Methods for treating immune dysfunction in liver cancer with toll-like receptor agonists

Abstract

Provided is a method for treating liver immune dysfunction in a subject. Liver immune dysfunction can be induced by at least one tumor arising from metastasis in the liver or at least one primary liver cancer. An exemplary method includes administering to a subject a Toll-like receptor 9 (TLR9) agonist, for example, a TLR9 agonist having the structure: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3'. The TLR9 agonist is administered to the liver, for example, the TLR9 agonist can be administered to the liver using local therapy through the vascular system.

Description

[Technical field]

[0001] Claiming priority This application claims priority to U.S. Provisional Application No. 63 / 302,004, entitled "Cancer Therapy Using Toll-Like Receptor Agonists," filed January 21, 2022, to U.S. Provisional Application No. 63 / 423,005, entitled "Cancer Therapy Using Toll-Like Receptor Agonists," filed November 6, 2022, and to U.S. Provisional Application No. 63 / 426,757, entitled "Cancer Therapy-Using Toll-Like Receptor Agonists," filed November 20, 2022, the entire contents of all of the above-identified applications are incorporated by reference herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. Said XML copy, created on Jan. 20, 2023, is named A372-509_WO_SL.xml and is 18,185 bytes in size.

[0003] The present disclosure relates generally to methods of treating cancer, particularly liver cancer, and to methods of delivering Toll-like receptor (TLR) agonists to solid tumors in the liver using localized therapy through the vasculature. [Background technology]

[0004] Cancer is a devastating disease involving the uncontrolled proliferation of cells, which can result in the growth of solid tumors in various organs, such as the skin, liver, and pancreas. Tumors may be present initially in any number of organs or may be the result of metastasis from another location or may be the result of spread.

[0005] The liver is a unique organ that is intrinsically immunosuppressive and drives the programming and proliferation of suppressive cells, such as myeloid-derived suppressive cells (MDSCs). In this regard, MDSCs proliferate in response to malignant tumors. MDSCs also drive the proliferation of other suppressive cell types, such as T regulatory cells (Tregs), tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs). MDSCs can downregulate immune cells and impede the efficacy of immunotherapy. Moreover, high MDSC levels generally predict poor prognosis in cancer patients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 9,422,564 [Patent Document 2] U.S. Patent No. 8,372,413 [Patent Document 3] U.S. Patent No. 8,500,775 [Patent Document 4] U.S. Patent No. 8,696,698 [Patent Document 5] U.S. Patent No. 8,696,699 [Patent Document 6] U.S. Patent No. 9,539,081 [Patent Document 7] U.S. Patent No. 9,808,332 [Patent Document 8] U.S. Patent No. 9,770,319 [Patent Document 9] U.S. Patent No. 9,968,740 [Patent Document 10] U.S. Patent No. 10,813,739 [Patent Document 11] U.S. Patent No. 10,588,636 [Patent Document 12] U.S. Patent No. 11,090,460 [Patent Document 13] U.S. Patent Publication No. 2018 / 0193591 [Patent Document 14] U.S. Patent Publication No. 2018 / 0250469 [Patent Document 15] U.S. Patent Publication No. 2019 / 0298983 [Patent Document 16] U.S. Patent Publication No. 2020 / 0038586 [Patent Document 17] U.S. Patent Publication No. 2020-0383688 Summary of the Invention [Problem to be solved by the invention]

[0007] Certain delivery challenges and immunosuppressive pathways may limit the success of immunotherapy in the liver. Thus, there remains a need for safe and effective therapies to treat hepatic immune dysfunction. [Means for solving the problem]

[0008] The present application relates to a method for treating hepatic immune dysfunction using a therapeutically effective amount of a Toll-like receptor (TLR) agonist. For example, the present application provides a method for treating hepatic immune dysfunction, comprising administering a therapeutically effective amount of a Toll-like receptor 9 agonist having the structure: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1) to a subject in need thereof. The hepatic immune dysfunction is induced by at least one tumor resulting from metastasis in the liver or at least one primary liver cancer.

[0009] In one aspect, the present application relates to a method of treating liver immune dysfunction, comprising administering a TLR agonist through a device by hepatic artery infusion (HAI). According to another example, treating liver immune dysfunction comprises administering a TLR agonist through a device by portal vein infusion (PVI). For example, the TLR9 agonist can be administered through a catheter device. The catheter device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow rate. In another example, the TLR9 agonist is administered through a catheter device via pressure-enabled drug delivery (PEDD).

[0010] In some cases, hepatic immune dysfunction may be induced as a result of a solid tumor in the liver, such as a tumor that is a metastasis of uveal melanoma, or may be induced as a result of at least one primary liver cancer, such as hepatocellular carcinoma or intrahepatic cholangiocarcinoma.

[0011] In some examples, the TLR agonist is administered through PEDD, which includes administering a therapeutic agent through a device such as a catheter device. In some examples, the catheter device includes a one-way valve that dynamically responds to local pressure changes. In some examples, the catheter device generates, causes, and / or contributes to a net increase in fluid pressure in a blood vessel and / or target tissue or tumor. In some examples, the catheter device generates, causes, and / or contributes to a net decrease in fluid pressure in a blood vessel and / or target tissue or tumor. In some examples, the catheter device generates, causes, and / or contributes to an initial decrease and then an increase in fluid pressure in a blood vessel and / or target tissue or tumor.

[0012] In some examples, the TLR agonist is administered through a pressure-enabled device, such as one that modulates vascular pressure.

[0013] In some examples, the amount of TLR agonist administered ranges from about 0.01 to 20 mg, or is selected from the group consisting of 2 mg, 4 mg, or 8 mg.

[0014] In some examples, the TLR agonist is administered for a period of about 10 to 200 minutes. In other embodiments, the TLR agonist is administered for a period of about 10 to 60 minutes. In other embodiments, the TLR agonist is administered for a period of about 25 minutes.

[0015] In some cases, administration of TLR9 agonist leads to gene expression changes in metastasis in liver. For example, gene expression changes include activation of immune cells in normal liver tissue and migration of activated immune cells to at least one tumor. In another example, gene expression changes include at least one of: improved TLR signaling; increased white blood cells; increased exhausted CD8 T cells; induction of Th1 programming; reduced Th2 programming; increased T cell receptor and improved T cell costimulatory signaling; increased IL9; IL15; CCL7; B cell activation; induction of mast cells; induction of NK cells; induction of IFNγ; improved interferon signaling; improved chemokine signaling; increased IL6 in liver without increased in blood; reduced M2 macrophages; increased M1 macrophages; reduced MDSC; reduced angiogenesis and VEGF; and reduced fatty acid oxidation.

[0016] In some examples, the TLR agonist is administered in combination with one or more checkpoint inhibitors. In some examples, the checkpoint inhibitor is administered systemically either simultaneously with, before or after the administration of the TLR9 agonist. In some examples, the checkpoint inhibitor comprises at least one of nivolumab, pembrolizumab and ipilimumab. In some examples, the administration of the TLR9 agonist in combination with one or more checkpoint inhibitors can result in a reduction in one of circulating tumor cell levels and circulating tumor DNA levels. In certain embodiments, the one or more checkpoint inhibitors are administered intraperitoneally or subcutaneously.

[0017] In some examples, the administration of the TLR agonist comprises a dosing regimen that includes cycles. In some examples, one or more of the cycles comprises the administration of the TLR9 agonist via a catheter device by HAI, followed by the systemic administration of one or more checkpoint inhibitors. Specifically, the checkpoint inhibitors include at least one of nivolumab, pembrolizumab, and ipilimumab.

[0018] In some cases, administration of TLR agonists through an intravascular device to the liver results in a reduction in myeloid-derived suppressor cells (MDSCs) or altered function of MDSCs to limit immunosuppression. In some cases, administration of TLR agonists through an intravascular device to the liver results in an anti-tumor effect.

[0019] In some examples, the TLR agonist is a TLR9 agonist, more specifically a class C TLR9 agonist. In some examples, the class C TLR9 agonist is SD-101.

[0020] In another example, the methods of the present application include methods for treating liver immune dysfunction and tumor-induced liver immune dysfunction, where administration of a TLR9 agonist, more specifically a class C TLR9 agonist, results in at least one of the following changes in gene expression in liver metastases (LM): activation of immune cells in normal liver (non-tumor liver of the same patient) with migration to liver metastases; activation and recruitment of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages); enhancement of TLR signaling; and activation of leukocytes (CD45 +); increase in exhausted CD8 T cells (critical for checkpoint responsiveness); induction of Th1 programming; reduction in Th2 programming; increase in T cell receptor and improved T cell costimulatory signaling; increase in IL9, IL15, CCL7; activation of B cells; induction of mast cells; induction of NK cells, induction of IFNg and related genes; improvement in interferon signaling; improvement in chemokine signaling; increase in IL6 in liver without increase in blood; reduction in M2 macrophages; increase in M1 / activation of macrophages and related genes: CD68, CD86 dendritic cell activation and migration to draining lymph nodes; reduction in MDSC and related genes: IDO1, T0D2, ARG-1, NOS2, TIMD4; reduction in angiogenesis and VEGF; reduction in fatty acid oxidation (preferred MDSC program).

[0021] In another example, the method of the present application includes a method for administering a TLR9 agonist, particularly a class C TLR9 agonist, more particularly in combination with a checkpoint inhibitor to a patient, where the administration of the TLR9 agonist in combination with the checkpoint inhibitor results in a reduction in circulating tumor cell levels and / or circulating tumor DNA levels. The patient may have liver immune dysfunction and / or tumor-induced liver immune dysfunction. For example, the patient may have metastatic uveal melanoma, hepatocellular carcinoma, or intrahepatic cholangiocarcinoma in the liver.

[0022] These and other objects, features and advantages of the exemplary embodiments of the present disclosure will become apparent from a reading of the following detailed description of the exemplary embodiments of the present disclosure when considered in conjunction with the entire specification.

