Cancer treatment using Toll-like receptor agonists

Abstract

Embodiments of the present invention provide methods of treating cancer and delivering Toll-like receptor (TLR) agonists to solid tumors in the liver using locoregional therapy through the vasculature. In one aspect, the present invention relates to a method of treating primary liver cancer, such as hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), comprising administering a TLR9 agonist to the liver.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 169,674, filed April 1, 2021, and U.S. Provisional Patent Application No. 63 / 298,589, filed January 11, 2022, both of which are incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 16, 2020, is named A372-502_SL.txt, and is 484 bytes in size.

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

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

[0005] Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common primary liver tumors and are among the solid organ malignancies whose intrahepatic progression leads to a rapidly progressive and often fatal condition.

[0006] HCC represents more than 90% of cases of primary liver tumors. Both HCC and ICC patients share many of the same characteristics and challenges with regard to treatment. For both diseases, the 5-year survival rate is <20%.

[0007] Surgery is the only potential treatment for primary liver cancer, such as HCC and ICC, but due to the lack of specific symptoms, about 70% of patients are diagnosed at advanced stages, and even after potentially curative resection, tumor recurrence is frequent. Considering its effectiveness in other malignant tumors, checkpoint inhibitors (CPIs) have also been tried in patients with HCC and ICC, but due to drug delivery challenges and suppression of immune response in the liver, their success to date has been limited.

[0008] Thus, there remains a need for safe and effective therapies for the treatment of primary liver cancers, such as HCC and ICC. Summary of the Invention

[0009] The present invention relates to a method of treating primary liver cancer using a therapeutically effective amount of a Toll-like receptor (TLR) agonist.

[0010] In one aspect, the present invention relates to a method for treating primary liver cancer, comprising administering a TLR agonist through a device by hepatic artery injection (HAI). According to another embodiment, the treatment of primary liver cancer comprises administering a TLR agonist through a device by portal vein injection (PVI).

[0011] In some embodiments, the TLR agonist is administered through pressure-enabled drug delivery (PEDD), which involves administration of a therapeutic agent through a device such as a catheter device. In some embodiments, the catheter device comprises a one-way valve that dynamically responds to local pressure changes. In some embodiments, the catheter device generates, causes, and / or contributes to a net increase in fluid pressure in the blood vessel and / or target tissue or tumor. In some embodiments, the catheter device generates, causes, and / or contributes to a net decrease in fluid pressure in the blood vessel and / or target tissue or tumor. In some embodiments, the catheter device generates, causes, and / or contributes to a first decrease and then an increase in fluid pressure in the blood vessel and / or target tissue or tumor.

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

[0013] In some embodiments, the amount of TLR agonist administered is within the range of about 0.01 to 20 mg, or at least one of 0.5 mg, 2 mg, 4 mg, or 8 mg.

[0014] In some embodiments, the TLR agonist is administered in a solution ranging from 1 to 100 mL, or at least one of 10 mL, 25 mL, or 50 mL.

[0015] In some embodiments, the TLR agonist is administered within the range of 0.0001-20 mg / mL, or at least one of 0.01 mg / mL, 0.04 mg / mL, 0.08 mg / mL, or 0.16 mg / mL.

[0016] In some embodiments, the TLR agonist is administered over a period of about 10 to 200 minutes. In other embodiments, the TLR agonist is administered over a period of about 10 to 60 minutes. In other embodiments, the TLR agonist is administered over a period of about 25 minutes.

[0017] In some embodiments, the TLR agonist is administered in combination with one or more CPIs. In some embodiments, the CPI is administered systemically either simultaneously with, before, or after the administration of the TLR agonist. In some embodiments, the CPI comprises at least one of nivolumab, pembrolizumab, and ipilimumab.

[0018] In some embodiments, the administration of the TLR agonist comprises a dosing regimen that includes cycles, in some embodiments, one or more of the cycles includes administration of the TLR agonist via a catheter device by HAI, followed by systemic administration of one or more CPIs.

[0019] In some embodiments, one cycle of the dosing regimen comprises administration of the TLR agonist once a week for three consecutive weeks, hi some embodiments, the dosing regimen comprises two cycles.

[0020] In some embodiments, administration of a TLR agonist through an intravascular device to the liver results in a reduction of myeloid-derived suppressor cells (MDSCs) or functional alteration of MDSCs to limit immune suppression, in some embodiments, administration of a TLR agonist through an intravascular device to the liver results in an anti-tumor effect.

[0021] In some embodiments, the TLR agonist is a TLR9 agonist. In some embodiments, the TLR9 agonist is SD-101.

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

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

[0024] [Figure 1] The structure of SD-101 is shown below. [Diagram 2] Illustrates an in vitro analysis of human PMBCs obtained from healthy donors and treated with IL6 and GM-CSF to induce MDSCs. [Diagram 3] Illustrates in vitro analysis of mouse bone marrow cells from mice with and without tumors, where GM-CSF was used to induce MDSCs. [Figure 4] 1 illustrates a cross-sectional image of a pig liver lobe following injection of SD-101. [Diagram 5] Illustrates the overall study design of a Phase 1b / 2 pressure-enabled local immuno-oncology study of HAI of SD-101 with systemic checkpoint blockade for HCC and ICC.

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

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

[0027] Toll-like receptor agonists Toll-like receptors are pattern recognition receptors that can detect microbial pathogen-associated molecular patterns (PAMPs). TLR stimulation, such as TLR9 stimulation, can not only provide a broad range of innate immune stimulation but also specifically address the predominant drivers of immune suppression in the liver. TLR1-10 are expressed in humans and recognize diverse microbial PAMPs. In this regard, TLR9 can respond to unmethylated CpG-DNA, including microbial DNA. CpG refers to the motif of cytosine and guanine dinucleotide l. TLR9 is constitutively expressed in B cells, plasmacytoid dendritic cells (pDCs), activated neutrophils, monocytes / macrophages, T cells, and MDSCs. TLR9 is also expressed in non-immune cells, including keratinocytes and intestinal, cervical, and respiratory epithelial cells. TLR9 can bind to its agonist in endosomes. Signaling can occur through MYD88 / IkB / NfκB to induce proinflammatory cytokine gene expression. Parallel signaling pathways through IRF7 induce type 1 and type 2 interferons (e.g., IFN-α, IFN-γ, etc.) that stimulate adaptive immune responses. Furthermore, TLR9 agonists can induce cytokine and IFN production, as well as functional maturation of antigen-presenting dendritic cells.

[0028] According to one embodiment, TLR9 agonists can reduce and reprogram MDSCs. MDSCs are the main driving force behind immune suppression in the liver. MDSCs also drive the proliferation of other suppressive cell types, such as T regulatory cells (Tregs), tumor-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs). MDSCs can downregulate immune cells and impede the effectiveness of immunotherapeutic drugs. Furthermore, high MDSC levels generally predict poor outcomes in cancer patients. In this regard, reducing, modifying, or eliminating MDSCs is believed to improve the host's immune system's ability to attack cancer and the ability of immunotherapy to induce more beneficial therapeutic responses. In one embodiment, TLR9 agonists can convert MDSCs into immune-stimulating M1 macrophages, convert immature dendritic cells into mature dendritic cells, and expand effector T cells to create a responsive tumor microenvironment that promotes antitumor activity.

[0029] According to one embodiment, synthetic CpG-oligonucleotides (CPG-ONs) that mimic the immune stimulatory properties of microbial CpG-DNA can be developed for therapeutic use. According to one embodiment, the oligonucleotides are oligodeoxynucleotides (ODNs). There are several different CpG-ODN class types, such as class A, class B, class C, class P, and class S, which share certain structural and functional characteristics. In this regard, class A type CPG-ODNs (or CPG-A ODNs) are associated with B cell and pDC maturation with little effect on maximal IFN-α induction, class B type CPG-ODNs (or CPG-B ODNs) strongly induce B cell proliferation, activate pDC and monocyte maturation, NK cell activation, and inflammatory cytokine production, and class C type CPG-ODNs (or CPG-C ODNs) can induce B cell proliferation and IFN-α production.

[0030] Furthermore, according to one embodiment, CPG-C ODNs may be associated with the following attributes: (i) an unmethylated dinucleotide CpG motif, (ii) a CpG motif juxtaposed with adjacent nucleotides (e.g., AACGTTCGAA), (iii) a perfect phosphorothioate (PS) backbone linking the nucleotides (as opposed to the natural phosphodiester (PO) backbone found in bacterial DNA), and (iv) a self-complementary palindromic sequence (e.g., AACGTT). In this regard, CPG-C ODNs, due to their palindromic nature, may link themselves, thereby generating double-stranded duplex or hairpin structures.

[0031] Further, according to one embodiment, the CPG-C ODN can include one or more 5'-TCG trinucleotides, in which the 5'-T is located 0, 1, 2, or 3 bases from the 5' end of the oligonucleotide, and at least one palindromic sequence of at least 8 bases in length, including one or more unmethylated CG dinucleotides. The one or more 5'-TCG trinucleotide sequences can be separated 0, 1, or 2 bases from the 5' end of the palindromic sequence, or the palindromic sequence can contain all or a portion of the one or more 5'-TCG trinucleotide sequences. In one embodiment, the CpG-C ODN is 12-100 bases in length, preferably 12-50 bases in length, preferably 12-40 bases in length, or preferably 12-30 bases in length. In one embodiment, the CpG-C ODN is 30 bases in length. In one embodiment, the ODN is at least (lower limit) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 50, 60, 70, 80, or 90 bases in length. In one embodiment, the ODN is at most (upper limit) 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 bases in length.

[0032] In one embodiment, the at least one palindromic sequence is 8 to 97 bases long, preferably 8 to 50 bases long, or preferably 8 to 32 bases long. In one embodiment, the at least one palindromic sequence is at least (lower limit) 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 bases long. In one embodiment, the at least one palindromic sequence is at most (upper limit) 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, or 10 bases long.

[0033] In one embodiment, the CpG-C ODN can comprise the sequence of SEQ ID NO:1.

[0034] According to one embodiment, the CpG-C ODN can include SD-101. SD-101 is a 30-mer phosphorothioate oligodeoxynucleotide having the following sequence: It has the following structure: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (sequence number 1).

[0035] SD-101 drug substance is isolated as the sodium salt. The structure of SD-101 is illustrated in Figure 1.

[0036] The molecular formula of SD-101 free acid is C 293 H 369 N 112 O 149 P 29 S 29 The molecular weight of SD-101 free acid is 9672 Daltons. The molecular formula of SD-101 sodium salt is C 293 H 340 N 112 O 149 P 29 S 29 Na 29 and the molecular weight of SD-101 sodium salt is 10,309 daltons.

[0037] Further, according to one embodiment, the CPG-C ODN sequence can correspond to SEQ ID NO: 172 as set forth in U.S. Patent No. 9,422,564, which is incorporated herein by reference in its entirety.

[0038] In one embodiment, the CpG-C ODN can include a sequence having at least 75% homology to any of the foregoing, such as SEQ ID NO:1.

