Methods of treating immune dysfunction of liver cancer with TOLL-like receptor agonists

Abstract

The present disclosure provides methods for treating liver immune dysfunction in a subject. The liver immune dysfunction may be induced by at least one tumor caused by liver metastasis, or at least one primary liver cancer. An exemplary method includes administering to the subject a Toll-like receptor 9 (TLR9) agonist, such as a TLR9 agonist having the following structure: 5 '-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3'. The TLR9 agonist can be administered to the liver, for example, the TLR9 agonist can be administered to the liver through the vasculature using local area therapy.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 302,004, filed on January 21, 2022, entitled “Cancer Therapy Using Toll-Like Receptor Agonists,” U.S. Provisional Application Serial No. 63 / 423,005, filed on November 6, 2022, entitled “Cancer Therapy Using Toll-Like Receptor Agonists,” and U.S. Provisional Application Serial No. 63 / 426,757, filed on November 20, 2022, entitled “Cancer Therapy-Using Toll-Like Receptor Agonists,” the entire contents of all of the above applications are hereby incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format, and the sequence listing is hereby incorporated by reference in its entirety. The XML copy created on January 20, 2023 is named A372-509_WO_SL.xml and is 18,185 bytes in size. Technical Field

[0005] The present disclosure relates generally to methods of treating cancer, particularly liver cancer, and methods of delivering Toll-like receptor (TLR) agonists to solid tumors in the liver using locoregional therapy via the vasculature. Background Art

[0006] Cancer is a devastating disease involving the unchecked growth of cells, which can result in the growth of solid tumors in various organs such as the skin, liver and pancreas. Tumors can first be present in any number of organs or can be the result of metastasis or spread from other locations.

[0007] The liver is a unique organ that has an immunosuppressive effect in nature and drives the programming and amplification of suppressor cells such as myeloid-derived suppressor cells (MDSC). In this regard, MDSC amplifies in response to malignant tumors. MDSC also drives the amplification of other suppressor cell types such as T regulatory cells (Treg), tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF). MDSC can lower immune cells and interfere with the effectiveness of immunotherapeutics. In addition, high MDSC levels generally indicate a poor prognosis for cancer patients.

[0008] Specific delivery challenges and immunosuppressive pathways may limit the success of immunotherapy in the liver. Therefore, there remains a need for safe and effective therapies for treating liver immune dysfunction. Summary of the invention

[0009] The present application relates to a method for treating liver immune dysfunction using a therapeutically effective amount of a Toll-like receptor (TLR) agonist. For example, the present application provides a method for treating liver immune dysfunction, the method comprising administering a therapeutically effective amount of a Toll-like receptor 9 agonist to a subject in need thereof, the Toll-like receptor 9 agonist having the following structure: 5'-TCGAAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO: 1). The liver immune dysfunction is induced by: at least one tumor caused by liver metastasis, or at least one primary liver cancer.

[0010] In one aspect, the present application relates to a method for treating liver immune dysfunction, the method comprising administering a TLR agonist by a device by hepatic artery infusion (HAI). According to another example, treating liver immune dysfunction comprises administering a TLR agonist by a device by portal vein infusion (PVI). For example, a TLR9 agonist can be administered by a catheter device. The catheter device includes a one-way valve that dynamically responds to local pressure and / or flow changes. In another example, a TLR9 agonist is administered by a catheter device via pressure-enabled drug delivery (PEDD).

[0011] In some examples, liver immune dysfunction may be induced by and / or caused by a solid tumor in the liver, such as a tumor that is a metastasis of uveal melanoma, or by at least one primary liver cancer, such as hepatocellular carcinoma or intrahepatic cholangiocarcinoma.

[0012] In some instances, the TLR agonist is administered by PEDD, which includes administering a therapeutic agent by a device such as a catheter device. In some instances, the catheter device includes a one-way valve that dynamically responds to local pressure changes. In some instances, the catheter device produces, causes and / or contributes to a net increase in fluid pressure in a blood vessel and / or target tissue or tumor. In some instances, the catheter device produces, causes and / or contributes to a net decrease in fluid pressure in a blood vessel and / or target tissue or tumor. In some instances, the catheter device produces, causes and / or contributes to a first decrease and then increase in fluid pressure in a blood vessel and / or target tissue or tumor.

[0013] In some examples, the TLR agonist is administered via a pressure-enabled device, such as a device that regulates vascular pressure.

[0014] In some examples, the amount of TLR agonist administered is in the range of about 0.01-20 mg, or is selected from the group consisting of: 2 mg, 4 mg, or 8 mg.

[0015] In some instances, the TLR agonist is administered for a period of about 10-200 minutes. In another embodiment, the TLR agonist is administered for a period of about 10-60 minutes. In another embodiment, the TLR agonist is administered for a period of about 25 minutes.

[0016] In some instances, administration of a TLR9 agonist causes changes in gene expression within liver metastasis. For example, changes in gene expression include activation of immune cells in normal liver tissue and migration of activated immune cells to at least one tumor. In another example, changes in gene expression include at least one of the following: TLR signaling increases; leukocytes increase; exhausted CD8 T cells increase; Th1 programming induction; Th2 programming reduction; T cell receptor and T cell co-stimulatory signaling increase; IL9, IL15, CCL7 increase; B cell activation; mast cell induction; NK cell induction; IFNγ induction; interferon signaling increases; chemokine signaling increases; IL6 increases in the liver, but not in the blood; M2 macrophages decrease; M1 macrophages increase; MDSC decreases; angiogenesis and VEGF decrease; and fatty acid oxidation decreases.

[0017] In some instances, a TLR agonist is administered in combination with one or more checkpoint inhibitors. In some instances, a checkpoint inhibitor is administered systemically while, before, or after the administration of a TLR agonist. In some instances, the checkpoint inhibitor includes at least one of the following: nivolumab, pembrolizumab, and ipilimumab. In some instances, the combined administration of a TLR9 agonist and one or more checkpoint inhibitors may cause a decrease in one of the levels of circulating tumor cells and circulating tumor DNA. In some aspects, one or more checkpoint inhibitors are administered intraperitoneally or subcutaneously.

[0018] In some instances, administering a TLR agonist includes a dosing regimen that includes cycles. In some instances, one or more of the cycles include administering a TLR agonist via a catheter device via HAI, followed by systemic administration of one or more checkpoint inhibitors. Specifically, the checkpoint inhibitors include at least one of the following: nivolumab, pembrolizumab, and ipilimumab.

[0019] In some examples, administration of a TLR agonist to the liver via an intravascular device results in a reduction in myeloid-derived suppressor cells (MDSCs) or functional changes in MDSCs to limit immunosuppression. In some examples, administration of a TLR agonist to the liver via an intravascular device results in an anti-tumor effect.

[0020] 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.

[0021] In another example, the method of the present application includes a method for treating liver immune dysfunction and tumor-induced liver immune dysfunction, wherein administration of a TLR9 agonist, more specifically a class C TLR9 agonist, causes at least one of the following changes in gene expression within liver metastases (LM): activation of immune cells in the normal liver (non-tumor liver of the same patient), which migrate into the liver metastases; activation and mobilization of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages); increased TLR signaling; increased leukocytes (CD45+); exhausted CD8 Increased T cells (very important for checkpoint responsiveness); induction of Th1 programming; decreased Th2 programming; increased T cell receptor and T cell co-stimulatory signaling; increased IL9, IL15, CCL7; B cell activation; induction of mast cells; induction of NK cells; induction of IFNg and related genes; increased interferon signaling; increased chemokine signaling; increased IL6 in the liver but not in the blood; decreased M2 macrophages; increased M1 / activated macrophages and related genes: CD68, CD86; dendritic cell activation and migration to draining lymph nodes; decreased MDSC and related genes: IDO1, TOD2, ARG-1, NOS2, TIMD4; decreased angiogenesis and VEGF; decreased fatty acid oxidation (preferred MDSC program).

[0022] In another example, the method of the present application includes a method for administering a TLR9 agonist, specifically a class C TLR9 agonist, and more specifically a method for administering in combination with a checkpoint inhibitor to a patient, wherein the combined administration of a TLR9 agonist and a checkpoint inhibitor causes a reduction in circulating tumor cell levels and / or circulating tumor DNA levels. The patient may suffer from liver immune dysfunction and / or tumor-induced liver immune dysfunction. For example, the patient may suffer from liver metastatic uveal melanoma, hepatocellular carcinoma, or intrahepatic bile duct carcinoma.

[0023] These and other objects, features and advantages of exemplary embodiments of the present disclosure will become apparent when the following detailed description of exemplary embodiments of the present disclosure is read in conjunction with the entire specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Additional objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings which show illustrative examples of the present disclosure.

[0025] Figure 1 The structure of SD-101 is shown.

[0026] Figure 2A In vitro experimental data are provided demonstrating the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of total MDSCs induced by treatment of human PBMCs harvested from healthy donors and treated with IL6 and GMCSF according to Example 1, compared to untreated controls.

[0027] Figure 2B In vitro experimental data are provided that demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of M-MDSCs and G-MDSCs induced by treating human PBMCs harvested from healthy donors and treated with IL6 and GMCSF according to Example 1, compared to untreated controls.

[0028] Figure 2C In vitro experimental data are provided demonstrating the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of M1 macrophages induced by treatment of human PBMCs harvested from healthy donors and treated with IL6 and GMCSF according to Example 1, compared to untreated controls.

[0029] Figure 2D In vitro experimental data are provided demonstrating the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of M2 macrophages induced by treatment of human PBMCs harvested from healthy donors and treated with IL6 and GMCSF according to Example 1, compared to untreated controls.

[0030] Figure 2E Images are provided of in vitro experiments demonstrating the effects of a TLR9 agonist on human MDSCs induced by treatment of human PBMCs harvested from healthy donors and treated with IL6 and GMCSF according to Example 1, compared to untreated controls.

[0031] Figure 3AIn vitro experimental data are provided that demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of MDSC (CD11b+Gr-1+) induced by mouse bone marrow cells treated with therapeutic GMCSF compared to untreated controls according to Example 2.

[0032] Figure 3B In vitro experimental data are provided that demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of macrophages (CD11b+F4 / CD80+) induced by murine bone marrow cells treated with therapeutic GMCSF compared to untreated controls according to Example 2.

[0033] Figure 3C In vitro experimental data are provided, which demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of M1 macrophages (CD11b+F4 / CD80+CD38+) induced by mouse bone marrow cells treated with therapeutic GMCSF compared to untreated controls according to Example 2.

[0034] Figure 4 According to Example 3, as shown by Changes in liver metastasis gene expression as measured by the immune cell panel, four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg via HAI and PEDD.

[0035] Figure 5 According to Example 3, as shown by Changes in liver metastasis gene expression four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg, as measured in the bone marrow group, via HAI and PEDD.

[0036] Figure 6 According to Example 3, as shown by Changes in liver metastasis gene expression four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg, as measured by HAI and PEDD in the bone marrow cell panel.

[0037] Figure 7 According to Example 3, as shown by Changes in liver metastatic gene expression as measured by the metabolome four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg via HAI and PEDD.

[0038] Figure 8 According to Example 3, as shown by Changes in liver metastasis gene expression four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg, as measured in the bone marrow group, via HAI and PEDD.

[0039] Fig. 9 According to Example 3, as shown by Changes in gene expression in normal adjacent liver tissue four weeks after the first cycle of three weekly SD-101 infusions at a dose of 2 mg, measured by HAI and PEDD, in the bone marrow group.

[0040] Fig.10 According to Example 3, as shown by Changes in PBMC gene expression measured in the bone marrow group following two cycles of three weekly SD-101 infusions at a dose of 2 mg via HAI and PEDD.

[0041] Fig.11A Experimental data are provided demonstrating that according to Example 4, TLR9 stimulation using SD-101 inhibits the generation of MDSCs from human PBMCs treated with IL6 and GMCSF for 7 days.