[0023] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description considered in conjunction with the accompanying drawings illustrating examples of the present disclosure. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 illustrates the structure of SD-101. [Figure 2A] FIG. 1 provides in vitro experimental data demonstrating the effect of a TLR4 agonist, a TLR7 agonist, a TLR9A agonist, a TLR9B agonist and SD-101, compared to an untreated control, on the amount of total MDSC induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF, according to Example 1. [Figure 2B] FIG. 1 provides in vitro experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101, compared to untreated controls, on the amount of M-MDSC and G-MDSC induced by treatment of human PBMC collected from healthy donors and treated with IL6 and GMCSF, according to Example 1. [Figure 2C] FIG. 1 provides in vitro experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101, compared to untreated controls, on the amount of M1 macrophages induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF, according to Example 1. [Figure 2D] FIG. 1 provides in vitro experimental data demonstrating the effect of a TLR4 agonist, a TLR7 agonist, a TLR9A agonist, a TLR9B agonist and SD-101, compared to an untreated control, on the amount of M2 macrophages induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF, according to Example 1. [Figure 2E] FIG. 1 provides images of an in vitro experiment demonstrating the effect of a TLR9 agonist on human MDSCs induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF, compared to an untreated control, according to Example 1. [Figure 3A]FIG. 1 provides in vitro experimental data demonstrating the effects of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 compared to untreated controls on the amount of MDSC (CD11b+Gr-1+) induced by treatment of mouse bone marrow cells treated with GMCSF according to Example 2. [Figure 3B] FIG. 1 provides in vitro experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 compared to untreated controls on the amount of macrophages (CD11b+F4 / CD80+) induced by treatment of mouse bone marrow cells treated with GMCSF according to Example 2. [Figure 3C] FIG. 1 provides in vitro experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 compared to untreated controls on the amount of M1 macrophages (CD11b+F4 / CD80+CD38+) induced by treatment of mouse bone marrow cells treated with GMCSF according to Example 2. [Figure 4] FIG. 1 illustrates changes in liver metastasis gene expression at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times, as measured by the NanoString® immune cell panel, according to Example 3. [Diagram 5] FIG. 1 illustrates the change in liver metastasis gene expression at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times as measured by the NanoString® Bone Marrow Panel according to Example 3. [Figure 6] FIG. 1 illustrates changes in liver metastasis gene expression at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times as measured by the NanoString® bone marrow cell panel according to Example 3. [Figure 7]FIG. 1 illustrates changes in liver metastasis gene expression at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times as measured by the NanoString® metabolic panel according to Example 3. [Figure 8] FIG. 1 illustrates the change in liver metastasis gene expression at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times as measured by the NanoString® Bone Marrow Panel according to Example 3. [Figure 9] FIG. 1 illustrates gene expression changes in normal adjacent liver tissue at 4 weeks following a first cycle of weekly SD-101 infusions via HAI and PEDD at a 2 mg dose for three times as measured by the NanoString® bone marrow panel according to Example 3. [Figure 10] FIG. 1 illustrates the changes in PBMC gene expression over two cycles of three weekly SD-101 infusions via HAI and PEDD, respectively, at a 2 mg dose, as measured by the NanoString® Bone Marrow Panel, according to Example 3. [Figure 11A] FIG. 13 provides experimental data demonstrating that TLR9 stimulation with SD-101 inhibits MDSC generation from human PBMCs treated with IL6 and GMCSF for 7 days according to Example 4. [Figure 11B] FIG. 13 provides experimental data demonstrating that TLR9 stimulation with SD-101 inhibits MDSC generation from human PBMCs treated with IL6 and GMCSF for 2 days according to Example 4. [Figure 11C] FIG. 13 provides experimental data demonstrating that TLR9 stimulation with SD-101 enhances M1 macrophage production from human PBMCs treated with IL6 and GMCSF for 7 days according to Example 4. [Figure 11D] FIG. 13 provides experimental data demonstrating that TLR9 stimulation with SD-101 enhances M1 macrophage production from human PBMCs treated with IL6 and GMCSF for 2 days according to Example 4. [Figure 12A] FIG. 1 provides experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9B agonist and SD-101 compared to untreated controls on the amount of MDSC (CD33+CD11b+HLADR-) induced by treatment of human PBMC collected from healthy donors and treated with IL6 and GMCSF according to Example 5. [Figure 12B] FIG. 1 provides experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9B agonist and SD-101, compared to untreated controls, on the amount of M1 macrophages (CD14+CD86+) induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF according to Example 5. [Figure 12C] FIG. 1 provides experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9B agonist and SD-101 compared to untreated controls on the amount of M / G-MDSC (M / G CD14+ / CD15+ MDSC) induced by treatment of human PBMC collected from healthy donors and treated with IL6 and GMCSF according to Example 5. [Figure 12D] FIG. 1 provides experimental data demonstrating the effect of TLR4 agonist, TLR7 agonist, TLR9B agonist and SD-101, compared to untreated controls, on the amount of monocytic dendritic cells (CD14+CD11c+CD123-) induced by treatment of human PBMC collected from healthy donors and treated with IL6 and GMCSF according to Example 5. [Figure 12E] FIG. 1 provides experimental data demonstrating the effect of a TLR4 agonist, a TLR7 agonist, a TLR9B agonist and SD-101 on the amount of mouse bone marrow MDSCs from mouse bone marrow (BM) treated in the absence of mouse GMCSF, compared to an untreated control, according to Example 5. [Figure 12F]FIG. 1 provides experimental data demonstrating the effect of a TLR4 agonist, a TLR7 agonist, a TLR9B agonist and SD-101 on the amount of mouse bone marrow MDSCs from mouse bone marrow (BM) treated in the presence of mouse GMCSF compared to untreated controls according to Example 5. [Figure 13A] FIG. 1 shows a volcano plot illustrating gene expression in human PBMCs following treatment with a TLR9B agonist versus SD-101 (a TLR9C agonist) for two days as determined using NanoString® mRNA analysis according to Example 6. [Figure 13B] FIG. 1 provides experimental data demonstrating the effect of TLR9B agonists and SD-101, compared to untreated controls, on the amount of MDSCs (CD33+CD11b+HLADR-) induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF according to Example 6. [Figure 13C] FIG. 1 provides experimental data demonstrating the effect of TLR9B agonists and SD-101, compared to untreated controls, on the amount of M1 macrophages (CD14+CD86+) induced by treatment of human PBMCs collected from healthy donors and treated with IL6 and GMCSF according to Example 6. [Figure 13D] FIG. 13B shows pathway scores for immune regulatory pathways affected by TLR9B versus SD-101, as determined by nSolver.4 software using data generated from the NanoString® analysis of FIG. 13A. [Figure 13E] FIG. 13B shows qRT-PCR data for PD-L1 expression for the same samples analyzed in FIG. 13A. [Figure 13F] FIG. 13B shows qRT-PCR data for IFNγ expression for the same samples analyzed in FIG. 13A. [Figure 13G] FIG. 13B shows qRT-PCR data for IP-10 expression for the same samples analyzed in FIG. 13A. [Figure 14]FIG. 1 illustrates the change in plasma SD-101 levels following single-agent SD-101 4 mg dosing in human patients after each of six doses administered over two cycles, according to Example 7. [Figure 15] FIG. 1 illustrates the change in serum IFNγ following increasing dosing levels of single agent SD-101 and SD-101 in combination with a checkpoint inhibitor in a human patient after each of the three doses administered over the first cycle, according to Example 7. [Figure 16] FIG. 1 illustrates the change in intratumoral monocytic MDSC levels following different dosing levels of single agent SD-101 in human patients according to Example 7. [Figure 17] FIG. 1 illustrates the changes in circulating tumor cells following dosing of human patients with 2 mg of SD-101 along with checkpoint inhibitors, according to Example 7. [Figure 18] FIG. 18 illustrates the changes in circulating tumor DNA following dosing of 2 mg SD-101 with checkpoint inhibitors in five of the human patients from FIG. 17, according to Example 7. [Figure 19A] FIG. 1 illustrates the changes in tumor progression of mouse liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle, according to Example 8. [Figure 19B] FIG. 1 illustrates the change in tumor burden of mouse liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle at day 10 post-treatment according to Example 8. [Figure 19C] FIG. 1 illustrates images captured by IVIS over various time points after initiation of treatment with PEDD administration of SD-101 in combination with an anti-PD-1 antibody delivered either IP or SQ compared to vehicle, according to Example 8. [Figure 19D]FIG. 13 provides uptake images of liver harvested 10 days after treatment with PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle, according to Example 8. [Figure 20A] FIG. 1 illustrates the changes in MDSCs present in LM in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle at day 10 post-treatment according to Example 8. [Figure 20B] FIG. 1 illustrates the change in B (B220+) cells among isolated CD45+ cells from liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle on day 10 post-treatment according to Example 8. [Figure 20C] FIG. 1 illustrates the changes in T (CD3+) cells among isolated CD45+ cells from liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle on day 10 post-treatment according to Example 8. [Figure 20D] Figure 1 illustrates the changes in M1 and M2 macrophages among isolated CD45+ cells from liver metastases. M1 (F4 / 80+CD38+Egr2-) and M2 (F4 / 80+CD38-Egr2+) macrophages in the tumor microenvironment quantified by FC in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle on day 10 post-treatment according to Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the present disclosure will now be described in detail with reference to the figures, it is done so in connection with the exemplary embodiments and is not limited by the specific embodiments illustrated in the figures and the accompanying paragraphs.

[0026] The following description of the embodiments provides non-limiting representative examples that refer to numbers to particularly describe the features and teachings of various aspects of the present invention. It should be recognized from the description of the embodiments that the described embodiments can be implemented separately or in combination with other embodiments. Those skilled in the art who review the description of the embodiments should be able to understand the various described aspects of the present invention. The description of the embodiments should facilitate understanding of the present invention to the extent that other implementations that are not specifically covered but are within the knowledge of those skilled in the art who read the description of the embodiments are understood to be consistent with the application of the present invention.

[0027] Unless otherwise specified, 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 belongs. Unless otherwise specified, certain terms used herein have the meanings set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0028] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0029] Unless otherwise indicated, all numerical values, such as concentrations or concentration ranges, described herein should be understood in all instances as being modified by the term "about." Such numerical values ​​may include ±1%, ±2%, ±3%, ±4% or ±5% of the stated value. As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within that range, including integers within such ranges and fractions of such values, unless the context clearly indicates otherwise.

[0030] As used herein, the term "subject" or "patient" refers to an animal, preferably a mammal, and more preferably a human. Examples of subjects include humans, as well as other animals such as rats, mice, and pigs. In one particular aspect, the subject is a human.

[0031] As used herein, the terms "treat", "treating" and "treatment" are all intended to refer to the improvement or reversal of at least one measurable physical parameter associated with a disease, disorder or condition. The terms "treat", "treating" and "treatment" can also refer to causing regression of a disease, disorder or condition, preventing its progression, or at least slowing its progression. In certain instances, "treat", "treating" and "treatment" refer to preventing the recurrence of a disease, disorder or condition. In other instances, "treat", "treating" and "treatment" refer to improving the survival rate of a subject having a disease, disorder or condition. In certain instances, "treat", "treating" and "treatment" refer to the elimination of a disease, disorder or condition in a subject.

[0032] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent that elicits a desired biological or medical response in a tissue system, animal or human, including improvement in one or more biomarkers of the disease, disorder or condition being treated or a reduction in the severity of one or more symptoms of the disease, disorder or condition being treated, e.g., a delay in the development and metastasis of tumors in a patient.

[0033] The term "agonist" as used herein refers to a compound that binds to a receptor and then enhances, enhances, sensitizes or upregulates the receptor.

[0034] The term "antagonist" as used herein refers to a compound that binds to a receptor and blocks or attenuates the receptor response to an agonist.

[0035] The term "pharmaceutically acceptable salt" refers to salts of compounds that are known to be non-toxic and are commonly used in the pharmaceutical arts. In some cases, pharmaceutically acceptable salts of compounds are those that retain their biological effectiveness and are not biologically or otherwise undesirable.

[0036] As used herein, "pharmaceutical acceptable excipient" refers to a substance that is non-toxic, biologically tolerable and otherwise biologically suitable for administration to a subject, e.g., an inert substance added to a pharmacological composition or used as a vehicle, carrier, or diluent to facilitate administration of an active compound or drug, and a substance that is compatible with an active compound or drug.

[0037] Toll-like receptor agonists Toll-like receptors are pattern recognition receptors that can detect microbial pathogen-associated molecular patterns (PAMPs). TLR stimulation, such as TLR9 stimulation, can not only result in a broad range of innate immune stimulation, but also specifically address dominant drivers of immune suppression in the liver. TLR1-10 are expressed in humans and recognize a variety of microbial PAMPs. In this regard, TLR9 can respond to unmethylated CpG-DNA, including microbial DNA. CpG refers to cytosine and guanine dinucleotide motifs. 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. Furthermore, TLR9 and the associated endosomal protein TLR7 are expressed in human liver metastatic tissues. TLR9 is also expressed in non-immune cells, including keratinocytes and intestinal, cervical, and respiratory epithelial cells. TLR9 can bind to its agonists in endosomes. Signaling can occur through MYD88 / IkB / NfκB to induce proinflammatory cytokine gene expression. A parallel signaling pathway through IRF7 induces type 1 and type 2 interferons (e.g., IFN-α, IFN-γ, etc.) that stimulate adaptive immune responses. Furthermore, TLR9 agonists can induce cytokine and IFN production, as well as functional maturation of antigen-presenting dendritic cells.

[0038] According to one example, TLR9 agonists can reduce and reprogram MDSCs. In this regard, reducing, altering or eliminating MDSCs is likely to improve the host's immune system's ability to attack cancer, as well as the ability of immunotherapy to induce more beneficial therapeutic responses. In one example, TLR9 agonists can convert MDSCs into immunostimulatory M1 macrophages, convert immature dendritic cells into mature dendritic cells, and expand effector T cells to create a responsive tumor microenvironment that promotes antitumor activity.

[0039] According to one example, synthetic CpG-oligonucleotides (CPG-ONs) that mimic the immune stimulatory properties of microbial CpG-DNA can be developed for therapeutic applications. According to one example, the oligonucleotide is an oligodeoxynucleotide (ODN). There are several different CpG-ODN class types, 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 type CPG-ODNs (or CPG-A ODNs) are associated with pDC maturation in the context of having little effect on B cells and maximal IFN-α induction; class B type CPG-ODNs (or CPG-B ODNs) strongly induce B cell proliferation and activate pDC and monocyte maturation, NK cell activation, and inflammatory cytokine production; class C type CPG-ODNs (or CPG-C ODNs) can induce B cell proliferation and IFN-α production.

[0040] Further, according to one example, a CPG-C ODN can be associated with the following attributes: (i) an unmethylated dinucleotide CpG motif, (ii) a CpG motif juxtaposed with adjacent nucleotides (e.g., AACGTTCGAA), (iii) a perfect phosphorothioate (PS) backbone linking the nucleotides (as opposed to the natural phosphodiester (PO) backbone found in bacterial DNA), and (iv) a self-complementary palindromic sequence (e.g., AACGTT). In this regard, CPG-C ODNs can bind to themselves due to their palindromic nature, thereby creating double-stranded duplexes (e.g., dimers) or hairpin structures.

[0041] Further, according to one example, the CPG-C ODN can include one or more 5'-TCG trinucleotides, in which the 5'-T is located 0, 1, 2, or 3 bases from the 5' end of the oligonucleotide, and at least one palindromic sequence of at least 8 bases in length, including one or more unmethylated CG dinucleotides. The one or more 5'-TCG trinucleotide sequences may be separated from the 5' end of the palindromic sequence by 0, 1, or 2 bases, or the palindromic sequence may contain all or part of the one or more 5'-TCG trinucleotide sequences. In one example, the CpG-C ODN is 12-100 bases long, preferably 12-50 bases long, preferably 12-40 bases long, or preferably 12-30 bases long. In one example, the CpG-C ODN is 30 bases long. In one example, the ODN is at least (lower limit) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80, or 90 bases in length. In one example, the ODN is at most (upper limit) 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 bases in length.

[0042] In one example, the at least one palindromic sequence is 8-97 bases long, preferably 8-50 bases long, or preferably 8-32 bases long. In a particular example, the at least one palindromic sequence is 8 bases long. In one example, the at least one palindromic sequence is at least (lower limit) 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 bases long. In one example, the at least one palindromic sequence is at most (upper limit) 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, or 10 bases long.