[0039] According to another embodiment, the CPG-C ODN sequence can correspond to any one of the other sequences described in U.S. Patent No. 9,422,564. Additionally, the CPG-C ODN sequence can also correspond to any of the sequences described in U.S. Patent No. 8,372,413, which is also incorporated by reference in its entirety.

[0040] According to one embodiment, any of the CPG-C ODNs discussed herein may be present in their pharma- ceutically acceptable salt form. Exemplary basic salts include alkali metal salts such as ammonium salts, sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, zinc salts, salts with organic bases (e.g., organic amines) such as N-Me-D-glucamine, salts with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, t-butylamine, and amino acids such as arginine and lysine. In one embodiment, the CpG-C ODN is in ammonium, sodium, lithium, or potassium salt form. In one preferred embodiment, the CpG-C ODN is in sodium salt form. The CpG-C ODN may be provided in a pharmaceutical solution that includes a pharma- ceutically acceptable excipient. Alternatively, CpG-C ODN can be provided as a lyophilized solid, which is then reconstituted in sterile water, saline, or a pharma- ceutically acceptable buffer solution prior to administration. Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, bulking agents, buffers, tonicity adjusters, and preservatives. In one embodiment, the pharmaceutical composition can include an excipient that functions as one or more of a solvent, bulking agent, buffer, and tonicity adjuster (e.g., sodium chloride in saline can function as both an aqueous vehicle and an isotonicity adjuster). The pharmaceutical composition of the present disclosure is suitable for parenteral and / or transdermal administration.

[0041] In one embodiment, 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 one embodiment, the composition is isotonic.

[0042] The pharmaceutical composition may include a bulking agent. The bulking agent is particularly useful when the pharmaceutical composition is freeze-dried before administration. In one embodiment, the bulking agent is a protective agent that helps stabilize and prevent the active agent from decomposing during freeze-drying or spray-drying and / or during storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbital, glucose, and raffinose.

[0043] The pharmaceutical composition may include a buffering agent. The buffering agent controls the pH to inhibit degradation of the active agent during processing, storage, and optionally reconstitution. Suitable buffers include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffering agents include amino acids, such as, for example, arginine, glycine, histidine, and lysine. The buffering agent may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 4 to 9. In one embodiment, the pH is greater than (lower limit) 4, 5, 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, 7, 6, or 5. That is, the pH is within the range of about 4 to 9, with the lower limit being less than the upper limit.

[0044] The pharmaceutical composition may include an isotonicity adjusting agent. Suitable isotonicity adjusting agents include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0045] The pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in one embodiment, the pharmaceutical composition is prepared under sterile conditions and is in a single-use container, and therefore does not require the inclusion of a preservative.

[0046] Table 1 illustrates the batch formula for SD-101 Drug-16g / L: [Table 1] 1 Amount based on measured content in solution (excluding water present in lyophilized powder) *SD-101 drug substance in Table 1 includes the sum of all oligonucleotide contents including SD-101.

[0047] In some embodiments, the unit dose strength may include from about 0.1 mg / mL to about 20 mg / mL. In one embodiment, the unit dose strength of SD-101 is 13.4 mg / mL.

[0048] In some embodiments, the amount of SD-101 administered is within the range of about 0.01 to 20 mg, or at least one of 0.5 mg, 2 mg, 4 mg, or 8 mg.

[0049] In some embodiments, SD-101 is administered in a solution ranging from 1 to 100 mL, or at least one of 10 mL, 25 mL, or 50 mL.

[0050] In some embodiments, the dosage of SD-101 is within the range of 0.0001-20 mg / mL, hi some embodiments, the dosage of SD-101 is one of 0.01 mg / mL, 0.04 mg / mL, 0.08 mg / mL, or 0.16 mg / mL.

[0051] CpG-C ODNs may contain modifications. Suitable modifications may include, but are not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar moiety, and modifications of the phosphate group. Modified bases may be included in palindromic sequences (e.g., the palindromic portion of a CpG-C ODN remains self-complementary) so long as the modified base maintains the same specificity to its natural complement through Watson-Crick base pairing. Examples of modifications of the 5'OH group may include biotin, cyanine 5.5, the cyanine family of dyes, Alexa Fluor 660, the Alexa Fluor family of dyes, IRDye 700, IRDye 800, IRDye 800CW, and the IRDye family of dyes.

[0052] CpG-C ODNs may be linear, circular, or may contain circular portions and / or hairpin loops. CpG-C ODNs may be single-stranded or double-stranded. CpG-C ODNs may be DNA, RNA, or a DNA / RNA hybrid.

[0053] CpG-C ODNs may contain naturally occurring or modified non-naturally occurring bases, and may contain modified sugars, phosphates, and / or termini. For example, in addition to phosphodiester bonds, phosphate modifications include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, and phosphorodithioates, and may be used in any combination. In one embodiment, CpG-C ODNs have only phosphorothioate bonds, only phosphodiester bonds, or a combination of phosphodiester and phosphorothioate bonds.

[0054] Sugar modifications known in the art, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras, may also be made and combined with any phosphate modification. Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and / or C-6 of cytosines (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) of CpG-C ODNs, and C-5 and / or C-6 of uracils (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil) of CpG-C ODNs. As mentioned above, the use of base modifications in palindromic sequences of CpG-C ODNs should not prevent the self-complementarity of bases involved in Watson-Crick base pairing. However, outside of palindromic sequences, modified bases can be used without this restriction. For example, 2'-O-methyl-uridine and 2'-O-methyl-cytidine may be used on the outside of the palindromic sequence, while 5-bromo-2'-deoxycytidine may be used both inside and outside of the palindromic sequence. Other modified nucleotides that may be used both inside and outside of the palindromic sequence include 7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0055] The duplex (i.e., double-stranded) and hairpin forms of most ODNs are often in dynamic equilibrium, with the hairpin form generally being favored at low oligonucleotide concentrations and high temperatures. Covalent interstrand or intrastrand crosslinks increase the stability of the duplex or hairpin, respectively, toward thermal, ionic, pH, and concentration-induced conformational changes. Chemical crosslinks can be used to lock polynucleotides into either duplex or hairpin forms for physicochemical and biological characterization. Crosslinked ODNs that are structurally homogeneous and "locked" in their most active form (either duplex or hairpin form) can potentially be more active than their non-crosslinked counterparts. Thus, some CpG-C ODNs of the present disclosure can contain covalent interstrand and / or interstrand crosslinks.

[0056] The techniques for making polynucleotides and modified polynucleotides are known in the art.Naturally occurring DNA or RNA containing phosphodiester bonds can generally be synthesized by sequentially attaching appropriate nucleoside phosphoramidites to the 5' hydroxyl group of the growing ODN that is attached to solid support at its 3' end, followed by oxidizing the intermediate phosphite triester to phosphate triester.Using this method, once the desired polynucleotide sequence is synthesized, the polynucleotide is removed from the support, the phosphate triester group is deprotected to phosphate diester, and the nucleoside base is deprotected using aqueous ammonia or other base.

[0057] CpG-C ODN may contain phosphate-modified oligonucleotides, some of which are known to stabilize ODN.Thus, some embodiments include stabilized CpG-C ODN.The phosphorus derivative (or modified phosphate group) that can be attached to the sugar or sugar analog moiety in ODN can be monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc.

[0058] CpG-C ODNs can contain one or more ribonucleotides (containing ribose as the sole or main sugar moiety), deoxyribonucleotides (containing deoxyribose as the main sugar moiety), modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and sugar analog cyclopentyl groups. The sugars can be in pyranosyl or furanosyl form. In CpG-C oligonucleotides, the sugar moiety is preferably a furanoside of ribose, deoxyribose, arabinose, or 2'-0-alkylribose, and the sugars can be linked to the respective heterocyclic bases in either anomeric configuration. The preparation of these sugars or sugar analogs, and the respective nucleosides in which such sugars or analogs are linked to the heterocyclic bases (nucleobases) themselves, is known and need not be described herein. Sugar modifications may also be made and combined with any phosphate modifications in preparing CpG-C ODNs.

[0059] The heterocyclic or nucleobases incorporated into the CpG-C ODNs can be the naturally occurring primary purine and pyrimidine bases (i.e., uracil, thymine, cytosine, adenine, and guanine, as described above), as well as naturally occurring and synthetic modifications of the primary bases. Thus, the CpG-C ODNs can include one or more of inosine, 2'-deoxyuridine, and 2-amino-2'-deoxyadenosine.

[0060] According to another embodiment, the CPG-ODN is one of a class A type CPG-ODN (CPG-A ODN), a class B type CPG-ODN (CPG-B ODN), a class P type CPG-ODN (CPG-P ODN), and a class S type CPG-ODN (CPG-S ODN). In this regard, the CPG-A ODN may be CMP-001.

[0061] In another embodiment, the CPG-ODN can be tilsotolimod (IMO-2125).

[0062] Furthermore, according to one embodiment, in vitro studies of human peripheral blood mononuclear cells (PMBCs) demonstrate that SD-101 is superior to class B TLR9 and TLR7 agonists in terms of reducing MDSCs, reducing M2 macrophages, and promoting M1 macrophages. For example, Figure 2 shows an in vitro analysis of human PMBCs taken from healthy donors and treated with IL6 and GM-CSF to induce MDSCs. In this regard, SD-101 showed (i) better elimination of dominant MDSC subsets in the liver, e.g., M-MDSCs, and (ii) a more favorable effect on the M1 / M2 macrophage ratio (i.e., induction of M1 and reduction of M2).

[0063] Moreover, according to one embodiment, in vitro studies of mouse bone marrow cells demonstrate that SD-101 is similarly superior to class B TLR9 and TLR7 agonists for programming mouse bone marrow cells. For example, Figure 3 shows an in vitro analysis of mouse bone marrow cells from mice with and without tumors, in which 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.

[0064] Checkpoint inhibitors According to one embodiment, the TLR agonist of the present invention may be used in combination with one or more CPIs. The CPI may include a programmed death 1 receptor (PD-1) antagonist. The PD-1 antagonist may be any chemical compound or biological molecule that blocks the binding of programmed cell death 1 ligand 1 (PD-L1) expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells, or NKT cells), and preferably also blocks the binding of PD-L2 programmed cell death 1 ligand 2 (PD-L2) expressed on cancer cells to immune cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include PDCD1, PD1, CD279, and SLEB2 for PD-1, PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1, and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of the therapeutic methods, medicaments and uses of the invention in which a human individual is treated, the PD-1 antagonist blocks the binding of human PD-L1 to human PD-1, and preferably blocks the binding of both human PD-L1 and PD-L2 to human PD-1.

[0065] According to one embodiment, the PD-1 antagonist may comprise a monoclonal antibody (mAb), or an antigen-binding fragment thereof, that specifically binds to PD-1 or PD-L1, preferably specifically binds to human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody and may comprise a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is a Fab, Fab'-SH, F(ab') 2 , scFv and Fv fragments.

[0066] According to one embodiment, the PD-1 antagonist may comprise an immunoadhesin that specifically binds to PD-1 or PD-L1, preferably human PD-1 or human PD-L1, e.g., a fusion protein comprising the 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.

