Silencing TGF-BETA 1 and COX2 using siRNA delivered in combination with immune checkpoint inhibitors to treat cancer

Abstract

The present invention provides certain drug molecules and compositions and methods of using the same to treat cancer. The molecules are small interfering RNA (siRNA) molecules that inhibit TGF-beta 1 and Cox2 in humans and other mammals, which are used alone or in combination with immune checkpoint inhibitors to treat cancer.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 785,647, filed on December 27, 2018, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 14, 2020, with the file name 4690_0016i_SL.txt and a size of 8,518 bytes. Technical Field

[0005] The present invention relates to certain drug molecules and compositions and their use in treating cancer, and in particular to the use of small interfering RNA (siRNA) molecules that inhibit TGF-beta 1 and Cox2, used alone or in combination with immune checkpoint inhibitors, for treating cancer. Background Art

[0006] Recent work has identified immune checkpoint inhibitors as an important target in the fight against cancer. Receptors such as PD-1 (on the surface of T cells) can interact with ligands (e.g., PD-L1) on the surface of tumor cells, and this binding between these molecules results in a signal to the T cells that the tumor should not be destroyed. As a result, this signaling mechanism has been thoroughly studied in an attempt to identify ways to block it, thereby promoting immune recognition of the tumor, which in turn results in increased tumor killing.

[0007] Many checkpoints have been discovered, including CTLA4, Lag3, Tim3, PD-1, and PD-L1. For example, anti-PD1 or anti-PD-L1 antibodies have been shown to block the interaction between the PD-1 receptor and the PD-L1 ligand, a blockade that inhibits the "do not eat me" signal from tumor cells to T cells that otherwise prevents T cells from responding normally to tumors and other foreign cells by releasing cell-killing enzymes.

[0008] As these antibodies (pembrolizumab, Keytruda, etc.) migrated to the clinic, it was discovered that treating patients with these agents could promote very strong immune responses in about 30% of patients, which led to long-term treatment of these patients. However, it was unclear why this response was only seen in 30% of treated patients, so research has focused extensively on other pathways and signaling mechanisms that may play a role in suppressing the immune response and tumor cell killing ability of T cells.

[0009] RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively simple and direct method of knocking down or theoretically silencing any gene containing a homologous sequence. In naturally occurring RNAi, double-stranded RNA (dsRNA) is cut into small interfering RNA (siRNA) molecules by RNase III / helicase protein Dicer, i.e., 19-27 nucleotides (nt) of dsRNA with a 2-nt overhang at the 3' end. Then, siRNA is integrated into a multi-component ribonuclease called RNA-induced silencing complex (RISC). One chain of siRNA remains associated with RISC to guide the complex to a homologous RNA having a sequence complementary to the guider ss-siRNA in RISC. This siRNA-guided endonuclease digests RNA, resulting in truncation and inactivation of the target RNA. Recent studies have revealed the utility of chemically synthesized 21-27-nt siRNAs in demonstrating RNAi effects in mammalian cells and have demonstrated that the thermodynamic stability of siRNA hybrids (at the termini or in the middle) plays a central role in determining the function of the molecule. More detailed features of RISC, siRNA molecules, and RNAi have been described in the scientific literature.

[0010] The utility of RNAi in downregulating gene expression in mammalian cells has been successfully demonstrated in the laboratory by utilizing chemically synthesized siRNA or endogenously expressed siRNA. Endogenous siRNA is first expressed as small hairpin RNA (shRNA) by an expression vector (plasmid or viral vector) and then processed by Dicer into functional siRNA.

[0011] In order to be active and to silence the target gene, siRNA can be delivered to cells that must be silenced by transfecting these cells. A method of delivering siRNA to these cells is to use nanoparticles, which can carry siRNA and allow siRNA to be taken in through the external cell membrane and approach the cytoplasm. siRNA is released into the cytoplasm so that these parts interact with the RISC complex, wherein the antisense strand is separated from the sense strand, and the sense strand is degraded, and the antisense strand is used by the RISC complex to monitor the sequence of the mRNA in the cell that is complementary to the antisense sequence. This allows two sequences to hybridize, and then the cutting of mRNA occurs by the action of the Dicer enzyme.

