality in many solid tumors. Uveal melanoma (UM) is a malignant disease of a solid organ in which metastatic spread to the liver causes rapidly progressive and often fatal symptoms, for which treatment options are limited. UM is a very rare condition, with fewer than 3,000 new cases diagnosed annually in the United States (US). Primary UM tumors can be effectively controlled with surgery or radiation therapy, but more than 50% of patients develop metastatic disease. These adverse outcome data are further complicated by the high predisposition of UM to spread to the liver via the bloodstream, an event that occurs in more than 90% of cases.

[0006] The liver is a unique organ that is immunosuppressive and drives the programming and expansion of suppressor cells, such as myeloid-derived suppressor cells (MDSCs). In this respect, MDSCs expand in response to malignancy. MDSCs also drive the expansion of other suppressor cell types, such as regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs). MDSCs can downregulate immune cells and interfere with the effectiveness of immunotherapeutic agents. Furthermore, high MDSC levels generally predict a poor prognosis in cancer patients. In the case of LM, MDSCs are a key driver of intrahepatic immunosuppression, enabling malignant tumor growth and progression. Specifically, MDSCs are associated with the pathogenesis of UM LM. As described below, suppressor immune cells in the liver not only suppress endogenous antitumor immunity but also limit the effectiveness of immuno-oncology therapies for LM.

[0007] UM has been shown to be highly refractory to immunotherapy. The prognosis for metastatic disease is very poor, with a 1-year overall survival (OS) rate of 43% and a response rate of less than 20% from initial diagnosis. Furthermore, the presence of LM has been shown to limit the effectiveness of checkpoint inhibitor therapy. Therefore, safe and effective treatments remain needed for UM LM. Summary of the Invention

[0008] This application relates to a method for treating liver metastases of uveal melanoma in a human subject. The subject may have stage IV uveal melanoma. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. In one example, the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), and The entire sequence is linked by phosphate thioester bonds. Specifically, the TLR9 agonist is SD-101 (also referred to herein as nelitolimod) or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is a sodium salt of SD-101, such as... Figure 1 As shown in the diagram. Specifically, the TLR9 agonist is administered locally to a region outside the liver metastasis of the uveal melanoma in the subject.

[0009] In another aspect of this application, a method for improving the survival rate of human subjects suffering from liver metastases of uveal melanoma is provided. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, said TLR9 agonist being an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. In one example, the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), and The entire sequence is linked by thiophosphate bonds. Specifically, the TLR9 agonist is SD-101 or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is a sodium salt of SD-101, such as... Figure 1 As shown in the illustration. Specifically, the TLR9 agonist is administered locally to a region outside the liver metastasis of the uveal melanoma in the subject. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the subject's progression-free survival (PFS).

[0010] In another aspect of this application, a method for treating uveal melanoma liver metastases in a human subject is provided. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, said TLR9 agonist being an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. In one example, the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), and The entire sequence is linked by thiophosphate bonds. Specifically, the TLR9 agonist is SD-101 or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is a sodium salt of SD-101, such as... Figure 1As shown in the illustration. Specifically, the TLR9 agonist is administered locally to a region outside the liver metastasis of the uveal melanoma in the subject. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the subject's progression-free survival (PFS). In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject.

[0011] In another aspect of this application, a method for reducing circulating tumor DNA (ctDNA) in a human subject with liver metastases from uveal melanoma is provided. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, said TLR9 agonist being an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. In one example, the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), and The entire sequence is linked by thiophosphate bonds. Specifically, the TLR9 agonist is SD-101 or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is a sodium salt of SD-101, such as... Figure 1 As shown in the diagram. Specifically, the TLR9 agonist is administered locally to a region outside the liver metastasis of the uveal melanoma in the subject.

[0012] In another aspect of this application, a method is provided for improving the response rate of human subjects with liver metastases from uveal melanoma to treatment with checkpoint inhibitors. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, said TLR9 agonist being an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. In one example, the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), and The entire sequence is linked by thiophosphate bonds. Specifically, the TLR9 agonist is SD-101 or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is a sodium salt of SD-101, such as... Figure 1 As shown in the illustration. Specifically, the TLR9 agonist is administered locally to a region outside the uveal melanoma liver metastasis in the subject's liver. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the subject's survival. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the subject's progression-free survival (PFS). In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject. In some instances, administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases immune cell activation within the uveal melanoma liver metastasis in the subject. In some instances, administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases systemic cytokine signaling in the subject's blood.

[0013] In all the foregoing aspects of this application, the one or more checkpoint inhibitors are administered systemically to the subject. The one or more checkpoint inhibitors may be administered intravenously, intraperitoneally, or subcutaneously. The one or more checkpoint inhibitors may be administered concurrently with, before, or after the TLR9 agonist.

[0014] In some instances, the one or more checkpoint inhibitors may be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, relatlimab, and crefmirlimab. In one instance, the one or more checkpoint inhibitors is nivolumab. In another instance, the one or more checkpoint inhibitors is pembrolizumab. In yet another instance, the one or more checkpoint inhibitors is ipilimumab. In still another instance, the one or more checkpoint inhibitors is crefmirlimab. In some instances, two checkpoint inhibitors are administered. For example, the two checkpoint inhibitors are nivolumab and ipilimumab. In another instance, the two checkpoint inhibitors are nivolumab and relatlimab.

[0015] The TLR9 agonist is administered locally to the liver of the subject via hepatic artery infusion at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor and to provide a plasma concentration of the TLR9 agonist below its concentration in the liver. In some instances, the plasma concentration of the TLR9 agonist is at a subtherapeutic dose when administered without the one or more checkpoint inhibitors. In other instances, the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject. For example, the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g. In some instances, the plasma concentration of the TLR9 agonist is below about 750 ng / mL, or below about 700 ng / mL, or below about 600 ng / mL. In some instances, the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is from about 1:2 to about 1:10. In another example, the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:7.

[0016] In some instances, the TLR9 agonist is administered via a catheter device. The catheter device may include a one-way valve that dynamically responds to changes in local pressure and / or flow. In some instances, the TLR9 agonist is administered via the catheter device using pressure-enabled drug delivery (PEDD).

[0017] In some instances, the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg. In other instances, the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg. In yet another instance, the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg. In still another instance, the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg. In some instances, the TLR9 agonist is administered weekly at a dose of about 2 mg.

[0018] In one specific example of a method for treating uveal melanoma (e.g., stage IV uveal melanoma), the method comprises locally administering a TLR9 agonist having the sequence of SEQ ID NO: 2 at a dose of about 2 mg, preferably by hepatic artery infusion (HAI) and more preferably by PEDD. The method also comprises systemic administration of a checkpoint inhibitor, specifically an anti-PD1 or anti-PDL1 antibody, and specifically nivolumab. The method may further comprise administration of an anti-CTLA4 antibody (e.g., ipilimumab) in addition to an anti-PD1 or anti-PDL1 antibody.

[0019] In some instances, the TLR9 agonist is administered weekly. In some instances, the TLR9 agonist may be administered weekly on a 3-week cycle with rest periods between cycles. The TLR9 agonist may be administered weekly for a duration of approximately 10 minutes to approximately 200 minutes. The TLR9 agonist may be administered via a catheter device. The catheter device may include a one-way valve that dynamically responds to changes in local pressure and / or flow. For example, the TLR9 agonist may be administered via the catheter device using pressure-enabled drug delivery (PEDD). In other instances, the TLR9 agonist is administered weekly for a duration of approximately 10 minutes to approximately 60 minutes. In yet another instance, the TLR9 agonist is administered for a duration of approximately 25 minutes.

[0020] These and other objects, features, and advantages of the exemplary embodiments of this disclosure will become apparent when read in conjunction with the following detailed description of the exemplary embodiments of this disclosure throughout the specification. Attached Figure Description

[0021] Other objects, features, and advantages of this disclosure will become clear from the following detailed description, taken in conjunction with the accompanying drawings illustrating illustrative embodiments of the present disclosure.

[0022] Figure 1 The chemical structure of the sodium salt of SD-101 is shown.

[0023] Figure 2A The Phase 1 / 1B clinical trial protocol described in Example 1 is presented.

[0024] Figure 2B Provided acceptance Figure 2A The characteristics of patients in the Phase 1 / 1B clinical trial, where LDH is an abbreviation for lactate dehydrogenase level and ULN is an abbreviation for the upper limit of normal.

[0025] Figure 2C Provided according to Figure 2AThe safety summary and adverse events of the Phase 1 / 1B clinical trial protocol, including groups A, B, and C who received SD-101 and / or CPI.

[0026] Figure 3 Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to profile the serum pharmacokinetics of SD-101 after administration of 2 mg.

[0027] Figure 4 Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to profile the serum pharmacokinetics of SD-101 after administration of 4 mg.

[0028] Figure 5 Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to profile the serum pharmacokinetics of SD-101 after administration of 8 mg.

[0029] Figure 6 Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the pharmacokinetic profile of SD-101 in liver tissue after administration of 8 mg.

[0030] Figure 7A Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the serum cytokine levels of CXCL10 (IP-10) after administration of a TLR9 agonist.

[0031] Figure 7B Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, serum cytokine analysis of IL-8 after administration of TLR9 agonist.

[0032] Figure 8A Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze serum cytokines of IL-2R after administration of TLR9 agonists.

[0033] Figure 8B Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, serum cytokine analysis of IFNγ after administration of TLR9 agonist.

[0034] Figure 9A Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol used MDSC-related genes to regulate TME in uveal melanoma liver metastases on day 57.

[0035] Figure 9B Showing according to Figure 2AThe Phase 1 / 1B clinical trial protocol used T-cell-related genes to regulate the TME of uveal melanoma liver metastases on day 57.

[0036] Figure 10 Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in intratumoral M-MDSCs 57 days after SD-101 administration.

[0037] Figure 11A Showing according to Figure 2A This study is based on the Phase 1 / 1B clinical trial protocol, analyzing circulating immune cells (RBCs) of changes in circulating NK cell protein expression profiles (day 1–day 36) after SD-101 administration. Data are presented as mean + SEM (n = 19).

[0038] Figure 11B Showing according to Figure 2A This study is based on the Phase 1 / 1B clinical trial protocol, and is an analysis of circulating immune cells showing changes in the protein expression profile of circulating CD8 T cells after SD-101 administration (day 1–day 36). Data are presented as mean + SEM (n = 23).

[0039] Figure 12A Showing according to Figure 2A This study is based on the Phase 1 / 1B clinical trial protocol, and is an analysis of circulating immune cells showing changes in the protein expression profile of circulating CD8 T cells after SD-101 administration (day 1–day 36). Data are presented as mean + SEM (n = 23).

[0040] Figure 12B Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol of SD-101 was used to analyze the changes in protein expression on circulating CD8+ T cells in groups A, B, and C (day 1-36) after administration of SD-101.

[0041] Figure 12C Provided separately according to the amount of SD-101 (also known as Netomod) applied. Figure 12B The data.

[0042] Figure 12D Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the changes in protein expression on circulating NK cells in groups A, B, and C after SD-101 administration (day 1–day 36) using circulating immune cells.

[0043] Figure 12E Provided separately according to the amount of SD-101 (also known as Netomod) applied. Figure 12D The data.

[0044] Figure 13A Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, the response and survival time of patients in groups A, B and C who had undergone extensive pretreatment.

[0045] Figure 13B Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, the response and survival time of patients in Group B who had undergone extensive pretreatment.

[0046] Figure 14A It shows that according to Figure 2A Changes in ctDNA levels in patients treated with the Phase 1 / 1B clinical trial protocol.

[0047] Figure 14B Explanation provided based on Figure 2A Additional data on changes in ctDNA levels in patients treated with the Phase 1 / 1B clinical trial protocol.

[0048] Figure 14C Explanation provided based on Figure 2A Additional data on changes in ctDNA levels in group B patients treated with the Phase 1 / 1B clinical trial protocol.

[0049] Figure 14D Explanation provided based on Figure 2A Data on changes in ctDNA levels in patients in groups B and C at time points after the first cycle of treatment (including day 36 and day 57, unless otherwise stated) in the Phase 1 / 1B clinical trial protocol.

[0050] Figure 14E Provided separate options for groups B and C Figure 14D The data.

[0051] Figure 14F Explanation provided based on Figure 2A Data on the reduction in ctDNA mutant allele fraction (MAF) relative to baseline in patients in groups B and C treated with the Phase 1 / 1B clinical trial protocol.

[0052] Figure 14G Provided separate options for groups B and C Figure 14F The data.

[0053] Figure 15A It is shown in accordance with Figure 2A Changes in CD8+ T cells in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0054] Figure 15B It is shown in accordance with Figure 2AChanges in CD4+ T cells in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0055] Figure 15C It is shown in accordance with Figure 2A Changes in NK cells in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0056] Figure 15D It is shown in accordance with Figure 2A Changes in M1 macrophages in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0057] Figure 16A It is shown in accordance with Figure 2A Changes in CD8+ T cells in patients in group B treated with the Phase 1 / 1B clinical trial protocol.

[0058] Figure 16B It is shown in accordance with Figure 2A Changes in CD4+ T cells in patients in group B treated with the Phase 1 / 1B clinical trial protocol.

[0059] Figure 16C It is shown in accordance with Figure 2A Changes in NK cells in patients in group B treated with the Phase 1 / 1B clinical trial protocol.

[0060] Figure 16D It is shown in accordance with Figure 2A Changes in M1 macrophages in patients in group B treated with the Phase 1 / 1B clinical trial protocol.

[0061] Figure 17A It is shown in accordance with Figure 2A The changes in tumor pathway scores from baseline to day 57 in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0062] Figure 17B It is shown in accordance with Figure 2A The changes in PBMC pathway scores from baseline to day 36 in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0063] Figure 17C It is shown in accordance with Figure 2A The changes in tumor pathway scores by group from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C.

[0064] Figure 17D It shows Figure 17CChanges in tumor pathway scores based on the administered SD-101 dose.

[0065] Figure 17E It is shown in accordance with Figure 2A The changes in PBMC pathway scores by group from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C.

[0066] Figure 17F It shows Figure 17E Changes in PBMC pathway scores based on the administered SD-101 dose.

[0067] Figure 17G It is shown in accordance with Figure 2A The changes in tumor gene expression levels from baseline to day 57 in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0068] Figure 17H It shows Figure 17G Changes in tumor gene expression levels categorized by the dose of SD-101 administered.

[0069] Figure 17I It shows the display according to Figure 2A Additional data on changes in tumor gene expression levels from baseline to day 57 in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0070] Figure 17J It shows Figure 17I Changes in tumor gene expression levels categorized by the dose of SD-101 administered.

[0071] Figure 17K It is shown in accordance with Figure 2A The changes in PBMC gene expression levels from baseline to day 36 in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol.

[0072] Figure 17L It shows Figure 17K Changes in PBMC gene expression levels based on the administered SD-101 dose.

[0073] Figure 17M It shows the display according to Figure 2A Additional data on changes in PBMC gene expression levels from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol, categorized by group: A, B, and C.