[0067] According to one embodiment, the PD-1 antagonist can block PD-L1 expressed by tumor cells and MDSCs, as well as other suppressive immune cells.

[0068] According to one embodiment, the PD-1 antagonist is capable of inhibiting the binding of PD-L1 to PD-1, and preferably also inhibits the binding of PD-L2 to PD-1. In some embodiments of the above methods, medicaments, and uses, the PD-1 antagonist is a monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to PD-1 or PD-L1 and blocks the binding of PD-L1 to PD-1. In one embodiment, the PD-1 antagonist is an anti-PD-1 antibody comprising a heavy chain and a light chain.

[0069] According to one embodiment, the PD-1 antagonist may be one of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0070] According to one embodiment, nivolumab is administered intravenously (IV) via a peripheral vein at a dose of 480 mg every 4 weeks ("Q4W") or 240 mg every 2 weeks ("Q2W"). According to another embodiment, nivolumab is administered intravenously (IV) via a peripheral vein at a dose of 360 mg nivolumab every 3 weeks ("Q3W"). In another embodiment, administration of nivolumab is weight-based at 3 mg / kg nivolumab Q2W or 10 mg / kg nivolumab Q2W. In another embodiment, administration of nivolumab is weight-based at 1 mg / kg nivolumab Q3W. In yet another embodiment, nivolumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101. In another embodiment, nivolumab is administered once a week, once every two weeks, once every three weeks, once every four weeks, or monthly following administration of one or more cycles of SD-101.

[0071] According to another embodiment, pembrolizumab is administered intravenously (IV) via a peripheral vein at a dose of 200 mg Q3W or 400 mg every six weeks ("Q6W"). In another embodiment, pembrolizumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101.

[0072] According to another embodiment, the CPI can include a PD-L1 antagonist. In this regard, the PD-L1 antagonist can be one of atezolizumab, avelumab, and durvalumab.

[0073] According to another embodiment, the CPI can include a CTLA-4 antagonist. In this regard, the CTLA-4 antagonist can be ipilimumab.

[0074] According to another embodiment, ipilimumab is administered intravenously (IV) via a peripheral vein at a dose of 3 mg / kg every 3 weeks. In yet another embodiment, ipilimumab is administered simultaneously, at the same time, at about the same time, or on the same day as SD-101 and / or nivolumab. In another embodiment, ipilimumab is administered weekly, every other week, every 3 weeks, every 4 weeks, or monthly after administration of one or more cycles of SD-101 and / or nivolumab.

[0075] Devices for achieving locoregional delivery According to one embodiment, any of the above devices may include any device useful for achieving locoregional delivery to a tumor, including the catheter itself, or may include a catheter along with other components that may be used in combination with the catheter (e.g., filter valves, balloons, pressure sensor systems, pump systems, syringes, outer delivery catheters, etc.) In certain embodiments, the catheter is a microcatheter.

[0076] In some embodiments, the device may have one or more attributes including, but not limited to, a self-centering capability that can provide an even distribution of the therapy in the downstream branching network of blood vessels, an anti-reflux capability that can block or inhibit retrograde flow of the TLR agonist (e.g., by use of valves and filters and / or balloons), a system for measuring pressure within the vessel, and a means for regulating pressure within the vessel, such as by causing a decrease in pressure upon placement and during TLR agonist infusion, and an increase in pressure during saline bolus or bolus infusion of TLR agonist. In some embodiments, the system is designed to continuously monitor real-time pressure or flow throughout the procedure.

[0077] In some embodiments, devices that may be used to practice the methods of the present invention are those disclosed in U.S. Patent No. 8,500,775, U.S. Patent No. 8,696,698, U.S. Patent No. 8,696,699, U.S. Patent No. 9,539,081, U.S. Patent No. 9,808,332, U.S. Patent No. 9,770,319, U.S. Patent No. 9,968,740, U.S. Patent No. 10,813,739, U.S. Patent No. 10,588,636, U.S. Patent No. 11,090,460, U.S. Patent Publication No. 2018 / 0193591, U.S. Patent Publication No. 2018 / 0250469, U.S. Patent Publication No. 2019 / 0298983, U.S. Patent Publication No. 2020 / 0038586, and U.S. Patent Publication No. 2020-0383688, all of which are incorporated herein by reference in their entireties.

[0078] In some embodiments, the device is a device disclosed in U.S. Patent No. 9,770,319. In certain embodiments, the device may be a device known as the Surefire Infusion System.

[0079] In some embodiments, the device assists in measuring intravascular pressure during use. In some embodiments, the device is a device disclosed in US 2020-0383688. In certain embodiments, the device may be a device known as the TriSalus infusion system. In certain embodiments, the device may be a device known as the TriNav® infusion system. TriNav® is a single-lumen catheter with a one-way valve that dynamically responds to local pressure and flow changes, such as those resulting from the cardiac cycle or 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.

[0080] In some embodiments, the TLR agonist can be administered through the device via PEDD. In some embodiments, the TLR agonist can be administered while monitoring the pressure in the blood vessel, which can be used to adjust and correct the position of the device at the injection site and / or to adjust the injection rate. The pressure can be monitored, for example, by a pressure sensor system including one or more pressure sensors.

[0081] The infusion rate may be adjusted to change vascular pressure or flow rate, which may facilitate penetration and / or binding of the TLR agonist into or at the surface of the target tissue or tumor. In some embodiments, the infusion rate may be adjusted and / or controlled using a syringe pump as part of the delivery system, or in any other manner (e.g., by an infusion rate adjustment device). In some embodiments, the infusion rate may be adjusted and / or controlled using a pump system. In some embodiments, the infusion rate using a pump system may be about 0.1 cc / min to about 40 cc / min, or about 0.1 cc / min to about 30 cc / min, or about 0.5 cc / min to about 25 cc / min, or about 0.5 cc / min to about 20 cc / min, or about 1 cc / min to about 15 cc / min, or about 1 cc / min to about 10 cc / min, or about 1 cc / min to about 8 cc / min, or about 1 cc / min to about 5 cc / min. Additionally, the infusion rate using a bolus infusion can be from about 30 cc / min to about 360 cc / min, or from about 120 cc / min to about 240 cc / min. In one embodiment, the SD-101 infusion procedure lasts for about 10 to 200 minutes. In another embodiment, the SD-101 infusion procedure lasts for about 10 to 60 minutes. In another embodiment, the SD-101 infusion procedure lasts for about 25 minutes.

[0082] Methods involving administration to the liver In one embodiment, the method of the present invention includes a method of treating a solid tumor of the liver, such as a tumor resulting from at least one primary liver cancer, such as HCC and ICC, or a tumor having characteristics of both HCC and ICC, comprising administering a Toll-like receptor agonist to a patient in need of treatment, the Toll-like receptor agonist being administered to such solid tumor of the liver through a device by HAI. HAI refers to the injection of a therapeutic agent into a branch of the hepatic artery or coronary artery of the liver. According to one embodiment, the one or more Toll-like receptor agonists are introduced through percutaneous introduction of a device into a branch of the hepatic artery or portal vein, such as a catheter and / or a device that facilitates pressure-enabled delivery. According to one embodiment, the Toll-like receptor agonist is a TLR9 agonist, and in some embodiments, the TLR9 agonist is SD-101. In one embodiment, the patient is a human patient. According to another embodiment, the method includes administration to a subject who is male or female and is 18 years of age or older.

[0083] According to another embodiment, the method of the present invention includes a method of treating a solid tumor of the liver, such as a tumor resulting from at least one primary liver cancer, such as HCC and ICC, or a tumor having characteristics of both HCC and ICC, comprising administering a toll-like receptor agonist to a patient in need of treatment, the toll-like receptor agonist being administered to such solid tumor of the liver through a device by PVI. PVI refers to the infusion of a therapeutic agent into the hepatic portal venous system. According to one embodiment, the toll-like receptor agonist is introduced through percutaneous introduction of a device into a branch of the hepatic portal venous system, such as a catheter and / or a device that facilitates pressure-enabled delivery. According to one embodiment, the one or more toll-like receptor agonists are TLR9 agonists, and in some embodiments, the TLR9 agonist is SD-101. In one embodiment, the patient is a human patient.

[0084] According to another embodiment, the method of the present invention includes a method for treating at least one primary liver cancer, such as HCC and ICC, or a tumor with characteristics of both HCC and ICC, and the subject has not received prior cytotoxic chemotherapy, targeted therapy, or external radiation therapy within 14 days prior to enrollment. According to yet another embodiment, the method of the present invention is administered to a subject who has not received prior therapy with SD-101. According to one embodiment of the present invention, the method also includes administration to a subject who has not received prior embolic HAI therapy with a permanent embolic material.

[0085] According to another embodiment, the method of the present invention includes a method for treating at least one primary liver cancer, such as HCC and ICC, or a tumor with characteristics of both HCC and ICC, and the subject does not have a history of previous disease or other concurrent malignancies, except when the malignancies are not clinically significant.In another embodiment, the subject treated according to the method of the present invention can not have ongoing treatment.In yet another embodiment, the subject is clinically stable.

[0086] In another embodiment, the method of the invention may include administration to a subject with measurable disease in the liver according to RECIST v.1.1 criteria. In an additional embodiment, the method of the invention may include administration to a subject exhibiting an Eastern Cooperative Oncology Group (ECOG) performance score (PS) of 0-1 at screening. In another embodiment, a subject administered treatment according to the method of the invention has a life expectancy of greater than 3 months at screening, as estimated by the investigator. In yet another embodiment, the subject has a QTc interval of 480 msec or less.

[0087] In another embodiment, all relevant clinically significant drug-related toxicity from previous cancer treatment is resolved prior to treatment.In this embodiment, resolution is less than or equal to grade 1 or the patient's pre-treatment level.In a further embodiment, the subject may have grade 2 alopecia and endocrine abnormalities that are controlled with replacement therapy.

[0088] In another embodiment, the method of the invention may include administration to a subject with adequate organ function at the time of screening. In one embodiment, a subject with adequate organ function may exhibit one or more of the following: (i) platelet count >100,000 / μL, (2) hemoglobin >8.0 g / dL, (3) white blood cell count (WBC) >2,000 / μL, (4) serum creatinine <2.0 mg / dL unless measured creatinine clearance is >30 mL / min as calculated by the Cockcroft-Gault formula, (5) total and direct bilirubin <2.0 x upper limit of normal (ULN) and alkaline phosphatase <5 x ULN. , (6) total bilirubin up to 3.0 mg / dL for patients with documented Gilbert's disease, (7) ALT and AST ≤ 5 × ULN, and (8) prothrombin time / international normalized ratio (INR) or activated partial thromboplastin time (aPTT) test result ≤ 1.5 × ULN at screening (this applies only to patients not receiving therapeutic anticoagulation; patients receiving therapeutic anticoagulation must be on a stable dose for at least 4 weeks before the first dose of the study intervention).