[0012] MRNA provides the template responsible for translating the mRNA sequence into the required living protein of the cell. The cutting of mRNA reduces the ability of the cell to produce the peptide or protein encoded by the mRNA. The silencing gene by siRNA can show a long-term effect, and depends on the turnover and balance between the degradation rate of synthesis rate, mRNA amount and mRNA and / or protein itself. It has been proved that siRNA has an effective silencing effect on the target gene, which can further cause the long-term reduction (several days to several weeks) of the protein product.

[0013] Elevated levels of TGF-beta 1 have been shown to be associated with inhibition of T cell infiltration into areas in proximity to tumors [1-3].

[0014] For example, in tumors from patients with colon cancer [1,2] or metastatic urothelial carcinoma treated with an anti-PD-L1 antibody (atezolizumab), the lack of response has been associated with transforming growth factor β (TGFβ) signaling in fibroblasts. This was initially observed in tumors that showed exclusion of CD8+ T cells from the tumor parenchyma and accumulation of these T cells in the peritumoral stroma rich in fibroblasts and collagen, a phenotype common in patients with metastatic urothelial carcinoma. Co-administration of a TGFβ-blocking antibody with an anti-PD-L1 antibody reduced TGFβ signaling in stromal cells, which in turn allowed T cells to infiltrate the center of the tumor—eliciting antitumor immunity and tumor regression in this model [3].

[0015] While [1] and [2] focused on colon cancer and [3] showed effects in urothelial carcinoma, no one has attempted to investigate the synergistic effects of TGFβ1 and PDL1 inhibition on immune responses when both targets are inhibited simultaneously. We used peptide nanoparticles (PNPs) to deliver siRNA targeting TGFβ1 and Cox2 to the liver to see if this effect would enhance the efficacy of PDL1 antibodies in this disease. Delivery to endothelial cells as well as normal hepatocytes showed that we could silence both genes in the vicinity of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Localization of intravenously injected STP707 in cells of the liver. AF647 fluorescently labeled siRNA was formulated with HKP to form nanoparticles. The nanoparticles were administered to mice by intravenous injection (tail vein administration). At the time points indicated in the figure, the animals were euthanized, the livers were removed and dissected, and the isolated cells were subjected to flow cytometry using labeled antibodies that can distinguish the different cell types shown. From left to right, the cell types are shown at each time point: Kupffer cells (KC), dendritic cells (DC), liver sinusoidal endothelial cells (LSEC), hepatocytes, high Ly6c (Ly6c High, inflammatory monocytes), low Ly6c (Ly6c Low), polymorphonuclear leukocytes (PMN), lymphocytes and astrocytes. The figure shows that hepatocytes, Kupffer cells and LSEC cells initially take up STP707 within 1 hour. The astrocyte population in the liver only accounts for 1.4% of the cell population. While flow cytometry showed some evidence of uptake, the signal was too low, so we validated uptake in primary human astrocytes and showed excellent uptake and gene silencing in these cells.

[0017] Figure 2 The amount of tumor was assessed by measuring the light efflux (measured using a digital imaging system) for each animal using a fluorescein substrate administered to anesthetized animals. Animals were assigned to cohorts based on normalization of tumors in all test groups. The graph shows the initial values ​​generated when assigned to each group - showing the uniformity of tumor size between cohorts before treatment was started.

[0018] Figure 3 Animal body weights were monitored after each treatment and averaged for all animals in the cohort. Data were plotted as a percentage (%) of initial body weight prior to dosing.

[0019] Sorafenib (red squares) alone induced mild changes in body weight. However, all other treatment regimens (STP707 alone or + anti-PDL1) were well tolerated and no significant body weight loss was observed in the treatment groups.