[0042] Fig. 11B Experimental data are provided demonstrating that according to Example 4, TLR9 stimulation using SD-101 inhibits the generation of MDSCs from human PBMCs treated with IL6 and GMCSF for 2 days.

[0043] Fig. 11C Experimental data are provided demonstrating that according to Example 4, TLR9 stimulation using SD-101 enhances the production of M1 macrophages from human PBMCs treated with IL6 and GMCSF for 7 days.

[0044] Fig.11D Experimental data are provided demonstrating that according to Example 4, TLR9 stimulation using SD-101 enhances the production of M1 macrophages from human PBMCs treated with IL6 and GMCSF for 2 days.

[0045] Fig. 12A Provide experimental data demonstrating that according to Example 5, TLR4 agonists, TLR7 agonists, TLR9B agonists and SD-101 have an inhibitory effect on MDSCs (CD33 + CD11b + HLADR - )’s effect on the amount.

[0046] Fig. 12BProvide experimental data demonstrating that according to Example 5, TLR4 agonists, TLR7 agonists, TLR9B agonists and SD-101 have an inhibitory effect on M1 macrophages (CD14 + CD86 + )’s quantity.

[0047] Fig. 12C Provide experimental data demonstrating that according to Example 5, TLR4 agonists, TLR7 agonists, TLR9B agonists and SD-101 have an inhibitory effect on M / G-MDSCs (M / G CD14 + / CD15 + The effect of the amount of MDSCs.

[0048] Fig.12D Provide experimental data demonstrating that, according to Example 5, TLR4 agonists, TLR7 agonists, TLR9B agonists and SD-101 have an inhibitory effect on monocytic dendritic cells (CD14 + CD11c + CD123 - )’s quantity.

[0049] Fig.12E Experimental data are provided demonstrating the effects of a TLR4 agonist, a TLR7 agonist, a TLR9B agonist and SD-101 on the amount of murine bone marrow MDSCs from the bone marrow (BM) of mice treated in the absence of murine GMCSF according to Example 5 compared to untreated controls.

[0050] Fig.12F Experimental data are provided demonstrating the effects of a TLR4 agonist, a TLR7 agonist, a TLR9B agonist and SD-101 on the amount of murine bone marrow MDSCs from the bone marrow (BM) of mice treated in the presence of murine GMCSF according to Example 5 compared to untreated controls.

[0051] Fig.13A A volcano plot is shown illustrating gene expression of human PBMCs after 2 days of treatment with a TLR9B agonist and SD-101 (TLR9C agonist) according to Example 6, as shown using Determined by mRNA analysis.

[0052] Fig. 13BExperimental data are provided demonstrating that, according to Example 6, TLR9B agonists and SD-101 have an effect on MDSCs (CD33 + CD11b + HLADR - )’s effect on the amount.

[0053] Fig. 13C Experimental data are provided demonstrating that, according to Example 6, TLR9B agonists and SD-101 have an effect on the expression of M1 macrophages (CD14 + CD86 + )’s effect on the amount.

[0054] Fig. 13C Experimental data are provided demonstrating that, according to Example 6, TLR9B agonists and SD-101 have an effect on the expression of M1 macrophages (CD14 + CD86 + )’s effect on the amount.

[0055] Fig.13D Pathway scores showing the immunomodulatory pathways affected by TLR9B and SD-101, as calculated by nSolver.4 software using Fig.13A of Determined by analyzing the data generated.

[0056] Fig.13E Show in Fig.13A qRT-PCR data of PD-L1 expression of the same samples analyzed in.

[0057] Fig.13F Show in Fig.13A qRT-PCR data of IFNγ expression of the same samples analyzed in .

[0058] Figure 13G Show in Fig.13A qRT-PCR data of IP-10 expression of the same samples analyzed in .

[0059] Fig.14 Shown is the change in SD-101 levels in plasma following administration of a single dose of 4 mg of SD-101 to a human patient following each of six doses administered over two cycles according to Example 7.

[0060] Fig.15Shown are changes in serum IFNγ following increasing dose levels of single agent SD-101 and combination use of SD-101 with a checkpoint inhibitor to human patients after each of the three doses administered in the first cycle according to Example 7.

[0061] Fig.16 Shown is the change in the level of mononuclear MDSCs within a tumor after administration of different levels of a single agent SD-101 to a human patient according to Example 7.

[0062] Fig.17 Shown are changes in circulating tumor cells after administration of 2 mg SD-101 with a checkpoint inhibitor to a human patient according to Example 7.

[0063] Fig.18 According to Example 7, Fig.17 Changes in circulating tumor DNA in 5 human patients after administration of 2 mg of SD-101 with checkpoint inhibitors.

[0064] Fig.19A Shown is the change in tumor progression of liver metastases in mice in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody according to Example 8 compared to vehicle.

[0065] Fig.19B Shown is the change in tumor burden of liver metastases in mice in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody 10 days after treatment according to Example 8 compared to vehicle.

[0066] Fig.19C Provided are images captured by IVIS at different time points after initiation of treatment by PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody according to Example 8, compared to vehicle.

[0067] Fig.19D Provided are images of harvested livers captured 10 days after treatment with PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody according to Example 8, compared to vehicle.

[0068] Fig. 20A Shown are changes in MDSCs present in LM in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody, according to Example 8, compared to vehicle, 10 days after treatment.

[0069] Fig. 20BAccording to Example 8, isolated CD45 from liver metastases were shown to be upregulated 10 days after treatment in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody compared to vehicle. + B (B220 + )Changes in cells.

[0070] Fig. 20C According to Example 8, isolated CD45 from liver metastases were shown to be upregulated 10 days after treatment in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody compared to vehicle. + T cells (CD3 + )Changes in cells.

[0071] Fig.20D Shown is the isolation of CD45 from liver metastasis + According to Example 8, compared with vehicle, at 10 days after treatment, in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibodies, M1 (F4 / 80 + CD38 + Egr2 - ) and M2(F4 / 80 + CD38 - Egr2 + ) Macrophages, as quantified by FC.

[0072] In all drawings, unless otherwise specified, the same reference numerals and characters are used to represent similar features, elements, components or parts of the illustrated embodiments. In addition, although the present disclosure will now be described in detail with reference to the accompanying drawings, this is done in conjunction with exemplary embodiments and is not limited to the specific embodiments shown in the drawings and the accompanying paragraphs. DETAILED DESCRIPTION

[0073] The following description of the embodiments provides non-limiting representative examples of reference numbers to specifically describe the features and teachings of different aspects of the present invention. The described embodiments should be considered to be able to be implemented separately or in combination with other embodiments from the description of the embodiments. Those of ordinary skill in the art who review the description of the embodiments should be able to understand the different described aspects of the present invention. The description of the embodiments should help to understand the present invention to such an extent that other implementations that are not specifically covered but within the knowledge of those skilled in the art who have read the description of the embodiments will be understood to be consistent with the application of the present invention.

[0074] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which this application belongs. In addition, certain terms used herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0075] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0076] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, will be understood to be modified in all cases by the term "about". The numerical value may include ±1%, ±2%, ±3%, ±4%, or ±5% of the recited value. As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0077] The term "subject" or "patient" as used herein refers to an animal, and preferably a mammal, and more preferably a human. Examples of subjects include humans, and may also include other animals, such as rats, mice, and pigs. In a specific aspect, the subject is a human.

[0078] 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, illness or condition. The terms "treat, treating and treatment" can also refer to causing the regression of a disease, illness or condition, preventing the progression of a disease, illness or condition, or at least slowing down the progression of a disease, illness or condition. In a specific example, "treat, treating and treatment" refers to preventing the recurrence of a disease, illness or condition. In another example, "treat, treating and treatment" refers to an increase in the survival rate of a subject suffering from a disease, illness or condition. In a specific example, "treat, treating and treatment" refers to eliminating a disease, illness or condition of a subject.

[0079] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or agent that elicits a desired biological or medical response in a tissue system, animal or human, which desired biological or medical response includes improvement of one or more biomarkers of the disease, disorder or condition being treated, reduction in severity of one or more symptoms of the disease, disorder or condition being treated, for example, slowing tumor development and metastasis in a patient.

[0080] As used herein, the term "agonist" refers to an agent that binds to a receptor and then increases, promotes, sensitizes, or upregulates the receptor.

[0081] As used herein, the term "antagonist" refers to a compound that binds to a receptor and blocks or reduces the response of the receptor to an agonist.

[0082] The term "pharmaceutically acceptable salt" refers to salts of compounds that are known to be non-toxic and commonly used in the pharmaceutical art. In some instances, a pharmaceutically acceptable salt of a compound retains its biological effectiveness and is not biologically or otherwise undesirable.

[0083] As used herein, a "pharmaceutically acceptable excipient" refers to a non-toxic, biologically tolerable and otherwise biologically suitable substance for administration to a subject, such as an inert substance added to a pharmacological composition or otherwise used as a vehicle, carrier or diluent to facilitate administration of an active compound or agent and is compatible therewith.

[0084] Toll-like receptor agonists

[0085] Toll-like receptors are pattern recognition receptors that can detect molecular patterns (PAMPs) associated with microbial pathogens. TLR stimulation, such as TLR9 stimulation, can not only provide a wide range of innate immune stimulation, but also can specifically address the main driver of immunosuppression in the liver. TLR1-10 is expressed in humans and recognizes a variety of different microbial PAMPs. In this regard, TLR9 can respond to unmethylated CpG-DNA, including microbial DNA. CpG refers to the motif of cytosine and guanine dinucleotides. TLR9 is constitutively expressed in B cells, plasmacytoid dendritic cells (pDC), activated neutrophils, monocytes / macrophages, T cells and MDSC. In addition, human MDSC expresses TLR9 on its surface. In addition, TLR9 and related endosomal protein TLR7 are expressed in human liver metastasis tissue. TLR9 is also expressed in non-immune cells, including keratinocytes and intestinal, cervical and respiratory epithelial cells. TLR9 can bind its agonist in the endosome. Signaling can be performed 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. In addition, TLR9 agonists can induce cytokine and IFN production and functional maturation of antigen-presenting dendritic cells.

[0086] According to the examples, TLR9 agonists can reduce and reprogram MDSCs. In this regard, reducing, altering or eliminating MDSCs is believed to improve the ability of the host immune system to attack cancer and the ability of immunotherapy to induce a more beneficial therapeutic response. In the examples, TLR9 agonists can convert MDSCs into immunostimulatory M1 macrophages, convert immature dendritic cells into mature dendritic cells, and expand effector T cells to generate a responsive tumor microenvironment to promote anti-tumor activity.

[0087] According to example, the synthetic CpG-oligonucleotide (CPG-ON) of the immunostimulatory property of simulation microorganism CpG-DNA can be developed for therapeutic use. According to example, oligonucleotide is oligodeoxynucleotide (ODN). There are many different CpG-ODN classification types, such as Class A, Class B, Class C, Class P and Class S, which share some structural and functional characteristics. In this respect, Class A CPG-ODN (or CPG-A ODN) is associated with pDC maturation, wherein B cell effect is very little, and IFN α induction degree is the highest; Class B CPG-ODN (or CPG-B ODN) strongly induces B cell proliferation, activates pDC and monocyte maturation, NK cell activation and inflammatory cytokine production; and Class C CPG-ODN (or CPG-C ODN) can induce B cell proliferation and IFN-α to produce.

[0088] Further, according to examples, CPG-C ODN can be associated with the following attributes: (i) unmethylated dinucleotide CpG motifs, (ii) CpG motifs juxtaposed with flanking nucleotides (e.g., AACGTTCGAA), (iii) a complete phosphorothioate (PS) backbone connecting nucleotides (contrary 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 ODN may self-attach due to its palindromic properties, thereby generating a double-stranded duplex (e.g., dimer) or a hairpin structure.