[0089] According to one or more embodiments, the method of the present invention includes a method for treating primary liver cancer, such as HCC or ICC, in which the subject has histologically or cytologically confirmed HCC or ICC with liver-only or liver-dominant disease.Liver-dominant disease may represent the majority of disease, or may present as intrahepatic disease when the progression of HCC or ICC represents a significant threat to the patient's life.

[0090] In one or more embodiments, the methods of the invention may include administration to a subject with a Class A designation on the Child-Pugh liver function scale (a three-category scale [A, B, or C], with C indicating the most severe impairment of liver function). In one or more additional embodiments, the methods of the invention may include administration to a subject with adequate hematological and organ function.

[0091] In one or more embodiments, the methods of the invention may include administration to a subject with persistent or progressive measurable disease as defined by RECIST version 1.1 who has previously received at least initial systemic therapy for liver cancer and is not amenable to curative therapy.

[0092] According to another embodiment, the tumor is unresectable.

[0093] According to another embodiment, the methods of the present invention may be administered in conjunction with other cancer therapeutics, such as immunomodulatory agents, tumor killing agents, and / or other targeted therapeutics.

[0094] According to one embodiment, TLR9 therapy may be administered in combination with cell therapy (thereby allowing cell therapy through modulation of the immune system), chemoembolization, or radioembolization.

[0095] In one embodiment, the above method of administration to the liver is intended to provide penetration of the Toll-like receptor agonist throughout the entire solid tumor, the entire organ, or substantially throughout the entire tumor. In one embodiment, such a method enhances the perfusion of the Toll-like receptor agonist in a patient in need of enhanced perfusion of the Toll-like receptor agonist, including overcoming interstitial fluid pressure and solid stress of the tumor. In another embodiment, perfusion throughout the entire organ or a portion thereof may provide benefits for the treatment of disease by fully exposing the tumor to the therapeutic agent. In one embodiment, such a method may better obtain delivery of the Toll-like receptor to areas of the tumor that have poor access to the systemic circulation. In another embodiment, such a method delivers a higher concentration of the Toll-like receptor agonist to such tumors, with less Toll-like receptor agonist being delivered to non-target tissues compared to conventional systemic delivery via peripheral veins. Non-target tissues are tissues that are directly perfused by the arterial network that is directly connected to the infusion device. In one embodiment, such a method results in a reduction in size, a reduction in growth rate, or shrinkage or elimination of the solid tumor.

[0096] The methods of the invention may also include mapping the blood vessels leading to the right and left lobes of the liver prior to performing the HAI or selective injection into specific sectors or segments, and optionally occluding blood vessels that do not lead to the liver or are otherwise not targeted. In some embodiments, prior to injection, the patient may undergo a mapping angiogram, for example, via a common femoral artery approach.

[0097] Methods for mapping blood vessels in the body and for delivery of therapeutic agents are well known to those skilled in the art. Occlusion can be achieved, for example, by the use of microcoil embolization, which allows the investigator to block off-target arteries or blood vessels, thereby optimizing delivery of modified cells to the liver. Microcoil embolization can be performed, if necessary, before administering the first dose of TLR9 agonist, for example, to facilitate optimal infusion of the pharmaceutical composition comprising the TLR9 agonist. In another embodiment, a sterile sponge (e.g., GELFOAM) can be used. In this regard, the sterile sponge can be cut and pushed into the catheter. In another embodiment, the sterile sponge can be provided as granules.

[0098] In some embodiments, the dose of a TLR9 agonist such as SD-101 can be about 0.01 mg, about 0.03 mg, about 0.05 mg, about 0.1 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, or about 8 mg. In some embodiments, SD-101 is administered in doses of 12 mg, 16 mg, and 20 mg. The administration of milligram amounts of SD-101 (e.g., about 2 mg) describes the administration of about 2 mg of the composition illustrated in FIG. 1. For example, such amounts of SD-101 (e.g., about 2 mg amounts) can also be present in compositions that contain materials such as other related and unrelated compounds in addition to such amounts of SD-101. Molar equivalent amounts of other pharma- ceutically acceptable salts are also contemplated.

[0099] In some embodiments, the dose of the TLR9 agonist, such as SD-101, can be about 0.01 mg to about 20 mg, about 0.01 mg to about 10 mg, about 0.01 mg to about 8 mg, and about 0.01 mg to about 4 mg. In some embodiments, the dose of the TLR9 agonist, such as SD-101, can be about 2 mg to about 10 mg, about 2 mg to about 8 mg, and about 2 mg to about 4 mg. In some embodiments, the dose of the TLR9 agonist, such as SD-101, can be less than about 10 mg, less than about 8 mg, less than about 4 mg, or less than about 2 mg. Such doses can be administered daily, weekly, or biweekly. In one embodiment, the dose of SD-101 is gradually increased, such as through administration of about 2 mg, followed by about 4 mg, then about 8 mg.

[0100] In some embodiments, the method of the present invention may include administering a dosing regimen that includes cycles, one or more of which include administering SD-101 via HAI and PEDD. As used herein, a "cycle" is a repeating dosing sequence. In one embodiment, a cycle includes three weekly doses per cycle (i.e., weekly administration of SD-101 for three consecutive weeks). In one embodiment, a cycle of treatment according to the present invention may include a period of SD-101 administration followed by an "off" period or rest period. In another embodiment, in addition to three weekly doses per cycle, the cycle further includes a 1-week, 2-week, 3-week, or 4-week rest period after the weekly administration of SD-101. In yet another embodiment, in addition to three weekly doses per cycle, the cycle further includes a rest period of about 38 days after the weekly administration of SD-101. In another embodiment, the entire cycle includes about 52 days. In another embodiment, the dosing regimen includes at least one, at least two, or at least three, or more cycles.

[0101] In some embodiments, the present invention relates to the use of a TLR9 agonist in the manufacture of a medicament for treating solid tumors of the liver, such as tumors resulting from primary liver cancers, such as HCC and ICC, the method comprising administering a TLR9 agonist to a patient in need of treatment, wherein the TLR9 agonist is administered to such solid tumors of the liver through a device by HAI.

[0102] In some embodiments, SD-101 is administered at a dose of 0.5 mg through the HAI for the treatment of primary liver cancer such as HCC and ICC, and in some embodiments, SD-101 is further administered through a pressure-regulating device (i.e., PEDD). In some embodiments, SD-101 is administered at a dose of 0.5 mg through the HAI and through a device that regulates vascular pressure in combination with a CPI, and the CPI is nivolumab. In other embodiments, SD-101 is administered at a dose of 0.5 mg through the HAI and through a device that regulates pressure in combination with ipilimumab. In some embodiments, SD-101 is administered at a dose of 0.5 mg through the HAI and through a device that regulates pressure in combination with ipilimumab and nivolumab. In other embodiments, SD-101 is administered at a dose of 0.5 mg through the HAI and through a device that regulates pressure in combination with pembrolizumab.

[0103] In some embodiments, SD-101 is administered at a dose of 2 mg through the HAI for the treatment of primary liver cancer such as HCC and ICC, and in some embodiments, SD-101 is further administered through a pressure-regulating device (i.e., PEDD). In some embodiments, SD-101 is administered at a dose of 2 mg through the HAI and through a device that regulates vascular pressure in combination with a CPI, and the CPI is nivolumab. In other embodiments, SD-101 is administered at a dose of 2 mg through the HAI and through a device that regulates pressure in combination with ipilimumab. In some embodiments, SD-101 is administered at a dose of 2 mg through the HAI and through a device that regulates pressure in combination with ipilimumab and nivolumab. In other embodiments, SD-101 is administered at a dose of 2 mg through the HAI and through a device that regulates pressure in combination with pembrolizumab.

[0104] In some embodiments, SD-101 is administered at a dose of 4 mg through the HAI for the treatment of primary liver cancer such as HCC and ICC, and in some embodiments, SD-101 is further administered through a pressure-regulating device (i.e., PEDD). In some embodiments, SD-101 is administered at a dose of 4 mg through the HAI and through a device that regulates vascular pressure in combination with a CPI, and the CPI is nivolumab. In other embodiments, SD-101 is administered at a dose of 4 mg through the HAI and through a device that regulates pressure in combination with ipilimumab. In some embodiments, SD-101 is administered at a dose of 4 mg through the HAI and through a device that regulates pressure in combination with ipilimumab and nivolumab. In other embodiments, SD-101 is administered at a dose of 4 mg through the HAI and through a device that regulates pressure in combination with pembrolizumab.

[0105] In some embodiments, SD-101 is administered at a dose of 8 mg through the HAI for the treatment of primary liver cancer such as HCC and ICC, and in some embodiments, SD-101 is further administered through a pressure-regulating device (i.e., PEDD). In some embodiments, SD-101 is administered at a dose of 8 mg through the HAI and through a device that regulates vascular pressure in combination with a CPI, and the CPI is nivolumab. In other embodiments, SD-101 is administered at a dose of 8 mg through the HAI and through a device that regulates pressure in combination with ipilimumab. In some embodiments, SD-101 is administered at a dose of 8 mg through the HAI and through a device that regulates pressure in combination with ipilimumab and nivolumab. In other embodiments, SD-101 is administered at a dose of 8 mg through the HAI and through a device that regulates pressure in combination with pembrolizumab.

[0106] In one or more embodiments, the methods of the invention may include administering to a subject up to six doses (up to two cycles of SD-101, three weekly doses per cycle). In one or more embodiments, fewer doses or cycles of SD-101 may be administered based on the subject's toxicity or tolerability.

[0107] In one or more embodiments, a solution of SD-101 can be administered to a subject via HAI using a PEDD device such as TriNav®. In some such embodiments, vascular access can be achieved using a femoral or radial artery approach. Hepatic hemangiomas, shunt vessels, or other vascular lesions that may impede therapeutic delivery can be embolized at the discretion of the treating interventional radiologist. In one or more embodiments, SD-101 can be prepared and delivered in a 50 mL syringe (therapeutic dose) and a 100 mL vial containing the amount required for a therapeutic flush (10 mL), both at therapeutic concentrations. The PEDD device can then be advanced into the target vessel.

[0108] In one or more embodiments, a 50 mL solution of SD-101 can be allocated per segment or sector of the liver. In one embodiment, a 50 mL therapeutic dose of SD-101 can be allocated as follows: 3 x 10 mL injections into the target vessels of the right liver lobe and 2 x 10 mL injections into the target vessels of the left liver lobe. Furthermore, the distribution of the 10 mL aliquots can be adjusted based on the location of measurable disease and the target vessel diameter. In one or more embodiments, the SD-101 injection can be expected to last about 10-60 minutes. For example, in some embodiments, the injection time can be about 25 minutes. Furthermore, in another embodiment, the entire interventional procedure can last 30-80 minutes. This involves all the handling time between injections at different locations. In some embodiments, the 50 mL solution of SD-101 can include one of 0.5 mg, 2 mg, 4 mg, or 8 mg of SD-101. In this regard, the injection dose of SD-101 can be one of 0.01 mg / mL, 0.04 mg / mL, 0.08 mg / mL, or 0.16 mg / mL.