[0074] Figure 17N It shows Figure 17MChanges in PBMC gene expression levels based on the administered SD-101 dose.

[0075] Figure 18A It is shown in accordance with Figure 2A The changes in Treg in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C as the dose of SD-101 increased.

[0076] Figure 18B It is shown in accordance with Figure 2A The changes in M-MDSC in groups A, B, and C of patients treated with the SD-101 dose were observed in the Phase 1 / 1B clinical trial protocol.

[0077] Figure 18C It is shown in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, changes in granzyme B were observed in groups A, B, and C with increasing SD-101 dose.

[0078] Figure 18D It is shown in accordance with Figure 2A The changes in IL-15 in groups A, B, and C of patients treated with the SD-101 dose were observed in the Phase 1 / 1B clinical trial protocol.

[0079] Figure 19A Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in Treg cell concentrations in Group B relative to baseline data at 2 mg, 4 mg, and 8 mg.

[0080] Figure 19B Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in cell concentration of M-MDSC cells in Group B relative to baseline data at 2 mg, 4 mg, and 8 mg.

[0081] Figure 19C Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in total MSDC cell concentration relative to baseline data in Group B at 2 mg, 4 mg, and 8 mg.

[0082] Figure 19D Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in cell concentration of M2 macrophages in Group B relative to baseline data at 2 mg, 4 mg, and 8 mg.

[0083] Figure 19E Showing according to Figure 2AThe Phase 1 / 1B clinical trial protocol was used to study the changes in cell concentrations of granzyme B relative to baseline data in Group B at 2 mg, 4 mg, and 8 mg.

[0084] Figure 19F Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study the changes in IL-15 cell concentrations in Group B relative to baseline at 2 mg, 4 mg, and 8 mg.

[0085] Figure 20 A shows according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized changes in the peripheral immune characteristic CXCL10 (IP-10) of patients treated with different doses of SD-101 via PEDD delivery.

[0086] Figure 20 B shows according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized changes in the peripheral immune characteristic IFNγ in patients treated with different doses of SD-101 via PEDD delivery.

[0087] Figure 20 C shows according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized changes in the peripheral immune characteristic TNFα in patients treated with different doses of SD-101 via PEDD delivery.

[0088] Figure 20 D shows according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized PEDD delivery of different doses of SD-101 to assess changes in SD-101-induced peripheral immune signature IL-2R in patients.

[0089] Figure 20 E shows according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized changes in the peripheral immune characteristic IL-15 of patients treated with different doses of SD-101 via PEDD delivery.

[0090] Figure 20 F shows that according to Figure 2A The Phase 1 / 1B clinical trial protocol utilized changes in the peripheral immune characteristic IL-18 of patients treated with different doses of SD-101 via PEDD delivery.

[0091] Figure 21 A shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in immune characteristics during the CXCL10 (IP-10) cycle in groups A, B, and C.

[0092] Figure 21 B shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in immune characteristics in the IFNγ circulation in groups A, B, and C.

[0093] Figure 21 C shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in immune characteristics of the TNFα circulation in groups A, B, and C.

[0094] Figure 21 D shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in immune characteristics in the SD-101-induced IL-2R circulation in groups A, B, and C.

[0095] Figure 21 E shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in the immune characteristics of circulating IL-15 in groups A, B, and C.

[0096] Figure 21 F shows that according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to study changes in the immune characteristics of circulating IL-18 in groups A, B, and C.

[0097] Figure 22 A shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in immune characteristics in the CXCL10 (IP-10) cycle of group B at 2 mg, 4 mg, and 8 mg.

[0098] Figure 22 B shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in the IFNγ circulating immune characteristics in group B at 2 mg, 4 mg, and 8 mg.

[0099] Figure 22 C shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in the immune characteristics of circulating TNFα in group B at 2 mg, 4 mg, and 8 mg.

[0100] Figure 22 D shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in immune characteristics in the SD-101-induced IL-2R cycle in group B at 2 mg, 4 mg, and 8 mg.

[0101] Figure 22 E shows according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in the immune profile of circulating IL-15 in group B at 2 mg, 4 mg, and 8 mg.

[0102] Figure 22 F shows that according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to investigate changes in the immune profile of circulating IL-18 in Group B at 2 mg, 4 mg, and 8 mg.

[0103] Figure 23A It is shown in accordance with Figure 2A Changes in circulating CD8+ T cells in all groups A, B, and C from day 1 to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol.

[0104] Figure 23B It is shown in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, changes in circulating NK+ cell protein expression profiles from day 1 to day 36 were observed in group B at 2 mg, 4 mg, and 8 mg.

[0105] Figure 23C It is shown in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, there was a systemic increase in NK cell proliferation in all groups A, B, and C.

[0106] Figure 23D It is shown in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, systemic NK cell proliferation was increased in group B at 2 mg, 4 mg, and 8 mg.

[0107] Figure 24A It is shown in accordance with Figure 2A Objective RECIST 1.1 response rates in patients treated with the Phase 1 / 1B clinical trial protocol in Group B at 2 mg, 4 mg, and 8 mg, and in Group C at 2 mg.

[0108] Figure 24B Showing from Figure 24A Patients and according to Figure 2A Objective RECIST 1.1 response rates in patients treated with the Phase 1 / 1B clinical trial protocol in Group B at 2 mg, 4 mg, and 8 mg, and in Group C at 2 mg and 4 mg.

[0109] Figure 25A Showing in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, the overall survival rate and survival probability in groups A, B, and C were compared by administering 2 mg SD-101 via PEDD and IV anti-PD-1 for several weeks.

[0110] Figure 25BShowing in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, the overall survival rate and survival probability in Group B were compared after administration of 2 mg, 4 mg, and 8 mg SD-101 for several weeks.

[0111] Figure 25C It shows the basis Figure 2A The overall survival rate and survival probability of patients treated for several weeks in the Phase 1 / 1B clinical trial protocol, groups A, B, and C. Figure 25 shows the data including... Figure 25A The data provided and other data.

[0112] Figure 25D Showing according to Figure 25C The data showed the overall survival rate of group B at 2 mg, 4 mg, and 8 mg SD-101 doses.

[0113] Figure 25E Showing according to Figure 25C The data showed the overall survival rates of groups B and C at 2 mg and 4 mg SD-101 doses.

[0114] Figure 26A Showing in accordance with Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0115] Figure 26B It shows Figure 26A Changes in intratumoral cell density categorized by the dose of SD-101 administered.

[0116] Figure 26C Provided display according to Figure 2A Additional data on changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol, categorized by group: A, B, and C.

[0117] Figure 26D It shows Figure 26C Further variations in intratumoral cell density based on the administered SD-101 dose.

[0118] Figure 26E Provided additional display according to Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0119] Figure 26F It shows Figure 26EFurther variations in intratumoral cell density based on the administered SD-101 dose.

[0120] Figure 26G Provided additional display according to Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0121] Figure 26H It shows Figure 26G Further variations in intratumoral cell density based on the administered SD-101 dose.

[0122] Figure 26I Provided additional display according to Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0123] Figure 26J It shows Figure 26I Further variations in intratumoral cell density based on the administered SD-101 dose.

[0124] Figure 26K Provided additional display according to Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0125] Figure 26L It shows Figure 26K Further variations in intratumoral cell density based on the administered SD-101 dose.

[0126] Figure 26M Provided additional display according to Figure 2A The changes in intratumoral cell density from baseline to day 57 in patients treated with the Phase 1 / 1B clinical trial protocol were analyzed by group, A, B, and C.

[0127] Figure 26N It shows Figure 26M Further variations in intratumoral cell density based on the administered SD-101 dose.

[0128] Figure 27A It is shown in accordance with Figure 2A Changes in plasma immune markers in groups A, B, and C from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol.

[0129] Figure 27B It shows Figure 27AChanges in plasma immune markers categorized by the dose of SD-101 administered.

[0130] Figure 27C Provided shown in accordance with Figure 2A Additional data on changes in plasma immune markers from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol, categorized by group: A, B, and C.

[0131] Figure 27D It shows Figure 27C Further variations in plasma immune markers based on the administered SD-101 dose.

[0132] Figure 27E Provided shown in accordance with Figure 2A Additional data on changes in plasma immune markers from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol, categorized by group.

[0133] Figure 27F It shows Figure 27E Further variations in plasma immune markers based on the administered SD-101 dose.

[0134] Figure 27G Provided shown in accordance with Figure 2A Additional data on changes in plasma immune markers from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C.

[0135] Figure 27H It shows Figure 27G Further variations in plasma immune markers based on the administered SD-101 dose.

[0136] Throughout the accompanying drawings, unless otherwise stated, the same reference numerals and characters are used to denote similar features, elements, components, or portions of the illustrated embodiments. Furthermore, while this disclosure will now be described in detail with reference to the accompanying drawings, it is described in conjunction with illustrative embodiments, and this disclosure is not limited to the specific embodiments shown in the drawings and appended paragraphs. Detailed Implementation

[0137] The following description of the embodiments provides non-limiting representative examples of reference figures to specifically describe the features and teachings of different aspects of the invention. The described embodiments should be considered as being practiceable separately or in combination with other embodiments described from the embodiments. Those skilled in the art who read the description of the embodiments should be able to understand the different aspects of the invention described herein. The description of the embodiments should help to understand the invention to such an extent that other implementations not specifically covered but within the knowledge of those skilled in the art upon reading the description of the embodiments will be understood to be consistent with the application of the invention.

[0138] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Additionally, certain terms used herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0139] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural indicators.

[0140] Unless otherwise stated, any numerical value, concentration, or concentration range as described herein, should be understood to be modified by the term “about” in all cases. The numerical value may include ±1%, ±2%, ±3%, ±4%, or ±5% of the stated value. As used herein, unless otherwise explicitly stated in the context, the use of a numerical range explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of the value within such ranges.

[0141] As used herein, the terms "subject" or "patient" refer to an animal, preferably a mammal, and more preferably a human. Examples of subjects include humans, but may also include other animals such as rats, mice, and pigs. In one particular aspect, the subject is a human.

[0142] As used herein, the term "treat / treating / treatment" is intended to refer to the improvement or reversal of at least one measurable physical parameter associated with a disease, condition, or symptom. The term "treatment" can also refer to the reduction of a disease, condition, or symptom, the prevention of its progression, or at least the slowing of its progression. In one specific instance, "treatment" refers to the prevention of recurrence of a disease, condition, or symptom. In another instance, "treatment" refers to increasing the survival of a subject suffering from a disease, condition, or symptom. In one specific instance, "treatment" refers to the elimination of a subject's disease, condition, or symptom.

[0143] As used herein, "therapeutic effective amount" means the amount of an active compound or pharmaceutical agent that elicits a desired biological or pharmaceutical response in an tissue system, animal, or human, including improvement of one or more biomarkers of the disease, condition, or symptom being treated, or reduction of the severity of one or more symptoms of the disease, condition, or symptom being treated, such as slowing tumor development and metastasis in a patient.

[0144] As used herein, the term "agonist" refers to a compound that binds to a receptor and then increases, promotes, sensitizes, or upregulates said receptor.

[0145] As used herein, the term "antagonist" refers to a compound that binds to a receptor to block or attenuate the receptor's response to an agonist.

[0146] The term "pharmaceutically acceptable salt" refers to a salt of a compound that is known to be non-toxic and is commonly used in the pharmaceutical field. In some instances, pharmaceutically acceptable salts of compounds retain their biological efficacy and are not biologically or otherwise undesirable.

[0147] As used herein, “pharmaceuticalally acceptable excipient” means a non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, that is added to a pharmacological composition or otherwise used as a medium, carrier, or diluent to facilitate the administration of an active compound or pharmaceutical preparation and is compatible with said active compound or pharmaceutical preparation.

[0148] This application relates to a method for treating, improving, or modulating liver metastases of uveal melanoma in a human subject, the method comprising administering to the subject, in the case of a human subject in need, a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist. The subject can be anyone requiring treatment for liver metastases of uveal melanoma. Specifically, the human subject may have stage IV uveal melanoma. In some instances, the human subject may have UM LM with liver involvement only or primarily. According to another instance, the human subject is male or female and is eighteen years of age or older. A therapeutically effective combination of one or more checkpoint inhibitors and a TLR9 agonist can provide improved disease control compared to administering one or more checkpoint inhibitors without a TL9 agonist or administering a toll-like receptor 9 (TLR9) agonist without one or more checkpoint inhibitors.

[0149] A therapeutically effective combination of one or more checkpoint inhibitors and a TLR9 agonist refers to a dosage of one or more checkpoint inhibitors and a TLR9 agonist administered together such that the combined effect produces the desired physiological or biological effect, such as treating, improving, or modulating UMLM in a human subject. For example, in a therapeutically effective combination, the dosage of the TLR9 agonist combined with one or more checkpoint inhibitors has a therapeutically effective effect, but either of them, when administered alone, may or may not be effective for treating, improving, or modulating UMLM in a human subject. Similarly, in a therapeutically effective combination, the dosage of one or more checkpoint inhibitors combined with a TLR9 agonist has a therapeutically effective effect, but either of them, when administered alone, may or may not be effective for treating, improving, or modulating UMLM in a human subject.

[0150] An effective combination of treatment with one or more checkpoint inhibitors and a TLR9 agonist can be administered to human subjects in parallel (e.g., at the same time), at different times, or intermittently. For example, an effective combination of treatment may include administering a TLR9 agonist within the same time period as administering one or more checkpoint inhibitors to a human subject. Specifically, a human subject may be receiving a therapy in which a TLR9 agonist and one or more checkpoint inhibitors are administered on the same day. In another instance, a human subject may be receiving a therapy in which a TLR9 agonist and one or more checkpoint inhibitors are administered separately. The length of time between administering a TLR9 agonist and one or more checkpoint inhibitors can be adjusted to achieve the desired therapeutic effect. The one or more checkpoint inhibitors may be administered before or after the TLR9 agonist.

[0151] For patients with multiple liver metastases (LM), direct needle injection is often not feasible and does not allow for uniform drug delivery within a single tumor. In some instances, the TLR9 agonist is administered locally to a region outside the uveal melanoma liver metastases in the subject's liver. As discussed further below, pressure-enabled drug delivery (PEDD) via hepatic artery infusion (PEDD / HAI) can be used to administer TLR9 agonists to the liver of human subjects, enabling efficient delivery of the TLR9 agonist to UM LM and penetration into high-pressure tumors while limiting systemic exposure. The intravascular route also allows for treatment of dysfunctional immune cells throughout the organ.