[0089] Further, according to example, CpG-C ODN can comprise one or more 5'-TCG trinucleotides, wherein 5'-T is positioned at 0,1,2 or 3 bases from the 5'-end of oligonucleotide, and at least one palindromic sequence of at least 8 bases in length, and this palindromic sequence comprises one or more unmethylated CG dinucleotides.One or more 5'-TCG trinucleotide sequences can be with 5'-end interval 0,1 or 2 bases of palindromic sequence, or palindromic sequence can contain all or part of one or more 5'-TCG trinucleotide sequences.In example, the length of CpG-C ODN is 12 to 100 bases, preferably 12 to 50 bases in length, preferably 12 to 40 bases in length, or preferably 12 to 30 bases in length.In example, the length of CpG-CODN is 30 bases. In an example, the length of 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 an example, the length of 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.

[0090] In an example, the length of at least one palindrome sequence is 8 to 97 bases, preferably 8 to 50 bases, or preferably 8 to 32 bases. In a specific example, the length of at least one palindrome sequence is 8 bases. In an example, the length of at least one palindrome sequence is at least (lower limit) 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 bases. In an example, the length of at least one palindrome 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.

[0091] In an example, the CpG-CODN may comprise the sequence of SEQ ID NO: 1, as shown below.

[0092] According to an example, the CpG-C ODN may comprise SD-101 or a pharmaceutically 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),

[0093] The entire 30-mer sequence is linked by phosphorothioate bonds. SD-101 can be in the form of a free acid. Specifically, the SD-101 drug substance is isolated as a sodium salt. The structure of the sodium salt of SD-101 is as follows Figure 1 shown.

[0094] The molecular formula of SD-101 in free acid form is C 293 H 369 N 112 O 149 P 29 S 29 , and the molecular weight of SD-101 in the free acid form is 9672 Daltons. Figure 1 As shown, the molecular formula of the sodium salt of SD-101 is C 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.

[0095] Further, according to an example, the CPG-C ODN sequence may correspond to SEQ ID NO: 172 as described in US Patent No. 9,422,564, which is herein incorporated by reference in its entirety.

[0096] In an example, the CpG-C ODN may comprise a sequence that is at least 75% homologous to any of the foregoing (such as SEQ ID NO: 1).

[0097] According to another example, the CPG-C ODN sequence may correspond to any of the other sequences described in U.S. Pat. No. 9,422,564. In addition, the CPG-C ODN sequence may also correspond to any sequence described in U.S. Pat. No. 8,372,413, which is also incorporated herein by reference in its entirety.

[0098] According to the example, any one of the CpG-C ODN discussed herein can exist in the form of its pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts of any one of the CpG-C ODN can include organic bases and inorganic bases. Exemplary alkaline salts include ammonium salts, alkali metal salts (such as sodium salts, lithium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts, zinc salts), with organic bases (for example, organic amines) (such as N-Me-D-glucosamine, N-[1-(2,3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, tert-butylamine) salts, and with amino acids (such as arginine, lysine, etc.) salts. In an example, CpG-C ODN is in the form of ammonium salts, sodium salts, lithium salts or potassium salts. In a preferred example, CpG-C ODN is in the form of sodium salts. CpG-C ODN can be provided in a pharmaceutical solution comprising one or more pharmaceutically acceptable excipients. Alternatively, the CpG-CODN can be provided as a lyophilized solid which is subsequently reconstituted in sterile water, saline or a pharmaceutically acceptable buffer prior to administration.

[0099] Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, fillers, buffers, tension regulators, and preservatives. In an example, the pharmaceutical composition may include an excipient that serves as one or more of a solvent, filler, buffer, and tension regulator (e.g., sodium chloride in saline can be used as both an aqueous vehicle and a tension regulator). The pharmaceutical composition of the present disclosure is suitable for parenteral and / or transdermal administration.

[0100] In an example, the pharmaceutical composition comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline solution, phosphate buffered saline and Ringer's solution. In an example, the composition is isotonic.

[0101] The pharmaceutical composition may include a filler. When the pharmaceutical composition needs to be lyophilized before administration, a filler is particularly useful. In an example, the filler is a protective agent that helps to stabilize and prevent degradation of the active agent during freezing or spray drying and / or during storage. Suitable fillers are sugars (monosaccharides, disaccharides and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose and raffinose.

[0102] The pharmaceutical composition may include a buffer. The buffer controls pH to inhibit degradation of the active agent during processing, storage and optional reconstitution. Suitable buffers include, for example, salts comprising acetate, citrate, phosphate or sulfate. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine and lysine. The buffer may further include hydrochloric acid or sodium hydroxide. In some instances, the buffer maintains the pH of the composition in the range of 4 to 9. In an example, the pH is greater than (lower limit) 4, 5, 6, 7 or 8. In some instances, the pH is less than (upper limit) 9, 8, 7, 6 or 5. That is, the pH is in the range of about 4 to 9, wherein the lower limit is less than the upper limit.

[0103] The pharmaceutical composition may contain a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerol and mannitol.

[0104] The pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in an example, the pharmaceutical composition is prepared under sterile conditions and in a disposable container, and therefore does not need to include a preservative.

[0105] Table 1 describes an exemplary batch formulation of SD-101 drug product - 16 g / L:

[0106] Table 1

[0107]

[0108] 1 Based on the amount measured in solution (excluding water present in lyophilized powder)

[0109] *The SD-101 drug substance in Table 1 reflects the sum of all oligonucleotide content, including SD-101.

[0110] In some examples, the unit dosage strength can include about 0.1 mg / mL to about 20 mg / mL. In one example, the unit dosage strength of SD-101 is 13.4 mg / mL.

[0111] CpG-CODN may contain modification.Suitable modification may include but is not limited to modification of 3'OH or 5'OH groups, modification of nucleotide bases, modification of sugar components and modification of phosphate groups.Modified bases may be included in palindromic sequences, as long as the modified bases maintain the same specificity to their natural complement (for example, the palindromic portion of CpG-CODN maintains self-complementarity) by Watson-Crick base pairing.The example of the modification of 5'OH groups may include biotin, cyanine 5.5, cyanine dyes, Alexa Fluor 660, Alexa Fluor dyes, IRDye 700, IRDye 800, IRDye800CW and IRDye dyes.

[0112] CpG-CODN can be linear, circular or include circular portion and / or hairpin loop.CpG-CODN can be single-stranded or double-stranded.CpG-CODN can be DNA, RNA or DNA / RNA hybrid.

[0113] CpG-C ODN can contain naturally occurring or non-naturally occurring bases of modification, and can contain sugar, phosphoric acid and / or the end of modification.For example, except phosphodiester bond, phosphate modification includes but is not limited to methylphosphonate, phosphorothioate, phosphoramidate (bridge joint or non-bridge joint), phosphotriester and phosphorodithioate, and can be used in any combination.In an example, CpG-C ODN only has thiophosphate bond, only has phosphodiester bond, or has the combination of phosphodiester bond and thiophosphate bond.

[0114] Sugar modifications known in the art can also be prepared, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA and 2'-alkoxy-or amino-RNA / DNA chimeras and other sugar modifications described herein, and combined with any phosphate modification. The example of base modification includes but is not limited to adding electron withdrawing moieties to C-5 and / or C-6 (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) of the cytosine of CpG-C ODN and C-5 and / or C-6 (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil) of the uracil of CpG-C ODN. As described above, the use of base modification in the palindromic sequence of CpG-C ODN should not interfere with the self-complementarity of the base involved in Watson-Crick base pairing. However, outside the palindromic sequence, modified bases can be used without such restrictions. For example, 2'-O-methyl-uridine and 2'-O-methyl-cytidine can be used outside of a palindromic sequence, while 5-bromo-2'-deoxycytidine can be used inside and outside of a palindromic sequence. Other modified nucleotides that can be used inside and outside of a palindromic sequence include 7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0115] The duplex (i.e. double-stranded) and hairpin forms of most ODNs are usually in dynamic equilibrium, wherein the hairpin form is usually favorable at low oligonucleotide concentrations and at higher temperatures. Covalent interchain or intrachain crosslinking increases the stability of duplex or hairpin to heat, ion, pH and concentration-induced conformational changes, respectively. Chemical crosslinking can be used to lock polynucleotides into duplex or hairpin forms for physical chemistry and biological characterization. The crosslinked ODN with uniform conformation and "locking" in its most active form (duplex or hairpin form) may be more active than its uncrosslinked counterpart. Therefore, some CpG-C ODNs disclosed herein may contain covalent interchain and / or intrachain crosslinking.

[0116] The technology of preparing polynucleotide and modified polynucleotide is known in the art.Naturally occurring DNA or RNA containing phosphodiester bond can be usually by being coupled to the 5'-hydroxyl group of the ODN in the growth that is attached to the solid support at 3'-end successively by suitable nucleoside phosphoramidite, then the intermediate phosphite triester is oxidized to phosphotriester and synthesized.Use this method, once synthesized required polynucleotide sequence, just remove polynucleotide from support, phosphotriester group is deprotected to phosphodiester, and use ammoniacal liquor or other alkali by nucleoside base deprotection.

[0117] CpG-C ODN can contain phosphoric acid-modified oligonucleotides, and it is known that some of them can stabilize ODN.Therefore, some examples include stable CpG-C ODN.The phosphorus derivative (or the phosphate group modified) that can be connected with the sugar or sugar analog part in the ODN can be monophosphate, diphosphate, triphosphate, alkyl phosphonate, thiophosphate, dithiophosphate, phosphoramidate etc.

[0118] CpG-C ODN can comprise one or more ribonucleotides (containing ribose as the only or main sugar component), deoxyribonucleotides (containing deoxyribose as the main sugar component), modified sugar or sugar analogs. Therefore, except ribose and deoxyribose, sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose and sugar analog cyclopentyl. Sugar can be pyranosyl or furanosyl form. In CpG-C oligonucleotide, sugar moiety is preferably the furanoside of ribose, deoxyribose, arabinose or 2'-O-alkyl ribose, and sugar can be connected to corresponding heterocyclic base with any anomeric configuration. The preparation of these sugars or sugar analogs and the corresponding nucleosides to which these sugars or analogs are attached to heterocyclic base (nucleic acid base) is known per se, therefore it is not necessary to describe here. Sugar modification can also be carried out in the preparation of CpG-C ODN and combined with any phosphate modification.

[0119] The heterocyclic base or nucleic acid base incorporated into the CpG-C ODN can be the naturally occurring major purine and pyrimidine bases (i.e. uracil, thymine, cytosine, adenine and guanine, as described above), as well as naturally occurring and synthetic modifications of the major bases. Therefore, the CpG-C ODN can include one or more of inosine, 2'-deoxyuridine and 2-amino-2'-deoxyadenosine.

[0120] According to another example, the CPG-ODN is one of a class A CPG-ODN (CPG-A ODN), a class B CPG-ODN (CPG-B ODN), a class P CPG-ODN (CPG-P ODN), and a class S CPG-ODN (CPG-S ODN). In this regard, the CPG-A ODN may be CMP-001.

[0121] In another example, the CPG-ODN can be tilsotolimod (IMO-2125).

[0122] Checkpoint inhibitors

[0123] According to an example, the TLR agonist of the present application can be used in combination with a checkpoint inhibitor (CPI). CPI may include a programmed death 1 receptor (PD-1) antagonist. PD-1 antagonists may be any compound or biomolecule that blocks the binding of programmed cell death 1 ligand 1 (PD-L1) expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells or NKT cells), and preferably also blocks the binding of programmed cell death 1 ligand 2 (PD-L2) expressed on cancer cells to PD-1 expressed by immune cells. 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 methods of treatment, medicaments and uses of the present application for treating human subjects, 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.

[0124] According to an 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, and preferably specifically binds to human PD-1 or human PD-L1. mAb may be a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some instances, the human constant region is selected from the group consisting of: IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred instances, the human constant region is IgG1 or IgG4 constant region. In some instances, the antigen binding fragment is selected from the group consisting of: Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0125] According to an example, a PD-1 antagonist may include an immunoadhesin that specifically binds to PD-1 or PD-L1, and preferably specifically binds to human PD-1 or human PD-L1, such as a fusion protein containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (such as the Fc region of an immunoglobulin molecule).