[0109] According to another embodiment, SD-101 can be prepared and delivered in a 25 mL solution. In some embodiments, the 25 mL solution of SD-101 can include one of 0.5 mg, 2 mg, 4 mg, or 8 mg of SD-101. In this regard, the injection dose of SD-101 can be one of 0.02 mg / mL, 0.08 mg / mL, 0.16 mg / mL, or 0.32 mg / mL.

[0110] According to another embodiment, SD-101 can be prepared and delivered in a 10 mL solution. In some embodiments, a 10 mL solution of SD-101 can contain one of 0.5 mg, 2 mg, 4 mg, or 8 mg of SD-101. In this regard, the injection dose of SD-101 can be one of 0.05 mg / mL, 0.2 mg / mL, 0.4 mg / mL, or 0.8 mg / mL.

[0111] In some embodiments, the method of the present invention results in the treatment of the target lesion. In this embodiment, the method of the present invention may result in a complete response, including the disappearance of all target lesions. In some embodiments, the method of the present invention may result in a partial response, including at least a 30% decrease in the sum of the longest diameters of the target lesions, based on the baseline sum longest diameter. In some embodiments, the method of the present invention may result in stable disease of the target lesions, with neither a sufficient decrease in the sum longest diameter since the start of treatment to indicate a partial response, nor an increase in the sum longest diameter since the start of treatment to indicate progressive disease. In such embodiments, progressive disease is characterized by at least a 20% increase in the sum longest diameter of the target lesions, based on the smallest sum longest diameter recorded since the start of treatment or the appearance of one or more new lesions. This sum must show an absolute increase of 5 mm.

[0112] In another embodiment, the method of the present invention results in the treatment of non-target lesions. Non-target lesions are lesions that are not directly perfused by an arterial network that is in direct communication with the infusion system. In this embodiment, the method of the present invention may result in a complete response, including the disappearance of all non-target lesions. In some embodiments, the method of the present invention results in the persistence of one or more non-target lesions, but does not result in a complete response or progressive disease. In such embodiments, progressive disease is characterized by a clear progression of existing non-target lesions and / or the appearance of one or more new lesions.

[0113] In some embodiments, the methods of the invention result in a beneficial overall response rate, such as an overall response rate by RECIST v.1.1. In these embodiments, the methods of the invention result in an overall response that is a complete response, where the subject shows a complete response of the target lesions, a complete response of the non-target lesions, and shows no new lesions. In other embodiments, the methods of the invention result in an overall response that is a partial response, where the subject shows a complete response to the target lesions, a non-complete response to the non-target lesions, and non-progressive disease, and shows no new lesions. In other embodiments, the methods of the invention result in an overall response that is a partial response, where the subject shows a partial response to the target lesions, non-progressive disease to the non-target lesions, and shows no new lesions. In another embodiment, the methods of the invention result in an overall response that is stable disease, where the subject shows stable disease of the target lesions, non-progressive disease to the non-target lesions, and shows no new lesions.

[0114] In some embodiments, the methods of the invention result in an increase in the duration of overall response. In some embodiments, the duration of overall response is measured from the time that the measurement criteria for complete response or partial response are met (whichever is recorded first) to the first date that recurrence or progressive disease is objectively documented (the minimum measurement recorded since the start of treatment is used as the criterion for progressive disease). The duration of overall complete response can be measured from the time that the measurement criteria for complete response are first met to the first date that progressive disease is objectively documented. In some embodiments, the duration of stable disease is measured from the start of treatment until the progression criterion is met, using the minimum measurement recorded since the start of treatment, including the baseline measurement.

[0115] In yet another embodiment, the method of the present invention results in improved overall survival. For example, overall survival can be calculated from the date of enrollment to the time of death. Patients who are alive before the data cutoff for the final efficacy analysis or who drop out before the end of the study are censored at the date of last known survival.

[0116] In other embodiments, the method of the present invention provides progression-free survival.For example, progression-free survival can be calculated from the date of enrollment to the time of CT scan documenting recurrence (or other clear indicator of disease onset), or to the date of death (whichever occurs first).Patients who do not have documented recurrence and are alive before the data cutoff for final efficacy analysis, or who drop out before the end of the study, are censored at the date of the last radiological evidence documenting no recurrence.

[0117] In some embodiments, the methods of the invention result in a beneficial overall response rate, such as an overall response rate by mRECIST. In these embodiments, the methods of the invention result in an overall response that is a complete response, where the subject shows a complete response of the target lesion, a complete response of the non-target lesion, and no new lesions. In other embodiments, the methods of the invention result in an overall response that is a partial response, where the subject shows a complete response to the target lesion, a non-complete response and an incomplete response to the non-target lesion, and no new lesions. In other embodiments, the methods of the invention result in an overall response that is a partial response, where the subject shows a partial response to the target lesion, non-progressive disease to the non-target lesion, and no new lesions. In another embodiment, the methods of the invention result in an overall response that is stable disease, where the subject shows stable disease of the target lesion, non-progressive disease to the non-target lesion, and no new lesions.

[0118] In some embodiments, the methods of the invention result in a beneficial overall response rate, such as an overall response rate by iRECIST.

[0119] According to another embodiment, the method of the present invention includes a method for treating primary liver cancer, such as HCC and ICC, wherein administration of SD-101 results in a reduction in tumor burden. In some embodiments, the 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%.

[0120] According to another embodiment, the method of the present invention includes a method for treating primary liver cancer, such as HCC and ICC, wherein administration of SD-101 results in a reduction in tumor progression or stabilization of tumor growth. In some embodiments, 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%.

[0121] According to another embodiment, the method of the present invention includes a method for treating primary liver cancer, such as HCC and ICC, and administration of SD-101 reprograms the hepatic MDSC compartment to allow immune control of liver cancer and / or improves responsiveness to systemic anti-PD-1 therapy through elimination of MDSC. In some embodiments, the method of the present invention is superior in controlling MDSC. In some embodiments, the method of the present invention includes a method for treating primary liver cancer, such as HCC and ICC, and administration of SD-101 reduces the frequency of MDSC cells (CD11b+Gr1+), monocytic MDSC (M-MDSC; CD11b+Ly6C+) cells, or granulocytic MDSC (G-MDSC; CD11b+LY6G+) cells, or human MDSC (CD33+CD11b+HLADR- (CD14+ for m-MDSC and CD15+ for G-MDSC)). According to another embodiment, the method of the present invention enhances M1 macrophages (CD14+CD86+). According to yet another embodiment, the method of the present invention reduces M2 macrophages (CD14+CD163+CD206+).

[0122] In another embodiment, the method of the invention increases NFκB activation. In yet a further embodiment, the method of the invention increases IL-6. In another embodiment, the method of the invention increases IL-10. In yet a further embodiment, the method of the invention increases IL-29. In another embodiment, the method of the invention increases IFNα. In a further embodiment, the method of the invention decreases STAT3 phosphorylation.

[0123] The present invention is further illustrated and / or demonstrated in the following examples, which are presented for illustrative / demonstrative purposes only and are not intended to limit the invention in any way.

[0124] Example 1 The aim of this study was to evaluate the distribution of injected TLR9 agonist SD-101 in and around primary liver tumors. In particular, the study was performed in a novel pig model that forms liver tumors in situ. To recapitulate the development of primary liver tumors, biopsies of liver tissue were collected from pigs, and cells were then exposed to the vector to induce cancerous growth. These cells were then implanted into liver tissue by local needle injection and allowed to develop tumors measuring approximately 2 cm in diameter.

[0125] TLR9 agonist SD-101 sequence oligo was synthesized and conjugated to IRD800CW (excitation wavelength 767 nm, emission wavelength 791 nm) fluorophore. IRD800CW-SD-101 (1 ml at 2.5 nmol) was then mixed with SD-101 (0.3 ml, containing 4 mg SD-101 drug) in 9 ml of sterile saline to produce a total volume of 10.3 ml for injection. The solution was injected via the HAI using a PEDD device, i.e., TriNav®.

[0126] Access was gained through the femoral artery of the subject pig using the Seldinger technique. A 5F introducer sheath was secured in place. An angiogram was performed using a 5F angiography catheter to identify the hepatic arterial anatomy. A vessel between 1.5 and 3.5 mm in diameter was then selected that supplied the entire left lateral lobe, left middle lobe, right middle lobe, or right lateral lobe containing the tumor. The TriNav® was then tracked to the target vessel location.

[0127] The syringe containing the solution was then placed in a syringe pump and delivered through the TriNav® at a rate of 2 ml / min. After the first 4 ml of solution was injected, a 1 ml high pressure bolus was delivered over a 0.5 second period at a rate of 2 cc / sec. The remainder of the solution was then administered at 2 ml / min.

[0128] Normal physiological conditions were maintained for 60 minutes, after which time the pigs were euthanized and their livers were removed. Each lobe of the liver was separated and near-infrared imaging was performed using a Pearl Trilogy Imaging System to identify the pattern of drug uptake. Each lobe was then cut into 1 cm thick sections and imaged separately on both tissue planes using the Pearl system (85 μm resolution, 700 nm, 800 nm, and white light channels). The entire volume of the liver was analyzed to visualize the distribution of fluorescently labeled SD-101, as shown in Figure 4. In this regard, A shows tumor tissue and areas of necrosis, B shows areas of growing primary tumor and surrounding normal tissue with high uptake of fluorescently labeled SD-101, and C shows normal tissue (showing little or no fluorescent signal of labeled SD-101).

[0129] This study demonstrated that pressure-enabled delivery of labeled SD-101 using TriNav® resulted in preferential accumulation of the fluorescent compound in the area surrounding the actively growing primary liver tumor periphery and surrounding normal tissue, an area that is expected to contain the majority of immune cells expressing TLR9, the target of SD-101. Furthermore, injection was performed through a blood vessel supplying the entire lobe containing the primary tumor, and the majority of tissue within the perfusion area was normal liver. Despite not injecting SD-101 through a specific tumor-supplying blood vessel, the use of PEDD resulted in preferential distribution of SD-101 in and around the tumor, with relatively low drug exposure in normal liver tissue.

[0130] Example 2 In this example, infusion of the TLR9 agonist SD-101 was administered with the aim of improving response rates to CPI therapy in patients with primary liver cancers such as HCC and ICC. SD-101 is a TLR9 agonist that can stimulate various immune cells in addition to favorably eliminating or reprogramming the inhibitory immune cells that drive tumor progression in the liver. Pressure-enabled drug delivery (PEDD) via hepatic artery infusion (PEDD / HAI) was applied in this study, which will allow for effective delivery of SD-101 to HCC tumors while limiting systemic exposure.