[0043] In one example, the CpG-C ODN can comprise the sequence of SEQ ID NO:1, as set forth below.

[0044] According to one example, the CpG-C ODN can include SD-101 or a pharma- ceutically acceptable salt thereof. SD-101 is a 30-mer phosphorothioate oligodeoxynucleotide having the following sequence: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1) having Here, the entire 30-mer sequence is linked by phosphorothioate bonds. SD-101 may be in the form of a free acid. In particular, SD-101 drug substance is isolated as a sodium salt. The structure of the sodium salt of SD-101 is illustrated in FIG. 1.

[0045] The molecular formula of SD-101 free acid is C 293 H 369 N 112 O 149 P 29 S 29 The molecular weight of SD-101 in the free acid form is 9672 Daltons. The molecular formula of the sodium salt of SD-101 is C, as shown in FIG. 293 H 340 N 112 O 149 P 29 S 29 Na 29 and the molecular weight of the sodium salt of SD-101 is 10,309 daltons.

[0046] Further, according to one example, the CPG-C ODN sequence can correspond to SEQ ID NO: 172 as set forth in US Pat. No. 9,422,564, the entirety of which is incorporated herein by reference.

[0047] In one example, a CpG-C ODN can include a sequence having at least 75% homology to any of the foregoing, such as SEQ ID NO:1.

[0048] According to another example, the CPG-C ODN sequence can correspond to any one of the other sequences described in U.S. Patent No. 9,422,564. Additionally, the CPG-C ODN sequence can also correspond to any of the sequences described in U.S. Patent No. 8,372,413, which is also incorporated by reference in its entirety.

[0049] According to one example, any of the CPG-C ODNs discussed herein can be present in their pharma- ceutically acceptable salt form. Suitable pharma- ceutically acceptable salts of any of the CPG-C ODNs can include organic and inorganic bases. Exemplary base salts include alkali metal salts such as ammonium salts, sodium salts, lithium salts, and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, zinc salts, salts with organic bases (e.g., organic amines) such as N-Me-D-glucamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, t-butylamine, and salts with amino acids such as arginine and lysine. In one example, the CpG-C ODN is in the form of an ammonium salt, a sodium salt, a lithium salt, or a potassium salt. In a preferred example, the CpG-C ODN is in the form of a sodium salt. The CpG-C ODN may be provided in a solution containing one or more pharma- ceutically acceptable excipients. Alternatively, the CpG-C ODN may be provided as a lyophilized solid, which is then reconstituted in sterile water, saline, or a pharma- ceutically acceptable buffer prior to administration.

[0050] The pharma- ceutically acceptable excipients of the present disclosure include, for example, solvents, bulking agents, buffers, tonicity adjusters, and preservatives.In one example, the pharmaceutical composition can include an excipient that functions as one or more of a solvent, bulking agent, buffer, and tonicity adjuster (e.g., sodium chloride in saline can act as both an aqueous vehicle and an isotonicity adjuster).The pharmaceutical composition of the present disclosure is suitable for parenteral and / or transdermal administration.

[0051] In one example, the pharmaceutical composition comprises an aqueous vehicle as a solvent. Suitable vehicles include, by way of example, sterile water, physiological saline, phosphate-buffered saline, and Ringer's solution. In one example, the composition is isotonic.

[0052] The pharmaceutical composition may include a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized prior to administration. In one example, the bulking agent is a protective agent that helps stabilize and prevent the active agent from decomposing during the lyophilization or spray drying process and / or during storage. Suitable bulking agents are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0053] The pharmaceutical composition may include a buffering agent. The buffering agent controls the pH and inhibits degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffers include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include, for example, amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further include hydrochloric acid or sodium hydroxide. In some examples, the buffering agent maintains the pH of the composition within the range of 4 to 9. In one example, the pH is greater than (lower limit) 4, 5, 6, 7, or 8. In some examples, 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, with the lower limit being less than the upper limit.

[0054] The pharmaceutical composition may include an isotonicity adjusting agent. Suitable isotonicity adjusting agents include, by way of example, dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0055] The pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antibacterial agents. However, in one example, the pharmaceutical composition is prepared under sterile conditions and is in a single-use container, and therefore does not require the inclusion of a preservative.

[0056] Table 1 lists an exemplary formulation of SD-101 drug - 16 g / L:

[0057] [Table 1]

[0058] In some examples, the unit dose strength can include from about 0.1 mg / mL to about 20 mg / mL. In one example, the unit dose strength of SD-101 is 13.4 mg / mL.

[0059] CpG-C ODNs can contain modifications. Suitable modifications can include, but are not limited to, modifications of the 3'OH or 5'OH groups, modifications of the nucleotide base, modifications of the sugar moiety, and modifications of the phosphate group. Modified bases can be included in palindromic sequences (e.g., the palindromic portion of the CpG-C ODN remains self-complementary) so long as the modified base maintains the same specificity to its natural complement through Watson-Crick base pairing. Examples of modifications of the 5'OH group can include biotin, cyanine 5.5, cyanine family of dyes, Alexa Fluor 660, Alexa Fluor family of dyes, IRDye 700, IRDye 800, IRDye 800CW, and IRDye family of dyes.

[0060] CpG-C ODNs may be linear, circular or may contain a circular portion and / or a hairpin loop. CpG-C ODNs may be single-stranded or double-stranded. CpG-C ODNs may be DNA, RNA or a DNA / RNA hybrid.

[0061] CpG-C ODN may contain naturally occurring or modified non-naturally occurring bases, and may contain modified sugars, phosphates and / or termini.For example, in addition to phosphodiester linkages, phosphate modifications include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters and phosphorodithioates, although phosphate modifications can be used in any combination.In one example, CpG-C ODN has only phosphorothioate linkages, only phosphodiester linkages, or a combination of phosphodiester linkages and phosphorothioate linkages.

[0062] 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 and others described herein, can also be made and combined with any phosphate modification. Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and / or C-6 of cytosines (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) of CpG-C ODNs, and electron-withdrawing moieties to C-5 and / or C-6 of uracils (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil) of CpG-C ODNs. As mentioned above, the use of base modifications in palindromic sequences of CpG-C ODNs must not interfere with the self-complementarity of bases involved in Watson-Crick base pairing. However, outside of palindromic sequences, modified bases can be used without these restrictions. For example, 2'-O-methyl-uridine and 2'-O-methyl-cytidine can be used outside of palindromic sequences, while 5-bromo-2'-deoxycytidine can be used both inside and outside of palindromic sequences. Other modified nucleotides that can be used both inside and outside of palindromic sequences include 7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0063] The duplex (i.e., double-stranded) and hairpin forms of most ODNs are often in dynamic equilibrium, but the hairpin form is generally favored at low oligonucleotide concentrations and high temperatures. Covalent interstrand or intrastrand crosslinks enhance the stability of duplexes or hairpins against thermally, ionically, pH- and concentration-induced conformational changes, respectively. Chemical crosslinks can be used to lock polynucleotides into either duplex or hairpin forms for physicochemical and biological characterization. Crosslinked ODNs that are structurally homogeneous and "locked" in their most active form (either duplex or hairpin) can potentially be more active than their non-crosslinked counterparts. Thus, some CpG-C ODNs of the present disclosure may contain covalent interstrand and / or intrastrand crosslinks.

[0064] The techniques for making polynucleotides and modified polynucleotides are known in the art.Naturally occurring DNA or RNA containing phosphodiester linkage can generally be synthesized by sequentially coupling suitable nucleoside phosphoramidites to the 5' hydroxyl group of the growing ODN attached to solid support at the 3' end, followed by oxidation of intermediate phosphite triester to phosphate triester.Using this method, once the desired polynucleotide sequence is synthesized, the polynucleotide is removed from support, and the phosphate triester group is deprotected to phosphate diester, and the nucleoside base is deprotected using aqueous ammonia or other base.

[0065] CpG-C ODN can contain phosphate-modified oligonucleotides, some of which are known to stabilize ODN.Thus, some examples include stabilized CpG-C ODN.The phosphorus derivative (or modified phosphate group) that can be attached to the sugar moiety or sugar analog moiety in ODN can be monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc.

[0066] CpG-C ODNs can contain one or more ribonucleotides (containing ribose as the sole or main sugar moiety), deoxyribonucleotides (containing deoxyribose as the main sugar moiety), modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and sugar analog cyclopentyl groups. The sugars can be in pyranosyl or furanosyl form. In CpG-C oligonucleotides, the sugar moiety is preferably a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugars can be attached to the respective heterocyclic bases in either anomeric configuration. The preparation of these sugars or sugar analogs, and the preparation of the respective nucleosides in which such sugars or analogs are attached to the heterocyclic bases (nucleobases) themselves, are known and therefore need not be described herein. Sugar modifications may also be made in preparing a CpG-C ODN and combined with any phosphate modifications.

[0067] The heterocyclic or nucleobases incorporated into the CpG-C ODNs can be the naturally occurring primary purine and pyrimidine bases (i.e., uracil, thymine, cytosine, adenine, and guanine, as described above), as well as naturally occurring and synthetic modifications of said primary bases. Thus, the CpG-C ODNs can include one or more of inosine, 2'-deoxyuridine, and 2-amino-2'-deoxyadenosine.

[0068] According to another example, the CPG-ODN is one of a class A type CPG-ODN (CPGP-A ODN), a class B type CPG-ODN (CPG-B ODN), a class P type CPG-ODN (CPG-P ODN), and a class S type CPG-ODN (CPG-S ODN). In this regard, the CPG-A ODN can be CMP-001.

[0069] In another example, the CPG-ODN can be tilsotolimod (IMO-2125).

[0070] Checkpoint inhibitors According to one example, the TLR agonist of the present application can be used in combination with a checkpoint inhibitor (CPI). The CPI can include a programmed death 1 receptor (PD-1) antagonist. A PD-1 antagonist can be any compound or biological molecule 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 immune cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. In any of the therapeutic methods, medicaments and uses of the present application in which a human individual is treated, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD-1.

[0071] According to one example, the PD-1 antagonist may include a monoclonal antibody (mAb), or an antigen-binding fragment thereof, that specifically binds to PD-1 or PD-L1, 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.

[0072] By way of example, the PD-1 antagonist may include an immunoadhesin that specifically binds to PD-1 or PD-L1, preferably that specifically binds to human PD-1 or human PD-L1, e.g., a fusion protein containing the extracellular portion 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.

[0073] According to one example, PD-1 antagonists can block PD-L1 expressed by tumor cells and MDSCs, as well as other suppressive immune cells.

[0074] According to one example, the 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 methods of treatment, medicaments and uses, the PD-1 antagonist is a monoclonal antibody, or an antigen-binding fragment thereof, 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.

[0075] According to one example, the PD-1 antagonist can be one of nivolumab, pembrolizumab, and cemiplimab. According to another example, nivolumab is administered intravenously (IV) via a peripheral vein at a dose of 480 mg every 4 weeks ("Q4W") or 240 mg every 2 weeks ("Q2W"). According to another example, nivolumab is administered intravenously (IV) via a peripheral vein at a dose of 360 mg nivolumab every 3 weeks ("Q3W"). In another example, nivolumab dosing is weight-based at nivolumab 3 mg / kg Q2W or 10 mg / kg Q2W. In another example, nivolumab dosing is weight-based at nivolumab 1 mg / kg Q3W. In yet another example, nivolumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101. In another example, nivolumab is administered once a week, once every other week, once every three weeks, once every four weeks, or once a month in conjunction with one or more cycles of SD-101 administration. A "cycle" of administration of SD-101 is further described below.

[0076] According to another example, pembrolizumab is administered intravenously (IV) via a peripheral vein at a dose of 200 mg Q3W or 400 mg every six weeks ("Q6W"). In another example, pembrolizumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101.

[0077] According to another example, the CPI can include a PD-L1 antagonist. In this regard, the PD-L1 antagonist can be one of atezolizumab, avelumab, and durvalumab.

[0078] According to another example, the CPI may include a CTLA-4 antagonist. In this regard, the CTLA-4 antagonist may be ipilimumab. According to another example, ipilimumab is administered intravenously (IV) via a peripheral vein at a dose of 3 mg / kg every 3 weeks. In yet another example, ipilimumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101 and / or nivolumab. In another example, ipilimumab is administered on a weekly, biweekly, 3-weekly, 4-weekly, or monthly basis following administration of one or more cycles of SD-101 and / or nivolumab.

[0079] Devices for localized delivery According to one example, any of the TLR agonists, alone or in combination with a CPI, can be administered using any device useful for localized delivery to a tumor, including the catheter itself, or any device that can include the catheter along with other components that can be used in combination with the catheter (e.g., filter valves, balloons, pressure sensor systems, pump systems, syringes, outer delivery catheters, implantable portions, etc.) In one particular example, the catheter is a microcatheter.