[0152] Toll-like receptor agonists Toll-like receptors (TLRs) are pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) in microorganisms. TLR stimulation, such as TLR9 stimulation, can provide broad innate immune stimulation and specifically address key drivers of immunosuppression in the liver. TLRs 1-10 are expressed in humans and recognize a variety of different microbial PAMPs. In this regard, TLR9 can respond to unmethylated CpG-DNA, including microbial DNA. CpG refers to the motifs of cytosine and guanine dinucleotides. TLR9 is constitutively expressed in B cells, plasmacytoid dendritic cells (pDCs), activated neutrophils, monocytes / macrophages, T cells, and MDSCs. Furthermore, human MDSCs express TLR9 on their surface. Additionally, TLR9 and its associated endosomal protein TLR7 are expressed in human liver metastases. TLR9 is also expressed in non-immune cells, including keratinocytes and intestinal, cervical, and respiratory epithelial cells. TLR9 can bind to its agonists in vivo. Signaling can be mediated via MYD88 / IkB / NfκB to induce the expression of pro-inflammatory cytokine genes. Type 1 and type 2 interferons (e.g., IFN-α, IFN-γ, etc.) stimulate adaptive immune responses through the parallel signaling pathway of IRF7. Furthermore, TLR9 agonists can induce cytokine and IFN production, as well as the functional maturation of antigen-presenting dendritic cells.

[0153] TLR9 agonists can include any suitable compound or biomolecule that binds to TLR9 and then increases, promotes, sensitizes, or upregulates TLR9 activity. In one example, TLR9 agonists include synthetic CpG-oligonucleotides (CPG-On) that mimic the immunostimulatory properties of microbial CpG-DNA. According to one example, the oligonucleotide is an oligodeoxynucleotide (ODN). Many different CpG-ODN classes exist, such as class A, class B, class C, class P, and class S, which share certain structural and functional characteristics. In this regard, class A CPG-ODN (or CPG-A ODN) is associated with pDC maturation, has little effect on B cells, and induces the highest levels of IFNα; class B CPG-ODN (or CPG-B ODN) strongly induces B cell proliferation, activates pDC and monocyte maturation, NK cell activation, and inflammatory cytokine production; while class C C CPG-ODN (or CPG-CODN) can induce B cell proliferation and IFN-α production.

[0154] According to one example, any CPG-C ODN discussed herein may be present in its pharmaceutically acceptable salt form. Suitable pharmaceutically acceptable salts of any CPG-C ODN may include organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts (such as sodium, lithium, and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts, zinc salts), salts with organic bases (e.g., organic amines) (such as N-Me-D-glucosamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, tert-butylamine), and salts with amino acids (such as arginine, lysine, etc.). In one example, CpG-C ODN is in the form of an ammonium salt, sodium salt, lithium salt, or potassium salt. In a preferred example, CpG-C ODN is in the form of a sodium salt. CpG-C ODN may be provided in a pharmaceutical solution containing one or more pharmaceutically acceptable excipients. Alternatively, CpG-C ODN can be provided as a lyophilized solid, which is subsequently reconstituted in sterile water, saline, or a pharmaceutically acceptable buffer prior to administration.

[0155] In one instance, the TLR9 agonist is CpG-C ODN, containing the sequence of SEQ ID NO: 1: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), Or a pharmaceutically acceptable salt thereof. More specifically, the TLR9 agonist is SD-101 (also referred to herein as netotinomycin) or a pharmaceutically acceptable salt thereof. SD-101 is a 30-meric oligodeoxynucleotide with the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), The entire 30-mer sequence is linked by thiophosphate bonds. The TLR agonist can be SD-101 in the form of a free acid or a pharmaceutically acceptable salt. In another example, the TLR9 agonist is a sodium salt. Figure 1 The structure of the sodium salt of SD-101 is shown.

[0156] The molecular formula of SD-101 in its free acid form is C0 293 H 369 N 112 O 149 P 29 S 29 Furthermore, SD-101 in its free acid form has a molecular weight of 9672 Daltons. For example... Figure 1 As shown, the molecular formula of the sodium salt of SD-101 is C2. 293 H340 N 112 O 149 P 29 S 29 Na 29 Furthermore, the molecular weight of the sodium salt of SD-101 is 10,309 Daltons. Note that all methods described herein related to TL9 agonists are particularly applicable to TLR9 C-type agonists and even more so to SD-101.

[0157] TLR9 agonists, specifically SD-101, can influence multiple cell types to initiate the tumor microenvironment (TME) in response to checkpoint inhibitor therapy. Administration of SD-101 may lead to MDSC depletion, T cell activation, and recruitment.

[0158] TLR9 agonists can be CpG-C ODNs containing naturally occurring or modified non-naturally occurring bases, and compared to SD-101, can contain modified sugars, phosphate esters, and / or terminals. For example, in addition to phosphodiester bonds, phosphate ester modifications include, but are not limited to, methylphosphonates, thiophosphates, aminophosphates (bridged or unbridged), triphosphates, and dithiophosphates, and can be used in any combination. In one example, the CpG-C ODN has only thiophosphate bonds, only phosphodiester bonds, or a combination of phosphodiester and thiophosphate bonds.

[0159] Sugar modifications known in the art, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras, as well as other sugar modifications described herein, can also be prepared and can be combined with any phosphate ester modifications. Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to the C-5 and / or C-6 of cytosine in CpG-C ODNs (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) and the C-5 and / or C-6 of uracil in CpG-C ODNs (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). As mentioned above, the use of base modifications in the palindromic sequence of CpG-C ODNs should not interfere with the self-complementarity of the bases involved in Watson-Crick base pairing. However, modified bases can be used outside palindromic sequences without this restriction. For example, 2'-O-methyluridine and 2'-O-methylcytidine can be used outside palindromic sequences, while 5-bromo-2'-deoxycytidine can be used both inside and outside palindromic sequences. Other modified nucleotides that can be used inside and outside palindromic sequences include 7-dea-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0160] Most ODNs exist in a dynamic equilibrium between their double-stranded (i.e., double-stranded) and hairpin forms, with the hairpin form typically favoring lower oligonucleotide concentrations and higher temperatures. Covalent inter- or intra-chain crosslinks increase the stability of the double-stranded or hairpin form to conformational changes induced by heat, ions, pH, and concentration, respectively. Chemical crosslinking can be used to lock polynucleotides into double-stranded or hairpin forms for physicochemical and biological characterization. Conformally homogeneous crosslinked ODNs “locked” in their most active form (double-stranded or hairpin form) may be more active than their uncrosslinked counterparts. Therefore, some CpG-C ODNs disclosed herein may contain covalent inter- and / or intra-chain crosslinks.

[0161] TLR9 agonists can be C-class type CPG-ODN (CpG-C) oligonucleotides (ODNs) formed by modifying SD-101. In CpG-C oligonucleotides, the sugar moiety is preferably a furanyl glycoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be linked to the corresponding heterocyclic base in any anomeric configuration. Sugar modification can also be performed in combination with any phosphate ester modification during the preparation of CpG-C ODN. The heterocyclic or nucleic acid bases incorporated into CpG-C ODN can be naturally occurring major purine and pyrimidine bases (i.e., uracil, thymine, cytosine, adenine, and guanine, as mentioned above), as well as naturally occurring and synthetic modifications of said major bases. Therefore, CpG-C ODN can include one or more of inosine, 2'-deoxyuridine, and 2-amino-2'-deoxyadenosine.

[0162] TLR9 agonists can be administered to human subjects in the form of pharmaceutical compositions suitable for parenteral and / or transdermal administration (e.g., aqueous or non-aqueous solutions or emulsions). The pharmaceutical composition may contain any pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients of this disclosure include, for example, solvents, compatibilizers, buffers, tonic modifiers, and preservatives. In one example, the pharmaceutical composition may contain an excipient that serves as one or more of a solvent, compatibilizer, buffer, and tonic modifier (e.g., sodium chloride in saline may serve as both an aqueous medium and a tonic modifier).

[0163] In one example, the pharmaceutical composition comprises an aqueous mediator as a solvent. Suitable mediators include, for example, sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In one example, the composition is isotonic.

[0164] In another example, a TLR9 agonist can be formulated into a pharmaceutical composition that is a lyophilized powder that will be reconstituted with solvents (as described above) prior to application. The pharmaceutical composition may contain a compatibilizer. Compatibilizers are particularly useful when the pharmaceutical composition is stored after lyophilization, freeze-drying, or spray drying and subsequently reconstituted with solvents prior to application. In one example, the compatibilizer is a protectant that helps stabilize the active agent and prevent its degradation during lyophilization or spray drying and / or storage. Suitable compatibilizers are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0165] Pharmaceutical compositions may contain buffers. Buffers control pH to inhibit degradation of the active agent during processing, storage, and optionally remodeling. Suitable buffers include, for example, salts comprising acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. Buffers may further contain hydrochloric acid or sodium hydroxide. In some instances, the buffer maintains the pH of the composition in the range of 4 to 9. In one instance, the pH is greater than (lower limit) 4, 5, 6, 7, or 8. In some instances, the pH is less than (upper limit) 9, 8, 7, 6, or 5. That is, the pH is in the range of about 4 to 9, where the lower limit is less than the upper limit.

[0166] The pharmaceutical composition may contain a tonic modulator. Suitable tonic modulators include, for example, dextran, glycerol, sodium chloride, glycerol, and mannitol.

[0167] Pharmaceutical compositions may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in one instance, the pharmaceutical composition is prepared under sterile conditions and in a single-use container, and therefore does not need to include preservatives.

[0168] Table 1 describes exemplary pharmaceutical compositions containing SD-101, wherein the concentration of SD-101 in the pharmaceutical composition is 16 g / L: Table 1

[0169] In some instances, SD-101 may be present in the pharmaceutical composition at concentrations of about 0.1 mg / mL to about 20 mg / mL. Specifically, SD-101 may be present in the pharmaceutical composition at a concentration of 13.4 mg / mL.

[0170] Local administration of TLR9 agonists to the liver As discussed above, TLR9 agonists, specifically SD-101 or its pharmaceutically acceptable salts (e.g., Figure 1The sodium salt shown is administered locally to the liver of a human subject via hepatic artery infusion (HAI). HAI refers to the infusion of a therapeutic agent into the hepatic artery or a branch of the hepatic artery. For example, a TLR9 agonist is introduced into a branch of the hepatic artery via percutaneous delivery of a device. The device can be any device for achieving local delivery to or near a tumor, including, for example, a catheter or a catheter together with other components that can be used in combination with the catheter (e.g., filter valve, balloon, pressure sensor system, pump system, syringe, external delivery catheter, implantable port, etc.). In some instances, the catheter is a microcatheter. In one instance, local administration of a TLR9 agonist provides penetration of the TLR9 agonist (more specifically, SD-101) through a UM-LM tumor of the liver, substantially through the entire UM-LM tumor of the liver, or through the entire liver of a human subject.

[0171] Specifically, a TLR9 agonist is infused into the hepatic artery or its branches under pressure (i.e., at a pressure increased relative to the unchanged intravascular pressure of the hepatic artery) via a hepatic artery intravascular injection (HAI). More specifically, the TLR9 agonist is infused via a HAI using a device having an expandable fluid pressure regulating structure to regulate the flow and / or pressure in the inserted vessel. For example, the fluid pressure regulating structure can regulate the flow and / or pressure in the inserted vessel in sync with the cardiac cycle of a human subject. The fluid pressure regulating structure can intermittently increase the pressure in the hepatic artery or its branches relative to the unchanged intravascular pressure of the hepatic artery. While regulating the pressure in the inserted hepatic artery or its branches, the fluid pressure regulating structure can intermittently close the hepatic artery or its branches. The fluid pressure regulating structure is configured to intermittently increase the pressure in the hepatic artery or its branches by an amount sufficient to overcome the interstitial fluid pressure and solid-state stress of a UM LM tumor in the liver of a human subject.

[0172] In some instances, devices for hepatic artery infusion (HAI) may have one or more properties, including but not limited to self-centering capability to provide uniform distribution of therapy in a downstream branching network of the vessel; anti-backflow capability (e.g., using valves and filters, and / or balloons) to block or inhibit retrograde flow of the infused pharmaceutical composition (e.g., a drug fluid containing a TLR9 agonist); a system for measuring pressure within the container; and devices or mechanisms for regulating intravascular pressure (e.g., one-way valves, intermittently closing valves, and / or porous balloons that dynamically respond to changes in local pressure), such as by inducing a pressure decrease upon placement and during TLR9 agonist infusion, and a pressure increase during saline bolus or during TLR9 agonist infusion. In some instances, the system is designed to continuously monitor real-time pressure or flow throughout the procedure. In one instance, the pressure regulating mechanism generates, causes, and / or contributes to a net increase in fluid pressure within the vessel and / or target tissue or tumor. In a specific instance, the pressure regulating mechanism may increase local vascular pressure at the target site, specifically by increasing the pressure beyond the baseline arterial pressure. The pressure regulation mechanism can operate synchronously with the cardiac cycle and / or facilitate anterograde flow. In some instances, the pressure regulation mechanism generates, causes, and / or promotes a net decrease in fluid pressure within blood vessels and / or target tissue or tumor. The pressure regulation mechanism can also redirect flow direction to improve the concentration of TLR9 agonists in tumor tissue while allowing for whole-liver treatment. In some instances, the pressure regulation mechanism generates, causes, and / or promotes an initial decrease followed by an increase in fluid pressure within blood vessels and / or target tissue or tumor. In some instances, the device supports the measurement of intravascular pressure during use.

[0173] In some instances, devices that can be used for HAI are as described in U.S. Patent Nos. 8,500,775, 8,696,698, 8,696,699, 9,539,081, 9,808,332, 9,770,319, 9,968,740, 10,813,739, and 10,580. The apparatus disclosed in U.S. Patent No. 8,636, U.S. Patent No. 11,090,460, U.S. Patent Publication No. 2018 / 0193591, U.S. Patent Publication No. 2018 / 0250469, U.S. Patent Publication No. 2019 / 0298983, U.S. Patent Publication No. 2020 / 0038586, and U.S. Patent Publication No. 2020 / 0383688, the entirety of which are incorporated herein by reference. In some instances, the apparatus is as disclosed in U.S. Patent No. 9,770,319. In some instances, the apparatus may be an apparatus referred to as the Surefire infusion system.

[0174] More specifically, TLR9 agonists are infused into the hepatic artery or a branch of the hepatic artery via a pressure-enabled drug delivery (PEDD) device using a HAI. The PEDD device is configured to overcome the intratumoral pressure of UM LM tumors by generating a favorable pressure gradient, while protecting non-target organs through anti-backflow properties.

[0175] In some instances, the PEDD device is the device disclosed in U.S. Patent Publication No. 2020-0383688. In some instances, the PEDD device may be a device referred to as the TriSalus infusion system. In some instances, the PEDD device may be a device referred to as TriNav. ® Devices for an infusion system. TriNav ® An infusion system is a single-lumen catheter equipped with a one-way valve that dynamically responds to changes in local pressure and flow, such as those caused by the cardiac cycle or by infusion. The valve structure regulates distal vascular pressure and blood flow. This, in turn, can alter therapeutic distribution and first-pass absorption due to increased contact time within the vascular system.