[0131] The study has two phases, namely Phase 1b and 2. In this regard, the primary objective of Phase 1b is to determine the safety and identify the maximum tolerated dose (MTD) or optimal dose of PEDD / HAI of SD-101 alone, the MTD or optimal dose of SD-101 in combination with pembrolizumab or both nivolumab and ipilimumab in patients with HCC or ICC or a mixture of HCC and ICC. Furthermore, the secondary objective is to evaluate the preliminary efficacy in terms of RECIST for immune-based therapy (iRECIST) ORR, modified RECIST (mRECIST) ORR, RECIST 1.1 liver-specific response rate (HRR), overall progression-free survival (PFS), and clinical benefit (complete response [CR] + partial response [PR] + stable disease [SD]). For Phase 2, the primary objective is to evaluate the overall response rate (ORR) of Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 to PEDD / HAI of SD-101 in combination with intravenous (IV) immune checkpoint blockade. The checkpoint regimen used in combination with SD-101 will be selected based on safety and response data from Phase 1b. Separate cohorts of HCC and ICC will be enrolled. Additionally, secondary objectives are to evaluate the duration of response (DOR), 12-month overall survival (OS), and safety / toxicity of the selected MTD or optimal dose of SD-101 in combination with CPI.Additionally, there are the following exploratory objectives: (i) to assess RECIST v1.1 liver-specific progression-free survival (HPFS); (ii) to assess pathologic response following PEDD / HAI of SD-101 with or without systemic IV CPI infusion, and correlation with imaging response scoring; (iii) to evaluate the intratumoral immunological effect of treatment on MDSC, lymphocyte, and cytokine profiles using paired baseline and on-treatment liver tumor and normal liver biopsies; (iv) to evaluate the peripheral immunopharmacodynamic effect of treatment using serial blood draws for circulating tumor cells (CTCs), circulating cytokines, and other immunological correlates; (v) to assess change from baseline in ECOG PS over time; and (vi) to assess change from baseline in quality of life using the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire for Cancer (EORTC-QLQ-C30) instrument.

[0132] The overall design of the study can be found in Figure 5. The study is open-label, multicenter, and non-randomized.

[0133] As described in more detail below, in Phase 1b, escalating doses of SD-101 can be administered alone (Cohort A), together with pembrolizumab (Cohort B), or together with ipilimumab and nivolumab in combination (Cohort C). Cohorts B and C will begin one dose level below the MTD or optimal dose from Cohort A to optimize safety when adding CPI to SD-101. Cohort B will not begin until completion of the DLT window of the last patient in Cohort A (Cohort B will proceed simultaneously with the expansion of Cohort A). Cohort C will not begin until completion of the DLT window of the last patient in Cohort B. A standard 3+3 dose escalation design will be employed to determine the MTD.

[0134] Overnight in-hospital observation or hospitalization is required after the first SD-101 infusion for each patient in Phase 1b. If the first SD-101 dose is well tolerated, further overnight observation or hospitalization will be at the discretion of the treating physician for subsequent SD-101 infusions. If subsequent infusions are performed on an outpatient basis, patients will be observed for a minimum of 6 hours after infusion before being discharged if clinically stable. If there is any event of grade 2 or greater related to SD-101 PEDD / HAI requiring inpatient therapy after the first infusion, patients will be retained for overnight observation or hospitalization after each subsequent SD-101 infusion.

[0135] After determining the recommended MTD or optimal dose of SD-101 for PEDD / HAI and which CPI regimen is tolerated, the study will proceed to Phase 2 to evaluate efficacy. Patients in Phase 2 will receive the SD-101 dose selected from Phase 1b along with single-agent or dual-agent checkpoint blockade. The choice of single or dual CPI therapy with SD-101 for Phase 2 will consider safety data in addition to response rates from cohorts B and C in Phase 1b. SD-101 will be administered at three weekly doses per cycle for two cycles.

[0136] Study Inclusion Criteria According to one embodiment, to be included in the study, patients must meet all of the following criteria: 1. Age 18 years or older with locally advanced, metastatic, or unresectable hepatocellular carcinoma or intrahepatic cholangiocarcinoma with a diagnosis confirmed by histological or cytological analysis or clinical features according to the American Association for the Study of Liver Diseases. 2.Has had persistent or progressive measurable disease as defined by RECIST version 1.1, having previously received at least first-line systemic therapy for liver cancer and is not suitable for curative therapy. 3. Have a performance status score of 0 or 1 on the ECOG scale (scores range from 0 to 5, with higher numbers reflecting greater disability). 4. Class A designation on the Child-Pugh Liver Function Scale (a three-category scale [A, B, or C], with C indicating the most severe impairment of liver function). 5. Adequate hematological and organ function. 6. Histologically or cytologically confirmed HCC or ICC with liver-only or liver-predominant disease. Liver-predominant disease will be defined as intrahepatic disease representing the greatest proportion of disease. 7. Able to understand the study and provide written informed consent prior to any study procedures. 8. No prior cytotoxic chemotherapy, targeted therapy, or external radiation therapy within 14 days prior to screening. 9. No prior embolic HAI therapy with permanent embolic material. NOTE: Previous embolic HAI therapy with permanent embolic material would not be exclusive if, after this therapy, the target vessel is not occluded and the tumor is perfused based on patient screening. 10. No previous medical history or other concurrent malignancies unless the malignancy is clinically significant, no ongoing treatment is required, and the patient is clinically stable. 11.Has measurable liver disease per RECIST v.1.1 criteria. 12.Has a life expectancy of >3 months at screening as estimated by the investigator. 13. Have a QTc interval of 480 msec or less. 14. All relevant clinically significant (in the investigator's judgment) drug-related toxicities from previous cancer treatment must have resolved (grade ≤ 1 or to the patient's pre-treatment levels) prior to study treatment administration (grade 2 alopecia and endocrinopathy controlled with replacement therapy are permitted). 15.Has adequate organ function at screening as evidenced by: ·Platelet count>100,000 / μL. Hemoglobin ≥ 8.0g / dL. ·White blood cell count (WBC)>2,000 / μL. · Serum creatinine ≤ 2.0 mg / dL unless the measured creatinine clearance is ≥ 30 mL / min as calculated by the Cockcroft-Gault formula. Total and direct bilirubin ≤ 2.0 x upper limit of normal (ULN) and alkaline phosphatase ≤ 5 x ULN. In patients with documented Gilbert's disease, a total bilirubin of up to 3.0 mg / dL is acceptable. ALT and AST ≤ 5 × ULN. Prothrombin time / international normalized ratio (INR) or activated partial thromboplastin time (aPTT) test result ≤ 1.5 x ULN at screening (this applies only to patients not receiving therapeutic anticoagulation; patients receiving therapeutic anticoagulation must be on a stable dose for at least 4 weeks prior to the first dose of the study intervention). Note: Laboratory tests with exclusionary results judged by the investigator to be incompatible with the patient's clinical condition may be repeated once for eligibility. 16. Females of childbearing potential must be non-pregnant and non-lactating or postmenopausal and have a negative serum human chorionic gonadotropin (hCG) pregnancy test result at screening and a negative urine or serum pregnancy test prior to the first dose of study intervention. Women of childbearing potential must agree to abstain from sexual activity with uncontraceptively treated male partners or, if sexually active with uncontraceptively treated male partners, agree to use highly effective contraception from screening through the duration of the study, and agree to continue such precautions for 100 days after the final dose of study intervention. Women of childbearing potential and sexually active, contraceptive-untreated men must agree to use effective contraception and to refrain from sperm donation during the study from Day 1 through 30 days after the final dose of study intervention.

[0137] Phase 1b The separate Phase 1b patient cohorts are: Cohort A - Dose escalation cohort of SD-101 monotherapy PEDD / HAI using a standard 3+3 design followed by an optional expansion arm to identify the MTD or optimal dose of SD-101 alone and estimate the response rate of monotherapy in patients with HCC and ICC (2 cycles, 1 dose per cycle per week x 3 weeks): Dose level 1: 4mg (n=3-6) Dose level 2: 8mg (n=3-6)

[0138] Enrollment of the first 2 patients at each dose level will be staggered by at least 48 hours. Additional incremental dose levels (in 4 mg increments) may be added based on safety and response data. An optional expansion cohort of 10 patients at the MTD or optimal dose of SD-101 monotherapy may proceed concurrently with Cohort B.

[0139] Cohort B - Standard 3+3 designed dose re-escalation cohort of PEDD / HAI of SD-101 with IV pembrolizumab 200 mg every 3 weeks (Q3W) to identify the MTD or optimal dose of SD-101 with single-agent pembrolizumab (2 cycles, once weekly dose per cycle x 3 weeks): · Dose level 1: Pembrolizumab with PEDD / HAI of SD-101 at a single dose level below the MTD or optimal dose from Cohort A (i.e., MTD-1 or optimal dose-1) (n=3-6). Dose level 2: Pembrolizumab with PEDD / HAI of SD-101 at the MTD or optimal dose from cohort A (n=3-6), or will taper to MTD-2 or optimal dose-2 if MTD-1 or optimal dose-1 is not tolerated.

[0140] Enrollment of the first 2 patients at each dose level will be staggered by at least 48 hours. An optional expansion group of up to 10 patients receiving SD-101 with pembrolizumab may proceed concurrently with Cohort C.

[0141] Cohort C - Standard 3+3 designed dose re-escalation cohort of PEDD / HAI of SD-101 with nivolumab at 1 mg / kg combined with ipilimumab 3 mg / kg every 3 weeks for 4 doses followed by single agent nivolumab 240 mg every 2 weeks to identify the MTD or optimal dose of SD-101 with dual agent CPI (2 cycles, 1 dose per week per cycle x 3 weeks): · Dose level 1: Nivolumab and ipilimumab with PEDD / HAI of SD-101 at a single dose level below the MTD or optimal dose from cohort B (n=3-6). Dose level 2: Nivolumab and ipilimumab with PEDD / HAI of SD-101 at the MTD or optimal dose of SD-101 from cohort B (n=3-6), or will taper to MTD-2 or optimal dose-2 if MTD-1 or optimal dose-1 is not tolerated.

[0142] Enrollment of the first 2 patients at each dose level will be staggered by at least 48 hours. If additional data is needed to decide between single-agent and double-agent CPI with SD-101 via PEDD / HAI for Phase 2, an optional expansion cohort of 10 patients may be enrolled.

[0143] Phase 2 Two separate cohorts will be enrolled for HCC and ICC. A two-stage design will be used in phase 2 to determine if the response rate at SD-101 MTD or optimal dose plus single-agent or double-agent CPI is high enough to justify further testing. The two-stage design with the smallest total sample size will be used for each tumor type.

[0144] Treatment period SD-101 Treatment Period (for Phase 1b and Phase 2 participants): Cohorts A, B, C of Phase 1b, and Phase 2 - up to 6 doses (maximum of 2 cycles of SD-101, 3 weekly doses per cycle). Fewer doses or cycles of SD-101 may be administered based on toxicity or tolerability. All patients with the presence of measurable disease at baseline, one post-baseline tumor assessment, receiving at least one dose of treatment, and no violations of key inclusion or study exclusion criteria will be considered evaluable. However, patients enrolled in violation of key inclusion or study exclusion criteria will be considered a major protocol violation and will not be considered evaluable.