[0080] In some examples, the device may have one or more attributes, including, but not limited to, the following: a self-centering function that can provide uniform distribution of therapy in the downstream branching network of blood vessels; an anti-reflux capability that can block or inhibit backflow of the injectable pharmaceutical composition, e.g., the injectable pharmaceutical liquid, including the TLR agonist (e.g., by using a valve and filter and / or a balloon); a system for measuring pressure inside the blood vessel; and a means or mechanism for modulating pressure inside the blood vessel, such as by causing a pressure drop during deployment and during the TLR agonist infusion process and a pressure increase during the saline bolus or TLR agonist bolus infusion process (e.g., a one-way valve that dynamically responds to local pressure changes, an intermittently occluding valve, and / or a porous balloon). In some examples, the system is designed to continuously monitor real-time pressure or flow throughout the entire procedure. In one example, the mechanism for modulating pressure generates, causes, and / or contributes to a net increase in fluid pressure within the blood vessel and / or the target tissue or tumor. In one particular example, the mechanism for modulating pressure can increase local vascular pressure at the target location, and in particular, the increased pressure is greater than baseline arterial pressure. The mechanism for modulating pressure can operate in synchrony with the cardiac cycle and / or promote antegrade flow. In some examples, the mechanism for modulating pressure generates, causes, and / or contributes to a net decrease in fluid pressure within the blood vessel and / or target tissue or tumor. In some examples, the mechanism for modulating pressure generates, causes, and / or contributes to an initial decrease and then an increase in fluid pressure within the blood vessel and / or target tissue or tumor.

[0081] In some examples, devices that can be used to practice the methods of the present application are those disclosed in U.S. Patent No. 8,500,775, U.S. Patent No. 8,696,698, U.S. Patent No. 8,696,699, U.S. Patent No. 9,539,081, U.S. Patent No. 9,808,332, U.S. Patent No. 9,770,319, U.S. Patent No. 9,968,740, U.S. Patent No. 10,813,739, U.S. Patent No. 10,588,636, 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, all of which are incorporated herein by reference in their entireties.

[0082] In some examples, the device is a device disclosed in U.S. Patent No. 9,770,319. In one particular example, the device can be a device known as the Surefire Infusion System.

[0083] In some examples, the device assists in measuring intravascular pressure during use. In some examples, the device is a device disclosed in U.S. Patent Publication No. 2020-0383688. In one particular example, the device can be a device known as the TriSalus Infusion System. In one particular example, the device can be a device known as the TriNav® Infusion System. TriNav® is a single-lumen catheter with a one-way valve that dynamically responds to local pressure and flow changes, such as those resulting from the cardiac cycle or those generated by injection. The valve structure modulates distal vascular pressure and blood flow. This can then alter the distribution and first-pass absorption of therapeutic agents due to extended contact time in the vasculature.

[0084] In some cases, the TLR agonist can be administered through a device via PEDD. In some cases, the TLR agonist can be administered while monitoring the pressure in the blood vessel, which can be used to adjust and correct the position of the device at the injection site and / or adjust the injection rate. Pressure can be monitored, for example, by a pressure sensor system that includes one or more pressure sensors.

[0085] The infusion rate can be adjusted to change vascular pressure or flow, which can facilitate penetration and / or binding of the TLR agonist to or at the surface of the target tissue or tumor. In some cases, the infusion rate can be adjusted and / or controlled using a syringe pump as part of the delivery system or by any other method (e.g., an infusion flow rate regulating device). In some cases, the infusion rate can be adjusted and / or controlled using a pump system. In some cases, the infusion rate using a pump system can be about 0.1 cc / min to about 40 cc / min, or about 0.1 cc / min to about 30 cc / min, or about 0.5 cc / min to about 25 cc / min, or about 0.5 cc / min to about 20 cc / min, or about 1 cc / min to about 15 cc / min, or about 1 cc / min to about 10 cc / min, or about 1 cc / min to about 8 cc / min, or about 1 cc / min to about 5 cc / min. Additionally, the infusion rate using a bolus infusion can be from about 30 cc / min to about 360 cc / min, or from about 120 cc / min to about 240 cc / min. In one example, the SD-101 infusion procedure lasts for about 10 to about 200 minutes. In another example, the SD-101 infusion procedure lasts for about 10 to about 60 minutes. In another example, the SD-101 infusion procedure lasts for about 25 minutes.

[0086] Methods involving administration to the liver In one example, the method of the present application includes a method of treating liver immune dysfunction, the method comprising administering a Toll-like receptor (TLR) agonist to a patient in need thereof, wherein the TLR agonist is administered to the liver through a device by HAI. HAI refers to the infusion of a treatment into the hepatic artery or a branch of the hepatic artery of the liver. According to one embodiment, the TLR agonist or agonists are introduced through percutaneous introduction of a device into a branch of the hepatic artery or portal vein, such as a catheter and / or a device that facilitates pressure-enabled delivery. According to one example, the TLR agonist is a TLR9 agonist, more specifically a class C TLR9 agonist, and in some examples, the class C TLR9 agonist is SD-101. In one example, the patient is a human patient.

[0087] According to another example, the method of the present application includes a method of treating hepatic immune dysfunction and tumor-induced immune dysfunction, comprising administering a toll-like receptor agonist to a patient in need thereof, wherein the toll-like receptor agonist is administered to the liver through a device by PVI. PVI refers to the infusion of a treatment into the hepatic portal system. According to one example, the toll-like receptor agonist or agonists are introduced through percutaneous introduction of a device into a branch of the hepatic portal system, such as a catheter and / or a device that facilitates pressure-enabled delivery. According to one example, the TLR agonist is a TLR9 agonist, more specifically a class C TLR9 agonist, and in some examples, the class C TLR9 agonist is SD-101. In one example, the patient is a human patient. According to another example, the method includes administration to a subject who is male or female and 18 years of age or older.

[0088] According to another example, the methods of the present application can be administered in conjunction with other cancer therapeutic agents, such as immune modulators, tumor killing agents, and / or other targeted therapeutic agents.

[0089] According to one example, TLR9 agonist therapy, particularly therapy using a class C TLR9 agonist, may be administered in combination with cell therapy (which allows cell therapy through modulation of the immune system), in combination with chemoembolization or radioembolization.

[0090] In one example, the above method of administration to the liver is intended to provide penetration of the Toll-like receptor agonist, particularly a TLR9 agonist, more particularly a class C TLR9 agonist, even more particularly SD-101, throughout a solid tumor, throughout an organ, or throughout substantially the entire tumor. In one example, such a method improves perfusion of the Toll-like receptor agonist to a patient in need thereof, including by overcoming interstitial fluid pressure and solid stress in the tumor. In another example, perfusion throughout an entire organ or a portion thereof can provide the benefit of treating a disease by thoroughly exposing the tumor to the therapeutic agent. In one example, such a method can provide more complete delivery of the Toll-like receptor to areas of the tumor that have poor access to the systemic circulation. In another example, such a method provides higher concentrations of the Toll-like receptor agonist to tumors in situations where the Toll-like receptor agonist is poorly delivered to non-target tissues, as compared to conventional systemic delivery via peripheral veins. Non-target tissues are tissues that are directly perfused by an arterial network that is directly connected to the infusion device. In one example, such methods result in a reduction in size, a reduction in the rate of growth, or the shrinkage or elimination of a solid tumor.

[0091] The methods of the present application may also include mapping blood vessels leading to the right and left lobes of the liver prior to performing the HAI or selective injection into specific sectors or segments, and optionally occluding blood vessels not leading to the liver or otherwise not targeted. In some instances, prior to injection, the patient may undergo a mapping angiogram, for example, via a femoral artery approach.

[0092] Methods for mapping blood vessels in the body and for delivery of therapeutic agents are well known to those skilled in the art. Occlusion can be achieved, for example, through the use of microcoil embolization, which allows the practitioner to block off-target arteries or blood vessels, thereby optimizing delivery of modified cells to the liver. Microcoil embolization can be performed as needed, such as before administering the first dose of a TLR9 agonist, particularly a class C TLR9 agonist, more particularly SD-101, to facilitate optimal infusion of a pharmaceutical composition comprising a TLR9 agonist. In another example, a sterile sponge (e.g., GELFOAM) can be used. In this regard, the sterile sponge can be cut and pushed into the catheter. In another example, the sterile sponge can be provided as granules.

[0093] In some examples, the dose of the TLR9 agonist, particularly the class C TLR9 agonist such as SD-101, can be about 0.01 mg, about 0.03 mg, about 0.05 mg, about 0.1 mg, about 0.3 mg, about 0.5 mg, about 1 mg, 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. In some examples, SD-101 is administered in doses of 12 mg, 16 mg, and 20 mg. The administration of milligram amounts of SD-101 (e.g., about 2 mg) describes the administration of about 2 mg of the composition illustrated in FIG. 1. For example, such amounts of SD-101 (e.g., amounts of about 2 mg) may also be present in compositions that contain such amounts of SD-101 plus materials such as other related and unrelated compounds. Equivalent molar amounts of other pharma- ceutically acceptable salts are also contemplated.

[0094] In some examples, the dose of the TLR9 agonist, particularly a class C TLR9 agonist such as SD-101, can be between about 0.01 mg and about 20 mg, between about 0.01 mg and about 10 mg, between about 0.01 mg and about 8 mg, and between about 0.01 mg and about 4 mg. In some examples, the dose of the TLR9 agonist, particularly a class C TLR9 agonist such as SD-101, can be between about 2 mg and about 10 mg, between about 2 mg and about 8 mg, and between about 2 mg and about 4 mg. In some examples, the dose of the TLR9 agonist, particularly a class C TLR9 agonist such as SD-101, can be less than about 10 mg, less than about 8 mg, less than about 4 mg, or less than about 2 mg. Such doses can be administered daily, weekly, or biweekly. In one example, the dose of SD-101 is gradually increased, for example, through administration of about 2 mg, followed by about 4 mg, then followed by about 8 mg.

[0095] In some examples, the method of the present application can include administering a dosing regimen that includes cycles, where one or more of the cycles include administering SD-101 via HAI and / or PEDD. As used herein, a "cycle" is a repetition of a dosing procedure. In one example, a cycle includes three weekly doses per cycle (i.e., administration of SD-101 once per week for three consecutive weeks). In one example, a cycle of treatment according to the present application can include a period of administration of SD-101, followed by an "off" period or rest period. In another example, in addition to three weekly doses per cycle, the cycle further includes a 1-week, 2-week, 3-week, or 4-week rest period after the weekly administration of SD-101. In yet another example, in addition to three weekly doses per cycle, the cycle further includes a rest period of about 38 days after the weekly administration of SD-101. In another example, the entire cycle includes about 52 days. In another example, the dosing regimen comprises at least 1 cycle, at least 2 cycles, or at least 3 cycles, or longer.

[0096] In some examples, the present application relates to the use of a TLR9 agonist, particularly a class C TLR9 agonist, in the manufacture of a medicament for treating hepatic immune dysfunction and tumor-induced immune dysfunction, the method comprising administering a TLR9 agonist, more particularly a class C TLR9 agonist, to a patient in need thereof, wherein the TLR9 agonist, more particularly a class C TLR9 agonist, is administered to the liver through a device by HAI.

[0097] In some examples, SD-101 is administered at a dose of 2 mg through a HAI for the treatment of liver immune dysfunction, and in some examples, SD-101 is further administered through a pressure modulating device (i.e., PEDD). In some examples, SD-101 is administered at a dose of 2 mg through a HAI and through a pressure modulating device in combination with a CPI, where the CPI is nivolumab. In other examples, SD-101 is administered at a dose of 2 mg through a HAI and through a pressure modulating device in combination with ipilimumab. In some examples, SD-101 is administered at a dose of 2 mg through a HAI and through a pressure modulating device in combination with ipilimumab and nivolumab. In other examples, SD-101 is administered at a dose of 2 mg through a HAI and through a pressure modulating device in combination with pembrolizumab.

[0098] In some examples, SD-101 is administered at a dose of 4 mg through an HAI for the treatment of liver immune dysfunction, and in some examples, SD-101 is further administered through a pressure modulating device (e.g., dynamically responding to local pressure changes, more specifically, a PEDD). In some examples, SD-101 is administered at a dose of 4 mg through an HAI, in combination with a CPI, through a vascular pressure modulating device (e.g., dynamically responding to local pressure changes, more specifically, a PEDD). The CPI may be administered systemically or locally. For example, the CPI may be administered intravenously (IV), subcutaneously (SQ), intraperitoneally (IP), or through an HAI, such as, for example, through a pressure modulating device (e.g., dynamically responding to local pressure changes, more specifically, a PEDD).

[0099] In some examples, SD-101 is administered at a dose of 4 mg through an HAI, through a device that modulates vascular pressure (e.g., dynamically responds to changes in local pressure, more specifically, PEDD), in combination with a CPI administered systemically (e.g., IV or SQ) or locally (e.g., through an HAI, such as via IP or PEDD), where the CPI is nivolumab. In other examples, SD-101 is administered at a dose of 4 mg through an HAI and through a device that modulates pressure (e.g., dynamically responds to changes in local pressure, more specifically, PEDD), in combination with ipilimumab administered systemically (e.g., IV or SQ) or locally (e.g., through an HAI, such as via IP or PEDD). In some examples, SD-101 is administered at a dose of 4 mg in combination with ipilimumab and nivolumab administered systemically (e.g., IV or SQ) or locally (e.g., through the HAI, such as via IP or PEDD) through a HAI and a pressure modulating device (e.g., dynamically responds to changes in local pressure, more specifically, PEDD). In other examples, SD-101 is administered at a dose of 4 mg in combination with pembrolizumab administered systemically (e.g., IV or SQ) or locally (e.g., through the HAI, such as via IP or PEDD) through a HAI and a pressure modulating device (e.g., dynamically responds to changes in local pressure, more specifically, PEDD).