[0126] By way of example, PD-1 antagonists can block PD-L1 expressed by tumor cells and MDSCs and other suppressive immune cells.

[0127] According to the examples, the PD-1 antagonist can inhibit the binding of PD-L1 to PD-1, and preferably can also inhibit the binding of PD-L2 to PD-1. In some examples of the above-mentioned treatment methods, drugs 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.

[0128] According to an 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 four weeks ("Q4W") or 240 mg every two weeks ("Q2W"). According to another example, nivolumab is administered intravenously (IV) via a peripheral vein at a dose of 360 mg nivolumab every three weeks ("Q3W"). In another example, nivolumab administration is based on body weight, with nivolumab 3 mg / kg Q2W or 10 mg / kg Q2W. In another example, nivolumab administration is based on body weight, with nivolumab 1 mg / kg Q3W. In yet another example, nivolumab is administered concomitantly, simultaneously, approximately simultaneously, or on the same day as SD-101. In another example, after administration of one or more cycles of SD-101, nivolumab is administered weekly, every other week, every three weeks, every four weeks, or monthly. A "cycle" of administration of SD-101 is further described below.

[0129] According to another example, pembrolizumab is administered intravenously (IV) via a peripheral vein at a dose of 200 mg Q3W or 400 mg every 6 weeks ("Q6W"). In another example, pembrolizumab is administered concomitantly, simultaneously, approximately simultaneously, or on the same day as SD-101.

[0130] According to another example, the CPI may include a PD-L1 antagonist. In this regard, the PD-L1 antagonist may be one of atezolizumab, avelumab, and durvalumab.

[0131] 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 three weeks. In yet another example, ipilimumab is administered concomitantly, simultaneously, approximately simultaneously, or on the same day as SD-101 and / or nivolumab. In another example, after administering one or more cycles of SD-101 and / or nivolumab, ipilimumab is administered once a week, every other week, every three weeks, every four weeks, or every month.

[0132] Devices for achieving localized regional delivery

[0133] According to an example, any TLR agonist (alone or in combination with a CPI) can be administered using any device that can be used to achieve localized delivery to a tumor, including the catheter itself, or can include a catheter along with other components that can be used in combination with the catheter (e.g., a filter valve, a balloon, a pressure sensor system, a pump system, a syringe, an external delivery catheter, an implantable port, etc.). In some examples, the catheter is a microcatheter.

[0134] In some instances, the device may have one or more properties, including but not limited to self-centering capability, which can provide uniform therapy distribution in the downstream branch network of the blood vessel; anti-reflux capability, which can block or inhibit the retrograde flow of the drug composition for infusion, such as a drug fluid for infusion containing a TLR agonist (e.g., using a valve and filter, and / or a balloon); a system for measuring the pressure in the container; and a device or mechanism for regulating intravascular pressure (e.g., a one-way valve, an intermittent occlusive valve, and / or a porous balloon that dynamically responds to local pressure changes), such as by causing a pressure reduction during placement and during TLR agonist infusion, and a pressure increase during saline bolus or during the bolus of TLR agonists. In some instances, the system is designed to continuously monitor real-time pressure or flow throughout the process. In one instance, a mechanism for regulating pressure produces, causes, and / or contributes to a net increase in fluid pressure in a blood vessel and / or a target tissue or tumor. In a specific instance, a mechanism for regulating pressure can increase the local vascular pressure at the target location, specifically, the increased pressure is greater than the baseline arterial pressure. The mechanism for regulating pressure can operate synchronously with the cardiac cycle and / or promote antegrade flow. In some instances, the mechanism for regulating pressure produces, causes and / or contributes to a net decrease in fluid pressure within a vessel and / or target tissue or tumor. In some instances, the mechanism for regulating pressure produces, causes and / or contributes to a decrease in fluid pressure within a vessel and / or target tissue or tumor first, and then an increase.

[0135] In some examples, the apparatus that can be used to perform the method of the present application is an apparatus as disclosed in the following: 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. 10,813,73 9, 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 Application No. 2020 / 0038586 and U.S. Patent Application No. 2020-0383688, which are incorporated herein by reference in their entirety.

[0136] In some instances, the device is a device as disclosed in US Pat. No. 9,770, 319. In certain instances, the device may be a device known as the Surefire Infusion System.

[0137] In some instances, the device supports measurement of intravascular pressure during use. In some instances, the device is a device as disclosed in U.S. Patent Publication No. 2020-0383688. In some instances, the device may be a device known as the TriSalus Infusion System. In some instances, the device may be a device known as the Device for infusion system. A single-lumen catheter equipped with a one-way valve that responds dynamically to local pressure and flow changes, such as those caused by the cardiac cycle or generated by infusion. The valve structure regulates distal vascular pressure and blood flow. This in turn may alter therapeutic distribution and first-pass absorption due to increased contact time within the vasculature.

[0138] In some instances, a TLR agonist can be administered via a PEDD through the device. In some instances, a TLR agonist can be administered while monitoring the pressure in the blood vessel, which can be used to adjust and correct the positioning of the device at the infusion site and / or adjust the infusion rate. The pressure can be monitored by, for example, a pressure sensor system comprising one or more pressure sensors.

[0139] Infusion rate can be adjusted to change vascular pressure or flow, which can promote TLR agonist to penetrate into target tissue or tumor or its surface and / or be attached to target tissue or tumor or its surface. In some instances, the injection pump as a part of the delivery system or any other method (e.g., infusion flow rate regulating device) can be used to adjust and / or control the infusion rate. In some instances, the pump system can be used to adjust and / or control the infusion rate. In some instances, the infusion rate using the pump system can be about 0.1cc / min to about 40cc / min, or about 0.1cc / min to about 30cc / min, or about 0.5cc / min to about 25cc / min, or about 0.5cc / min to about 20cc / min, or about 1cc / min to about 15cc / min, or about 1cc / min to about 10cc / min, or about 1cc / min to about 8cc / min, or about 1cc / min to about 5cc / min. Further, the infusion rate using a bolus infusion can be about 30cc / min to about 360cc / min, or about 120cc / min to about 240cc / min. In one example, the SD-101 infusion process lasts for about 10-200 minutes. In another example, the SD-101 infusion process lasts for about 10-60 minutes. In another example, the SD-101 infusion process lasts for about 25 minutes.

[0140] Methods comprising administration to the liver

[0141] In an example, the method of the present application includes a method for 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 by a HAI device. HAI refers to infusion of treatment into the hepatic artery of the liver or in a branch of the hepatic artery. According to an embodiment, one or more TLR agonists are introduced by percutaneously introducing a device into a branch of the hepatic artery or portal vein, the device being a device such as a catheter and / or promoting pressure to enable delivery. According to an example, the TLR agonist is a TLR9 agonist, more specifically, a C-class TLR9 agonist, and in some instances, a C-class TLR9 agonist is SD-101. In an example, the patient is a human patient.

[0142] According to another example, the method of the present application includes a method for treating liver immune dysfunction and tumor-induced immune dysfunction, the method comprising administering a Toll-like receptor agonist to a patient in need thereof, wherein the Toll-like receptor agonist is administered to the liver by a device by PVI. PVI refers to infusing the treatment into the hepatic portal vein system. According to an example, one or more Toll-like receptor agonists are introduced by percutaneously introducing a device into a branch of the hepatic portal vein system, the device being a device such as a catheter and / or facilitating pressure-enabled delivery. According to an example, the Toll-like receptor 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 administering to a male or female subject aged eighteen years or older.

[0143] According to another example, the methods of the present application can be administered with other cancer therapeutics such as immunomodulators, tumor killing agents, and / or other targeted therapeutics.

[0144] According to an example, TLR9 agonist therapy, in particular therapy with a class C TLR9 agonist, can be administered in combination with cell therapy (thereby achieving cell therapy by modulating the immune system), chemoembolization, or radioembolization.

[0145] In one example, the above method of administering to the liver is intended to cause a Toll-like receptor agonist, specifically a TLR9 agonist, more specifically a C-class TLR9 agonist, and even more specifically SD-101 to penetrate throughout a solid tumor, throughout an entire organ, or substantially throughout the entire tumor. In an example, such methods enhance the perfusion of a Toll-like receptor agonist to a patient in need thereof, including by overcoming the interstitial fluid pressure and solid stress of the tumor. In another example, perfusion throughout an entire organ or a portion thereof can provide benefits for the treatment of a disease by completely exposing the tumor to the therapeutic agent. In an example, such methods are better able to deliver a Toll-like receptor agonist to a tumor region that is difficult to access the systemic circulation. In another example, such methods deliver a higher concentration of a Toll-like receptor agonist to such a tumor, while delivering less Toll-like receptor agonists to non-target tissues, compared to conventional systemic delivery via a peripheral vein. Non-target tissues are tissues that are directly perfused through an arterial network directly connected to an infusion device. In one example, such methods result in a reduction in size, a reduction in growth rate, or a reduction or elimination of a solid tumor.

[0146] The method of the present application may also include mapping the blood vessels leading to the right and left lobes of the liver before performing HAI, or selectively infusing into specific sectors or segments, and when necessary, occluding blood vessels that do not lead to the liver or are not targets. In some examples, prior to infusion, the patient may undergo a mapping angiogram, for example, via a femoral artery approach.

[0147] Methods for drawing blood vessels in vivo and delivering therapeutic agents are well known to those of ordinary skill in the art. For example, occlusion can be achieved by using micro-coil embolization, which allows practitioners to block off-target arteries or blood vessels, thereby optimizing the delivery of modified cells to the liver. Micro-coil embolization can be performed as needed, such as before applying the first dose of TLR9 agonists, particularly C-class TLR9 agonists, more particularly SD-101, to promote the best infusion of the pharmaceutical composition comprising TLR9 agonists. In another example, sterile sponges (e.g., GELFOAM) can be used. In this regard, sterile sponges can be cut and pushed into catheters. In another example, sterile sponges can be provided as particles.

[0148] In some examples, a TLR9 agonist, particularly a class C TLR9 agonist, such as SD-101, can be administered at a dose of 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 at doses of 12 mg, 16 mg, and 20 mg. Administration of milligram amounts of SD-101 (e.g., about 2 mg) describes administration of about 2 mg. Figure 1 For example, such an amount of SD-101 (e.g., an amount of about 2 mg) may also be present in a composition containing materials other than such an amount of SD-101, such as other related and unrelated compounds. Equivalent molar amounts of other pharmaceutically acceptable salts are also contemplated.

[0149] In some instances, TLR9 agonists, specifically C-class TLR9 agonists, such as SD-101, can be between about 0.01mg and about 20mg, between about 0.01mg and about 10mg, between about 0.01mg and about 8mg, and between about 0.01mg and about 4mg. In some instances, TLR9 agonists, specifically C-class TLR9 agonists, such as SD-101, can be between about 2mg and about 10mg, between about 2mg and about 8mg, and between about 2mg and about 4mg. In some instances, TLR9 agonists, specifically C-class TLR9 agonists, such as SD-101, can be less than about 10mg, less than about 8mg, less than about 4mg, or less than about 2mg. Such dosages can be administered every day, weekly, or every other week. In one example, the dosage of SD-101 increases incrementally, such as by administering about 2mg, followed by about 4mg, and then followed by about 8mg.

[0150] In some examples, the method of the present application may include administering a dosing regimen including a cycle, wherein one or more cycles in the cycle include administering SD-101 via HAI and / or PEDD. As used herein, a "cycle" is a repetition of an administration sequence. In one example, a cycle includes three weekly doses per cycle (i.e., SD-101 is administered once a week for three consecutive weeks). In one example, a treatment cycle according to the present application may include an SD-101 administration phase, followed by an "off" phase or a rest phase. In another example, in addition to three weekly doses per cycle, the cycle further includes one week, two weeks, three weeks, or four weeks as a rest phase after weekly administration of SD-101. In yet another example, in addition to three weekly doses per cycle, the cycle further includes about thirty-eight days as a rest phase after weekly administration of SD-101. In another example, the entire cycle includes about fifty-two days. In another example, the dosing regimen includes at least one, at least two, or at least three cycles or longer.