[0145] Duration of CPI administration Phase 1b, Cohort A: Not applicable. Phase 1b, Cohort B, and optional expansion cohort: · Pembrolizumab 200 mg Q3W for up to 6 months. Phase 1b, Cohort C, and optional expansion cohorts: · 1 mg / kg IV nivolumab Q3W for 4 doses, then 240 mg every 2 weeks (Q2W) for up to 12 months. · 3mg / kg IV ipilimumab Q3W for 4 doses. Phase 2: CPI regimen determined by up to 6 months of Phase 1b data. Phase 1, Cohort B, and Optional Expansion Cohort: Nivolumab 480mg Q4W for up to 12 months. Phase 1, optional cohort B1: Systemic IV ipilimumab 3mg / kg Q3W for 4 doses. Phase 1, Cohort C, and optional expansion cohorts: systemic IV nivolumab at 1 mg / kg Q3W for four doses, then 480 mg / kg Q4W for up to 12 months, and (ii) systemic IV ipilimumab at 3 mg / kg Q3W for four doses. Phase 1b: CPI regimen determined by up to 12 months of Phase 1 data.

[0146] Intervention administered The interventions and planned dose levels are summarized in Table 2 below. [Table 2] Abbreviations: HAI = hepatic artery infusion, IMP = investigational drug, IV = intravenous, ODN = oligodeoxynucleotide, PEDD = pressure-enabled drug delivery device. a Each segment, sector, or lobe injection must be completed within that time to ensure that SD-101 does not remain in the TriNav device for longer than 60 minutes. This does not limit the overall procedure time. c A vial will be discarded after preparation of a dose for one patient. In no event will more than one dose be drawn from a single vial.

[0147] PEDD / HAI of SD-101 A PEDD device, e.g., TriNav®, can be used to inject the SD-101 solution via the hepatic arterial system. Vascular access can be achieved using femoral or radial approaches. Hepatic hemangiomas, shunt vessels, or other vascular lesions that may impede therapeutic delivery can be embolized at the discretion of the treating interventional radiologist. For the SD-101 injection procedure, the drug will be prepared at a local pharmacy and delivered to the interventional radiology suite in a 50 mL syringe (therapeutic dose) and a 100 mL vial containing the amount needed for the therapeutic flush (10 mL), both at therapeutic concentrations. The pharmacy can be notified one day prior to the planned injection time and again when the patient is on the table. The PEDD device, TriNav®, will be advanced into the target vessel.

[0148] In one embodiment, the volume of 50 mL administered is allocated per segment or sector of the liver. In one embodiment, a 50 mL therapeutic dose can be allocated as follows: 3 x 10 mL injections into the target vessels of the right liver lobe and 2 x 10 mL injections into the target vessels of the left liver lobe. Furthermore, the distribution of the 10 mL aliquots can be adjusted based on the location of measurable disease and target vessel diameter. In one embodiment, the SD-101 injection can be expected to last about 10-60 minutes. For example, the injection time can be about 25 minutes. Furthermore, in one embodiment, the entire interventional procedure can last 30-80 minutes. This includes all handling time between injections at different locations.

[0149] Tumor response assessment All patients will undergo imaging with magnetic resonance imaging (MRI) or CT to assess disease in the liver and other sites, as well as liver biopsies and assays for CTCs, circulating cytokines, and other immunological correlates. Tumor response will be measured radiologically using standard RECIST v1.1 criteria. Formal response scoring (according to RECIST v1.1) will be preliminarily assessed at day 84. An additional response assessment will be obtained at day 168 to confirm the initial scoring in case of pseudoprogression. Thereafter, imaging procedures will be performed every 90 days. Liver imaging using MRI with Eovist® contrast should be used for evaluation whenever possible. Regional image readout will be utilized for response assessment during phase 1b and phase 2. An independent central review for response assessment may be performed during phase 2.

[0150] Up to four liver biopsies will be performed: A baseline biopsy will be obtained on Day 1 prior to the first infusion of SD-101, in addition to the post-infusion biopsy on Day 1. A pre-infusion biopsy will be performed at the start of the second cycle of SD-101 (prior to the fourth SD-101 infusion), and a final biopsy procedure will be performed on Day 100. · Pathologic response will be assessed based on review by local site pathologists using scoring of necrosis and fibrosis in tumor and normal tissue samples.

[0151] Quality of life Global patient status and quality of life can be assessed using the ECOG PS scale and the EORTC-QLQ-C30 questionnaire.

[0152] Pharmacokinetics Blood samples will be collected to characterize systemic exposure to SD-101 following PEDD / HAI. No sampling or testing will be performed for pembrolizumab, nivolumab, or ipilimumab concentrations. Phase 1b: Serial venous blood samples will be collected for measurement of plasma SD-101 concentrations pre-dose and after completion of each dose level for each dose level in Cohorts A, B, and C. Sample times may be adjusted based on results observed after the first dose of Cohort A in Phase 1b of the study. · Phase 2: If the concentrations are largely below the lower limit of quantification of the assay in Phase 1b, these measurements may be omitted in Phase 2.

[0153] Tumor levels of SD-101 will be measured in pre- and post-infusion biopsy specimens obtained on the following days: Day 1 and Day 57 (pre-infusion only) for Cohorts A, B, and C.

[0154] Each plasma sample can be divided into two aliquots of approximately 2-3 mL each (one for the SD-101 measurement and one as a backup). Samples collected for analysis of SD-101 may also be used to evaluate aspects of safety or efficacy related to concerns that arise during or after the study.

[0155] Pharmacodynamics Blood samples can be collected for measurement of CTCs, circulating cytokines, and other immunological correlates including IFN-α and IFN-γ associated gene signatures, which may be more informative than pharmacokinetic evaluation for this class of therapeutics.

[0156] safety Safety assessments included adverse events (AEs), clinical laboratory tests, vital signs, physical examination, and electrocardiogram (ECG).

[0157] The following will be considered DLTs during Phase 1b when observed within 2 weeks after the last SD-101 dose in Cycle 1 and will be considered attributable to the study intervention (SD-101 or CPI therapy) and / or the TriNav device. Note, however, that the same adjustment or stopping rules will apply to any DLTs (defined below) that may occur during the infusion period of Cycle 2. Cytokine release syndrome (CRS) ≥ Grade 3 according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Autoimmune AEs ≥ Grade 3 according to NCI CTCAE Allergic reactions according to NCI CTCAE AE ≥ Grade 3 Grade 4 hematologic AE by NCI CTCAE that does not resolve to ≤ Grade 2 within 7 days Grade 4 AEs according to NCI CTCAEs in any organ system

[0158] Patients who develop DLT will be permanently discontinued from the study intervention. Patients can be treated according to clinical practice and monitored for resolution of toxicity.

[0159] SD-101 and / or CPI therapy will be permanently discontinued for severe or life-threatening infusion-related reactions. Dose interruption, delay, or discontinuation of SD-101 and / or CPI therapy is necessary if a patient has an immune-mediated reaction of grade 3 or greater. Discontinuation of SD-101 and / or CPI therapy for abnormal liver tests is required if a patient meets one of the conditions outlined below, or in the presence of abnormal liver chemistry that does not meet the stopping rules prescribed in the protocol, if the investigator believes it is in the patient's best interest. · The patient is clinically jaundiced. Patient has evidence of coagulopathy. Patient has clinical evidence of portal hypertension, including but not limited to ascites or variceal bleeding.

[0160] All patients can be followed in this study for safety for at least 6 months after initiation of treatment and for 1 year of disease status.

[0161] Patients who discontinue study treatment for reasons other than disease progression (e.g., toxicity, withdrawal of consent) will continue to have scheduled tumor evaluations every 90 days until the patient dies, experiences disease progression (intrahepatic or extrahepatic), or begins further systemic cancer treatment, whichever occurs first.

[0162] Rescue Medications and Treatments The study site will provide locally obtained immunomodulatory rescue medications. The following rescue medications may be used: Drugs · IL6 receptor antibody tocilizumab 4 to 8 mg / kg systemic IV over 60 minutes for CRS may be repeated as clinically indicated. For CRS or neurologic dysfunction greater than Grade 2, methylprednisolone 2 mg / kg systemic IV bolus followed by 0.5 mg / kg IV every 6 to 12 hours. The first dose may be administered without consultation with the investigator or clinical investigator, but subsequent doses should be administered after consultation with the investigator or clinical investigator. Consider 1-2 doses of an anti-TNFα agent (infliximab or etanercept). Benefit is unclear, but TNFα can rise rapidly so it may be worth considering early in the disease process. Etanercept 25 mg SC twice weekly for two doses given 3 to 4 days apart (0.4 mg / kg twice weekly, maximum 25 mg per dose). · Infliximab dose 10mg / kg systemic IV, once weekly for 2 doses. intervention · Endoscopic cholangiography and stent placement. ·Percutaneous cholangiography and stent placement.

[0163] The use of rescue medication is acceptable at any time during the study, but if possible and clinically appropriate, rescue medication should be postponed for at least 6 hours after administration of the study intervention. The date and time of rescue medication administration, as well as the name and dosing regimen of the rescue medication, must be recorded.

[0164] Adverse events clearly attributable to disease progression, unrelated to the investigational drug, or expected in the eligible patient population of the study are not considered DLTs.

[0165] Evaluation of cytokine release syndrome The investigator or investigator will assess each patient for the presence of CRS. CRS grading will be determined based on the NCI CTCAE v5.0 and will be administered as shown in Table 3. [Table 3] Abbreviations: 2D = two-dimensional, ALT = alanine aminotransferase, AST = aspartate aminotransferase, ASTCT = American Society for Transplantation and Cellular Therapy, CRS = cytokine release syndrome, CTCAE = Common Terminology Criteria for Adverse Events, ICU = intensive care unit, IL = interleukin, IV = intravenous, NCI = National Cancer Institute, NS = saline, SBP = systolic blood pressure, ULN = upper limit of normal. a Fever is defined as a body temperature ≧38° C. not attributable to any other cause. Source: Adapted from NCI CTCAE and ASTCT (Lee DW, Santomasso BD, Locke FL, Ghobadi A, Turtle CJ, Brudno JN, et al. ASTCT consensus grading for cytokine release syndrome and neurologic toxicity associated with immune effector cells. Biol Blood Marrow Transplant. 2019;25;625-38).

[0166] image The extent of disease will be measured radiologically at the time points of Cohort A, Cohort B, and Cohort C. Screening evaluations must include MRI of the abdomen and pelvis (with oral / IV Eovist contrast unless contraindicated) and a brain scan (CT or MRI with IV contrast). In addition to the MRI of the abdomen and pelvis, a spiral CT scan of the chest should be obtained. If MRI is medically contraindicated or at the physician's discretion, a CT scan of the chest, abdomen, and pelvis may be performed with triple-phase IV contrast. If the site does not allow for a triple-phase CT of the chest, abdomen, and pelvis, a CT of the chest, abdomen, and pelvis may be performed with IV contrast and arterial and portal venous phase images may be obtained. If a PET / CT scan is performed (not required), the CT portion of the study must be consistent with the standard of a full contrast CT scan. Liver metastasis response will be assessed with abdominal CT or MRI, while extrahepatic disease will be assessed with whole-body PET / CT or CT / MRI scans covering the chest, abdomen, and pelvis. Liver imaging using MRI with Eovist contrast should be used to evaluate liver tumors whenever possible. The same imaging methodology used at screening should be used throughout the study.