[0176] In some instances, TLR9 agonists can be administered while monitoring pressure in the blood vessel, which can be used to adjust and correct the device's positioning at the infusion site and / or adjust the infusion rate. Pressure can be monitored, for example, by a pressure sensor system comprising one or more pressure sensors. The infusion rate can be adjusted to alter vascular pressure or flow, which can facilitate the penetration of the TLR9 agonist into and / or binding to the target tissue or tumor on its surface. In some instances, an infusion pump can be used as part of the delivery system or by other methods (e.g., infusion flow rate regulating devices) to adjust and / or control the infusion rate. In some instances, a pump system can be used to adjust and / or control the infusion rate.

[0177] In one instance, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered weekly using an infusion schedule lasting approximately 10–200 minutes. In another instance, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered weekly using an infusion schedule lasting approximately 10–60 minutes. In yet another instance, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered weekly using an infusion schedule lasting approximately 25 minutes.

[0178] In some instances, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered locally to the liver of the subject via hepatic artery infusion at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor and to provide a plasma concentration of the TLR9 agonist below that concentration in the liver. Specifically, the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject. In one instance, the plasma concentration of the TLR9 agonist is at a subtherapeutic level, i.e., when used alone or in combination with one or more checkpoint inhibitors to treat UM LM, it is not therapeutically effective.

[0179] A TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, may be administered at a dose below the maximum tolerated dose, specifically when administered topically via a HAI, and more specifically when administered via a PEDD using a HAI. In some instances, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered topically to the liver of a subject via a HAI at a dose sufficient to provide a concentration in the liver of about 750 ng / g to about 3000 ng / g while simultaneously providing plasma concentrations of the TLR9 agonist below those in the liver, for example, below about 750 ng / mL, below about 700 ng / mL, or below about 600 ng / mL. In other instances, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered topically to the liver of a subject via a HAI, providing a plasma concentration of TLR9 agonist at a ratio of about 1:2 to about 1:10, or about 1:3 to about 1:7.

[0180] In some instances, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, may be administered weekly at doses of about 0.5 mg to about 20 mg, about 1 mg to about 10 mg, about 2 mg to about 8 mg, or about 4 mg to about 8 mg. In some instances, a TLR9 agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, may be administered weekly at doses of about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, or about 8 mg. Unless otherwise stated, the milligram amount of SD-101 described in this application (e.g., about 2 mg) refers to... Figure 1 The amount of sodium salt of SD-101 shown is approximately 2 mg. Equivalent molar amounts of free acid or other pharmaceutically acceptable salts are also envisioned.

[0181] In some instances, UMLM in human subjects was treated with SD-101 administered at a weekly dose of 2 mg via HAI, and in some instances, SD-101 was further administered via a pressure-regulating device (i.e., PEDD). The pressure can be regulated, for example, using the TriNav® infusion system as described above, in synchronization with the cardiac cycle of the human subject. In some instances, UMLM in human subjects was treated with SD-101 administered at a weekly dose of 4 mg via HAI, and in some instances, SD-101 was further administered via a pressure-regulating device (i.e., PEDD), wherein the pressure can be regulated, for example, using the TriNav® infusion system as described above, in synchronization with the cardiac cycle of the human subject. In some instances, UMLM in human subjects was treated with SD-101 administered at a weekly dose of 8 mg via HAI, and in some instances, SD-101 was further administered via a pressure-regulating device (i.e., PEDD), wherein the pressure can be regulated, for example, using the TriNav® infusion system as described above, in synchronization with the cardiac cycle of the human subject.

[0182] In some instances, TLR9 agonists, specifically SD-101 or a pharmaceutically acceptable salt thereof, can be administered in a dosing regimen comprising cycles, wherein one or more cycles comprise administration of SD-101 via HAI and / or PEDD. As used herein, a “cycle” is a repetition of the dosing sequence. In one instance, a cycle comprises three weekly doses per cycle (i.e., SD-101 administered weekly for three consecutive weeks). In one instance, a treatment cycle according to this application may comprise a period of SD-101 administration followed by a “stop” period or rest period. In another instance, in addition to three weekly doses per cycle, the cycle further comprises one, two, three, or four weeks as rest periods following weekly administration of SD-101. In yet another instance, in addition to three weekly doses per cycle, the cycle further comprises approximately thirty-eight days as rest periods following weekly administration of SD-101. In yet another instance, the entire cycle comprises approximately fifty-two days. In yet another instance, the dosing regimen comprises at least one, at least two, or at least three cycles or longer. The weekly doses as described herein can be modified to be administered weekly within a cycle.

[0183] Checkpoint inhibitors As discussed above, this application describes a therapeutically effective combination of administering one or more checkpoint inhibitors (CPIs) and toll-like receptor 9 (TLR9) agonists to a subject. The CPI may be a programmed cell death 1 receptor (PD-1) antagonist. The PD-1 antagonist may be any compound or biomolecule that blocks the binding of programmed cell death 1 ligand 1 (PD-L1) expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and preferably also blocks the binding of programmed cell death 1 ligand 2 (PD-L2) expressed on cancer cells to PD-1 expressed on immune cells. Alternative names or synonyms for PD-1 and its ligands include: for PD-1, PDCD1, PD1, CD279, and SLEB2; for PD-L1, PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H; and for PD-L2, PDCD1L2, PDL2, B7-DC, Btdc, and CD273. Specifically, in the treatment methods, pharmaceutical products, and uses of this application for treating human subjects, PD-1 antagonists block the binding of human PD-L1 to human PD-1, preferably blocking the binding of human PD-L1 and PD-L2 to human PD-1.

[0184] According to one example, a PD-1 antagonist may comprise a monoclonal antibody (mAb) or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some examples, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred examples, the human constant region is an IgG1 or IgG4 constant region. In some examples, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0185] According to one example, a PD-1 antagonist may include an immunoadhesin that specifically binds to PD-1 or PD-L1, preferably an immunoadhesin that specifically binds to human PD-1 or human PD-L1, such as a fusion protein containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (such as the Fc region of an immunoglobulin molecule).

[0186] According to one example, PD-1 antagonists can block the expression of PD-L1 in tumor cells, MDSCs, and other suppressive immune cells.

[0187] According to one example, a PD-1 antagonist can inhibit the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some examples of the above-described treatment methods, pharmaceuticals, and uses, the PD-1 antagonist is a monoclonal antibody or its antigen-binding fragment that specifically binds to PD-1 or PD-L1 and blocks the binding of PD-L1 to PD-1. In one example, the PD-1 antagonist is an anti-PD-1 antibody comprising a heavy chain and a light chain.

[0188] According to one example, the PD-1 antagonist may be one of nivolumab, pembrolizumab, and cimiprilmab. According to another example, nivolumab is administered intravenously (IV) via peripheral vein at a dose of 480 mg every four weeks (“Q4W”) or 240 mg every two weeks (“Q2W”). According to another example, nivolumab is administered intravenously (IV) via peripheral vein at a dose of 360 mg every three weeks (“Q3W”). In yet another example, nivolumab administration is based on body weight, at 3 mg / kg Q2W or 10 mg / kg Q2W. In yet another example, nivolumab administration is based on body weight, at 1 mg / kg Q3W. In yet another example, nivolumab is administered concurrently with SD-101, approximately concurrently, or on the same day. In another instance, nivolumab is administered weekly, every other week, every three weeks, every four weeks, or monthly, following one or more cycles of SD-101 administration. The “cycle” of SD-101 administration is as described above.

[0189] According to another example, pembrolizumab is administered intravenously (IV) via peripheral vein at a dose of 200 mg Q3W or 400 mg every 6 weeks (“Q6W”). In another example, pembrolizumab is administered concurrently with, approximately concurrently with, or on the same day as SD-101.

[0190] According to another example, a CPI may include a PD-L1 antagonist. In this regard, a PD-L1 antagonist may be one of atezolizumab, avelumab, and durvalumab.

[0191] According to another example, a CPI may include a CTLA-4 antagonist. In this regard, a CTLA-4 antagonist may be ipilimumab. According to another example, ipilimumab is administered intravenously (IV) at a dose of 3 mg / kg every three weeks via a peripheral vein. In yet another example, ipilimumab is administered concurrently, approximately concurrently, or on the same day as SD-101 and / or nivolumab. In yet another example, ipilimumab is administered weekly, every other week, every three weeks, every four weeks, or monthly after one or more cycles of SD-101 and / or nivolumab.

[0192] According to another example, CPIs can include inhibitors of lymphocyte activation gene-3 (LAG-3). In this regard, a LAG-3 inhibitor could be renalalimab.

[0193] According to another example, a combination of one or more checkpoint inhibitors and TLR9 agonists can be administered together with other cancer treatment agents such as 1ressu-modulators, tumor killers, and / or other targeted therapies.

[0194] According to one example, a combination of one or more checkpoint inhibitors and TLR9 agonists can be administered in combination with cell therapy (which achieves cell therapy through modulation of the immune system), chemoembolization therapy, or radioembolization therapy.

[0195] In some instances, a toll-like receptor 9 (TLR9) agonist, specifically SD-101 or a pharmaceutically acceptable salt thereof, is administered to human subjects, and one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. SD-101 may be administered in combination with nivolumab to human subjects. SD-101 may be administered in combination with pembrolizumab to human subjects. SD-101 may be administered in combination with ipilimumab to human subjects. SD-101 may be administered in combination with camrelimab to human subjects. SD-101 may be administered in combination with two checkpoint inhibitors to human subjects. For example, SD-101 may be administered in combination with nivolumab and ipilimumab to human subjects. In another instance, SD-101 may be administered in combination with nivolumab and renalalimab to human subjects.

[0196] In some instances, SD-101 was administered to human subjects at a weekly dose of 2 mg via a HAI, and in others, SD-101 was further administered via a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD). As described herein, pressure can be regulated, for example, using the TriNav® infusion system as described above, in sync with the cardiac cycle of the human subject. In some instances, SD-101 was administered at a dose of 2 mg via a HAI in combination with a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD) and a CPI. The CPI, for example, can be administered intravenously (IV) or subcutaneously (SQ). In some instances, SD-101 was administered at a weekly dose of 2 mg via a HAI in combination with a pressure-regulating device, wherein the CPI is nivolumab. In other instances, SD-101 was administered at a weekly dose of 2 mg via a HAI in combination with pembrolizumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 2 mg via a HAI and in combination with ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 2 mg via a HAI and in combination with nivolumab and ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 2 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device. In yet another example, SD-101 was administered at a dose of 2 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device.

[0197] In some instances, SD-101 was administered to human subjects at a weekly dose of 4 mg via a HAI, and in others, SD-101 was further administered via a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD). As described herein, pressure can be regulated, for example, using the TriNav® infusion system as described above, in sync with the cardiac cycle of the human subject. In some instances, SD-101 was administered at a dose of 4 mg via a HAI in combination with a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD) and a CPI. The CPI, for example, can be administered intravenously (IV) or subcutaneously (SQ). In some instances, SD-101 was administered at a weekly dose of 4 mg via a HAI in combination with a pressure-regulating device, wherein the CPI is nivolumab. In other instances, SD-101 was administered at a weekly dose of 4 mg via a HAI in combination with pembrolizumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 4 mg via a HAI and in combination with ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 4 mg via a HAI and in combination with nivolumab and ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 4 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device. In yet another example, SD-101 was administered at a dose of 4 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device.

[0198] In some instances, SD-101 was administered to human subjects at a weekly dose of 8 mg via a HAI, and in others, SD-101 was further administered via a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD). As described herein, pressure can be regulated, for example, using the TriNav® infusion system as described above, in sync with the cardiac cycle of the human subject. In some instances, SD-101 was administered at a dose of 8 mg via a HAI in combination with a pressure-regulating device (e.g., dynamically responsive to changes in local pressure, more specifically, PEDD) and a CPI. The CPI, for example, can be administered intravenously (IV) or subcutaneously (SQ). In some instances, SD-101 was administered at a weekly dose of 8 mg via a HAI in combination with a pressure-regulating device, wherein the CPI is nivolumab. In other instances, SD-101 was administered at a weekly dose of 8 mg via a HAI in combination with pembrolizumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 8 mg via a HAI and in combination with ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 8 mg via a HAI and in combination with nivolumab and ipilimumab via a regulated device. In another example, SD-101 was administered at a weekly dose of 8 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device. In yet another example, SD-101 was administered at a dose of 4 mg via a HAI and in combination with nivolumab and pillarizumab via a regulated device.

[0199] In one aspect of this application, a method for treating uveal melanoma liver metastases in a human subject is provided. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a TLR9 agonist. For example, the TLR9 agonist is a TLR9 C-type agonist, specifically SD-101 or SD-101 hydrochloride, such as... Figure 1As shown. In one example, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, and more specifically, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the entire sequence is linked by a phosphate thioester bond. The TLR9 agonist may be administered locally to the liver of the subject, as by means of a HAI (specifically, HAI and PEDD), at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor and to provide a plasma concentration of the TLR9 agonist lower than its concentration in the liver. Specifically, the TLR9 agonist is administered locally to a region of the liver of the subject outside the uveal melanoma liver metastasis. In some instances, the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject. The one or more checkpoint inhibitors may comprise any of the checkpoint inhibitors described above, such as anti-PD-1 antibodies or anti-PD-L1 antibodies. Specifically, the one or more checkpoint inhibitors comprise nivolumab. The one or more checkpoint inhibitors can be administered to the human subjects in any suitable manner. In one instance, the one or more checkpoint inhibitors are administered systemically to the subjects. The one or more checkpoint inhibitors can be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. In one specific instance, the TLR9 agonist is SD-101, administered in combination with nivolumab at a dose of approximately 2 mg weekly over a 3-week cycle, with rest periods between cycles. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases progression-free survival (PFS) in the subjects. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subjects.

[0200] In another aspect of this application, a method is provided for improving the survival rate of human subjects with liver metastases from uveal melanoma. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist. Administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist can increase the progression-free survival (PFS) of the subject. The TLR9 agonist is, for example, a TLR9 C-type agonist, specifically SD-101 or the hydrochloride salt of SD-101, such as... Figure 1As shown. In one example, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, and more specifically, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the entire sequence is linked by a phosphate thioester bond. The TLR9 agonist can be applied locally to the liver of the subject, as by means of HAI (specifically HAI and PEDD). Specifically, the TLR9 agonist is administered locally to a region outside the uveal melanoma liver metastasis in the liver of the subject. In some instances, the TLR9 agonist is infused while modulating distal vascular pressure and blood flow in the subject. The one or more checkpoint inhibitors may comprise any of the checkpoint inhibitors described above, such as anti-PD-1 antibodies, anti-PD-L1 antibodies. Specifically, the one or more checkpoint inhibitors comprise nivolumab. The one or more checkpoint inhibitors can be administered to the human subject in any suitable manner. In one example, the one or more checkpoint inhibitors are administered systemically to the subject. The one or more checkpoint inhibitors may be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. In one specific example, the TLR9 agonist is SD-101, which is combined with nivolumab and administered weekly at a dose of approximately 2 mg over a 3-week cycle, with rest periods between cycles.