[0100] In some examples, SD-101 is administered at a dose of 8 mg through an HAI for the treatment of liver immune dysfunction, and in some embodiments, SD-101 is further administered through a pressure modulating device (e.g., dynamically responding to local pressure changes, more specifically, a PEDD). In some examples, SD-101 is administered through an HAI in combination with a CPI administered systemically (e.g., IV or SQ) or locally (e.g., through an HAI, such as via IP or PEDD) through a device that modulates vascular pressure (e.g., dynamically responding to local pressure changes, more specifically, a PEDD), where the CPI is nivolumab, at a dose of 8 mg. In other examples, SD-101 is administered at a dose of 8 mg in combination with ipilimumab administered systemically (e.g., IV or SQ) or locally (e.g., through the HAI, such as via IP or PEDD) through a HAI and through a pressure modulating device (e.g., dynamically responds to changes in local pressure, more specifically, PEDD). In some examples, SD-101 is administered at a dose of 8 mg in combination with ipilimumab and nivolumab administered systemically (e.g., IV or SQ) or locally (e.g., through the HAI, such as via IP or PEDD) through a HAI and through a pressure modulating device (e.g., dynamically responds to changes in local pressure, more specifically, PEDD). In other examples, SD-101 is administered at a dose of 8 mg in combination with pembrolizumab administered systemically (e.g., IV or SQ) or locally (e.g., through the HAI, such as IP or via PEDD) through an HAI and through a pressure modulating device (e.g., a PEDD that dynamically responds to changes in local pressure).

[0101] In some examples, the method of the present application results in the treatment of the target lesion, e.g., the target liver cancer lesion or tumor. In this example, the method of the present application can result in a complete response, including the total disappearance of the target lesion, e.g., the target liver cancer lesion or tumor. In some examples, the method of the present application can result in a partial response, including at least a 30% decrease in the sum of the longest diameters of the target lesion, e.g., the target liver cancer lesion or tumor, based on the sum of the longest diameters of the baseline. In some examples, the method of the present application can result in the stabilization of the target lesion, e.g., the target liver cancer lesion or tumor, based on the smallest sum of the longest diameters since the start of treatment, without either a sufficient shrinkage to qualify as a partial response or a sufficient worsening to qualify as progression. In such examples, progression is characterized by at least a 20% worsening of the sum of the longest diameters of the target lesion, e.g., the target liver cancer lesion or tumor, based on the smallest sum of the longest diameters recorded since the start of treatment or the appearance of one or more new lesions. This sum must demonstrate an absolute worsening of 5 mm or about 5 mm. In some cases, the response in the form of a reduction in size may be offset by tumor killing and inflammation that leads to tumor swelling (known as pseudoprogression).

[0102] In another example, the method of the present application results in the treatment of non-target lesions. Non-target lesions are lesions that are not directly perfused by an arterial network that is in direct communication with the infusion system. In this example, the method of the present application can result in a complete response, including disappearance of all non-target lesions or elimination of all viable tumors. In some examples, the method of the present application results in the persistence of one or more non-target lesions while not resulting in a complete response or progression. In such examples, progression is characterized by a clear progression of existing non-target lesions and / or the appearance of one or more new lesions.

[0103] In some cases, the methods of the present application result in an extended duration of overall response. In some cases, the duration of overall response is measured from the time that the measurement criteria are met for a complete response or partial response (whichever is first recorded) to the first date of relapse or progression, e.g., the first date that relapse or progression is objectively documented (using the minimum measurement recorded since the start of treatment as the progression criterion). The duration of overall complete response can be measured from the time that the measurement criteria are first met for a complete response to the first date of relapse or progression, e.g., the first date that progression is objectively documented. In some cases, the duration of stable disease is measured from the start of treatment until progression is observed, e.g., progression criteria are met using the minimum measurement recorded since the start of treatment, including the baseline measurement.

[0104] In yet other instances, the methods of the present application result in improved overall survival. For example, overall survival can be calculated from the date of treatment initiation to the time of death. In certain instances, in a clinical trial of a treatment, it can be calculated from the date of enrollment to the time of death. Patients who are still alive before the data cutoff for the final efficacy analysis of the clinical trial study or who drop out before the end of the study are censored at the date they were last known to be alive.

[0105] In other examples, the methods of the present application provide progression-free survival. Progression-free survival can be determined from the date of treatment initiation to recurrence or death, whichever occurs first. In particular examples, for example, progression-free survival can be calculated from the date of enrollment in a treatment trial to the time a CT scan documents recurrence (or other unambiguous indicator of disease progression) or the date of death, whichever occurs first. Patients who do not have a documented recurrence and are still alive before the data cutoff of the final efficacy analysis or who drop out before the end of the study are censored at the date of the last radiological evidence documenting the absence of recurrence.

[0106] According to another example, the method of the present application includes a method for treating liver immune dysfunction. Liver immune dysfunction refers to immune dysfunction in the liver tumor microenvironment (TME), which reduces the ability of the patient's immune system to attack cancer cells and / or the ability of immunotherapy to induce a beneficial therapeutic response. Liver immune dysfunction can be characterized by several different biological features, such as, for example, increased levels of MDSCs present in the liver TME, decreased levels of T cells and / or macrophages (e.g., M1 macrophages and / or M2 macrophages) present in the liver TME, decreased levels of mature dendritic cells, reduced immune control of liver cancer, and / or reduced responsiveness to anti-PD-1 therapy. Other biological features of liver immune dysfunction include: activation of immune cells in normal liver (non-tumor liver of the same patient) with migration to liver metastases; activation and recruitment of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages); enhanced TLR signaling; leukocytes (CD45 + ); increase in exhausted CD8 T cells (critical for checkpoint responsiveness); induction of Th1 programming; reduction in Th2 programming; increase in T cell receptors and improved T cell costimulatory signaling; increase in IL9, IL15, CCL7; activation of B cells; induction of mast cells; induction of NK cells, induction of IFNg and related genes; improved interferon signaling; improved chemokine signaling; increase in IL6 in the liver without increase in the blood; increase in M1 / activation of macrophages and related genes: CD68, CD86 dendritic cell activation and migration to draining lymph nodes; decrease in MDSC-related genes: IDO1, T0D2, ARG-1, NOS2, TIMD4; decrease in angiogenesis and VEGF; decrease in fatty acid oxidation (preferred MDSC program); and / or increase in PD-L1, IFNγ, IP10 gene expression.

[0107] In one example, the method of the present application includes a method for treating liver immune dysfunction, wherein administration of SD-101 results in a reduction in tumor burden. Tumor burden can be determined using any suitable method, such as the length of the longest diameter of a tumor lesion or the volume of a tumor lesion. In some examples, tumor burden is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In one example, tumor burden is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as determined by the length of the longest diameter of a tumor lesion. In another example, the tumor is reduced by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, or by about 100%, as determined by the volume of the tumor lesion.

[0108] According to another example, the method of the present application includes a method for treating liver immune dysfunction, wherein administration of SD-101 results in a reduction in tumor progression or stabilization of tumor growth. In some examples, tumor progression is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. Tumor burden can be determined using any suitable method, such as the length of the longest diameter of tumor lesions or the volume of tumor lesions. In one example, tumor progression is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as determined by the length of the longest diameter of tumor lesions. In another example, tumor progression is reduced by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, or by about 100%, as determined by the length of the longest diameter of the tumor lesion and by the volume of the tumor lesion.

[0109] According to another example, the method of the present application includes a method for treating liver immune dysfunction, in which administration of SD-101 reduces MDSCs in the liver TME, allowing for improved immune control of liver cancer and / or improving responsiveness to systemic anti-PD-1 therapy. For example, a method for treating liver immune dysfunction includes administration of SD-101, which reprograms the liver MDSC compartment to allow for improved immune control of liver cancer and / or improving responsiveness to systemic anti-PD-1 therapy through the elimination of MDSCs from the liver TME. In some examples, the method of the present application is superior in controlling the amount of MDSCs in the liver TME. In some examples, the method of the present application includes a method for treating liver immune dysfunction, in which administration of SD-101 reduces MDSCs in the liver TME, allowing for improved immune control of liver cancer and / or improving responsiveness to systemic anti-PD-1 therapy through the elimination of MDSCs from the liver TME. + Grl + ), monocytic MDSCs (M-MDSCs, e.g., CD11b + Ly6C + ) cells, granulocytic MDSC (G-MDSC, e.g. CD11b + LY6G + ) cells, and / or human MDSCs (e.g., CD33 + CD11b + HLADR - (e.g., CD14 for m-MDSCs + , and CD15 for G-MDSCs. + In another example, the method of the present application reduces the frequency of M1 macrophages (e.g., CD14 + CD86 + In yet another example, the methods of the present application increase the amount of M2 macrophages (e.g., CD14 + CD163 + CD206 + According to yet another example, the methods of the present application reduce monocytic dendritic cells (e.g., CD14 + CD11c + CD123 -According to one example, the effects of MDSC, M1, M2 and dendritic cells described in this paragraph can be achieved by, but not limited to, human M-MDSC: (e.g., CD33 + CD11b + HLA-DR - CD14 + ); Human G-MDSC: (e.g., CD33 + CD11b + HLA-DR - CD15 + ); Human M1 macrophages: (e.g., CD14 + CD86 + ); Human M2 macrophages: (e.g., CD14 + CD163 + CD206 + ); and human monocytic dendritic cells (e.g., CD14 + CD11c + CD123 - ) and other human phenotypes.

[0110] In another example, the methods of the present application increase other molecules associated with NFκB activation and TLR9 signaling, for example in the liver TME. In yet a further example, the methods of the present application increase IL-6, for example in the liver TME. In another example, the methods of the present application increase IL10, for example in the liver TME. In yet a further example, the methods of the present application increase IL-29, for example in the liver TME. In another embodiment, the methods of the present application increase IFNα, for example in the liver TME. As a further embodiment, the methods of the present application decrease STAT3 phosphorylation, for example in the liver TME.

[0111] In another example, the methods of the present application include a method for treating liver immune dysfunction, where administration of a TLR9 agonist, particularly a class C TLR9 agonist, more particularly SD-101, results in at least one of the following changes in gene expression in liver metastases: · Activation of immune cells in normal liver (non-tumorous liver of the same patient), with migration to liver metastases; Activation and mobilization of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages); Enhanced TLR signaling; white blood cells (CD45 + ) increase; · Increase in exhausted CD8 T cells (critical for checkpoint responsiveness); · Induction of Th1 programming; ·Reduced Th2 programming; ·Increased T cell receptors and improved T cell costimulatory signaling; Increased IL9, IL15, CCL7; · B cell activation; ·Mast cell induction; Induction of NK cells; Induction of IFNg and related genes; ·Improved interferon signaling; ·Improved chemokine signaling; Increased IL6 in the liver without an increase in blood; Increase in M1 / macrophage gene activation: CD68, CD86 dendritic cell activation and migration to draining lymph nodes; · Decreased MDSC-associated genes: IDO1, T0D2, ARG-1, NOS2, TIMD4; ·Reduction of angiogenesis and VEGF; Reduced fatty acid oxidation (a prioritized MDSC program); and / or Increased expression of PD-L1, IFNγ and IP10 genes.

[0112] In another embodiment, the methods of the present application include methods for administering a TLR9 agonist, particularly a class C TLR9 agonist, especially SD-101, in combination with a checkpoint inhibitor to a patient, where administration of the TLR9 agonist, particularly a class C TLR9 agonist, more particularly SD-101, in combination with a CPI results in a reduction in circulating tumor cell levels and / or circulating tumor DNA levels, thereby resulting in a reduction in tumor burden. In one example, the CPI can be administered systemically (e.g., IV or SQ) or locally (e.g., through a HAI, such as via IP or PEDD).

[0113] The present application is further illustrated and / or demonstrated in the following examples, which are provided for illustrative / demonstrative purposes only and are not intended to limit the invention in any way. EXAMPLES

[0114] Example 1 In Example 1, peripheral blood mononuclear cells (PBMCs) obtained from human donors were cultured in vitro in the presence or absence of IL6 and GMCSF. The presence of IL6 and GMCSF is used to induce MDSCs. The treated cells were then evaluated using flow cytometry to determine the effect of various TLR agonists on MDSCs (M-MDSCs and G-MDSCs) and macrophages (M1 macrophages and M2 macrophages). Data generated by flow cytometry are shown in Figures 2A-2D.

[0115] 2A-2E illustrate in vitro analysis of human PMBCs collected from healthy donors and treated with IL6 and GM-CSF to induce MDSCs according to Example 1.