[0020] Figure 4 Tumor Associated BioLuminescence (TABL) measurements were performed by administering a luciferase substrate (luciferin) to anesthetized animals and then imaging the light produced using the IVIS Live Animal Imaging System. Animals were treated throughout the dosing period (grey highlighted areas) and monitored without treatment at other times.

[0021] Control (vehicle) treated animals showed rapid growth of tumors as determined by larger light signals. During the dosing phase, sorafenib and anti-PDL1 mAb treatment showed a static effect on tumor growth. STP707 alone (40ug or about 2mg / kg per injection) or with anti-PDL1 mAb after 5 to 6 doses showed a substantial reduction in tumor cells. No tumors were visible in these treatment groups.

[0022] Figure 5 .Control (vehicle treated) animals showed uncontrolled tumor growth with a substantial impact on animal viability. Due to increased tumor burden during the dosing phase of the experiment, 50% of the untreated animals died or were euthanized. One animal was euthanized after 37 days of sorafenib treatment. No animals died in any of the other treatment groups.

[0023] Figure 6 The data showed that the control sample (PNP loaded with non-silencing (NS) siRNA) showed a large increase in tumor cell growth, as measured by flow (Flux) read from outside the animal using an IVIS imaging system. The PDL1 antibody showed a weak inhibitory effect on tumor growth. STP707 (1 mg / kg) alone showed an even greater tumor growth inhibition effect than the PDL1 Ab, and in the presence of the anti-PDL1 mAb, STP707 completely eliminated tumors after 6 doses - indicating some additive effect with the antibody.

[0024] Figure 7 STP707 was administered to animals with syngeneic orthotopic HCC tumors at 1 mg / kg for 3 doses, and the livers were then excised, sectioned, and stained (H&E) to show tumor location and size. Note the dramatic reduction in tumor size when STP707 was administered for such a short period of time. The amount of CD4+ and CD8+ T cells was quantified by staining and staining point counting in the areas shown in white boxes. These areas are magnified on the right side of each image, and it can be clearly seen that STP707 treatment produced a dramatic increase in the number of CD4+ and CD8+ T cells present within the liver-tumor margins - indicating that STP707 treatment allowed more T cells to infiltrate the tumor.

[0025] Figure 8 .Use with Figure 7The images shown in Figure 1 are similar to the images shown in Figure 2, with T cell quantification performed in each segment measured away from the edge of the tumor (either inward toward the tumor or outward toward the liver). The number of T cells was quantified within each segment (50um thick) and plotted on the graph shown. Compared to vehicle treatment alone, treatment with STP707 together with anti-PDL1 Ab showed a 2-fold increase in CD8+T cells at a depth of 500um within the tumor. It also showed an increase in CD8+T cells in the liver close to the tumor - indicating that a decrease in the TGF-beta "wall" surrounding the tumor may induce the recruitment of T cells. DETAILED DESCRIPTION

[0026] The present invention relates to the use of small interfering RNA (siRNA) molecules that inhibit TGF-beta 1 and Cox2 in a subject, which are used alone or in combination with immune checkpoint inhibitors for treating cancer in a subject. As used herein, the term "subject" refers to any mammal, including humans. The subject can be a laboratory animal, such as a rodent, a ferret, or a non-human primate. Preferably, the subject is a human.

[0027] In one embodiment, the present invention relates to a method of killing cancer cells in a subject by administering to the subject a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered intravenously to the subject. In another aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered to a tumor of the subject. In yet another aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered near the tumor or systemically in a carrier that allows delivery to the tumor.

[0028] In another embodiment, the present invention relates to a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered intravenously to the subject. In another aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered to a tumor of the subject. In yet another aspect of this embodiment, the anti-TGF-beta 1 siRNA is administered near the tumor or systemically in a carrier that allows delivery to the tumor.