[0201] In another aspect of this application, a method for treating uveal melanoma liver metastases in a human subject is provided. The method comprises administering a therapeutically effective amount of one or more checkpoint inhibitors to the subject; and locally administering (e.g., using HAI (specifically HAI and PEDD) toll-like receptor 9 (TLR9) agonists. In some instances, the TLR9 agonist is infused while modulating distal vascular pressure and blood flow in the subject. The one or more checkpoint inhibitors and the TLR9 agonist are administered in a therapeutically effective combination to reduce the tumor burden on the subject. For example, the TLR9 agonist is a TLR9 type C agonist, specifically SD-101 or SD-101 hydrochloride, such as... Figure 1As shown. In one example, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, and more specifically, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the entire sequence is linked by a phosphate thioester bond. The one or more checkpoint inhibitors may comprise any of the checkpoint inhibitors described above, such as anti-PD-1 antibodies or anti-PD-L1 antibodies. Specifically, the one or more checkpoint inhibitors comprise nivolumab. The one or more checkpoint inhibitors may be administered to the human subject in any suitable manner. In one example, the one or more checkpoint inhibitors are administered systemically to the subject. The one or more checkpoint inhibitors may be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and cliradilimab. In a specific example, the TLR9 agonist is SD-101, which is combined with nivolumab and administered weekly at a dose of approximately 2 mg over a 3-week cycle, with rest periods between cycles. In one instance, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the survival rate of the subject. In another instance, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the progression-free survival (PFS) of the subject. In yet another instance, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject.

[0202] In another aspect of this application, a method for reducing circulating tumor DNA (ctDNA) in a human subject with liver metastases from uveal melanoma is provided. The method comprises administering to the subject a therapeutically effective combination of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist. For example, the TLR9 agonist is a TLR9 type C agonist, specifically SD-101 or SD-101 hydrochloride, such as... Figure 1As shown. In one example, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, and more specifically, the TLR9 agonist is an oligonucleotide having the sequence SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the entire sequence is linked by a phosphate thioester bond. The TLR9 agonist may be administered locally to the liver of the subject, such as by means of a HAI (specifically, HAI and PEDD), at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor. In some instances, the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject. The one or more checkpoint inhibitors may comprise any of the checkpoint inhibitors described above, such as anti-PD-1 antibodies, anti-PD-L1 antibodies. Specifically, the one or more checkpoint inhibitors comprise nivolumab. The one or more checkpoint inhibitors may be administered to the human subject in any suitable manner. In one example, the one or more checkpoint inhibitors are administered systemically to the subject. The one or more checkpoint inhibitors may be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. In one specific example, the TLR9 agonist is SD-101, which is combined with nivolumab and administered weekly at a dose of approximately 2 mg over a 3-week cycle, with rest periods between cycles.

[0203] In another aspect of this application, a method is provided for improving the response rate of human subjects with liver metastases from uveal melanoma to treatment with a checkpoint inhibitor. The method comprises locally administering a combination of a toll-like receptor 9 (TLR9) agonist and a checkpoint inhibitor. For example, the TLR9 agonist is a TLR9 type C agonist, specifically SD-101 or the hydrochloride salt of SD-101, such as... Figure 1As shown. In one example, the TLR9 agonist is an oligonucleotide having the sequence of SEQ ID NO:1, or a pharmaceutically acceptable salt thereof, and more specifically, the TLR9 agonist is an oligonucleotide having the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof, wherein the entire sequence is linked by a phosphate thioester bond. The TLR9 agonist may be administered locally to the liver of the subject, such as by means of a HAI (specifically, HAI and PEDD), at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor. In some instances, the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject. The one or more checkpoint inhibitors may comprise any of the checkpoint inhibitors described above, such as anti-PD-1 antibodies, anti-PD-L1 antibodies. Specifically, the one or more checkpoint inhibitors comprise nivolumab. The one or more checkpoint inhibitors may be administered to the human subject in any suitable manner. In one example, the one or more checkpoint inhibitors are administered systemically to the subject. The one or more checkpoint inhibitors may be selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. In one specific example, the TLR9 agonist is SD-101, administered in combination with nivolumab at a dose of approximately 2 mg weekly over a 3-week cycle, with rest periods between cycles. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the survival rate of the subject. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the progression-free survival (PFS) of the subject. In some instances, administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject. In some instances, administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases immune cell activation within the uveal melanoma liver metastases of the subject. In some instances, the administration of the combination of the one or more checkpoint inhibitors with the TLR9 agonist increases the density of immune cells (e.g., CD4 T cells, CD8 T cells, and / or MDSCs) in the subject. In some instances, the administration of the combination of the TLR9 agonist with the checkpoint inhibitor increases systemic cytokine signaling in the subject's blood.

[0204] In some instances of the above methods, when the TLR9 agonist is administered without the one or more checkpoint inhibitors, the plasma concentration of the TLR9 agonist is at a subtherapeutic dose. In some instances, the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject. In some instances, the subject has stage IV uveal melanoma. In some instances, the TLR9 agonist is administered locally to a region outside the liver metastasis of the uveal melanoma in the subject. In some instances, the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously. In some instances, the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:10. In some instances, the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:7. In some instances, the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g. In some instances, the plasma concentration of the TLR9 agonist is less than about 750 ng / mL. In some instances, the plasma concentration of the TLR9 agonist is less than about 700 ng / mL. In some instances, the plasma concentration of the TLR9 agonist is less than about 600 ng / mL. In some instances, the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg. In some instances, the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg. In some instances, the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg. In some instances, the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg. In some instances, the TLR9 agonist is administered weekly at a dose of about 2 mg. In some instances, the TLR9 agonist is administered weekly for a duration of approximately 10 minutes to approximately 200 minutes. In some instances, the TLR9 agonist is administered weekly for a duration of approximately 10 minutes to approximately 60 minutes. In some instances, the TLR9 agonist is administered for a duration of approximately 25 minutes. In some instances, the one or more checkpoint inhibitors are administered concurrently with the TLR9 agonist. In some instances, the one or more checkpoint inhibitors are administered before the TLR9 agonist. In some instances, the one or more checkpoint inhibitors are administered after the TLR9 agonist. In some instances, the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab. In some instances, the one or more checkpoint inhibitors are nivolumab.In some instances, the one or more checkpoint inhibitors are pembrolizumab. In some instances, the one or more checkpoint inhibitors are ipilimumab. In some instances, the one or more checkpoint inhibitors are cliradimab. In some instances, two checkpoint inhibitors are administered.

[0205] This application will be further illustrated and / or demonstrated in the following examples, which are given for illustrative / shown purposes only and are not intended to limit the invention in any way.

[0206] Example Example 1: Metastatic uveal melanoma liver metastases (MUM-LM) are resistant to immune checkpoint inhibitors (ICIs) for several reasons, including the prevalence of myeloid-derived suppressor cells (MDSCs). Progression-free survival (PFS) is limited, and even with approved therapies such as tebentafusp (median 3.3 months), the incidence of grade 3 / 4 adverse events is typically >30%. In Example 1, in a dose-escalation clinical study (NCT04935229), the class C TLR9 agonist SD-101 was administered to human patients via pressure-enabled hepatic artery infusion (PEDD / HAI) using the TriNav® infusion system to enhance response rates to checkpoint inhibitor (CPI) therapies such as ipilimumab and nivolumab in human patients with uveal melanoma liver metastases (MUM). SD-101 was delivered in 3 weekly doses / cycles over 2 outpatient cycles.

[0207] This study included groups A, B, and C, with group A receiving a single agent of SD-101 (where n = 3–12), group B receiving a combination of SD-101 with an anti-PD-1 CPI, nivolumab (where n = 3–12), and group C receiving a combination of SD-101 with nivolumab and an anti-CTLA-4 CPI, ipilimumab (where n = 3–12). The doses of SD-101 administered to human patients in groups A, B, and C spanned a dose escalation range from approximately 2 mg per dose (the minimum expected biological effect level, MABEL) to approximately 8 mg (i.e., 2 mg, 4 mg, and 8 mg).

[0208] The dosing regimen consisted of SD-101 administration via PEDD over two cycles, each cycle comprising three weekly doses of SD-101 (i.e., weekly administration of SD-101 over three consecutive weeks) and a five-week rest period following the first cycle of weekly SD-101 administration. Plasma SD-101 levels were analyzed by LC-MS, and plasma cytokine levels were analyzed by Luminex. Gene expression levels within PBMCs and LMs were analyzed using NanoString. Advanced nSolver analysis was performed for pathway scoring. DAPI, CD14, HLA-DR, CD79a, CD163, CD68, CD11b, CD8, CD15, CD56, CD3e, CD4, CD20, FOXP3, gp100, and CD45 were stained using an Akoya Bioscience PhenoCycler-Fusion system, and FFPE tissue sections were analyzed by multiplex IF. Quantitative cell density data were generated using Qupath 0.4.3. Cytoflex was used to perform flow cytometry to evaluate CD8+ T cell proliferation (CD45+CD3+CD8+Ki67+), activation (CD45+CD3+CD8+ICOS+), and NK cell proliferation (CD45+CD3-CD56+Ki67+).

[0209] Figure 2A This study presents a Phase 1 / 1B clinical trial protocol comprising groups A, B, and C, and optionally group D. The study was conducted in two phases. In phase 1, a sentinel group was enrolled to determine the safety of SD-101 delivered via PEDD / HAI with dose escalation across two dose groups. Sentinel group patients received two infusions (2 weeks apart) and underwent toxicity assessment before escalating from the first dose level (0.5 mg) to the second dose level (2 mg). Patients in the sentinel group were staggered by 7 days. In the absence of dose-limiting toxicities (DLTs), each patient was eligible to switch to group A at the second infusion time point to receive dose level 1 (i.e., group A, day 8 dose). After completion of the sentinel group, escalating doses of SD-101 were administered alone (group A), with nivolumab (group B), and with a combination of ipilimumab and nivolumab (group C).

[0210] Group A is a dose-escalation 3+3 design and dose expansion study, starting with a 2 mg (lowest expected biological effect level, MABEL) dose of SD-101, followed by a 4 mg dose, and then an 8 mg dose. An optional dose expansion study is conducted after determining the maximum tolerated dose (MTD) once daily (OD). Group B is a further dose-escalation 3+3 study, starting with a 2 mg MABEL dose of SD-101 and a 1 mg / kg dose of nivolumab, followed by a 4 mg dose of SD-101 and a 1 mg / kg dose of nivolumab. After determining the MTD / OD, an 8 mg dose of SD-101 and a 1 mg / kg dose of nivolumab are administered, and an optional Group B1 study can be initiated, which includes SD-101, 2 mg (MABEL), a 1 mg / kg dose of nivolumab, and a 3 mg / kg dose of ipilimumab. Group C consists of 3+3 dose-escalation studies involving a combination of 2 mg MABEL dose of SD-101 with 1 mg / kg nivolumab and 3 mg / kg ipilimumab, followed by a combination of 4 mg SD-101 with 1 mg / kg nivolumab and 3 mg / kg ipilimumab. Optional dose-expansion studies of SD-101, nivolumab, and ipilimumab can be conducted after determining the MTD / OD. Alternatively, optional Group C1 can be conducted, which includes SD-101, nivolumab, and piracetamab. Group D consists of optional 3+3 dose-escalation studies that determine the MTD / OD of a total of four doses of PEDD / HAI SD-101 + CPI over two cycles.

[0211] In summary, in groups A, B, and C, SD-101 was administered once weekly for two cycles, each lasting three weeks. The first cycle consisted of weekly administration for three weeks, followed by a five-week off-cycle, and then the second cycle of weekly SD-101 administration for three weeks. In groups B and C, human subjects were also administered the CPIs as indicated above. Specifically, in group B, nivolumab 480 mg IV was administered every four weeks (Q4W). For group C, a dual CPI was administered as follows: ipilimumab IV 3 mg / kg and nivolumab IV 1 mg / kg Q3W for four consecutive cycles, each followed by nivolumab 480 mg IV Q4W. In Group D (to be implemented), SD-101 may be administered at a dosing schedule of only 2 weekly SD-101 infusions per cycle for 2 cycles, in combination with one or more of the following three CPI regimens: (1) a single-dose nivolumab IV at 480 mg every 4 weeks; (2) a CPI two-dose regimen of IV ipilimumab 3 mg / kg and IV nivolumab 1 mg / kg every 3 weeks (Q3W) for 4 doses, each followed by nivolumab 480 mg IV Q4W; or (3) nivolumab 480 mg and renalalimab 160 mg IV Q4W. As described above for Groups B, C, and D, human subjects may be administered checkpoint inhibitors concurrently with SD-101 for two cycles, and checkpoint inhibitors may continue to be administered to human subjects for up to 12 months.

[0212] SD-101 solution is administered via hepatic artery infusion, optionally using TriNav. ® Infusion system. Femoral artery or brachial / radial artery access can be used. For the SD-101 infusion procedure, the drug is prepared and delivered in a 50 mL syringe (therapeutic dose) and a 100 mL vial containing the required volume (10 mL) for therapeutic flushing, both at therapeutic concentrations. The 50 mL volume to be administered is dispensed according to each segment or sector of the liver.

[0213] Figure 2B This demonstrates the patient characteristics of patients tested during the Phase 1 clinical trial. Overall, in many cases, this is a heavily pre-treated population with a heavy disease burden.

[0214] Figure 2CSafety summaries and adverse events (Ae) for groups A, B, and C are presented, including dose-limiting toxicities (DLTs) and serious adverse events (SAEs). The most common adverse events across all groups were gastrointestinal symptoms (41%), fatigue (30%), and skin-related events (27%). The following conditions observed during an SD-101 cycle or within 4 weeks after the last SD-101 dose in cycle 1 are considered DLTs and are considered attributable to the study intervention (SD-101 or CPI therapy) and / or the PEDD device: ●According to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), ≥ Grade 4 cytokine release syndrome does not recover to ≤ Grade 2 within 7 days. ●According to NCI CTCAE, a level 3 CRS that has not recovered to level ≤ 2 within 7 days ●According to NCI CTCAE, autoimmune AE ≥ grade 3 ●According to NCI CTCAE, allergic reaction Ae ≥ 3 ● Hematological grade 4 hematology Ae that has not recovered to grade 2 or below within 7 days ●Any Level 4 AE in any organ system according to NCI CTCAE Figure 2C The data showed that adding SD-101 delivered to the liver via HAI and PEDD to CPI treatment regimens was well tolerable and had a similar safety profile to SPI in the absence of SD-101.

[0215] Figure 3-5 Serum pharmacokinetic profiles of SD-101 at doses of 2 mg, 4 mg, and 8 mg following each hepatic infusion are presented. Plasma was collected at specified time points on the infusion day, and SD-101 levels were analyzed by LC-MS. Notably, TriNav... ® The infusion system is a PEDD device that allows for high liver SD-101 levels with limited and safe systemic exposure. Figure 3-5 Following all doses of SD-101 shown, SD-101 was transiently detected in serum (<2 hours). Furthermore, the pharmacokinetic profile of SD-101 was unaffected by CPI administration. Figure 6 The concentration of SD-101 in liver tissue after infusion is shown. At an 8 mg dose, liver tissue levels as high as 2,720 ng / g were observed. Figure 3-6 The study showed that plasma levels of SD-101 were transient and remained low after PEDD, while high drug levels were observed in the liver. This data demonstrates that SD-101 delivered via HAI and PEDD concentrates the drug in the target organ while minimizing systemic exposure.