[0116] In particular, FIG. 2A shows the amount of total MDSC induced in in vitro samples (i.e., NT (untreated) or treated with TLR4 agonist, TLR7 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist, or SD101). The data in FIG. 2A demonstrates the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist, and SD-101, compared to untreated controls, on the amount of total MDSC induced by treatment of human PBMC with IL6 and GMCSF. In FIG. 2A, * indicates p<0.05 compared to NT, *** indicates p<0.01 compared to NT, # indicates p<0.05 compared to SD-101, and & indicates p=0.006 compared to SD-101. Figure 2B demonstrates the effect of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 compared to untreated controls on the amount of monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs) induced by treatment of human PBMCs with IL6 and GMCSF. In Figure 2B, *** indicates p<0.001 compared to NT, **** indicates p<0.0001 compared to NT, % indicates p<0.01 compared to SD-101, and ^ indicates p<0.001 compared to SD-101. As can be seen in Figures 2A and 2B, SD-101 demonstrated (i) better resolution of the predominant MDSC subsets in the liver, e.g., M-MDSC, and (ii) a more favorable effect on the M1 / M2 macrophage ratio (i.e., induction of M1 and reduction of M2). Figures 2C and 2D demonstrate the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist, and SD-101, compared to untreated controls, on the amount of M1 and M2 macrophages, respectively, induced by treatment of human PBMC with IL6 and GMCSF. In Figure 2C, * indicates p<0.05 compared to NT, ** indicates p<0.01 compared to NT, # indicates p<0.05 compared to SD-101, and @ indicates p<0.00001 compared to SD-101. In Figure 2D, * indicates p<0.05 compared to NT.For each of the data sets shown in Figures 2A-2D, the number of different human donors is n=5.

[0117] FIG. 2E shows images of MDSC cells induced from PMBCs when the cells were treated with a TLR9 agonist.

[0118] In view of the data shown in Figures 2A-2E, these in vitro studies of human PMBCs treated with IL6 and GM-CSF to induce MDSCs demonstrate that SD-101 is superior to class B TLR9 and TLR7 agonists in reducing MDSCs, reducing M2 macrophages, and promoting M1 macrophages.

[0119] Example 2 In Example 2, mouse bone marrow cells from mice with or without tumors were cultured in vitro in the presence or absence of GMCSF. The presence of GMCSF is used to induce MDSCs. The treated cells were then evaluated using flow cytometry to determine the effect of various TLR agonists on total MDSCs, total macrophages, and M1 macrophages in this mouse model. Data generated by flow cytometry are shown in Figures 3A-3C.

[0120] FIG. 3A demonstrates that treatment of mouse bone marrow cells with GMCSF induced MDSCs (CD11b + Gr-1 + 3A and 3B show the effects of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 on the amount of macrophages (CD11b ) induced by treatment of mouse bone marrow cells with GMCSF. In Fig. 3A, ** indicates p<0.01 compared to NT and **** indicates p<0.0001 compared to NT. Fig. 3B demonstrates that the amount of macrophages (CD11b ) induced by treatment of mouse bone marrow cells with GMCSF was significantly increased by 100%. + F4 / CD80 +3B, the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 compared to untreated control on the amount of M1 macrophages (CD11b + F4 / CD80 + CD38 + 3C, the effect of TLR4 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist and SD-101 compared to untreated control on the amount of IgG1 (IgG1-IgG2) in the IL-16 / IL-2+ ...

[0121] As can be seen from the data in Figures 3A-C, in vitro studies of mouse bone marrow cells demonstrate that SD-101 is similarly superior to class B TLR9 and TLR7 agonists for programming mouse bone marrow cells. In particular, Figures 3A-C illustrate in vitro analysis of mouse bone marrow cells from mice with and without tumors, where GM-CSF was used to induce MDSCs. In this regard, SD-101 was superior to class A TLR9, class B TLR9, and TLR7 agonists in reducing MDSCs and M2 macrophages, in addition to inducing M1 macrophages.

[0122] Example 3 In Example 3, the class C TLR9 agonist SD-101 was administered via hepatic artery infusion (PEDD / HAI) to improve response rates to CPI therapy in patients with uveal melanoma liver metastases. Four patients were enrolled and treated with 2 mg of SD-101 per each of three weekly SD-101 infusions via HAI and PEDD in the first cycle. Pharmacokinetic (PK) data collected in this study suggest that the PEDD / HAI delivery approach achieved its goal of achieving high concentrations of SD-101 in liver tissue and transient and low levels (<2 hours, <150 ng / ml) of systemic exposure to SD-101, as determined based on plasma levels. Cell-mediated antitumor immunity (Th1, TCR / costimulatory, CD8 T cells, cytotoxic cells, B cells, NK cells) was improved in uveal melanoma liver metastases. Concomitant declines in normal liver tissue suggested migration to adjacent tumors, demonstrating the importance of treating the entire immunosuppressed organ. CD8 T, Th1, B, and NK were increased in peripheral blood at later time points along with serum IFNγ induction. With regard to cytokine programming, IFNγ-related genes were increased in liver metastases and serum, and Th1 genes were increased in liver metastases with a decline in Th2. MDSC-related genes were decreased (NOS-2, ARG-1, IDO-1, TDO-2). Changes in the liver were seen 4 weeks after three injections of 2 mg SD-101 via PEDD.

[0123] Post-treatment liver metastasis samples from these patients (collected 4 weeks after the first cycle of 3 weekly injections of SD-101 via HAI and PEDD) were tested with the NanoString® Immune Cell Panel to profile the expression of gene signatures for immune cells, including cytotoxic cells, dendritic cells (DCs), CD45, macrophages, T cells, mast cells, neutrophils, exhausted CD8, Th1 cells, and CD8 T cells. Data from the NanoString® Immune Cell Panel is shown in FIG. 4, where BL-T refers to baseline time and D57-T refers to 4 weeks after the first cycle of 3 weekly injections. As illustrated in Figure 4, NanoString® immune cell panels of post-treatment liver metastasis samples (collected 4 weeks after the first cycle of weekly SD-101 infusions via HAI and PEDD for three times) showed, among other things, enhanced gene signatures including cytotoxic cells, macrophages, T cells, and exhausted CD8 T cells, and a reduced gene signature including dendritic cells likely due to migration to draining lymph nodes. In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0124] Post-treatment liver metastasis samples from these patients (collected 4 weeks after the first cycle of weekly SD-101 infusions for three times via HAI and PEDD) were tested with the NanoString® Bone Marrow Panel to profile the expression of myeloid gene signatures, including angiogenesis, antigen presentation, cell cycle and apoptosis, cell migration and adhesion, chemokine signaling, complement activation, cytokine signaling, myeloid differentiation and maintenance, ECM remodeling, Fc receptor signaling, growth factor signaling, interferon signaling, lymphocyte activation, metabolism, pathogen response, T cell activation and checkpoint signaling, Th1 activation, Th2 activation, and TLR signaling. Data from the NanoString® Bone Marrow Panel are shown in Figure 5, where BL-T refers to the baseline time and D57-T refers to the time 4 weeks after the first cycle of weekly infusions for three times. As illustrated in Figure 5, NanoString® Bone Marrow Panel of post-treatment liver metastasis samples (collected 4 weeks after the first cycle of weekly SD-101 infusions via HAI and PEDD for three times) showed, among other things, improved gene signatures including lymphocyte activation, Th1 activation, interferon and TLR signaling, antigen presentation, exhausted CD8 T cells, and reduced gene signatures including Th2 activation and angiogenesis. In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0125] Post-treatment liver metastasis samples from these patients (collected 4 weeks after the first cycle of weekly SD-101 infusions for 3 times via HAI and PEDD) were tested with the NanoString® Myeloid Cell Panel to profile the expression of myeloid cell gene signatures, including mast cells, exhausted CD8, macrophages, B cells, CD8 T cells, DCs, T cells, neutrophils, and CD45. Data from the NanoString® Myeloid Cell Panel are shown in Figure 6, where BL-T refers to the baseline time and D57-T refers to the time 4 weeks after the first cycle of weekly infusions for 3 times. As illustrated in Figure 6, NanoString® bone marrow cell panels of post-treatment liver metastasis samples (collected 4 weeks after the first cycle of HAI and PEDD-mediated weekly SD-101 infusions for three times) showed, among other things, enhanced gene signatures including B cells, CD8 T cells, cytotoxic cells and macrophages, and a reduced gene signature including dendritic cells likely due to migration to draining lymph nodes. In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0126] Post-treatment liver metastasis samples from these patients (collected 4 weeks after the first cycle of weekly SD-101 infusions via HAI and PEDD for three times) were tested with the NanoString® Metabolic Panel to profile the expression of metabolic gene signatures, including amino acid transporters, AMPK, antigen presentation, arginine metabolism, autophagy, cell cycle, cytokine and chemokine signaling, DNA damage repair, endocytosis, epigenetic regulation, fatty acid oxidation, fatty acid synthesis, glutamine metabolism, glycolysis, hypoxic IDH12 activity, KEAP1NBRF2 pathway, lysosomal degradation, MAPK, mitochondrial respiration, mTOR, Myc, NF-κB, nucleotide salvage, nucleotide synthesis, p53 pathway, pentose phosphate pathway, P13K, reactive oxygen species response, TCR and costimulatory signaling, TLR signaling, transcriptional regulation, tryptophan kynurenine metabolism, and vitamin and cofactor metabolism. Data from the NanoString® metabolic panel is shown in Figure 7, where BL-T refers to the baseline time and D57-T refers to the time 4 weeks after the first cycle of three weekly injections. As illustrated in Figure 7, the NanoString® metabolic panel of post-treatment liver metastasis samples (collected 4 weeks after the first cycle of three weekly injections of SD-101 via HAI and PEDD) showed, among other things, improved gene signatures including antigen presentation, chemokine and cytokine signaling, and TCR and costimulatory signaling, and reduced gene signatures including cell cycle and fatty acid oxidation, and reduced MDSC-related genes including ARG1, NOS2, and GM-CSF-R. In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0127] Post-treatment liver metastasis samples from these patients (collected 4 weeks after the first cycle of HAI and 3 weekly SD-101 infusions via PEDD) were tested with the NanoString® Bone Marrow Panel to profile the expression of myeloid gene signatures, including genes associated with inflammation and immune cell function, as well as genes associated with immunosuppression and tumor progression. Data from the NanoString® Bone Marrow Panel are shown in FIG. 8. As illustrated in FIG. 8, a NanoString® Bone Marrow Panel of post-treatment liver metastasis samples (collected 4 weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) showed increased expression of genes associated with inflammation and immune cell function (e.g., SERPINB2, TREM2, CD18, IL9, CD68, ELANE, IFNA2, TREM1, TNFRSF11A, CD86, PDZKHP1, IL15RA, IL6, CCL7, IL2, CCL2, CCL8, PRG2, and IFNGR1) and decreased expression of genes associated with immune suppression and tumor progression (e.g., CSF2, RSGRF2, JUN, PROk2, GKS, VEGA, MET, CXCL1, TIMD4). In particular, the figure illustrates the enhancement of gene signatures including TREM2 (associated with TMB and MSI), IFNA2 and IFNGR1, CD68, CD86, IL2, IL9, IL15 and CCL7, and the reduction of gene signatures including CSF2 (GM-CSF), IDO-1, TDO-2, VEGFA and TIMD4 (role in MDSC suppressor function). In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0128] Post-treatment normal liver tissue samples from these patients (collected 4 weeks after the first cycle of 3 weekly SD-101 infusions via HAI and PEDD) were tested with the NanoString® Myeloid Cell Panel to profile the expression of myeloid cell gene signatures, including cytotoxic cells, DC, CD45, macrophages, T cells, mast cells, neutrophils, NK cells, exhausted CD8, Th1 cells, and CD8 T cells. Data from the NanoString® Myeloid Cell Panel are shown in Figure 9, where BL-T refers to the baseline time and D57-T refers to the time 4 weeks after the first cycle of 3 weekly infusions. As illustrated in FIG. 9, NanoString® bone marrow panels of normal liver tissue samples (collected 4 weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) showed that SD-101 infusion was associated with a decline in Th1 cells, CD8 T cells, and cytotoxic cells in normal liver parenchyma. The opposite changes were seen in tumor cells. The lack of immune cell proliferation / activation supports the safety of the effect of SD-101 on normal liver tissue. We hypothesize that SD-101 treats immune dysfunction in the liver and tumor microenvironment and promotes the recruitment of activated T cells from normal liver to "cold" tumors. In other words, the NanoString® data of normal liver tissue in FIG. 9 suggests intrahepatic immune cell migration.

[0129] Post-treatment peripheral blood mononuclear cell (PBMC) samples from these patients were tested with the NanoString® Myeloid Cell Panel to profile the expression of gene signatures of myeloid cells, including NK cells, NK CD56dim cells, exhausted CD8, macrophages, B cells, CD8 T cells, cytotoxic cells, T cells, neutrophils, CD45, and Th1 cells. Data from the NanoString® Myeloid Cell Panel are shown in FIG. 10. FIG. 10 illustrates the NanoString® Myeloid Panel of a post-treatment peripheral blood mononuclear cell (PBMC) sample. In this regard, a single injection of SD-101 .5 mg was followed by two cycles of SD-101 2 mg injections once a week for three cycles, with tissue collection occurring during the first week of each of the two cycles. As illustrated in the figure, hepatic arterial infusion of SD-101 with PEDD was associated with an increase in peripheral blood of NK cells, Th1 cells, B cells, CD8 T cells, and cytotoxic cells. These increases were recorded after favorable immune changes within liver metastases, including delivery of four to six doses of the second cycle. We hypothesize that SD-101 treats immune dysfunction in the liver and tumor microenvironment and promotes the emigration of activated dendritic cells and other immune cells from the reprogrammed liver. These changes in PBMC gene expression suggest a systemic effect following liver-targeted SD-101 infusion.