[0029] Cancer (and cancer cells) is any cancer that afflicts a subject. Such cancers include liver cancer, colon cancer, pancreatic cancer, lung cancer, and bladder cancer. Liver cancer can be a primary liver cancer or a cancer that has metastasized to the liver from another tissue. Primary liver cancer includes hepatocellular carcinoma and hepatoblastoma. Metastatic cancers include colon cancer and pancreatic cancer.

[0030] The anti-TGF-beta 1 siRNA molecules included the sequences identified in Table 1.

[0031] Table 1: Anti-TGF-beta 1 siRNA sequences

[0032]

[0033] In one aspect, the anti-TGF-beta 1 siRNA comprises the following sequences: sense: 5'-cccaagggcuaccaugccaacuucu-3' (SEQ ID NO: 3); antisense: 5'-agaaguuggcaugguagcccuuggg-3' (SEQ ID NO: 4).

[0034] The anti-TGF-beta 1 siRNA is administered to a subject in a pharmaceutically acceptable carrier. Such a carrier includes a branched histidine-lysine polymer. In one embodiment of such a polymer, the polymer has the formula (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17) or R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 18), X=C(O)NH2, K=lysine, H=histidine. Such a polymer forms nanoparticles with the anti-TGF-beta 1 siRNA. The nanoparticles can be administered to a subject intravenously or intratumorally.

[0035] In another embodiment, the present invention relates to a method of killing cancer cells in a subject by administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor together with a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, administering an immune checkpoint inhibitor together with an anti-TGF-beta 1 siRNA increases the efficacy of the anti-TGF-beta 1 siRNA.

[0036] In another embodiment, the present invention relates to a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, administering an immune checkpoint inhibitor and an anti-TGF-beta 1 siRNA increases the efficacy of the anti-TGF-beta 1 siRNA.

[0037] As described above, the immune checkpoint inhibitor and anti-TGF-beta 1 siRNA are administered intravenously to the subject, into the subject's tumor, in the vicinity of the tumor, or systemically in a vector that allows delivery to the tumor.

[0038] In one aspect of this embodiment, the immune checkpoint inhibitor is a monoclonal antibody that blocks the interaction between receptors (such as PD-1, PD-L1, CTLA4, Lag3 and Tim3) on mammalian cells (such as human cells) and the ligands of these receptors. In a particular aspect, the monoclonal antibody is a monoclonal antibody against PD1 or PDL1. Examples of monoclonal antibodies include Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab and Ipilimumab.

[0039] In another aspect of this embodiment, the immune checkpoint inhibitor is a small molecule that blocks the interaction between receptors (such as PD-1, PD-L1, CTLA4, Lag3 and Tim3) on mammalian cells (such as human cells) and the ligands of these receptors. In a particular aspect, the small molecule blocks the binding between PD1 and PDL1. BMS202 and similar ligands are examples of such small molecules.

[0040] In a further embodiment, the present invention relates to a method of killing cancer cells in a subject by administering to the subject a therapeutically effective amount of an anti-Cox-2 siRNA and a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, the combination is administered intravenously to the subject. In another aspect of this embodiment, the combination is administered to a tumor of the subject. In yet another aspect of this embodiment, the combination is administered near the tumor or systemically in a carrier that allows delivery to the tumor.

[0041] In a further embodiment, the invention relates to a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of an anti-Cox-2 siRNA and a therapeutically effective amount of an anti-TGF-beta 1 siRNA. In one aspect of this embodiment, the combination is administered intravenously to the subject. In another aspect of this embodiment, the combination is administered to a tumor in the subject. In yet another aspect of this embodiment, the combination is administered near the tumor or systemically in a carrier that allows delivery to the tumor.

[0042] The anti-Cox2 siRNA molecules include the sequences identified in Table 2.

[0043] Table 2: Anti-Cox2 siRNA sequences

[0044]

[0045]

[0046] In one aspect, the anti-Cox2 siRNA comprises the following sequences: sense: 5'-ggucuggugccuggucugaugaugu-3' (SEQ ID NO: 19); antisense: 5'-acaucaucagaccaggcaccagacc-3' (SEQ ID NO: 20).