[0216] Figure 7A Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the serum cytokine levels of CXCL10 (IP-10) after administration of a TLR9 agonist. Figure 7B Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, serum cytokine analysis of IL-8 after administration of TLR9 agonist. Figure 8A Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze serum cytokines of IL-2R after administration of TLR9 agonists. Figure 8B Provided according to Figure 2A The Phase 1 / 1B clinical trial protocol, serum cytokine analysis of IFNγ after administration of TLR9 agonist. Figure 7A , 7B Data from 8A and 8B showed that serum cytokine levels were increased despite low systemic exposure to SD-101.

[0217] Figure 9A Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol used MDSC-related genes to regulate TME in uveal melanoma liver metastases on day 57. Figure 9B Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol used T-cell-related genes to regulate the TME of uveal melanoma liver metastases on day 57. Figure 9A and 9B This study demonstrates that SD-101 induced extensive and favorable changes in gene expression after 57 days. This data indicates improved TME regulation in uveal melanoma liver metastases. Figure 10 The changes in M-MDSC within the tumor were shown after 57 days.

[0218] Figure 11A Showing according to Figure 2A This study is based on the Phase 1 / 1B clinical trial protocol, analyzing circulating immune cells (RBCs) of changes in circulating NK cell protein expression profiles (day 1–day 36) after SD-101 administration. Data are presented as mean + SEM (n = 19). Figure 11B Showing according to Figure 2A This study is based on the Phase 1 / 1B clinical trial protocol, and is an analysis of circulating immune cells showing changes in the protein expression profile of circulating CD8 T cells after SD-101 administration (day 1–day 36). Data are presented as mean + SEM (n = 23). Figure 11A and 11B This demonstrates intraoperative changes in flow cytometry despite low systemic exposure to SD-101. For Figure 11A and 11BThe data shown indicates that on day 36, 13 out of 16 subjects had an increase in NK cells.

[0219] Figures 11C-11F provide data on circulating immune signatures associated with tumor regression. Figure 11C illustrates the data based on... Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the changes in protein expression on circulating CD8+ T cells (day 1–day 36) in groups A, B, and C after SD-101 administration using circulating immune cell analysis. Figure 11D provides the data from Figure 11C separated based on the amount of SD-101 (also known as netomod) administered. Figures 11C and 11D show the systemic increase in CD8+ T cell proliferation and activation in all groups. Figure 11E shows the data based on… Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the changes in protein expression on circulating NK cells in groups A, B, and C (day 1–day 36) after SD-101 administration using circulating immune cell analysis. Figure 11F provides data from Figure 11E separated based on the amount of SD-101 (also known as netomod) administered. Figures 11E and 11F illustrate the systemic increase in NK cell proliferation.

[0220] Figure 12A The changes observed during flow cytometry were demonstrated despite the low systemic exposure to SD-101. Figure 12B-12E Data on circulating immune signatures associated with tumor regression were provided. Figure 12B Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol of SD-101 was used to analyze the changes in protein expression on circulating CD8+ T cells in groups A, B, and C (day 1-36) after administration of SD-101. Figure 12C Provided separately according to the amount of SD-101 (also known as Netomod) applied. Figure 12B The data. Figure 12B and 12C The study showed a systemic increase in CD8+ T cell proliferation and activation across all groups. Figure 12D Showing according to Figure 2A The Phase 1 / 1B clinical trial protocol was used to analyze the changes in protein expression on circulating NK cells in groups A, B, and C after SD-101 administration (day 1–day 36) using circulating immune cells. Figure 12E Provided separately according to the amount of SD-101 (also known as Netomod) applied. Figure 12D The data. Figure 12D and 12E The data provided show a systemic increase in NK cell proliferation.

[0221] Figure 13AThe study presents swimlane plots of patients, showing excellent outcomes in 16 out of 25 patients with SD-101, mild response (MR) in 1 out of 25 patients, and demonstrating the potential to prolong survival in progressive disease (PD). Figure 13B yes Figure 13A This is a continuation of the previous study, and additional study days are shown for group B alone. Data showed that patients receiving the 2 mg SD-101 plus nivolumab combination had superior progression-free survival (PFS) compared to those receiving the 4 mg or 8 mg SD-101 plus nivolumab (Nivo). The median PFS in the 2 mg SD-101 group was 11.7 months (~356 days), with a 1-year overall survival (OS) of 86%. Figure 13B The data showed that 70% of patients in group B had a 2L or higher disease level, including 4L and 6L patients. The data also showed a 27% ctDNA clearance rate, and 5 out of 7 patients with 2 mg + nivolumab showed a ctDNA reduction of >50%, including 2 complete ctDNA responders. This data suggests that even patients with progressive disease and reduced ctDNA may show improved survival.

[0222] Figure 14A , 14B 14C showed changes in ctDNA levels by dose level in patients who received large doses of treatment. Figure 14A It shows that according to Figure 2A Changes in ctDNA levels in patients treated with the Phase 1 / 1B clinical trial protocol. Figure 14A The study showed that ctDNA levels were reduced in 8 of the 13 patients, with ctDNA being cleared in 3 of them. Furthermore, 61% of patients with measurable ctDNA at baseline showed a favorable decrease in ctDNA levels. Figure 14B Explanation provided based on Figure 2A Additional data on changes in ctDNA levels in patients treated with the Phase 1 / 1B clinical trial protocol. Figure 14C Explanation provided based on Figure 2A Additional data on changes in ctDNA levels in group B patients treated with the Phase 1 / 1B clinical trial protocol. Figure 14B and 14C The study presented changes in ctDNA levels in additional patients (specifically, patients in group B), showing decreased ctDNA levels in 16 of the 26 patients, with ctDNA clearance in 7 of them. Figure 14B and 14CctDNA was quantified during cycle 1 (during treatment, weeks 1-3) and at the most recent available time point after cycle 1 (post-treatment, week 6 or 9). Group B was identified by next-generation sequencing. Figure 14C ) and groups B and C (by dosage combination) Figure 14B Changes in ctDNA levels.

[0223] Figures 14D to 14G The data provided show a reduction in ctDNA in patients who underwent extensive pretreatment. Figure 14D Explanation provided based on Figure 2A Data on changes in ctDNA levels in patients in groups B and C at time points after the first cycle of treatment (including day 36 and day 57, unless otherwise stated) in the Phase 1 / 1B clinical trial protocol. Figure 14E Provided based on groups B and C Figure 14D The data. Figure 14D and 14E The data showed a 69.6% (16 / 23) reduction in ctDNA and a 21.7% (5 / 23) clearance of ctDNA. At the 2 mg dose level of SD-101, the ctDNA reduction was 81.8% (9 / 11) and the ctDNA clearance was 36% (4 / 11). Figure 14D and 14E In this context, the time points following the first cycle include day 36 and day 57. 27 patients were not included. Figure 14D and 14E They are not evaluable because ctDNA was not detected at baseline or baseline data was unavailable. Figure 14F Explanation provided based on Figure 2A Data on the reduction in ctDNA mutant allele fraction (MAF) relative to baseline in patients in groups B and C treated with the Phase 1 / 1B clinical trial protocol. Figure 14G Provided based on groups B and C Figure 14F The data. Figure 14F and 14G The data showed a molecular response rate of 76.9% (20 / 26) (MAF reduction ≥ 50% relative to baseline) and 50% (13 / 26) ctDNA clearance. At the 2 mg dose level of SD-101, the molecular response rate was 90.9% (10 / 11) (MAF reduction ≥ 50% relative to baseline) and 54.5% (6 / 11) ctDNA clearance.

[0224] Figure 15A The changes in CD8+ T cell concentrations in groups A, B, and C are shown. Figure 15B The changes in CD4+ T cell concentrations in groups A, B, and C are shown. Figure 15CThe changes in NK cell concentrations in groups A, B, and C are shown. Figure 15D The figures show changes in M1 macrophage concentrations in groups A, B, and C. For group B, these figures demonstrate a sustained increase in CD8+ T cells, CD4+ T cells, NK cells, and M1 macrophages in the tumor at day 57 compared to baseline. Figure 15A -D data are represented as mean ± SEM.

[0225] Figure 16A The changes in CD8+ T cell concentrations in group B are shown at 2 mg, 4 mg, and 8 mg. Figure 16B The changes in CD4+ T cell concentrations in group B are shown at 2 mg, 4 mg, and 8 mg. Figure 16C The changes in CD8+ T cell concentrations in group B are shown at 2 mg, 4 mg, and 8 mg. Figure 16D The changes in M1 macrophage concentrations in group B are shown at 2 mg, 4 mg, and 8 mg. Data are presented as mean ± SEM.

[0226] Figure 17A The changes in tumor pathway scores from baseline to day 57, determined by advanced analysis of NanoString gene expression data, are shown for groups A, B, and C (group A (2 mg n = 3, 4 mg n = 2, 8 mg n = 2); group B (2 mg n = 2, 4 mg n = 5, 8 mg n = 6); group C (2 mg n = 4)). Figure 17A This study demonstrates SD-101 hepatic artery infusion via PEDD in groups A, B, and C at baseline and day 57, which were associated with the induction of T cell activation and cytokine signaling in liver metastases. Figure 17A The data showed a consistent positive change in tumor pathway scores in group B, and increased immune cell activation (e.g., T cell activation and cytokine signaling) within liver metastases. Data are presented as mean ± SEM.

[0227] Figure 17B The changes in PBMC pathway scores from baseline to day 36, as determined by advanced analysis of NanoString gene expression data, are shown for groups A, B, and C (group A (2 mg n = 4, 4 mg n = 3, 8 mg n = 3); group B (2 mg n = 1, 4 mg n = 7, 8 mg n = 7); group C (2 mg n = 7)). Figure 17BThis study demonstrates that despite low extrahepatic SD-101 levels, hepatic arterial infusion of SD-101 via PEDD was associated with the induction of T cell activation and cytokine signaling in the blood at baseline and day 36 for groups A, B, and C. Data are presented as mean ± SEM.

[0228] Figure 17C It is shown in accordance with Figure 2A The changes in tumor pathway scores by group from baseline to day 57 were determined by advanced analysis of NanoString gene expression data in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C. Figure 17D It shows Figure 17C Changes in tumor pathway scores based on the administered SD-101 dose. Figure 17C and 17D In the data, dose-response changes in intratumoral pathway scores were observed, with 8 mg netomod producing the greatest increase in immune signaling and activation pathway scores after cycle 1.

[0229] Figure 17E It is shown in accordance with Figure 2A The changes in PBMC pathway scores from baseline to day 36, determined by advanced analysis of NanoString gene expression data, in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol. Figure 17F It shows Figure 17E Changes in tumor pathway scores based on the administered SD-101 dose. Figure 17E and 17F The data showed that 4 mg netomod produced the greatest increase in the score of immune signaling and activation pathways in PBMCs after cycle 1.

[0230] Figure 17G It is shown in accordance with Figure 2A The changes in tumor gene expression levels from baseline to day 57 were determined by advanced analysis of NanoString gene expression data in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol. Figure 17H It shows Figure 17G Changes in tumor gene expression levels categorized by the dose of SD-101 administered. Figure 17G and 17H The data showed that in all groups, the gene expression levels of pro-inflammatory genes in tumors increased after cycle 1.

[0231] Figure 17I It shows the display according to Figure 2AAdditional data on changes in tumor gene expression levels from baseline to day 57, determined by advanced analysis of NanoString gene expression data in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol. Figure 17J It shows Figure 17I Changes in tumor gene expression levels categorized by the dose of SD-101 administered. Figure 17I and 17J The data showed that within tumors, an 8 mg dose level increased the expression of most MDSC-related genes, while a 2 mg dose level decreased the expression of most MDSC-related genes.

[0232] Figure 17K It is shown in accordance with Figure 2A The changes in PBMC gene expression levels from baseline to day 36 were determined by advanced analysis of NanoString gene expression data in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol. Figure 17L It shows Figure 17K Changes in PBMC gene expression levels based on the administered SD-101 dose. Figure 17K and 17L The data showed that IFNB1 expression increased with PBMC after cycle 1 in all groups, and that CXCL10, granzyme A, and ICOS increased in PBMC after cycle 1 in groups B and C.

[0233] Figure 17M It shows the display according to Figure 2A Additional data on changes in PBMC gene expression levels from baseline to day 36, determined by advanced analysis of NanoString gene expression data in groups A, B, and C of patients treated with the Phase 1 / 1B clinical trial protocol. Figure 17N It shows Figure 17M Changes in PBMC gene expression levels based on the administered SD-101 dose. Figure 17M and 17N The data showed that, at all doses, IFNA2 and IFNB1 expression increased in PBMCs after cycle 1, and that 2 mg and 4 mg netomod dose levels increased granzyme A and ICOS in PBMCs after cycle 1.

[0234] Figure 18A The changes in the concentration of regulatory T cells (Tregs) in groups A, B, and C relative to baseline data are shown. Figure 18B The changes in cell concentration of myeloid monocyte suppressor cells (M-MDSCs) in groups A, B, and C relative to baseline data are shown. Figure 18C The changes in total MDSC cell concentrations in groups A, B, and C relative to baseline data are shown. Figure 18D The changes in M2 macrophage concentrations relative to baseline data are shown in groups A, B, and C. The reduction in Treg cells and MDSC cells in liver tumors leads to a decrease in the cells driving CPI failure. Figure 18A -D data are represented as mean ± SEM.

[0235] Figure 19A The changes in cell concentration of Treg cells in group B relative to baseline data are shown at 2 mg, 4 mg, and 6 mg. Figure 19B The changes in cell concentration of M-MDSC cells in group B relative to baseline data are shown at 2 mg, 4 mg, and 6 mg. Figure 19C The changes in total MSDC cell concentration in group B relative to baseline data are shown at 2 mg, 4 mg, and 6 mg. Figure 19D The changes in cell concentration of M2 macrophages in group B relative to baseline data are shown at 2 mg, 4 mg, and 6 mg. Figure 19E The changes in granzyme B cell concentrations relative to baseline data are shown at 2 mg, 4 mg, and 6 mg. Figure 19F The changes in IL-15 concentration in group B relative to baseline are shown at 2 mg, 4 mg, and 6 mg. Increased granzyme B contributes to T cell killing of tumor cells. Increased IL-15 increases T cell and NK cell activation and plays a role in the persistence of CD8+ memory T cells. Decreased MDSCs and Tregs in liver tumors correspond to a reduction in cells driving CPI failure. Figure 19A -F data are represented as mean ± SEM.

[0236] Clinical data from Example 1 described in this article show that 2 mg SD-101 + nivolumab with the aid of PEDD provided a >80% ctDNA response rate, a >80% disease control rate, and 11.7 months of progression-free survival (PFS).