[0151] In some examples, the present application relates to the use of a TLR9 agonist, specifically a class C TLR9 agonist, in the preparation of a medicament for treating liver immune dysfunction and tumor-induced immune dysfunction, the method comprising administering a TLR9 agonist, more specifically a class C TLR9 agonist, to a patient in need thereof, wherein the TLR9 agonist, more specifically a class C TLR9 agonist, is administered to the liver by a HAI through a device.

[0152] In some instances, SD-101 is administered at a dose of 2 mg by HAI for the treatment of liver immune dysfunction, and in some instances, is further administered by a device that regulates pressure (i.e., PEDD). In some instances, SD-101 is combined with a CPI and administered at a dose of 2 mg by HAI through a device that regulates vascular pressure, wherein the CPI is nivolumab. In other instances, SD-101 is administered in combination with ipilimumab by HAI and by a device that regulates pressure at a dose of 2 mg. In some instances, SD-101 is administered in combination with ipilimumab and nivolumab by HAI and by a device that regulates pressure at a dose of 2 mg. In other instances, SD-101 is administered in combination with pembrolizumab by HAI and by a device that regulates pressure at a dose of 2 mg.

[0153] In some instances, SD-101 is administered at a dose of 4 mg by HAI SD-101 is used to treat liver immune dysfunction, and in some instances, is further administered by a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD). In some instances, SD-101 is administered in combination with CPI at a dose of 4 mg by HAI by a device that regulates vascular pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD). CPI can be administered systemically or locally. For example, CPI can be administered intravenously (IV), subcutaneously (SQ), intraperitoneally (IP), or by HAI, such as, for example, by a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD).

[0154] In some instances, SD-101 is administered at a dose of 4 mg in combination with a CPI by HAI through a device that regulates vascular pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD), wherein the CPI is nivolumab. In other instances, SD-101 is administered at a dose of 4 mg in combination with ipilimumab by HAI and through a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD). In some instances, SD-101 is administered at a dose of 4 mg in combination with ipilimumab and nivolumab by HAI and through a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD). In other examples, SD-101 is administered at a dose of 4 mg in combination with pembrolizumab via a HAI and via a device that modulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), either systemically (e.g., IV or SQ) or locally (e.g., IP or via a HAI, such as via PEDD).

[0155] In some examples, SD-101 is administered at a dose of 8 mg by HAI for the treatment of liver immune dysfunction, and in some embodiments, SD-101 is further administered by a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD). In some examples, SD-101 is administered at a dose of 8 mg by HAI through a device that regulates vascular pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD) in combination with a CPI, wherein the CPI is nivolumab, administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD). In other examples, SD-101 is administered at a dose of 8 mg by HAI and by a device that regulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD) in combination with ipilimumab, administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD). In some examples, SD-101 is administered at a dose of 8 mg in combination with ipilimumab and nivolumab by HAI and by a device that modulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD). In other examples, SD-101 is administered at a dose of 8 mg in combination with pembrolizumab by HAI and by a device that modulates pressure (e.g., dynamically responds to local pressure changes, more particularly, PEDD), systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD).

[0156] In some instances, the method of the present application causes treatment of target lesions, such as target liver cancer lesions or tumors. In this instance, the method of the present application may cause a complete response, including the disappearance of all target lesions (e.g., target liver cancer lesions or tumors). In some instances, the method of the present application may cause a partial response, including a reduction of at least 30% in the sum of the longest diameters of the target lesions (e.g., target liver cancer lesions or tumors), with the baseline sum of the longest diameters as a reference. In some instances, the method of the present application may cause a disease of a stable target lesion (e.g., target liver cancer lesions or tumors), including neither sufficient shrinkage to meet the conditions of a partial response nor sufficient increase to meet the conditions of a progressive disease, with the smallest sum of the longest diameters since the start of treatment as a reference. In such instances, the progressive disease is characterized by an increase of at least 20% in the sum of the longest diameters of the target lesions (e.g., target liver cancer lesions or tumors), with the smallest sum of the longest diameters recorded since the start of treatment or the appearance of one or more new lesions as a reference. The sum must be demonstrated to be or be about an absolute increase of 5 mm. In some instances, the response in the form of size reduction may be offset by tumor killing and inflammation leading to tumor swelling (termed pseudoprogression).

[0157] In another example, the method of the present application leads to the treatment of non-target lesions. Non-target lesions are lesions that are not directly perfused through an arterial network that is directly connected to an infusion system. In this example, the method of the present application may cause a complete response, including the disappearance of all non-target lesions or the reduction of all viable tumors. In some examples, the method of the present application causes the persistence of one or more non-target lesions without causing a complete response or progressive disease. In such examples, progressive disease is characterized by a clear progression of existing non-target lesions, and / or the appearance of one or more new lesions.

[0158] In some instances, the method of the present application causes the duration of overall response to increase. In some instances, the duration of overall response is measured from the time when complete response or partial response (based on the first recorded) meets the measurement criteria, until objectively recording the first day of recurrent or progressive disease (with the minimum measured value recorded since the start of treatment as a reference for progressive disease). The duration of overall complete response can be measured from the time when complete response first meets the measurement criteria, until objectively recording the first day of recurrent or progressive disease, for example, objectively recording the first day of progressive disease. In some instances, the duration of stable disease is measured from the start of treatment until progressive disease is observed, for example, the standard of progress is met, with the minimum measured value (including baseline measurement value) recorded since the start of treatment as a reference.

[0159] In other examples, the method of the present application causes an improvement in overall survival. For example, the overall survival can be calculated from the start of treatment date to the time of death. In a specific example, in a clinical trial for treatment, the registration date is to the time of death. Patients who are still alive before the data cutoff of the final efficacy analysis or who withdraw before the end of the study in a clinical trial study will be examined on the day they are last known to survive.

[0160] In other examples, the method of the application causes progression-free survival. Progression-free survival can be determined from the start-up treatment date until recurrence or death (whichever occurs first). In a specific example, for example, progression-free survival can be calculated from the clinical trial enrollment date for treatment to the CT scan time or death date (whichever occurs first) of recording recurrence (or other clear indications of disease development). Patients who do not record recurrence and are still alive before the final efficacy analysis data cutoff or withdraw before the end of the study will be reviewed on the last radiological evidence date of recording no recurrence.

[0161] 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) that reduces the ability of the patient's immune system to attack cancer cells and / or the ability of immunotherapy to induce beneficial therapeutic responses. The characteristics of liver immune dysfunction can be many different biological features, such as, for example, increased levels of MDSC present in liver TME, reduced levels of T cells and / or macrophages (e.g., M1 and / or M2 macrophages) present in liver TME, reduced 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), where migration to liver metastases; activation and mobilization of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages); increased TLR signaling; increased leukocytes (CD45+); exhausted CD8 Increased T cells (which is very important for checkpoint responsiveness); induction of Th1 programming; decreased Th2 programming; increased T cell receptor and T cell co-stimulatory signaling; increased IL9, IL15, CCL7; B cell activation; induction of mast cells; induction of NK cells; induction of IFNg and related genes; increased interferon signaling; increased chemokine signaling; increased IL6 in the liver but not in the blood; increased M1 / activated macrophage genes: CD68, CD86; dendritic cell activation and migration to draining lymph nodes; decreased MDSC-related genes: IDO1, TOD2, ARG-1, NOS2, TIMD4; decreased angiogenesis and VEGF; decreased fatty acid oxidation (preferred MDSC program); and / or increased PD-L1, IFNγ, and IP10 gene expression.

[0162] In one example, the method of the present application includes a method for treating liver immune dysfunction, wherein the administration of SD-101 causes a reduction in tumor load. Any suitable method can be used to determine the tumor load, such as the length of the longest diameter of the tumor lesion or the volume of the tumor lesion. In some examples, the tumor load 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, the tumor load 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 the tumor lesion. In another 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 volume of tumor lesions.

[0163] According to another example, the method of the present application includes a method for treating liver immune dysfunction, wherein the administration of SD-101 causes a reduction in tumor progression or a stabilization of tumor growth. In some instances, 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%. Any suitable method can be used to determine tumor load, such as the length of the longest diameter of a tumor lesion or the volume of a tumor lesion. 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 shortest diameter of a tumor lesion. In another 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 the tumor lesions, as determined by the volume of the tumor lesions.

[0164] According to another example, the method of the present application includes a method for treating liver immune dysfunction, wherein the administration of SD-101 reduces MDSC in the liver TME, enabling increased immune control of liver cancer and / or improved responsiveness to systemic anti-PD-1 therapy. For example, the method for treating liver immune dysfunction includes administering SD-101 to reprogram the liver MDSC compartment to enable immune control of liver cancer and / or improve responsiveness to systemic anti-PD-1 therapy by eliminating MDSC from the liver TME. In some instances, the method of the present application is superior in controlling the amount of MDSC in the liver TME. In some examples, the method of the present application includes a method for treating liver immune dysfunction, wherein the administration of SD-101 reduces the frequency of MDSC cells (e.g., CD11b+Gr1+), mononuclear MDSC (M-MDSC; e.g., CD11b+Ly6C+) cells, granulocyte MDSC (G-MDSC; e.g., CD11b+LY6G+) cells and / or human MDSC (e.g., CD33+CD11b+HLADR- (e.g., CD14+ for m-MDSC, CD15+ for G-MDSC)) in the liver TME. According to another example, the method of the present application increases the amount of M1 macrophages (e.g., CD14+CD86+) in the liver TME. According to yet another example, the method of the present application reduces M2 macrophages (e.g., CD14+CD163+CD206+) in the liver TME. According to yet another example, the method of the present application increases mononuclear dendritic cells (e.g., CD14+CD163+CD206+) in the liver TME. + CD11c +CD123 - ). According to an example, the MDSC, M1, M2 and dendritic cell effectors as described in this paragraph are applicable to human phenotypes, including 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 - ).

[0165] In another example, the method of the present application increases NFκB activation and other molecules associated with TLR9 signaling in, for example, liver TME. In yet another additional example, the method of the present application increases IL-6 in, for example, liver TME. In another example, the method of the present application increases IL10 in, for example, liver TME. In yet another additional example, the method of the present application increases IL-29 in, for example, liver TME. In another example, the method of the present application increases IFNα in, for example, liver TME. As another example, the method of the present application reduces STAT3 phosphorylation in, for example, liver TME.

[0166] In another example, the method of the present application includes a method for treating liver immune dysfunction, wherein administration of a TLR9 agonist, particularly a class C TLR9 agonist, and more particularly SD-101, causes at least one of the following changes in gene expression within liver metastases:

[0167] Activation of immune cells in the normal liver (non-tumor liver of the same patient) that migrate to liver metastases;

[0168] ●Activation and mobilization of peripheral blood immune cells (T, NK, B, cytotoxic T, CD4 Th1 T, exhausted T, macrophages);

[0169] ●Increased TLR signaling; increased leukocytes (CD45+);

[0170] ● Increase in exhausted CD8 T cells (which are important for checkpoint responsiveness);

[0171] ●Induction of Th1 programming;

[0172] ●Reduction of Th2 programming;

[0173] Increased T cell receptor and T cell co-stimulatory signaling

[0174] Increased IL9, IL15, and CCL7

[0175] B cell activation

[0176] ●Induction of mast cells;

[0177] ●NK cell induction;

[0178] ●Induction of IFNg and related genes;

[0179] ●Increased interferon signaling;

[0180] ●Increased chemokine signaling;

[0181] IL6 increases in the liver but not in the blood.

[0182] ● Increase in M1 / activated macrophage genes: CD68, CD86 dendritic cells activate and migrate to draining lymph nodes;

[0183] ●Decrease in MDSC-related genes: IDO1, TOD2, ARG-1, NOS2, TIMD4;

[0184] ● Decreased angiogenesis and VEGF;

[0185] ● Reduced fatty acid oxidation (preferred MDSC procedure); and / or

[0186] ●Increased expression of PD-L1, IFNγ, and IP10 genes.