[0130] In summary, SD-101 and PEDD provide a tumor microenvironment modulation approach that addresses a key immune suppressive target (MDSC) with broad and additional CPI enhancing effects while treating immune dysfunction in the surrounding non-tumorous liver.

[0131] Example 4 In Example 4, we evaluated the impact of delivery of various TLR agonists (TLR4, TLR7, a class B TLR9 (TLR9B) agonist, and a class C TLR9 (TLR9C) agonist) to bone marrow cells, specifically investigating their differential effects on MDSCs and their ability to immunomodulate the hepatic tumor microenvironment (TME).

[0132] There are three classes of TLR9 agonists, with differential effects on plasmacytoid DC (pDC) IFNα production, B cell and NK cell activation, and other immune cell populations. TLR9A agonists stimulate plasmacytoid DC (pDC) to produce IFNα. TLR9B agonists activate B cells and NK cells. TLR9C has broader immunological effects. Specific delivery challenges and immunosuppressive pathways, including unique programming of myeloid-derived suppressor cells (MDSC), may limit the success of immunotherapy in the liver.

[0133] The effects of different TLR agonists were assessed in vitro by flow cytometry using different cell types obtained from healthy human donors, as well as murine bone marrow (BM) MDSCs.

[0134] Peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were cultured in vitro in the presence or absence of IL6+GMCSF (20 ng / ml). The presence of IL6+GMCSF is used to induce MDSCs. The treated cells were then assessed using flow cytometry to identify MDSCs (e.g., CD33 + CD11b + HLADR - / lo ) and M1-macrophages (e.g., CD86 + The effect of SD-101 on IL-16 expression was determined. Data generated by flow cytometry using such cells are presented in Figures 11A-11D.

[0135] Figures 11A-11D show that TLR9 stimulation with SD-101 inhibits MDSC generation and enhances M1 macrophage production. Data in Figures 11A-11D were generated using human PBMCs treated with increasing concentrations of SD-101 (0.1-3 μM) and Ctrl ODN5328 (1 μM) in the presence / absence of IL6+GMCSF (20 ng / ml, respectively) for 2 days (Figures 11A and 11C) and 7 days (Figures 11B and 11D). Figures 11A and 11B show that MDSC populations (CD33 + CD11b + HLADR - / lo ) were evaluated. In Figures 11C and 11D, M1 macrophages (CD14 + CD86 + The effect of treatment with SD-101 or Ctrl ODN5328 on IL6+GMCSF stimulated 1 μM Ctrl ODN-treated cells was evaluated. As shown in Figures 11A-11D, a 0.3 μM dose of SD-101 resulted in inhibition of MDSC generation and enhanced M1 macrophage production after 7 days of treatment. One-way ANOVA followed by Tukey's post-hoc test was performed on the data shown in Figures 11A-11D, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 vs. IL6+GMCSF stimulated 1 μM Ctrl ODN-treated cells, n=number of healthy donors.

[0136] Example 5 In Example 5, PBMCs obtained from healthy human donors were cultured in vitro in IL6+GMCSF (20 ng / ml) to induce MDSCs, followed by treatment with TLR agonists. Flow cytometry was performed to detect MDSCs (CD33 + CD11b + HLADR - ), MDSC subtypes (monocytic / granulocytic CD14 + / CD15 + MDSC), M1 macrophages (CD14 + CD86 + ), and monocytic dendritic cells (CD14+ CD11c + CD123 - The effects of various TLR agonists on TLR4 agonist (TLR5 agonist) and TLR7 agonist (TLR9 ​​agonist) were evaluated. The TLR agonists evaluated included a TLR4 agonist, a TLR7 agonist, a class B TLR9 agonist, and SD-101, a class C TLR9 agonist. In addition, bone marrow (BM) murine MDSCs (CD11b + GR-1 + ) were treated with GMCSF for 72 hours and the effect of various TLR agonists on mouse BM MDSCs was assessed by flow cytometry. Data generated from flow cytometry of various types of human and mouse cells are provided in Figures 12A-F.

[0137] Figures 12A-12F show the effect of various TLR agonists on human MDSCs and other immune cells (i.e., mouse immune cells). Data in Figures 12A-12D are generated using PBMCs obtained from healthy human donors stimulated with IL6+GMCSF (20 ng / ml each) and treated with vehicle or 0.3 μM of TLR4 agonist, TLR7 agonist, class B TLR9 agonist, or class C TLR9 agonist SD-101 for 7 days. In Figure 12A, the effect of treatment with various TLR agonists on MDSCs was evaluated. In Figure 12B, the effect of treatment with various TLR agonists on M1 macrophages was evaluated. In Figure 12C, the effect of treatment with various TLR agonists on M / G-MDSCs was evaluated. In Figure 12D, the effect of treatment with various TLR agonists on dendritic cells (CD33 + CD11c + ) were evaluated for the effects of treatment with various TLR agonists.

[0138] In addition, Figures 12E and 12F show the effect of various TLR agonists on mouse BM MDSCs. The data in Figure 12E was generated using cells obtained from mouse bone marrow (BM) treated in the absence of mouse GMCSF. The data in Figure 12F was generated using cells obtained from mouse bone marrow (BM) treated in the presence of mouse GMCSF. Student's t-test and one-way ANOVA followed by Tukey's post-hoc test were performed on the data shown in Figures 12A-F, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 vs. Veh. Additionally, % indicates p<0.01 vs. TLR4, & indicates p<0.01 vs. TLR7, ^ indicates p<0.001 vs. TLR9B, # indicates p<0.05 and ## indicates p<0.01 vs. SD-101.

[0139] As shown in the data in Figures 12A-F, the TLR9C agonist SD-101 significantly inhibited MDSC proliferation at day 2 compared to vehicle (Veh), TLR9B, TLR4 and TLR7 agonists (p<0.01; n=11). Similar results were obtained with mouse BM-derived MDSCs (p<0.001; n=5). SD-101 significantly reduced the human M / G-MDSC ratio compared to Veh, TLR4, TLR7 and TLR9B agonists (p<0.05; n=11). In addition, SD-101 shifted dendritic cells toward a myeloid program compared to Veh, TLR4 and TLR7 agonists (p<0.05; n=6). In summary, SD-101 significantly inhibited MDSC proliferation compared to vehicle (Veh), TLR9B, TLR4 and TLR7 agonists at day 2, and SD-101 significantly reduced the human M / G-MDSC ratio compared to Veh, TLR4, TLR7 and TLR9B agonists.

[0140] Example 6 In Example 6, PBMCs from healthy human donors (n=6) were cultured in vitro in IL6+GMCSF (20ng / ml) to induce MDSCs and subsequently treated with either the TLR9B agonist or the TLR9C agonist SD-101 at a concentration of 0.3μM for 2 days. Total RNA was isolated for each half of the samples (n=3) and assessed using NanoString® analysis. Samples for NanoString® analysis were also analyzed by qRT-PCR for validation. The remaining half of the samples were assessed using flow cytometry to identify MDSCs (CD33 + CD11b + HLADR - / lo ) and M1 macrophages (CD14 + CD86 + The effects of TLR9B agonists and SD-101 on TLR9B-associated leukemia (LE) ...

[0141] Figures 13A-G demonstrate that TLR9 stimulation with the TLR9C agonist SD-101 promotes enhanced pro-inflammatory and anti-tumor signaling compared to TLR9B agonists. Specifically, Figure 13A shows a volcano plot illustrating gene expression in human PBMCs following 2 days of treatment with a TLR9B agonist versus SD-101 (a TLR9C agonist) as determined using NanoString® mRNA analysis on samples prepared as described above.

[0142] Data generated using NanoString® analysis is shown in Table 2 and demonstrates that SD-101 upregulates a greater number of genes than TLR9B agonists.

[0143] [Table 2]

[0144] Data generated using NanoString® analysis is shown in Table 3 and demonstrates that SD-101 downregulates other genes beyond the TLR9B agonists.

[0145] [Table 3]

[0146] In addition, Figures 13B and 13C show the effect of TLR9B and TLR9C agonists on MDSCs and M1 macrophages, respectively, as determined by flow cytometry on samples prepared as described above. Figure 13D shows the pathway scores of immune regulatory pathways affected by TLR9B versus SD-101, as determined by nSolver.4 software using data generated from NanoString® analysis. Figures 13E, 13F, and 13G show qRT-PCR data for the same samples analyzed in Figure 13A. The data in Figures 13E, 13F, and 13G show the beneficial effect of SD-101 in improving PD-L1, IFNγ, and IP-10 expression.

[0147] A multiple t-test was performed for the pathway score analysis shown in Figure 13D. One-way ANOVA followed by Tukey's post-hoc test was performed for the qRT-PCR analysis shown in Figures 13E-G. For both Figures 13B through 13E-G, * indicates p<0.05 and ***** indicates p<0.0001 vs. Veh. Additionally, ^ indicates p<0.05, ^^ indicates p<0.01 and ^^^ indicates p<0.001 vs. TLR9B.

[0148] As shown above, NanoString® gene expression analysis revealed that SD-101 induced higher adaptive, innate, and immunometabolic scores compared to Veh and TLR9B agonists (p<0.05, n=3). SD-101 induced greater TLR, Th1, NFκB, and lymphocyte activation signatures compared to Veh and TLR9B agonists (p<0.05, n=3). SD-101 induced more PD-L1, IFNγ, and IP10 gene expression compared to Veh and TLR9B (p<0.05, n=6), as well as more B cell and NK cell proliferation compared to Veh (p<0.05, n=6). In summary, SD-101 induced greater innate immune signaling pathways, adaptive immune signaling pathways, TLR, Th1, NFκB and lymphocyte activation signatures compared to Veh and TLR9B agonists, and SD-101 induced greater PD-L1, IFNγ and IP10 gene expression compared to Veh and TLR9B agonists.

[0149] Overall, SD-101 inhibited MDSC proliferation and enhanced polarization towards M1 macrophages. In addition to a broad range of immune stimulatory effects, the favorable impact on MDSCs suggests that TLR9C agonists, if effectively delivered, have the potential to enable better performance of other immunotherapeutic agents within the hostile liver tumor microenvironment (TME).

[0150] Example 7 In Example 7, infusion of SD-101, a class C TLR9 agonist, was administered to human patients via pressure-enabled hepatic arterial infusion (PEDD / HAI) using the TriNav® infusion system to improve response rates to CPI therapy in human patients with uveal melanoma liver metastases, advanced hepatocellular carcinoma, or advanced intrahepatic cholangiocarcinoma. The study included cohorts A, B, and C. Cohort A received SD-101 as a single agent (here n=9), cohort B received SD-101 via TriNav plus systemic administration of anti-PD-1 (i.e., nivolumab or pembrolizumab) (here n=6-12), and cohort C received SD-101 via TriNav plus systemic administration of anti-PD-1 (i.e., nivolumab) plus systemic administration of anti-CTLA-4 (i.e., ipilimumab) (here n=6-12). The amount of SD-101 administered to human patients in cohorts A, B, and C can range from about 0.5 mg to about 8 mg per dose (i.e., 0.5 mg, 2 mg, 4 mg, and 8 mg). The dosing regimen includes administering SD-101 via PEDD for two cycles, where each cycle includes three weekly doses of SD-101 (i.e., administration of SD-101 once per week for three consecutive weeks) and a five-week washout period following the weekly administration of SD-101.

[0151] Figure 14 illustrates the pharmacokinetic profile of plasma SD-101 levels for five different patients in Cohort A receiving a single 4 mg dose of SD-101 over a 6 hour period following infusion for each of the six doses administered over the two cycles discussed above. Specifically, following infusion with TriNav, transient levels of SD-101 (<2 hours) were detected in plasma as shown in the figure. At all doses (not shown), systemic exposure levels detected were transient (<4 hours).

[0152] High SD-101 levels were observed in the liver of patients receiving SD-101 infusion using TriNav. In patients receiving SD-101 at 8 mg per dose, SD-101 was detected in liver tissue up to 2340 ng / ml. This human clinical data demonstrates that TriNav can be used to achieve higher liver SD-101 levels with limited systemic exposure.

[0153] In addition, PMBC cells from patients receiving SD-101 treatment and administered SD-101 in the above manner were analyzed by NanoString® for gene expression levels at multiple time points. Specifically, NanoString® analysis of gene expression levels in liver metastases from three patients in the SD-101 2mg single agent cohort revealed increased ISG15, IL-9, IFNα, and IL-2 transcripts and decreased ARG1 and IDO transcripts, with improved scores for macrophages, exhausted CD8 T cells, Th1 cells, and Th1 activation. NanoString® gene expression data showed that SD-101 delivered via TriNav was associated with evidence of proliferation of antitumor natural killer cells in the blood, consistent with systemic immune activation.