[0167] Any evaluable or measurable disease should be documented at screening and reassessed at each subsequent tumor assessment. For patients with measurable disease, response will be assessed according to RECIST v1.1. Local image reads will be utilized for response assessment during Phase 1b. Independent central review (ICR) for response assessment may be considered during Phase 2.

[0168] At the investigator's discretion, imaging may be performed whenever PD is suspected. In addition, mRECIST and iRECIST assessments will be performed for secondary endpoint data collection but will not be incorporated into the official response score.

[0169] ECOG performance status The ECOG PS scale is used to assess how the disease is affecting the patient's activities of daily living and their ability to care for themselves. At each designated time point, qualified facility personnel will assess the patient according to the following scale: Fully active, able to perform all pre-illness activities without limitations · Physically strenuous activity is limited, but walking is possible and work of a light or sedentary nature (e.g., light housework, clerical work) is possible Able to walk and perform all self-care activities, but unable to perform any work activities and able to get up and move around for at least 50% of waking hours Limited self-care possible, confined to bed or chair for more than 50% of waking time Completely disabled, unable to perform any self-care activities and completely confined to a bed or chair ·death

[0170] Changes, ie, worsening, are not recorded as AEs unless reported during non-prompted questioning of the AE.

[0171] Definitions of RECIST v1.1 Measurable Disease - the presence of at least one measurable lesion. If measurable disease is limited to a solitary lesion, its neoplastic nature should be confirmed by cytology / histology.

[0172] Measurable lesion - a lesion that can be accurately measured in at least one dimension with a longest diameter ≥ 10 mm (CT scan section thickness ≤ 5 mm).

[0173] Non-measurable lesions - All other lesions including small lesions (<10 mm in longest diameter) as well as truly non-measurable lesions (leptomeningeal disease, ascites, pleural / pericardial effusion, inflammatory breast disease, lymphatic involvement of the skin or lung, abdominal masses that cannot be measured by reproducible imaging techniques, etc.).

[0174] Baseline Documentation All measurable lesions, up to a maximum of two per organ and a total of five, should be identified as target lesions, recorded and measured at baseline.

[0175] Target lesions should be selected based on their size (lesion with longest diameter) and suitability for accurate repeated measurement with consistent imaging techniques.

[0176] The sum of the longest diameter (LD) of all target lesions (non-nodular) is calculated and reported as the baseline sum LD, which is used as a reference to characterize objective tumor response in measurable dimensions of disease.

[0177] All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence or absence of each should be noted during follow-up.

[0178] Target Lesion Evaluation Complete response (CR): Disappearance of all target lesions

[0179] Partial response (PR): At least a 30% reduction in the sum of the LD of target lesions, based on baseline sum LD.

[0180] Progressive Disease (PD): At least a 20% increase in the sum of the LD of the target lesions based on the smallest recorded sum LD since the start of treatment or the appearance of one or more new lesions. This sum must show an absolute increase of 5 mm.

[0181] Stable Disease (SD): Based on the smallest total LD ​​since the start of treatment, there is neither a sufficient shrinkage to allow PR nor a sufficient increase to allow PD.

[0182] Evaluation of non-target lesions Complete response (CR): Disappearance of all non-target lesions

[0183] Non-CR / Non-PD: Persistence of one or more non-target lesions

[0184] Progressive Disease (PD): Definite progression of existing non-target lesions and / or appearance of one or more new lesions.

[0185] Assessment of success Responses will be assessed at Cohort A, Cohort B, and Cohort C time points according to Table 4. [Table 4] Abbreviations: CR = complete response, PD = progressive disease, PR = partial response, SD = stable disease.

[0186] Duration of overall response Duration of overall response is measured from the time that the metrics are met for CR or PR (whichever is recorded first) to the first date of objective documentation of relapse or PD (PD criterion is the smallest measurement recorded since treatment initiation). Overall CR duration is measured from the time that the metrics are first met to the first date of objective documentation of PD. Duration of SD: Stable disease is measured from the start of treatment until progression criteria are met, based on the sum of the smallest measurements recorded since treatment initiation, including baseline measurements.

[0187] overall survival rate For all patients, OS will be calculated from the date of enrollment to the time of death. Patients who are still alive before the data cutoff for the final efficacy analysis or who drop out before the end of the study will be censored at the date of their last known survival.

[0188] Progression-free survival For all patients, PFS will be calculated from the date of enrollment to the time of a CT scan documenting recurrence (or other clear indicator of disease development) or the date of death, whichever occurs first. Patients without documented recurrence and still alive prior to the data cutoff for the final efficacy analysis, or patients who drop out before the end of the study, will be censored at the date of the last radiological evidence documenting freedom from recurrence.

[0189] Modified RECIST (mRECIST) The mRECIST definitions for hepatocellular carcinoma are as follows:

[0190] Complete response (CR) = disappearance of any intratumoral arterial enhancement in all target lesions

[0191] Partial Response (PR) = At ​​least a 30% reduction in the sum of the diameters of viable (enhancing in the arterial phase) target lesions compared to the baseline sum of the diameters of the target lesions

[0192] Stable disease (SD) = cases not corresponding to PR or progressive disease

[0193] Progressive Disease (PD) = At ​​least a 20% increase in the sum of the diameters of viable (enhancing) target lesions based on the smallest sum of the diameters of viable (enhancing) target lesions recorded since the start of treatment

[0194] Responses will also be assessed by mRECIST, as shown in Table 5. [Table 5] Abbreviations: CR=complete response, PR=partial response, IR=incomplete response, SD=stable disease, PD=progressive disease. Source: Lencioni R, Llovet JM. Modified RECIST (mRECIST) assessment for hepatocellular carcinoma.Sem Liver Dis.2010;30:52-60.

[0195] RECIST 1.1 for immune-based therapies (iRECIST) Responses will also be assessed by iRECIST, as shown in Table 6. In summary, the main differences between RECIST and iRECIST are described in Seymour et al 2017 as follows: "The principles used to determine objective tumor response remain largely unchanged from RECIST 1.1, but the major change in iRECIST is the concept of "resetting the bar" if RECIST 1.1 progression continues at the next assessment with tumor shrinkage. iRECIST defines iUPD based on the principles of RECIST 1.1, but iUPD requires confirmation, which is based on observing either a further increase in size (or number of new lesions) of the lesion category in which progression was initially identified (i.e., target, non-target disease) or progression of a lesion category (as defined by RECIST 1.1) that did not previously meet RECIST 1.1 progression criteria." However, if progression is not confirmed, but instead tumor shrinkage (compared to baseline) is confirmed that meets the criteria for iCR, iPR, or iSD, the bar is reset so that the iUPD occurs again (compared to the nadir) and must then be confirmed (by further growth) at the next assessment for an iCPD to be assigned. If no change in tumor size or extent from the iUPD occurs, the response time point will again be the iUPD. This approach allows atypical responses, such as delayed responses occurring after pseudoprogression, to be identified, further understood, and better characterized." [Table 6] Abbreviations: CR = complete response, iCPD = confirmed immune PD, iCR = immune complete response, iPR = immune partial response, iRECIST = Response Evaluation Criteria in Solid Tumors for Immune-Based Therapies, iSD = immune stable disease, iUPD = immune unconfirmed PD, NA = not applicable, NL = new lesion, NLT = new lesion target, NLNT = new lesion non-target, NT = not target, PD = progressive disease, PR = partial response, RECIST = Response Evaluation Criteria in Solid Tumors, SD = stable disease, SOM = sum of measurements, TP = time point *Using RECIST 1.1 principles. RECIST 1.1 and iRECIST categories of CR, PR, and SD can be the same if pseudoprogression does not occur. **In any lesion category. ***Previously specified at the most recent evaluation of this TP.

[0196] The above merely illustrates the principles of the present disclosure. In view of the teachings herein, various modifications and alterations to the described embodiments will be apparent to those skilled in the art. Thus, it will be appreciated that those skilled in the art can devise numerous systems, arrangements, and procedures that, although not explicitly shown or described herein, embody the principles of the present disclosure and thus are within the spirit and scope of the present disclosure. Various different exemplary embodiments may be used together and interchangeably with each other, as should be understood by those skilled in the art. In addition, certain terms used in this disclosure, including this specification, may be used synonymously in certain instances, including, but not limited to, data and information. It should be understood that these words, and / or other words that may be synonymous with each other, may be used synonymously herein, although there may be cases where such words are not intended to be used synonymously. Furthermore, to the extent that prior art knowledge has not been expressly incorporated by reference herein above, it is expressly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entirety.

Claims

1. 1. A pharmaceutical composition for the treatment of primary liver cancer, comprising a therapeutically effective amount of a Toll-like receptor 9 (TLR9) agonist having the structure: 5'-TCG AAC GTT CGA ACG TTC GAA CGT TCG AAT-3' (SEQ ID NO:1).

2. The pharmaceutical composition of claim 1 , wherein the primary liver cancer is hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICC).

3. The pharmaceutical composition of claim 1, administered through a device by hepatic arterial infusion (HAI).

4. The pharmaceutical composition of claim 1, administered through a device by portal vein injection (PVI).

5. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount of the TLR9 agonist is selected from the group consisting of 0.01 mg / mL, 0.04 mg / mL, 0.08 mg / mL, or 0.16 mg / mL.

6. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount of the TLR9 agonist is selected from the group consisting of 0.5 mg, 2 mg, 4 mg, or 8 mg.

7. The pharmaceutical composition of claim 1, administered through a catheter device.

8. The pharmaceutical composition of claim 7 , wherein the catheter device comprises a one-way valve that dynamically responds to local pressure and / or flow changes.

9. The pharmaceutical composition of claim 7, administered through the catheter device via pressure-enabled drug delivery.

10. The pharmaceutical composition of claim 7, administered over a period of about 10 to 200 minutes.

11. The pharmaceutical composition of claim 10, administered over a period of about 10 to 60 minutes.

12. The pharmaceutical composition of claim 11, administered over a period of about 25 minutes.

13. The pharmaceutical composition of claim 1, administered in combination with one or more checkpoint inhibitors (CPIs), wherein the CPIs are administered systemically either simultaneously with, prior to, or following administration of the TLR9 agonist.

14. 14. The pharmaceutical composition of claim 13, wherein the one or more CPIs comprise at least one of nivolumab, pembrolizumab, and ipilimumab.

15. The pharmaceutical composition of claim 1, administered by a dosing regimen comprising cycles, one or more of the cycles comprising administration of the TLR9 agonist via a catheter device by hepatic artery infusion, followed by systemic administration of the one or more checkpoint inhibitors (CPIs).

16. 16. The pharmaceutical composition of claim 15, wherein one cycle comprises administration of said therapeutically effective amount of a TLR9 agonist once a week for three consecutive weeks.

17. 17. The pharmaceutical composition of claim 16, wherein the dosing regimen comprises two cycles.

18. 16. The pharmaceutical composition of claim 15, wherein the one or more CPIs comprise at least one of nivolumab, pembrolizumab, and ipilimumab.

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