[0187] In another embodiment, the method of the present application includes a method for administering a TLR 9 agonist, specifically a class C TLR9 agonist, specifically SD-101, and a checkpoint inhibitor to a patient in combination, wherein the combined administration of a TLR9 agonist, specifically a class C TLR9 agonist, and more specifically SD-101 and a CPI causes a decrease in circulating tumor cell levels and / or circulating tumor DNA levels, thereby causing a decrease in tumor burden. In one example, the CPI can be administered systemically (e.g., IV or SQ) or locally (e.g., IP or by HAI, such as via PEDD).

[0188] The present application will be further described and / or illustrated in the following examples, which are given for the purpose of illustration / illustration only and are not intended to limit the present application in any way.

[0189] Examples

[0190] Example 1:

[0191] 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 was used to induce MDSCs. The treated cells were then evaluated using flow cytometry to determine the effects of different TLR agonists on MDSCs (M-MDSCs and G-MDSCs) and macrophages (M1 and M2 macrophages). The data generated by flow cytometry is shown in Figure 1. Figures 2A to 2D shown.

[0192] Figures 2A to 2E Shown is an in vitro analysis of human PMBCs harvested from healthy donors and treated with IL6 and GM-CSF to induce MDSCs according to Example 1.

[0193] Specifically, Figure 2A The amount of total MDSCs induced in the in vitro samples (ie, NT (untreated) or treated with TLR4 agonist, TLR7 agonist, TLR7 agonist, TLR9A agonist, TLR9B agonist or SD101) is shown. Figure 2A The data demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists, and SD-101 on the amount of total MDSCs induced by treatment of human PBMCs with IL6 and GMCSF compared to untreated controls. Figure 2A In the table, * indicates p<0.05 compared with NT, ** indicates p<0.01 compared with NT, # indicates p<0.05 compared with SD-101, and & indicates p=0.006 compared with SD-101. Figure 2B To demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs) induced by treatment of human PBMCs with IL6 and GMCSF compared to untreated controls. Figure 2B *** indicates p<0.001 compared with NT, **** indicates p<0.0001 compared with NT, % indicates p<0.01 compared with SD-101, and ^ indicates p<0.001 compared with SD-101. Figure 2A and 2B As shown, SD-101 demonstrated (i) better elimination of major MDSC subsets in the liver, such as M-MDSCs, and (ii) a more favorable effect on the M1 / M2 macrophage ratio (ie, induction of M1 and reduction of M2). Figure 2C and 2DTo demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the number of M1 macrophages and M2 macrophages induced by treatment of human PBMCs with IL6 and GMCSF, respectively, compared to untreated controls. Figure 2C In the table, * indicates p<0.05 compared with NT, ** indicates p<0.01 compared with NT, # indicates p<0.05 compared with SD-101, and @ indicates p<0.00001 compared with SD-101. Figure 2D In the table, * indicates p < 0.05 compared with NT. Figures 2A to 2D The number of different human donors was n=5 for each of the datasets shown in .

[0194] Figure 2E Images showing MDSC cells induced from PMBCs when cells were treated with a TLR9 agonist.

[0195] Given that Figures 2A to 2E The data shown in these in vitro studies of MDSC induction in human PMBC treated with IL6 and GM-CSF demonstrated that SD-101 was superior to class B TLR9 agonists and TLR7 agonists in terms of reducing MDSC, reducing M2 macrophages, and promoting M1 macrophages.

[0196] Example 2:

[0197] 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 was used to induce MDSCs. The treated cells were then evaluated using flow cytometry to determine the effects of different TLR agonists on total MDSCs, total macrophages, and M1 macrophages in this mouse model. The data generated by flow cytometry are shown in Figure 2. Figures 3A to 3C shown.

[0198] Figure 3A The effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of MDSCs (CD11b+Gr-1+) induced by treatment of murine bone marrow cells with GMCSF were demonstrated compared to untreated controls. Figure 3A In the table, ** indicates p<0.01 compared with NT, and **** indicates p<0.0001 compared with NT. Figure 3B To demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of macrophages (CD11b+F4 / CD80+) induced by treatment of murine bone marrow cells with GMCSF compared to untreated controls. Figure 3BIn the table, *** indicates p<0.001 compared with NT, * indicates p<0.05 compared with NT, and @ indicates p<0.05 compared with SD-101. Figure 3C To demonstrate the effects of TLR4 agonists, TLR7 agonists, TLR9A agonists, TLR9B agonists and SD-101 on the amount of M1 macrophages (CD11b+F4 / CD80+CD38+) induced by treatment of murine bone marrow cells with GMCSF compared to untreated controls. Figure 3C In the table, *** indicates p<0.001 compared with NT, ** indicates p<0.01 compared with NT, * indicates p<0.05 compared with NT, and @ indicates p<0.05 compared with SD-101. Figures 3A to 3C The number of different human donors was n=3 for each of the datasets shown in .

[0199] like Figures 3A to 3C As shown in the data, in vitro studies of mouse bone marrow cells have demonstrated that SD-101 is also superior to class B TLR9 agonists and TLR7 agonists for mouse bone marrow cell programming. Specifically, Figures 3A to 3C Depicts in vitro analysis of murine bone marrow cells from mice with or 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.

[0200] Example 3:

[0201] In Example 3, the infusion of the class C TLR9 agonist, SD-101, was administered via hepatic artery infusion (PEDD / HAI) with the goal of enhancing the response rate to CPI therapy in patients with uveal melanoma liver metastases. Four patients were enrolled, and in the first cycle, the patients were treated at a dose of 2 mg SD-101 for each of three weekly SD-101 infusions via HAI and PEDD. The pharmacokinetic (PK) data collected in this study showed that the PEDD / HAI delivery route was achieving its goal of obtaining high concentrations of SD-101 in liver tissue and transient and low levels of systemic exposure to SD-101, as determined by plasma levels (<2 hours, <150 ng / ml). Cell-mediated anti-tumor immunity (Th1, TCR / co-stimulation, CD8 T cells, cytotoxic cells, B cells, NK cells) increased in uveal melanoma liver metastases. The simultaneous reduction of normal liver tissue indicates metastasis to adjacent tumors, thereby showing the importance of treating the entire immunosuppressive organ. With the induction of serum IFNγ, CD8 T, Th1, B and NK in peripheral blood increased at a later time point. For cytokine programming, IFNγ-related genes increased in liver metastases, and serum increased; and with Th2 reduction, Th1 genes increased in liver metastases. MDSC-associated genes decreased (NOS-2, ARG-1, IDO-1, TDO-2). Changes in the liver were observed four weeks after three infusions of 2mg SD-101 via PEDD.

[0202] Post-treatment liver metastasis samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The immune cell panel was tested to analyze the expression of immune cell gene signatures including cytotoxic cells, dendritic cells (DC), CD45, macrophages, T cells, mast cells, neutrophils, exhausted CD8, Th1 cells, and CD8 T cells. The data of immune cell group are shown in Figure 4 , 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. Figure 4 Figure 2. Post-treatment liver metastasis samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The immune cell panel showed, among others, an increased gene signature including cytotoxic cells, macrophages, T cells, and exhausted CD8 T cells; and a decreased gene signature including dendritic cells, probably 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 before treatment and two after treatment).

[0203] Post-treatment liver metastasis samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The bone marrow panel was tested to analyze the expression of myeloid gene signatures including angiogenesis, antigen expression, 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. The data from the bone marrow group showed Figure 5 , 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. Figure 5 Figure 2. Post-treatment liver metastasis samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The bone marrow group showed, among others, an increased gene signature including lymphocyte activation, Th1 activation, interferon and TLR signaling, antigen presentation, exhausted CD8 T cells; and a decreased gene signature including Th2 activation and angiogenesis. In this regard, liver metastasis samples were taken from four patients (average relative gene expression levels from four before treatment and two after treatment).

[0204] Post-treatment liver metastasis samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The bone marrow cell panel was tested to analyze the expression of gene signatures of myeloid cells, including mast cells, exhausted CD8, macrophages, B cells, CD8 T cells, DCs, T cells, neutrophils, and CD45. The data from the bone marrow cell group 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 3 weekly infusions. Figure 6 Figure 2. Post-treatment liver metastasis samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The myeloid cell panel showed, among others, an increased gene signature including B cells, CD8 T cells, cytotoxic cells and macrophages, and a decreased gene signature including dendritic cells, probably due to migration to draining lymph nodes. In this regard, liver metastasis samples were obtained from four patients (average relative gene expression levels from four before treatment and two after treatment).

[0205] Post-treatment liver metastasis samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The metabolome was tested to analyze 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, hypoxia IDH12 activity, KEAP1NBRF2 pathway, lysosomal degradation, MAPK, mitochondrial respiration, mTOR, Myc, NF-κB, nucleotide recycling, nucleotide synthesis, p53 pathway, pentose phosphate pathway, P13K, reactive oxygen species response, TCR and co-stimulatory signaling, TLR signaling, transcriptional regulation, tryptophan kynurenine metabolism, and vitamin and cofactor metabolism. Metabolomics data are 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 3 weekly infusions. Figure 7 Figure 2. Post-treatment liver metastasis samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The metabolome showed, among others, an increase in gene signatures including antigen presentation, chemokine and cytokine signaling, and TCR and co-stimulatory signaling, a decrease in gene signatures including cell cycle and fatty acid oxidation, and a decrease in MDSC-associated genes including ARG1, NOS2, and GM-CSF-R. In this regard, liver metastasis samples were obtained from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0206] Post-treatment liver metastasis samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The bone marrow panel was tested to analyze the expression of a bone marrow gene signature, including genes associated with inflammation and immune cell function as well as genes associated with immune suppression and tumor progression. The data from the bone marrow group showed Figure 8 In. Figure 8 Figure 2. Post-treatment liver metastasis samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The bone marrow group shows that the gene expression associated with inflammation and immune cell function increases (e.g., SERPINB2, TREM2, CD18, IL9, CD68, ELANE, IFNA2, TREM1, TNFRSF11A, CD86, PDZK1IP1, IL15RA, IL6, CCL7, IL2, CCL2, CCL8, PRG2 and IFNGR1), and the gene expression associated with immunosuppression and tumor progression decreases (e.g., CSF2, RSGRF2, JUN, PROk2, GKS, VEGA, MET, CXCL1, TIMD4). Specifically, the figure shows that gene signatures have been increased, including TREM2 (associated with TMB and MSI), IFNA2 and IFNGR1, CD68, CD86, IL2, IL9, IL15 and CCL7; and gene signatures have been reduced, including CSF2 (GM-CSF), IDO-1, TDO-2, VEGFA and TIMD4 (roles in MDSC suppressive function). In this regard, liver metastasis samples were obtained from four patients (average relative gene expression levels from four pre-treatment and two post-treatment).

[0207] Post-treatment normal liver tissue samples from these patients (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD) were collected at The myeloid cell panel was tested to analyze the expression of gene signatures of myeloid cells, including cytotoxic cells, DCs, CD45, macrophages, T cells, mast cells, neutrophils, NK cells, exhausted CD8, Th1 cells, and CD8 T cells. The data from the bone marrow cell group are shown in Fig. 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. Fig. 9 Figure 2. Post-treatment normal liver tissue samples (collected four weeks after the first cycle of three weekly SD-101 infusions via HAI and PEDD). The bone marrow panel showed that SD-101 infusion was associated with a decrease in Th1 cells, CD8 T cells, and cytotoxic cells in the normal liver parenchyma. The opposite changes were observed in tumor cells. The lack of immune cell expansion / activation supports the safety of SD-101 with respect to its effects on normal liver tissue. It is hypothesized that SD-101 treats immune dysfunction in the liver and tumor microenvironment, thereby promoting the recruitment of activated T cells from the normal liver into the “cold” tumor. In other words, Fig. 9 Normal liver tissue Data indicate intrahepatic immune cell motility.