[0154] Dose-response trends were observed in serum cytokines with increases across the 2 mg, 4 mg, and 8 mg single-agent dose levels for IL-18, IFNγ, IP-10, and soluble CD25. In particular, FIG. 15 illustrates the changes in serum IFNγ following increasing dosing levels of SD-101 single agent (Cohort A) and SD-101 in combination with a CPI (i.e., nivolumab or pembrolizumab) (Cohort B) after each of the three doses administered over the first cycle. Specifically, the figure illustrates the trend of increasing serum IFNγ levels following infusion of SD-101 via TriNav for Cohort A (at doses of .5 mg, 2 mg, 4 mg, and 8 mg of SD-101) and Cohort B (at a dose of 2 mg of SD-101 in combination with pembrolizumab or nivolumab). Serum IFNγ was determined by Luminex assay. The data in Figure 15 demonstrate that injection of SD-101 with TriNav results in IFNγ cytokine induction.

[0155] Figure 16 illustrates the change in monocytic MDSC concentration in tumors receiving different dose levels of single agent SD-101 (cohort A) in the manner described above. Specifically, the figure illustrates the reduction in monocytic MDSC concentration in tumors for (i) three patients receiving a 2 mg dose of SD-101, and (ii) one patient receiving an 8 mg dose of SD-101. Monocytic MDSC concentration was determined by multiplex immunofluorescence microscopy. The percentage reduction in monocytic MDSC concentration in tumors was calculated from the earliest available time point (day 1 or day 57) to the latest available time point (day 57 or day 100). The data in Figure 16 demonstrate that injection of SD-101 using TriNav results in MSDC resolution, as shown by the reduction in monocytic MDSC levels in liver tumors.

[0156] For patients receiving SD-101 2 mg plus a CPI with liquid biopsy data available, 4 of 7 demonstrated a decline in circulating tumor cells after one cycle of treatment, however, in this case, 3 of 5 patients showed a decline in circulating tumor DNA (ctDNA) after the first cycle, as shown below in Figures 17 and 18.

[0157] Figure 17 illustrates the changes in circulating tumor cells receiving SD-101 2 mg with a CPI after the first cycle of treatment. Specifically, the figure illustrates that 4 of 7 patients receiving SD-101 2 mg alone had a decrease in circulating tumor cell levels, thereby resulting in a decrease in tumor burden.

[0158] Figure 18 illustrates the change in circulating tumor DNA (ctDNA) after the first cycle of treatment with a single dose of 2 mg SD-101. Specifically, the figure illustrates that 3 out of 5 patients had a decrease in circulating tumor DNA (ctDNA) levels with a single dose of 2 mg SD-101, resulting in a decrease in tumor burden. The data provided in Figure 18 is for the same first 5 patients as in Figure 17.

[0159] Example 8 In Example 8, TLR9C agonist SD-101 delivered via PEDD in combination with anti-PD-1 antibody delivered SQ or IP significantly reduced LM in mice. Tumor-infiltrating lymphocytes (CD45 + Further analysis of the hepatic MDSCs revealed that anti-PD-1 in combination with SD-101 significantly reduced hepatic MDSCs. Percentage of B cells; CD3 +The ratio of T cells and M1 / M2 macrophages was significantly improved compared to Veh. There was no significant difference between SQ and IP CPI delivery in controlling tumor progression or modulating the tumor microenvironment. The data demonstrate that TLR9 stimulation via PEDD in combination with anti-PD-1 resulted in equivalent control of LM, regardless of the delivery route. PEDD, a class C TLR9 agonist, also has the ability to prime the TME to reduce immunosuppression in LM, which can improve the anti-tumor efficacy of anti-PD-1, regardless of the administration route. In some cases, the checkpoint inhibitor is administered systemically or subcutaneously prior to administration of the TLR agonist.

[0160] In this example, SD-101 was delivered in combination with an anti-PD-1 antibody administered either intraperitoneally (IP) or subcutaneously (SQ) in a mouse model of PEDD to evaluate whether the CPI administration route impacts the ability of intrahepatic TLR9 stimulation to control liver metastases. To generate liver metastases, C57 / BL6 mice were challenged with MC38-Luc tumor cells via the intrasplenic route, followed by splenectomy. One week later, mice were treated with 10 μg SD-101 via PEDD and twice weekly anti-PD-1 antibody delivered either IP or SQ. Tumor burden was monitored by IVIS and livers were harvested at D10 to assay for CD45 + Cells were isolated. Tumor burden was monitored by IVIS and livers were harvested on DIO to identify CD45 + Myeloid-derived suppressor cells (MDSCs) blunt the activity of immunotherapy through promoting an immunosuppressive tumor microenvironment (TME) in the setting of liver metastasis. Flow cytometry analysis was performed to identify MDSCs (CD11b + Grl + ), B cells (B220), T (CD3 + ) cells, M1(F4 / 80 + CD38 + Egr2 - ) Macrophages and M2 (F4 / 80 + CD38 - Egr2 +) Macrophages were quantified.

[0161] SD-101 delivered via PEDD in combination with anti-PD-1 antibody delivered either via SQ or IP significantly (p<0.0001) reduced progression of liver metastases (fold over D0) compared to controls (Veh: 87.46 vs. SD-101: 13.90 vs. SQ: 0.002 vs. IP: 0.04). CD45 isolated from tumor-bearing liver + Flow cytometric analysis of the cells revealed that CPI in combination with SD-101 significantly reduced hepatic MDSCs (Veh: 37.57% vs. SQ: 7.18% vs. IP: 10.18%; p<0.05). The percentage of B cells (Veh: 8.32% vs. SQ: 18.09% vs. IP: 15.65%: p<0.05); T cells (Veh: 7.14% vs. SD-101: 15.18% vs. SQ: 17.81% vs. IP: 19.13%: p<0.05) and the ratio of M1 / M2 macrophages (Veh: 2.25 vs. SD-101: 12.8 vs. SQ: 12.99 vs. IP: 12.90: p<0.05) were significantly improved compared to vehicle (Veh). There was no significant difference between SQ and IP CPI delivery in controlling tumor progression or in modulating TEM.

[0162] Figure 19A illustrates the changes in tumor progression of mouse liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ, as described in this example. C57 / BL6 mice were challenged with MC38-Luc tumor cells via the intrasplenic route, followed by splenectomy to generate LM. One week later, mice were treated with 10 μg SD-101 via PEDD and anti-PD-1 antibody delivered either IP or SQ at DO, D2, D4, and D7. Tumor burden was monitored by an in vivo imaging system (IVIS) and reported as fold change relative to DO tumor burden. Two-way ANOVA followed by Tukey's post-hoc test was performed on the data shown in Figure 19, where **** indicates p<0.001 vs. D10 Veh.

[0163] Figure 19B illustrates the change in tumor burden of mouse liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibodies delivered either IP or SQ compared to vehicle at day 10 post-treatment. IVIS values ​​are reported as photons / sec. Two-way ANOVA followed by Tukey's post-hoc test was performed on the data shown in Figure 19B, where # indicates p<0.001 vs. D10 Veh and ^ indicates p<0.0001 vs. D10 Veh.

[0164] Figure 19C illustrates images captured by IVIS over various time points after initiating treatment with PEDD administration of SD-101 in combination with an anti-PD-1 antibody delivered either IP or SQ compared to vehicle. Figure 19D provides images captured of livers harvested 10 days after treatment with PEDD administration of SD-101 in combination with an anti-PD-1 antibody delivered either IP or SQ compared to vehicle according to Example 8.

[0165] Figure 20A illustrates the changes in MDSCs present in the LM in response to PEDD administration of SD-101 in combination with anti-PD-1 antibodies delivered either IP or SQ compared to vehicle on day 10 post-treatment, as described in this example. On day 10 (D10), livers were harvested and analyzed for CD45 + Cells were isolated. Specifically, livers were harvested on D10 and NPCs were isolated after collagenase digestion; CD45 + Cells were separated using magnetic beads. Flow cytometry (FC) was performed using antibodies against CD11b; GR1, NK1.1, B220, and PD-L1. FC analysis was performed to identify MDSCs (CD11b + Grl + ) was quantified.

[0166] FIG. 20B shows CD45 expression levels isolated from liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle 10 days after treatment. + Among the cells, B (B220 + ) cell changes were compared to vehicle 10 days after treatment. Figure 20C shows CD45+ cells isolated from liver metastases in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ. + Among the cells, T (CD3 + Figure 20D illustrates the changes in M1 and M2 macrophages among CD45+ cells isolated from liver metastases. Figure 20C illustrates the changes in M1 (F4 / 80) and M2 macrophages among CD45+ cells isolated from liver metastases. Figure 20D illustrates the changes in M1 and M2 macrophages in the tumor microenvironment as quantified by FC in response to PEDD administration of SD-101 in combination with anti-PD-1 antibody delivered either IP or SQ compared to vehicle at day 10 post-treatment. + CD38 + Egr2 - ) Macrophages and M2 (F4 / 80 + CD38 - Egr2 +) macrophages. In view of the data in Example 8 above, SD-101 administered via PEDD in combination with CPI delivered IP or SQ resulted in control of liver metastases, where intrahepatic TLR9 stimulation rendered CPI equally effective via either route. PEDD, a class C TLR9 agonist, is believed to prime the TME to reduce immune suppression in liver metastases, thereby improving the antitumor efficacy of CPI regardless of the route of administration.

[0167] The above merely illustrates the principles of the disclosure. In light of the teachings herein, various modifications and alterations of the described embodiments will become apparent to those skilled in the art. Those skilled in the art will appreciate that numerous systems, configurations, and procedures can be devised that embody the principles of the disclosure even if not explicitly shown or described herein, and thus fall within the spirit and scope of the disclosure. As should be understood by those skilled in the art, various different exemplary embodiments may be used together with each other and interchangeably with each other. In addition, certain terms used in this disclosure, including this specification, may be used synonymously in certain instances, including, but not limited to, for example, data and information. It should be understood that such words and / or other words that may be synonymous with each other may be used synonymously herein, while there may be instances where such words are not intended to be used synonymously. Furthermore, to the extent that prior art knowledge has not been expressly incorporated herein by reference above, the prior art knowledge is expressly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entirety.

Claims

1. Structure: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1) 1. A method for treating liver immune dysfunction comprising administering to a subject in need thereof a therapeutically effective amount of a Toll-like receptor 9 agonist having the formula: The liver immune dysfunction is induced by at least one tumor resulting from metastasis in the liver or at least one primary liver cancer; method.

2. The TLR9 agonist is administered through a device by hepatic artery infusion (HAI); The method of claim 1.

3. The TLR9 agonist is administered through a device by portal vein infusion (PVI); The method of claim 1.

4. The therapeutically effective amount of the TLR9 agonist administered ranges from about 0.01 to 20 mg. The method of claim 1.

5. the therapeutically effective amount of the TLR9 agonist administered is selected from the group consisting of 2 mg, 4 mg, or 8 mg; The method of claim 4.

6. The TLR9 agonist may be administered through a catheter device.

6. The method according to any one of claims 1 to 5.

7. the catheter device comprises a one-way valve that dynamically responds to changes in local pressure and / or flow; The method of claim 6.

8. The TLR9 agonist is administered through a catheter device via pressure-enabled drug delivery. The method of claim 6.

9. The TLR9 agonist is administered for a period of about 10 to 200 minutes. The method of claim 6.

10. The TLR9 agonist is administered for a period of about 10 to 60 minutes. The method of claim 9.

11. The TLR9 agonist is administered for a period of about 25 minutes. The method of claim 10.

12. The method of any one of claims 1 to 5, wherein administration of the TLR9 agonist results in altered gene expression within the metastases in the liver.

13. The changes in gene expression include activation of immune cells in normal liver tissue and migration of activated immune cells to the at least one tumor. The method of claim 12.

14. The change in gene expression may include: Improved TLR signaling; increased white blood cells; increased exhausted CD8 T cells; induction of Th1 programming; reduced Th2 programming; increased T cell receptor and improved T cell costimulatory signaling; increased IL9, IL15, CCL7; B cell activation; induction of mast cells; induction of NK cells; induction of IFNγ; improved interferon signaling; improved chemokine signaling; increased IL6 in the liver without an increase in the blood; reduced M2 macrophages; increased M1 macrophages; reduced MDSCs; reduced angiogenesis and VEGF; and reduced fatty acid oxidation. At least one of the following is included: The method of claim 12.

15. wherein the TLR9 agonist is administered in combination with one or more checkpoint inhibitors.

6. The method according to any one of claims 1 to 5.

16. the checkpoint inhibitor is administered systemically either simultaneously with, before, or after the administration of the TLR9 agonist; The method of claim 15.

17. The one or more checkpoint inhibitors include at least one of nivolumab, pembrolizumab, and ipilimumab. The method of claim 16.

18. wherein said administering of said TLR9 agonist comprises a dosing regimen comprising cycles; wherein one or more of said cycles comprises said administration of said TLR9 agonist via a catheter device by hepatic artery infusion, followed by systemic administration of said one or more checkpoint inhibitors. The method of claim 16.

19. wherein said administering said TLR9 agonist in combination with said one or more checkpoint inhibitors results in a reduction in one of circulating tumor cell levels and circulating tumor DNA levels. The method of claim 16.

20. The one or more checkpoint inhibitors are administered intraperitoneally or subcutaneously. The method of claim 15.

Images