[0237] Figure 20 AF demonstrates the peripheral immune signature induced by SD-101 delivered via PEDD. Data are presented as mean ± SEM. Specifically, Figure 20 AF showed that SD-101 delivered via PEDD induced systemic immune activation and an increase in pro-inflammatory cytokines (peripheral immune signatures) on day 36. Figure 20 A demonstrates activation induced by the pro-inflammatory cytokine CXCL10 (IP-10). Figure 20B demonstrates activation induced by the pro-inflammatory cytokine IFNγ. Figure 20 C demonstrates activation induced by the pro-inflammatory cytokine TNFα. Figure 20 D demonstrates activation induced by the pro-inflammatory cytokine IL-2R. Figure 20 E demonstrates activation induced by the pro-inflammatory cytokine IL-15. Figure 20 F demonstrates the activation of the pro-inflammatory cytokine IL-18.

[0238] Figure 21 AF shows circulating immune signatures associated with tumor regression in groups A, B, and C. Data are presented as mean ± SEM. Figure 21 A demonstrates activation induced by the pro-inflammatory cytokine CXCL10 (IP-10). Figure 21 B demonstrates activation induced by the pro-inflammatory cytokine IFNγ. Figure 21 C demonstrates activation induced by the pro-inflammatory cytokine TNFα. Figure 21 D demonstrates activation induced by the pro-inflammatory cytokine IL-2R. Figure 21 E demonstrates activation induced by the pro-inflammatory cytokine IL-15. Figure 21 F demonstrates the activation of the pro-inflammatory cytokine IL-18.

[0239] Figure 22 AF shows circulating immune signatures associated with tumor regression in group B. Data are presented as mean ± SEM. Figure 22 A demonstrates activation induced by the pro-inflammatory cytokine CXCL10 (IP-10). Figure 22 B demonstrates activation induced by the pro-inflammatory cytokine IFNγ. Figure 22 C demonstrates activation induced by the pro-inflammatory cytokine TNFα. Figure 22 D demonstrates activation induced by the pro-inflammatory cytokine IL-2R. Figure 22 E demonstrates activation induced by the pro-inflammatory cytokine IL-15. Figure 22 F demonstrates the activation of the pro-inflammatory cytokine IL-18.

[0240] Figure 22 AD shows changes in protein expression profiles on circulating cells. Data are presented as mean ± SEM. Figure 22 A shows a systemic increase in CD8+ T cell proliferation and activation across all groups A, B, and C. Figure 22 Group B showed a systemic increase in CD8+ T cell proliferation and activation at 2 mg, 4 mg, and 8 mg. Figure 22 C shows a systemic increase in NK cell proliferation across all groups A, B, and C. Figure 22D shows a systemic increase in NK cell proliferation in group B at 2 mg, 4 mg, and 8 mg.

[0241] Figure 20 Data from AF, 21A-F, 22A-F, and 23A-D showed a systemic increase in CD8+ T cell proliferation and activation, as well as a systemic increase in NK cell proliferation, across all groups.

[0242] The overall response rate (ORR) and 12-month overall success (OS) of patients receiving a combination of PEDD / HAI SD 101 and systemic (i.e., intravenous, IV) immune checkpoint blockade were also evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.

[0243] Figure 24A Objective RECIST 1.1 response rates are presented for patients in Group B at 2 mg, 4 mg, and 8 mg, and in Group C at 2 mg. Lesser response was reduced by approximately 10-29%. Partial response was reduced by approximately ≥ 30%. Progressive disease was defined as an increase of at least 20% in the sum of the diameters of live (enhanced) target lesions, referenced to the minimum sum of the diameters of live (enhanced) target lesions recorded since the start of treatment. Stable disease was defined as any case that did not meet the criteria for partial response, lesser response, or progressive disease. Results showed a disease control rate (DCR) of 81% at the optimal biological dose of SD-101 at 2 mg, and a DCR of 58% for all SD-101 doses when combined with an IV CPI. Some subjects experienced delayed responses, including conversion of PD to SD or MR. Figure 24B Showing from Figure 24A Patients and according to Figure 2A Objective RECIST 1.1 response rates in patients treated with the Phase 1 / 1B clinical trial protocol in Group B at 2 mg, 4 mg, and 8 mg, and in Group C at 2 mg and 4 mg. Figure 24A and 24B The data shown highlight the importance of ctDNA and PFS for disease control measurements.

[0244] Figure 25A The Kaplan-Meier curves show the overall survival (OS) proportions for groups A, B, and C. Figure 25B The overall survival rates for group B are shown at 2 mg, 4 mg, and 8 mg. For overall survival (OS), the optimal biological dose for group B is 2 mg. Figure 25BAs shown, the 1-year overall survival (OS) rate in Group B patients who received 2 mg of SD-101 weekly (in a 3-week cycle with 5 weeks between each cycle) and nivolumab was 86%.

[0245] Following the regimen of Group B, three (3) patients were administered 2 mg or 4 mg of SD-101 weekly (in 3-week cycles with 5-week intervals between each cycle) and nivolumab. Serial MRI / CT scans were obtained, and tumor sizes are provided in Table 2 below. Two of the patients showed a response to treatment that reduced tumor size.

[0246] Table 2

[0247] The treatment described in Example 1 resulted in high intrahepatic drug levels and low transient levels in peripheral blood. Following SD-101, widespread changes in immunostimulatory gene expression were observed in both tumor and normal liver tissue. Increased serum IL-18 and IFNγ were observed. Increased infiltration of CD8+ T cells, NK cells, and M1 macrophages was observed in the tumor. Decreased immunosuppressive Tregs, M-MDSCs, and M2 macrophages were observed in the tumor. Gene expression levels in the LM and PBMCs showed increased expression of genes associated with CD8+ cytotoxic T lymphocyte activity, Th1 activation, cytokine and chemokine signaling. Systemic immune activation induced by increased pro-inflammatory cytokines was observed. Increased systemic proliferation and activation of NK cells and CD8+ T cells were observed. The median PFS for 2 mg SD-101 + nivolumab was 11.7 months.

[0248] Based on the data provided above, the TriNav PEDD device achieved high hepatic SD-101 levels with limited and safe systemic exposure. SD-101 delivered via hepatic artery infusion (HAI) and PEDD was well-tolerated (with or without systemic CPI) and associated with enhanced immune activity. Evidence of the biological effects of lower doses of SD-101 in combination with nivolumab was observed. Results also showed MDSC depletion and reduction of MDSC-associated genes in liver metastases, with notable widespread immune effects in both liver metastases and the periphery. Similarly, a reduction in ctDNA was observed in patients with extensive pretreatment. In MUM-LM patients, SD-101 delivered via PEDD plus systemic ICI resulted in clinical activity with a median PFS of 11.7 months, MDSC reprogramming, and evidence of peripheral and intratumoral immune activation.

[0249] Figure 25C The Kaplan-Meil curves show the overall survival (OS) proportions for groups A, B, and C. Figure 25 shows the data including... Figure 25AThe data provided and other data. Figure 25D Showing according to Figure 25C The data showed the overall survival rate of group B at doses of 2 mg, 4 mg, and 8 mg SD-101. Figure 25E Showing according to Figure 25C The data showed the overall survival rates of groups B and C at 2 mg and 4 mg SD-101 doses.

[0250] Figures 26A to 26N Showing in accordance with Figure 2A In patients treated with the Phase 1 / 1B clinical trial protocol, changes in intratumoral cell density from baseline to day 57 were measured using multiplex immunofluorescence (IF). Figures 26A to 26N The data showed that, on average, all cell types increased after treatment, but the increase was currently greatest for CD4 and CD8 T cells. Additionally, the data showed that, on average, all cell types increased after treatment, but the increase was currently greatest for CD4 and CD8 T cells. Although the data showed an increase in total MDSCs within the tumor at the 2 mg dose level, the expression levels of MDSC-related genes within the tumor decreased at this dose level, suggesting a possible reduction in MDSC activity after cycle 1.

[0251] Figures 27A to 27H Showing in accordance with Figure 2A The changes in plasma cytokine immunomarkers from baseline to day 36 in patients treated with the Phase 1 / 1B clinical trial protocol in groups A, B, and C, as analyzed by Luminex. Figure 27A Data up to 27N showed that plasma levels of IL-2R, CXCL10, and granzyme B increased after cycle 1 in all doses and groups.

[0252] The foregoing merely illustrates the principles of this disclosure. Various modifications and alterations to the described embodiments will be readily apparent to those skilled in the art in light of the teachings herein. Therefore, it should be understood that those skilled in the art will be able to design numerous systems, arrangements, and programs that, while not expressly shown or described herein, embody the principles of this disclosure and are therefore within the spirit and scope of this disclosure. Those skilled in the art will understand that various different exemplary embodiments can be used together and interchangeably with each other. Furthermore, certain terms used in this disclosure, including the specification, may be used synonymously in certain circumstances, including, for example, data and information. It should be understood that while these terms and / or other terms that may be synonymous with each other may be used synonymously herein, there may be situations where these terms are not intended to be used synonymously. Moreover, the foregoing prior art, to the extent that it is not expressly incorporated herein by reference, is expressly incorporated in its entirety. All referenced publications are incorporated herein by reference in their entirety.

Claims

1. A method for treating uveal melanoma liver metastases in human subjects, the method comprising: The subject was given an effective combination of treatment with one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, The one or more checkpoint inhibitors are administered systemically to the subject, and The TLR9 agonist is administered locally to the liver of the subject by means of hepatic artery infusion at a dose sufficient to provide a therapeutically effective concentration in the liver when combined with the checkpoint inhibitor and to provide a plasma concentration of the TLR9 agonist below its concentration in the liver.

2. The method of claim 1, wherein the TLR9 agonist has the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), wherein the entire sequence is linked by thiophosphate bonds.

3. The method according to claim 2, wherein the TLR9 agonist is a sodium salt of the oligonucleotide of SEQ ID NO:

2.

4. The method according to any one of claims 1 to 3, wherein when the TLR9 agonist is administered without the administration of the one or more checkpoint inhibitors, the plasma concentration of the TLR9 agonist is administered at a subtherapeutic dose.

5. The method according to any one of claims 1 to 3, wherein the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject.

6. The method according to any one of claims 1 to 3, wherein the subject has stage IV uveal melanoma.

7. The method according to any one of claims 1 to 3, wherein the TLR9 agonist is applied locally to a region outside the liver metastasis of the uveal melanoma in the liver of the subject.

8. The method according to any one of claims 1 to 3, wherein the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously.

9. The method according to any one of claims 1 to 3, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:

10.

10. The method of claim 9, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:

7.

11. The method according to any one of claims 1 to 3, wherein the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g.

12. The method of claim 11, wherein the plasma concentration of the TLR9 agonist is less than about 750 ng / mL.

13. The method of claim 12, wherein the plasma concentration of the TLR9 agonist is less than about 700 ng / mL.

14. The method of claim 13, wherein the plasma concentration of the TLR9 agonist is less than about 600 ng / mL.

15. The method according to any one of claims 1 to 3, wherein the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg.

16. The method of claim 15, wherein the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg.

17. The method of claim 16, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg.

18. The method of claim 17, wherein the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg.

19. The method of claim 17, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg.

20. The method of claim 19, wherein the TLR9 agonist is administered weekly in a 3-week cycle with rest periods between cycles.

21. The method according to any one of claims 1 to 20, wherein the TLR9 agonist is administered via a catheter device.

22. The method of claim 21, wherein the conduit device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow.

23. The method of claim 22, wherein the TLR9 agonist is administered via the catheter device by means of pressure-enabled drug delivery (PEDD).

24. The method of claim 21, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 200 minutes.

25. The method of claim 24, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 60 minutes.

26. The method of claim 25, wherein the TLR9 agonist is administered over a period of approximately 25 minutes.

27. The method according to any one of claims 1 to 3, wherein the one or more checkpoint inhibitors are administered in parallel with the TLR9 agonist.

28. The method according to any one of claims 1 to 3, wherein the one or more checkpoint inhibitors are administered prior to the TLR9 agonist.

29. The method according to any one of claims 1 to 3, wherein the one or more checkpoint inhibitors are administered after the TLR9 agonist.

30. The method according to any one of claims 1 to 3, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, relatlimab, and crefmirlimab.

31. The method of claim 30, wherein the one or more checkpoint inhibitors is nivolumab.

32. The method of claim 30, wherein the one or more checkpoint inhibitors is pembrolizumab.

33. The method of claim 30, wherein the one or more checkpoint inhibitors is ipilimumab.

34. The method of claim 30, wherein one or more checkpoint inhibitors are cialidomide.

35. The method of claim 30, wherein two checkpoint inhibitors are applied.

36. The method of claim 35, wherein the two checkpoint inhibitors are nivolumab and ipilimumab.

37. The method of claim 35, wherein the two checkpoint inhibitors are nivolumab and renalalimab.

38. A method for improving the survival rate of human subjects with liver metastases from uveal melanoma, the method comprising: An effective combination of treatment, administered to the subject, consisting of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof.

39. The method of claim 38, wherein the TLR9 agonist has the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), wherein the entire sequence is linked by thiophosphate bonds.

40. The method of claim 39, wherein the TLR9 agonist is a sodium salt of the oligonucleotide of SEQ ID NO:

2.

41. The method according to any one of claims 38 to 40, wherein the one or more checkpoint inhibitors are administered systemically to the subject.

42. The method according to any one of claims 38 to 41, wherein when the TLR9 agonist is administered without the administration of the one or more checkpoint inhibitors, the plasma concentration of the TLR9 agonist is at a subtherapeutic dose.

43. The method according to any one of claims 38 to 41, wherein the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject.

44. The method according to any one of claims 38 to 41, wherein the subject has stage IV uveal melanoma.

45. The method according to any one of claims 38 to 41, wherein the TLR9 agonist is applied locally to a region outside the liver metastasis of the uveal melanoma in the liver of the subject.

46. ​​The method of claim 41, wherein the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously.

47. The method according to any one of claims 38 to 41, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:

10.

48. The method of claim 47, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:

7.

49. The method according to any one of claims 38 to 41, wherein the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g.

50. The method of claim 49, wherein the plasma concentration of the TLR9 agonist is less than about 750 ng / mL.

51. The method of claim 50, wherein the plasma concentration of the TLR9 agonist is less than about 700 ng / mL.

52. The method of claim 51, wherein the plasma concentration of the TLR9 agonist is less than about 600 ng / mL.

53. The method according to any one of claims 38 to 41, wherein the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg.

54. The method of claim 53, wherein the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg.

55. The method of claim 54, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg.

56. The method of claim 55, wherein the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg.

57. The method of claim 55, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg.

58. The method of claim 57, wherein the TLR9 agonist is administered weekly in a 3-week cycle with rest periods between cycles.

59. The method according to claims 38 to 58, wherein the TLR9 agonist is administered locally to the liver of the subject by means of hepatic artery infusion.

60. The method of claim 59, wherein the TLR9 agonist is administered via a catheter device.

61. The method of claim 59, wherein the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject.

62. The method of claim 59, wherein the conduit device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow.

63. The method of claim 60, wherein the TLR9 agonist is administered to the liver of the subject by means of pressure-enabled drug delivery (PEDD).