[0208] Post-treatment peripheral blood mononuclear cell (PBMC) samples from these patients were The bone marrow cell panel was tested to analyze the gene signature expression 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. The data from the bone marrow cell group are shown in Fig.10 middle. Fig.10 The peripheral blood mononuclear cell (PBMC) samples after treatment are shown. Bone marrow group. In this regard, a single infusion of .5mg SD-101 was followed by two cycles of three weekly 2mg SD-101 infusions with tissue collection performed in the first week of each of the two cycles. As shown, hepatic arterial infusion of SD-101 with PEDD was associated with increases in NK cells, Th1 cells, B cells, CD8 T cells, and cytotoxic cells in the peripheral blood. These increases were noted following favorable immune changes within liver metastases, which included the delivery of four to six doses in the second cycle. It is hypothesized that SD-101 treats immune dysfunction in the liver and tumor microenvironment, thereby promoting the migration of activated dendritic cells and other immune cells from the reprogrammed liver. These PBMC gene expression changes suggest systemic activity following liver-directed SD-101 infusion.

[0209] In summary, SD-101 and PEDD provide a tumor microenvironment modulation pathway that addresses an important immunosuppressive target (MDSC) with additional broad CPI promotion while treating immune dysfunction in the surrounding non-tumor liver.

[0210] Example 4:

[0211] In Example 4, the effects of delivering various TLR agonists (TLR4, TLR7, class B TLR9 (TLR9B) agonists, and class C TLR9 (TLR9C) agonists) to bone marrow cells were evaluated, specifically investigating their differential effects on MDSCs and their potential to immunomodulate the liver tumor microenvironment (TME).

[0212] 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 a broader range of immunological effects. Specific delivery challenges and immunosuppressive pathways, including the unique programming of myeloid-derived suppressor cells (MDSC), may limit the success of immunotherapy in the liver.

[0213] The effects of various TLR agonists were evaluated in vitro by flow cytometry using different cell types obtained from healthy human donors and also using murine bone marrow (BM) MDSCs.

[0214] 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 was used to induce MDSCs. The treated cells were then evaluated using flow cytometry to determine the effects of SD-101 on MDSCs (e.g., CD33 + CD11b + HLADR - / lo ) and M1 macrophages (e.g. CD86 + ). Data generated by flow cytometry using these cells provide Figures 11A to 11D middle.

[0215] Figures 11A to 11D TLR9 stimulation with SD-101 was shown to inhibit MDSC generation and enhance M1 macrophage production. Figures 11A to 11D Data were generated using human PBMC treated with increasing concentrations (0.1-3 μM) of SD-101 and Ctrl ODN5328 (1 μM) for 2 days in the presence / absence of IL6+GMCSF (20 ng / ml each) ( Fig.11A and 11C ) and 7 days ( Fig. 11B and 11D ).exist Fig.11A and 11B The effects of SD-101 or Ctrl ODN5328 treatment on the MDSC population (CD33 + CD11b + HLADR - / lo ). Fig. 11C and 11D In this study, the effects of SD-101 or Ctrl ODN5328 treatment on M1 macrophages (CD14 + CD86 + ) Figures 11A to 11D As shown, 0.3 μM SD-101 dose inhibited MDSC generation and increased M1 macrophage production after 7 days of treatment. Figures 11A to 11D The data shown in were subjected to one-way ANOVA followed by Tukey's post hoc test, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 relative to IL6+GMCSF stimulated cells treated with 1 μM Ctrl ODN, n=number of healthy donors. Example 5:

[0216] In Example 5, PBMCs obtained from healthy human donors were cultured in vitro in IL6+GMCSF (20 ng / ml) to induce MDSCs, and then treated with TLR agonists. Flow cytometry was performed to evaluate the effects of different TLR agonists on MDSCs (CD33 + CD11b + HLADR - ), MDSC subtypes (monocyte / granulocyte CD14 + / CD15 + MDSC), M1 macrophages (CD14 + CD86 + ) and mononuclear dendritic cells (CD14 + CD11c + CD123 - ). The TLR agonists evaluated included TLR4 agonists, TLR7 agonists, class B TLR9 agonists, and SD-101 (which is a class C TLR9 agonist). In addition, bone marrow (BM) mouse MDSCs (CD11b + GR-1 + ) for 72 hours and the effects of different TLR agonists on mouse BM MDSCs were evaluated by flow cytometry. The data generated from flow cytometry of different types of human and mouse cells are provided in Figures 12A to 12F middle.

[0217] Figures 12A to 12F The effects of different TLR agonists on human MDSCs and other immune cells (ie, murine immune cells) are shown. Figures 12A to 12D The data were generated using PBMC obtained from healthy human donors that had been stimulated with IL6+GMCSF (20 ng / ml each) and with vehicle or 0.3 μM of a TLR4 agonist, a TLR7 agonist, a class B TLR9 agonist, or SD-101 (which is a class C TLR9 agonist) for 7 days. Fig. 12A In this study, the effects of treatment with different TLR agonists on MDSCs were evaluated. Fig. 12B In the present study, the effects of treatment with different TLR agonists on M1 macrophages were evaluated. Fig. 12C In this study, the effects of treatment with different TLR agonists on M / G-MDSCs were evaluated. Fig.12D In this study, we evaluated the effects of different TLR agonists on dendritic cells (CD33 + CD11c + )’s role.

[0218] in addition, Fig.12E and 12FShown are the effects of different TLR agonists on murine BM MDSCs. Fig.12E The data were generated using cells obtained from the bone marrow (BM) of mice treated in the absence of murine GMCSF. Fig.12F The data were generated using cells obtained from the bone marrow (BM) of mice treated in the presence of murine GMCSF. Figures 12A to 12F The data shown in were subjected to Student's t-test and one-way ANOVA, followed by Tukey's post-hoc test, where * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 relative to vehicle. In addition, % indicates p<0.01 relative to TLR4, & indicates p<0.01 relative to TLR7, ^ indicates p<0.001 relative to TLR9B, # indicates p<0.05, and ## indicates p<0.01 relative to SD-101.

[0219] like Figures 12A to 12F As shown in the data of , SD-101 (TLR9C agonist) significantly inhibited MDSC expansion on 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 vehicle, TLR4, TLR7 and TLR9B agonists (p < 0.05; n = 11). In addition, SD-101 shifted dendritic cells toward the myeloid program compared to vehicle, TLR4 and TLR7 agonists (p < 0.05; n = 6). In conclusion, SD-101 significantly inhibited MDSC expansion at day 2 compared to vehicle (Veh), TLR9B, TLR4 and TLR7 agonists, and SD-101 significantly reduced the human M / G-MDSC ratio compared to vehicle, TLR4, TLR7 and TLR9B agonists.

[0220] Example 6:

[0221] In Example 6, PBMCs obtained from healthy human donors (n=6) were cultured in vitro in IL6+GMCSF (20 ng / ml) to induce MDSCs, and then treated with TLR9B agonist or SD-101 (which is a TLR9C agonist) at a concentration of 0.3 μM for 2 days. Analysis Total RNA from each half of the samples (n=3) was isolated and evaluated. The analyzed samples were also analyzed by qRT-PCR for validation. The other half of the samples were evaluated using flow cytometry to determine the effects of TLR9B agonists and SD-101 on MDSCs (CD33+ CD11b + HLADR - / lo ) and M1 macrophages (CD14 + CD86 + )’s role.

[0222] Figures 13A to 13G demonstrated that TLR9 stimulation with the TLR9C agonist SD-101 promoted enhanced pro-inflammatory and anti-tumorigenic signaling compared to the TLR9B agonist. Fig.13A Shown is a volcano plot illustrating gene expression in human PBMCs after 2 days of treatment with a TLR9B agonist and SD-101 (TLR9C agonist), as measured using mRNA analysis was determined on samples prepared as described above.

[0223] Table 2 shows the use The generated data were analyzed and demonstrated that SD-101 upregulated many genes to a greater extent than TLR9B agonists.

[0224] Table 2:

[0225]

[0226]

[0227] Table 3 shows the use The generated data were analyzed and demonstrated that SD-101 downregulated other genes to a greater extent than TLR9B agonists.

[0228] Table 3:

[0229]

[0230] in addition, Fig. 13B and 13C Shown are the effects of TLR9B and TLR9C agonists on MDSCs and M1 macrophages, respectively, as determined by flow cytometry on samples prepared as described above. Fig.13D Displays the results as determined by nSolver.4 software, using the The data generated by the analysis were used to generate pathway scores for immunomodulatory pathways affected by TLR9B and SD-101. Fig.13E , 13F and 13G display Fig.13A qRT-PCR data of the same samples analyzed in . Fig.13E , 13F The data from 13G and 13D showed the excellent effect of SD-101 in enhancing the expression of PD-L1, IFNγ and IP-10.

[0231] Multiple t tests were performed to Fig.13D One-way ANOVA followed by Tukey's post hoc test was performed for Figures 13E to 13G The qRT-PCR analysis shown in Figures 13B to 13E To 13G, * indicates p<0.05 and ***** indicates p<0.0001 relative to the vehicle. In addition, ^ indicates p<0.05, ^^ indicates p<0.01, and ^^^ indicates p<0.001 relative to TLR9B.

[0232] As shown above, Gene expression analysis showed that SD-101 induced higher adaptive immunity, innate immunity, and immunometabolism scores compared with vehicle and TLR9B agonist (p < 0.05; n = 3). SD-101 induced more TLR, Th1, NFκB, and lymphocyte activation signatures compared with vehicle and TLR9B agonist (p < 0.05; n = 3). SD-101 induced more PD-L1, IFNγ, and IP10 gene expression compared with vehicle and TLR9B (p < 0.05; n = 6), in addition to B cell and NK cell expansion compared with vehicle (p < 0.05; n = 6). In summary, SD-101 better induced innate adaptive immune signaling pathways, TLR, Th1, NFκB, and lymphocyte activation signatures compared with vehicle and TLR9B agonist, and SD-101 induced more PD-L1, IFNγ, and IP10 gene expression compared with vehicle and TLR9B agonist.

[0233] Overall, SD-101 inhibited MDSC expansion and enhanced polarization toward M1 macrophages. In addition to the broad immune activation effects, the favorable effects on MDSCs suggest that TLR9C agonists, if delivered effectively, have the potential to enable other immunotherapeutic agents to perform better in the hostile liver tumor microenvironment (TME).

[0234] Example 7:

[0235] In Example 7, the infusion of a class C TLR9 agonist (SD-101) was performed using The infusion system is administered to human patients via pressure-enabled hepatic arterial infusion (PEDD / HAI) with the goal of enhancing the response rate to CPI therapy in human patients with liver metastases of uveal melanoma, advanced hepatocellular carcinoma, or advanced intrahepatic cholangiocarcinoma. The study includes cohorts A, B, and C. SD-101 (where n=9) is administered as a single agent to cohort A, SD-101 (where n=6-12) is administered to cohort B via TriNav+systemic administration of anti-PD-1 (i.e., nivolumab or pembrolizumab), and SD-101 (where n=6-12) is administered to cohort C via TriNav+systemic administration of anti-PD-1 (i.e., nivolumab)+systemic administration of anti-CTLA-4 (i.e., ipilimumab). The amount of SD-101 administered to human patients in cohorts A, B, and C can span a dose escalation range of 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 included administration of SD-101 via PEDD in two cycles, wherein each cycle included three weekly doses of SD-101 (ie, weekly administration of SD-101 for three consecutive weeks) and a five-week rest period following weekly administration of SD-101.

[0236] Fig.14 The pharmacokinetic profiles of SD-101 levels in plasma of five different patients in Cohort A are shown following administration of a single agent of 4 mg of SD-101 six hours after infusion of each of the six doses administered in two cycles as described above. Specifically, as shown, transient SD-101 levels (<2 hours) were detected in plasma following infusion with TriNav. At all doses (not shown), detected systemic exposure levels were transient (<4 hours).

[0237] High SD-101 levels were observed in the liver of patients following infusion of SD-101 with TriNav. In patients administered 8 mg of SD-101 per dose, up to 2340 ng / ml of SD-101 was detected in liver tissue. This human clinical data demonstrates that TriNav can be used to achieve higher liver SD-101 levels with limited systemic exposure.

[0238] In addition, after SD-101 treatment, PMBC cells from patients administered SD-101 in the manner described above were analyzed for gene expression levels at multiple time points. Specifically, gene expression levels in liver metastases from three patients in the 2 mg SD-101 single agent cohort were analyzed. Analysis showed an increase in ISG15, IL-9, IFNα, and IL-2 transcripts and a decrease in ARG1 and IDO transcripts, with increased scores for macrophages, exhausted CD8 T cells, Th1 cells, and Th1 activation. Gene expression data showed that SD-101 delivered via TriNav was associated with expansion of anti-tumor natural killer cells in the blood, consistent with systemic immune activation.

[0239] Dose-response trends were observed in serum cytokines, with IL-18, IFNγ, IP-10, and soluble CD25 increasing across 2 mg, 4 mg, and 8 mg single-agent dose levels. Fig.15 The change in serum IFNγ after increasing the dose level of single agent SD-101 (cohort A) and SD-101 in combination with CPI (i.e., nivolumab or pembrolizumab) (cohort B) after each of the three doses administered in the first cycle is shown. Specifically, the figure depicts the trend of increasing serum IFNγ levels after TriNav infusion of SD-101 in cohort A (doses of .5 mg, 2 mg, 4 mg, and 8 mg of SD-101) and cohort B (doses of 2 mg of SD-101 in combination with pembrolizumab or nivolumab). Serum IFNγ was determined by Luminex assay. Fig.15 The data demonstrate that infusion of SD-101 with TriNav results in IFNγ cytokine induction.

[0240] Fig.16 The change of the concentration of monocyte MDSC in the tumor after changing the dosage level of the single agent SD-101 (cohort A) in the above manner is shown. Specifically, the figure depicts the reduction of the concentration of monocyte MDSC in the following tumors: (i) three patients with 2 mg dose of SD-101, and (ii) one patient with 8 mg dose of SD-101. The concentration of monocyte MDSC is determined by multiple immunofluorescence microscopy. The reduction of the concentration of monocyte MDSC in the tumor is calculated from the earliest available time point (day 1 or day 57) to the latest available time point (day 57 or day 100). Fig.16 The data demonstrate that infusion of SD-101 with TriNav results in MSDC depletion, as shown by a decrease in MDSC levels in liver tumor mononuclear cells.

[0241] For patients receiving 2 mg SD-101 + CPI with available liquid biopsy data, 4 of 7 patients demonstrated a reduction in circulating tumor cells after 1 cycle of treatment, and 3 of 5 patients showed a reduction in circulating tumor DNA (ctDNA) after the first cycle, as follows Fig.17 and 18 shown.

[0242] Fig.17 Shown is the change in circulating tumor cells following administration of 2 mg SD-101 with a CPI following the first cycle of treatment. Specifically, the graph depicts that following administration of 2 mg of a single agent SD-101, four out of seven patients had reduced levels of circulating tumor cells, resulting in a reduction in tumor burden.

[0243] Fig.18 Shown is the change in circulating tumor DNA (ctDNA) after administration of 2 mg of a single agent SD-101 after the first cycle of treatment. Specifically, the figure depicts that after administration of 2 mg of a single agent SD-101, three out of five patients had reduced circulating tumor DNA (ctDNA) levels, resulting in a reduction in tumor burden. Fig.18 The data provided in this article are for Fig.17 The same first 5 patients.

[0244] Example 8:

[0245] In Example 8, the combination of SD-101, a TLR9C agonist delivered via PEDD, and an anti-PD-1 antibody delivered via SQ or IP significantly reduced LM in mice. Further analysis of tumor infiltrating lymphocytes (CD45+ cells) isolated from tumor-bearing livers showed that the combination of anti-PD-1 and SD-101 significantly reduced liver MDSC. The percentage of B cells; the ratio of CD3+T cells and M1 / M2 macrophages was significantly increased compared to the vehicle. There was no significant difference in the control of tumor progression or regulation of the tumor microenvironment between SQ and IP CPI delivery. The data show that TLR9 stimulation via PEDD combined with anti-PD-1 can provide equivalent control of LM regardless of the delivery route. In addition, PEDD of class C TLR9 agonists has the potential to initiate TME to reduce immunosuppression in LM, which may improve the anti-tumor efficacy of anti-PD-1, regardless of the route of administration. In some instances, checkpoint inhibitors are administered systemically or subcutaneously before administering TLRC agonists.

[0246] In this example, SD-101 was delivered in combination with an anti-PD-1 antibody administered intraperitoneally (IP) or subcutaneously (SQ) into a PEDD mouse model to evaluate whether the CPI administration route affects the ability of intrahepatic TLR9 stimulation to control liver metastasis. To develop 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 anti-PD-1 antibodies were delivered twice a week by IP or SQ. Tumor burden was monitored by IVIS, and livers were harvested at D10 to isolate CD45 +Myeloid-derived suppressor cells (MDSCs) impair the activity of immunotherapy by promoting an immunosuppressive tumor microenvironment (TME) in the setting of liver metastases. Flow cytometric analysis was performed to quantify MDSCs (CD11b + Gr1 + ), B cells (B220), T (CD3 + ) cells, M1(F4 / 80 + CD38 + Egr2 - ) and M2(F4 / 80 + CD38 - Egr2 + )Macrophages.

[0247] SD-101 delivered via PEDD in combination with anti-PD-1 antibody delivered via SQ or IP significantly (p<0.0001) reduced liver metastasis progression (D0 fold) compared to controls (vehicle: 87.46 vs. SD-101: 13.90 vs. SQ: 0.002 vs. IP: 0.04). + Flow cytometry analysis of cells showed that the combination of CPI and SD-101 significantly reduced liver MDSC (vehicle: 37.57% vs. SQ: 7.18% vs. IP: 10.18%; p < 0.05). The percentage of B cells (vehicle: 8.32% vs. SQ: 18.09% vs. IP: 15.65%: p < 0.05); T cells (vehicle: 7.14% vs. SD-101: 15.18% vs. SQ: 17.81% vs. IP: 19.13%: p < 0.05) and M1 / M2 macrophages (vehicle: 2.25 vs. SD-101: 12.8 vs. SQ: 12.99 vs. IP: 12.90: p < 0.05) ratios were significantly increased compared to vehicle (Veh). There was no significant difference between SQ and IP CPI delivery in controlling tumor progression or TME regulation.

[0248] Fig.19A Shown are changes in tumor progression of liver metastases in mice in response to PEDD administration of SD-101 in combination with an anti-PD-1 antibody delivered 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 for the development of LM. One week later, mice were treated with 10 μg SD-101 via PEDD and anti-PD-1 antibody delivered IP or SQ on D0, D2, D4, and D7. Tumor burden was monitored by an in vivo imaging system (IVIS) and D0 fold changes in tumor burden have been reported. Fig.19A Data shown in were subjected to two-way ANOVA followed by Tukey's post hoc test, where **** indicates p<0.001 relative to D10 vehicle.

[0249] Fig.19B Shown is the change in tumor burden of liver metastases in mice in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody 10 days after treatment compared to vehicle. IVIS values ​​have been reported in photons / s. Fig.19B Data shown in were subjected to two-way ANOVA followed by Tukey's post hoc test, where # indicates p<0.001 relative to D10 vehicle and ^ indicates p<0.0001 relative to D10 vehicle.

[0250] Fig.19C Provided are images captured by IVIS at different time points after initiation of treatment with PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody compared to vehicle. Fig.19D Provided are images of harvested livers captured 10 days after treatment with PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody according to Example 8, compared to vehicle.

[0251] Fig. 20A As shown in this example, changes in MDSCs present in the LM in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody 10 days after treatment compared to vehicle. On day 10 (D10), livers were harvested to isolate CD45 + Cells. Specifically, livers were harvested at D10, followed by collagenase digestion of NPCs; and CD45+ cells were isolated using magnetic beads. Flow cytometry (FC) was performed using antibodies against: CD11b; GR1, NK1.1, B220, PD-L1. FC analysis was performed to quantify MDSC (CD11b + Gr1 + ).

[0252] Fig. 20B Figure 2 shows that isolated CD45 from liver metastases increased in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody 10 days after treatment compared to vehicle. + B (B220 + )Changes in cells. Fig. 20C Figure 2 shows that isolated CD45 from liver metastases increased in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 antibody 10 days after treatment compared to vehicle.+ T cells (CD3 + )Changes in cells. Fig.20D Shown is the isolation of CD45 from liver metastasis + Changes in M1 and M2 macrophages in the tumor microenvironment in response to PEDD administration of SD-101 in combination with IP or SQ delivered anti-PD-1 compared to vehicle 10 days after treatment. + CD38 + Egr2 - ) and M2(F4 / 80 + CD38 - Egr2 + ) macrophages (as quantified by FC). In view of the data of Example 8 above, SD-101 administered via PEDD in combination with CPI delivered by IP or SQ provides control of liver metastasis, where intrahepatic TLR9 stimulation equalizes CPI via either route. It is believed that PEDD of class C TLR9 agonists may initiate the TME to reduce immunosuppression in liver metastasis, which may improve the anti-tumor efficacy of CPI regardless of the route of administration.

[0253] The above only illustrates the principle of the present disclosure. In view of the teachings of this article, various modifications and changes to the described embodiments will be obvious to those skilled in the art. Therefore, it should be understood that those skilled in the art will be able to design many systems, arrangements and programs, which, although not explicitly shown or described herein, embody the principle of the present disclosure, and therefore can be within the spirit and scope of the present disclosure. It should be understood by those skilled in the art that various different exemplary embodiments can be used together or interchangeably with each other. In addition, certain terms used in the present disclosure, including the specification, can be used synonymously in some cases, including but not limited to, for example, data and information. It should be understood that although these words and / or other words that can be synonymous with each other can be used synonymously in this article, there may be such a situation that these words may not be intended to be used synonymously. In addition, to the extent that the above-mentioned prior art knowledge is not explicitly incorporated herein by reference, it is incorporated herein by reference in its entirety. All publications referenced are incorporated herein by reference in their entirety.

Claims

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

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

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

4. The method of claim 1, wherein the therapeutically effective amount of the TLR9 agonist administered is in the range of about 0.01-20 mg.

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

6. The method according to any one of claims 1 to 5, wherein the TLR9 agonist can be administered by a catheter device.

7. A method according to claim 6, wherein the catheter device includes a one-way valve that responds dynamically to local pressure and / or flow changes.

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

9. The method of claim 6, wherein the TLR9 agonist is administered for a period of about 10-200 minutes.

10. The method of claim 9, wherein the TLR9 agonist is administered for a period of about 10-60 minutes.

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

12. The method of any one of claims 1 to 5, wherein administration of the TLR9 agonist causes changes in gene expression within the liver metastasis.

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

14. The method of claim 12, wherein the changes in gene expression comprise at least one of: increased TLR signaling; increased leukocytes; increased exhausted CD8 T cells; induction of Th1 programming; decreased Th2 programming; increased T cell receptor and T cell co-stimulatory signaling; increased IL9, IL15, CCL7; B cell activation; induction of mast cells; induction of NK cells; induction of IFNγ; increased interferon signaling; increased chemokine signaling; increased IL6 in the liver but not in the blood; decreased M2 macrophages; increased M1 macrophages; decreased MDSCs; decreased angiogenesis and VEGF; and decreased fatty acid oxidation.

15. The method of any one of claims 1 to 5, wherein the TLR9 agonist is administered in combination with one or more checkpoint inhibitors.

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

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

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

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

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

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