64. The method of claim 59, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 200 minutes.

65. The method of claim 64, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 60 minutes.

66. The method of claim 65, wherein the TLR9 agonist is administered over a period of approximately 25 minutes.

67. The method according to any one of claims 38 to 41, wherein the one or more checkpoint inhibitors are administered in parallel with the TLR9 agonist.

68. The method according to any one of claims 38 to 41, wherein the one or more checkpoint inhibitors are administered prior to the TLR9 agonist.

69. The method according to any one of claims 38 to 41, wherein the one or more checkpoint inhibitors are administered after the TLR9 agonist.

70. The method according to any one of claims 38 to 41, wherein the one or more checkpoint inhibitors comprise an anti-PD1 or anti-PD-L1 antibody or an antigen-binding fragment thereof.

71. The method of claim 70, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, pillalimab, and camrelimab.

72. The method of claim 71, wherein the one or more checkpoint inhibitors is nivolumab.

73. The method of claim 71, wherein the one or more checkpoint inhibitors is pembrolizumab.

74. The method of claim 71, wherein the one or more checkpoint inhibitors is ipilimumab.

75. The method of claim 71, wherein the one or more checkpoint inhibitors is cialilimab.

76. The method according to any one of claims 38 to 41, wherein two checkpoint inhibitors are applied.

77. The method of claim 76, wherein the two checkpoint inhibitors are nivolumab and ipilimumab.

78. The method of claim 76, wherein the two checkpoint inhibitors are nivolumab and renalalimab.

79. The method of any one of claims 38 to 41, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the progression-free survival (PFS) of the subject.

80. A method for treating uveal melanoma liver metastases in a human subject, the method comprising: Administer a therapeutically effective amount of one or more checkpoint inhibitors to the subject; and Topical application of a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, The one or more checkpoint inhibitors and the TLR9 agonist are administered in a therapeutically effective combination to reduce the tumor burden on the subject.

81. The method of claim 80, wherein the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), wherein the entire sequence is linked by thiophosphate bonds.

82. The method of claim 81, wherein the TLR9 agonist is a sodium salt of the oligonucleotide of SEQ ID NO:

2.

83. The method according to any one of claims 80 to 82, wherein when the TLR9 agonist is administered without the administration of the one or more checkpoint inhibitors, the plasma concentration of the TLR9 agonist is at a subtherapeutic dose.

84. The method according to any one of claims 80 to 82, wherein the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject.

85. The method according to any one of claims 80 to 82, wherein the subject has stage IV uveal melanoma.

86. The method according to any one of claims 80 to 82, wherein the TLR9 agonist is applied locally to a region outside the uveal melanoma liver metastasis in the liver of the subject.

87. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously.

88. The method according to any one of claims 80 to 82, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:

10.

89. The method of claim 88, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:

7.

90. The method according to any one of claims 80 to 82, wherein the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g.

91. The method of claim 90, wherein the plasma concentration of the TLR9 agonist is less than about 750 ng / mL.

92. The method of claim 91, wherein the plasma concentration of the TLR9 agonist is less than about 700 ng / mL.

93. The method of claim 92, wherein the plasma concentration of the TLR9 agonist is less than about 600 ng / mL.

94. The method according to any one of claims 80 to 82, wherein the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg.

95. The method of claim 94, wherein the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg.

96. The method of claim 95, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg.

97. The method of claim 96, wherein the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg.

98. The method of claim 96, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg.

99. The method of claim 96, wherein the TLR9 agonist is administered weekly in a 3-week cycle with rest periods between cycles.

100. The method according to any one of claims 80 to 82, wherein the TLR9 agonist is administered locally to the liver of the subject by means of hepatic artery infusion.

101. The method of claim 100, wherein the TLR9 agonist is administered via a catheter device.

102. The method of claim 101, wherein the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject.

103. The method of claim 101, wherein the conduit device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow.

104. The method of claim 100, wherein the TLR9 agonist is administered to the liver of the subject by means of pressure-enabled drug delivery (PEDD).

105. The method of claim 100, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 200 minutes.

106. The method of claim 105, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 60 minutes.

107. The method of claim 106, wherein the TLR9 agonist is administered over a period of approximately 25 minutes.

108. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors are administered systemically to the subject.

109. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors are administered in parallel with the TLR9 agonist.

110. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors are administered prior to the TLR9 agonist.

111. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors are administered after the TLR9 agonist.

112. The method according to any one of claims 80 to 82, wherein the one or more checkpoint inhibitors comprise an anti-PD1 or anti-PD-L1 antibody or an antigen-binding fragment thereof.

113. The method of claim 112, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimumab, and camrelimab.

114. The method of claim 113, wherein the one or more checkpoint inhibitors is nivolumab.

115. The method of claim 113, wherein the one or more checkpoint inhibitors is pembrolizumab.

116. The method of claim 113, wherein the one or more checkpoint inhibitors is ipilimumab.

117. The method of claim 113, wherein the one or more checkpoint inhibitors is cialilimab.

118. The method according to any one of claims 80 to 82, wherein two checkpoint inhibitors are applied.

119. The method of claim 118, wherein the two checkpoint inhibitors are nivolumab and ipilimumab.

120. The method of claim 118, wherein the two checkpoint inhibitors are nivolumab and renalalimab.

121. The method according to any one of claims 80 to 82, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the survival rate of the subject.

122. The method according to any one of claims 80 to 82, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the progression-free survival (PFS) of the subject.

123. The method according to any one of claims 80 to 82, wherein the administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject.

124. A method for reducing circulating tumor DNA (ctDNA) in human subjects with liver metastases from uveal melanoma, the method comprising: An effective combination of treatment, administered to the subject, consisting of one or more checkpoint inhibitors and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof.

125. The method of claim 124, wherein the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), wherein the entire sequence is linked by thiophosphate bonds.

126. The method of claim 125, wherein the TLR9 agonist is a sodium salt of the oligonucleotide of SEQ ID NO:

2.

127. The method according to any one of claims 124 to 126, wherein when the TLR9 agonist is administered without the administration of the one or more checkpoint inhibitors, a subtherapeutic TLR9 agonist is present in plasma concentrations.

128. The method of claim 127, wherein the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject.

129. The method according to any one of claims 124 to 126, wherein the subject has stage IV uveal melanoma.

130. The method according to any one of claims 124 to 126, wherein the TLR9 agonist is applied locally to a region outside the uveal melanoma liver metastasis in the liver of the subject.

131. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously.

132. The method according to any one of claims 124 to 126, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:

10.

133. The method of claim 132, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:

7.

134. The method according to any one of claims 124 to 126, wherein the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g.

135. The method of claim 134, wherein the plasma concentration of the TLR9 agonist is less than about 750 ng / mL.

136. The method of claim 135, wherein the plasma concentration of the TLR9 agonist is less than about 700 ng / mL.

137. The method of claim 136, wherein the plasma concentration of the TLR9 agonist is less than about 600 ng / mL.

138. The method according to any one of claims 124 to 126, wherein the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg.

139. The method of claim 138, wherein the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg.

140. The method of claim 139, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg.

141. The method of claim 140, wherein the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg.

142. The method of claim 140, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg.

143. The method of claim 140, wherein the TLR9 agonist is administered weekly in a 3-week cycle with rest periods between cycles.

144. The method according to claims 124 to 126, wherein the TLR9 agonist is administered locally to the liver of the subject by means of hepatic artery infusion.

145. The method of claim 144, wherein the TLR9 agonist is administered via a catheter device.

146. The method of claim 144, wherein the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject.

147. The method of claim 145, wherein the conduit device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow.

148. The method of claim 147, wherein the TLR9 agonist is administered to the liver of the subject by means of pressure-enabled drug delivery (PEDD).

149. The method of claim 144, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 200 minutes.

150. The method of claim 149, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 60 minutes.

151. The method of claim 150, wherein the TLR9 agonist is administered over a period of approximately 25 minutes.

152. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors are administered systemically to the subject.

153. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors are administered in parallel with the TLR9 agonist.

154. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors are administered prior to the TLR9 agonist.

155. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors are administered after the TLR9 agonist.

156. The method according to any one of claims 124 to 126, wherein the one or more checkpoint inhibitors comprise an anti-PD1 or anti-PD-L1 antibody or an antigen-binding fragment thereof.

157. The method of claim 156, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and camrelimab.

158. The method of claim 157, wherein the one or more checkpoint inhibitors is nivolumab.

159. The method of claim 157, wherein the one or more checkpoint inhibitors is pembrolizumab.

160. The method of claim 157, wherein the one or more checkpoint inhibitors is ipilimumab.

161. The method of claim 157, wherein the one or more checkpoint inhibitors is cialilimab.

162. The method according to any one of claims 124 to 126, wherein two checkpoint inhibitors are applied.

163. The method of claim 162, wherein the two checkpoint inhibitors are nivolumab and ipilimumab.

164. The method of claim 162, wherein the two checkpoint inhibitors are nivolumab and renalalimab.

165. The method according to any one of claims 124 to 126, wherein the administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the subject's response rate to the one or more checkpoint inhibitors.

166. The method according to any one of claims 124 to 126, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the survival rate of the subject.

167. The method according to any one of claims 124 to 126, wherein the TLR9 agonist is locally applied to the liver of the subject by means of hepatic artery infusion.

168. A method for improving the response rate of human subjects with liver metastases from uveal melanoma to treatment with checkpoint inhibitors, the method comprising: Topical application of a combination of a toll-like receptor 9 (TLR9) agonist and the checkpoint inhibitor, wherein the TLR9 agonist is an oligonucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof.

169. The method of claim 168, wherein the TLR9 agonist has the sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 2), wherein the entire sequence is linked by thiophosphate bonds.

170. The method of claim 169, wherein the TLR9 agonist is a sodium salt of the oligonucleotide of SEQ ID NO:

2.

171. The method according to any one of claims 168 to 170, wherein when the TLR9 agonist is administered without the administration of the one or more checkpoint inhibitors, a subtherapeutic TLR9 agonist is present in plasma concentrations.

172. The method according to any one of claims 168 to 170, wherein the plasma concentration of the TLR9 agonist is at a level that is non-toxic to the human subject.

173. The method according to any one of claims 168 to 170, wherein the subject has stage IV uveal melanoma.

174. The method according to any one of claims 168 to 170, wherein the TLR9 agonist is applied locally to a region outside the uveal melanoma liver metastasis in the liver of the subject.

175. The method according to any one of claims 168 to 170, wherein the one or more checkpoint inhibitors are administered intravenously, intraperitoneally, or subcutaneously.

176. The method according to any one of claims 168 to 170, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:2 to about 1:

10.

177. The method of claim 176, wherein the ratio of the plasma concentration of the TLR9 agonist to the concentration of the TLR9 agonist in the liver is about 1:3 to about 1:

7.

178. The method according to any one of claims 168 to 170, wherein the concentration of the TLR9 agonist in the liver is from about 750 ng / g to about 3000 ng / g.

179. The method of claim 178, wherein the plasma concentration of the TLR9 agonist is less than about 750 ng / mL.

180. The method of claim 179, wherein the plasma concentration of the TLR9 agonist is less than about 700 ng / mL.

181. The method of claim 180, wherein the plasma concentration of the TLR9 agonist is less than about 600 ng / mL.

182. The method according to any one of claims 168 to 170, wherein the TLR9 agonist is administered weekly at a dose of about 0.5 mg to about 20 mg.

183. The method of claim 182, wherein the TLR9 agonist is administered weekly at a dose of about 1 mg to about 10 mg.

184. The method of claim 183, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg to about 8 mg.

185. The method of claim 184, wherein the TLR9 agonist is administered weekly at a dose of about 4 mg to about 8 mg.

186. The method of claim 184, wherein the TLR9 agonist is administered weekly at a dose of about 2 mg.

187. The method of claim 184, wherein the TLR9 agonist is administered weekly in a 3-week cycle with rest periods between cycles.

188. The method according to any one of claims 168 to 170, wherein the TLR9 agonist is locally applied to the liver of the subject by means of hepatic artery infusion.

189. The method of claim 188, wherein the TLR9 agonist is administered via a catheter device.

190. The method of claim 189, wherein the TLR9 agonist is infused while regulating distal vascular pressure and blood flow in the subject.

191. The method of claim 189, wherein the conduit device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow.

192. The method of claim 189, wherein the TLR9 agonist is administered to the liver of the subject by means of pressure-enabled drug delivery (PEDD).

193. The method of claim 188, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 200 minutes.

194. The method of claim 193, wherein the TLR9 agonist is administered weekly for a period of about 10 minutes to about 60 minutes.

195. The method of claim 194, wherein the TLR9 agonist is administered over a period of approximately 25 minutes.

196. The method according to any one of claims 168 to 170, wherein the one or more checkpoint inhibitors are administered systemically to the subject.

197. The method according to any one of claims 168 to 170, wherein the one or more checkpoint inhibitors are administered in parallel with the TLR9 agonist.

198. The method according to any one of claims 168 to 170, wherein the one or more checkpoint inhibitors are administered prior to the TLR9 agonist.

199. The method according to any one of claims 168 to 170, wherein the one or more checkpoint inhibitors are administered after the TLR9 agonist.

200. The method of any one of claims 168 to 170, wherein the one or more checkpoint inhibitors comprise an anti-PD1 or anti-PD-L1 antibody or an antigen-binding fragment thereof.

201. The method of claim 200, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, renalalimab, and keratalimab.

202. The method of claim 201, wherein the one or more checkpoint inhibitors is nivolumab.

203. The method of claim 201, wherein the one or more checkpoint inhibitors is pembrolizumab.

204. The method of claim 201, wherein the one or more checkpoint inhibitors is ipilimumab.

205. The method of claim 201, wherein the one or more checkpoint inhibitors is cialilimab.

206. The method according to any one of claims 80 to 82, wherein two checkpoint inhibitors are applied.

207. The method of claim 119, wherein the two checkpoint inhibitors are nivolumab and ipilimumab.

208. The method of claim 119, wherein the two checkpoint inhibitors are nivolumab and renalalimab.

209. The method according to any one of claims 168 to 170, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist improves the survival rate of the subject.

210. The method of any one of claims 168 to 170, wherein administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist increases the progression-free survival (PFS) of the subject.

211. The method according to any one of claims 168 to 170, wherein the administration of the combination of the one or more checkpoint inhibitors and the TLR9 agonist reduces circulating tumor DNA (ctDNA) in the subject.

212. The method of any one of claims 168 to 170, wherein the administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases the activation of immune cells within the uveal melanoma liver metastases of the subject.

213. The method of any one of claims 168 to 170, wherein the administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases systemic cytokine signaling in the blood of the subject.

214. The method of any one of claims 168 to 170, wherein the administration of the combination of the TLR9 agonist and the checkpoint inhibitor increases the density of one or more immune cells within the uveal melanoma liver metastasis of the subject.

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