[0047] Anti-TGF-beta 1 siRNA and anti-Cox2 siRNA are administered in a pharmaceutically acceptable carrier. Such a carrier includes a branched histidine-lysine polymer. In one embodiment of such a polymer, the polymer has the formula (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17) or R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 18), X=C(O)NH2, K=lysine, H=histidine, N=asparagine. Such a polymer forms nanoparticles with anti-TGF-beta 1 siRNA and anti-Cox2 siRNA. The nanoparticles can be administered to a subject intravenously or intratumorally.

[0048] As described above, with respect to anti-TGF-beta 1 siRNA, the cancer (and cancer cells) targeted by the combination of anti-TGF-beta 1 siRNA and anti-Cox2 siRNA can be any cancer that afflicts a subject. Such cancers include liver cancer, colon cancer, pancreatic cancer, lung cancer, and bladder cancer. Liver cancer can be primary liver cancer or a cancer that has metastasized to the liver from another tissue. Primary liver cancer includes hepatocellular carcinoma and hepatoblastoma. Metastatic cancers include colon cancer and pancreatic cancer.

[0049] In a further embodiment, the present invention relates to a method of killing cancer cells in a subject by administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor with a therapeutically effective amount of an anti-TGF-beta 1 siRNA and a therapeutically effective amount of an anti-Cox2 siRNA. The cancer cells and cancers targeted by the method are any cancers that afflict the subject, including those described above. In one aspect of this embodiment, administering an immune checkpoint inhibitor with the anti-TGF-beta 1 siRNA and anti-Cox2 siRNA increases the efficacy of either siRNA alone.

[0050] In a further embodiment, the present invention relates to a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor with a therapeutically effective amount of an anti-TGF-beta 1 siRNA and a therapeutically effective amount of an anti-Cox2 siRNA. The cancer cells and cancers targeted by the method are any cancers that afflict the subject, including those described above. In one aspect of this embodiment, administration of an immune checkpoint inhibitor with the anti-TGF-beta 1 siRNA and anti-Cox2 siRNA increases the efficacy of either siRNA alone.

[0051] As described above, the immune checkpoint inhibitor, anti-TGF-beta 1 siRNA, and anti-Cox2 siRNA are administered intravenously to a subject, into a tumor of a subject, in the vicinity of a tumor, or systemically in a vector that allows delivery to a tumor.

[0052] The immune checkpoint inhibitors administered in combination with the siRNA molecules are monoclonal antibodies or small molecules as described above. They can be administered before, after or simultaneously with the siRNA molecule combination.

[0053] The present invention relates to certain pharmaceutical compositions. In one embodiment, the composition comprises an anti-TGF-beta 1 siRNA as described herein in a pharmaceutically acceptable carrier as described herein.

[0054] In another embodiment, the pharmaceutical composition is used in combination with an immune checkpoint inhibitor as described herein. Thus, this embodiment of the invention relates to a combination of therapeutic drugs comprising an immune checkpoint inhibitor and a pharmaceutical composition comprising an anti-TGF-beta 1 siRNA in a pharmaceutically acceptable carrier as described herein.

[0055] In another embodiment, the present invention relates to a combination of therapeutic drugs, which includes an immune checkpoint inhibitor and a pharmaceutical composition comprising anti-TGF-beta 1 siRNA, anti-Cox2 siRNA as described herein, and a pharmaceutically acceptable carrier.

[0056] The therapeutic drug combination described herein can also be used to enhance the anti-tumor efficacy of immune checkpoint inhibitors in subjects with cancer. A therapeutically effective amount of a pharmaceutical composition comprising anti-TGF-beta 1 siRNA and anti-Cox2 siRNA and a therapeutically effective amount of a checkpoint inhibitor are administered to a subject. Anti-TGF-beta 1 siRNA reduces the inflammatory response of the subject to cancer and allows T cells and other immune cells to better penetrate into the tumor. Compared with the immune response caused by the checkpoint inhibitor alone, it also causes a stronger immune response to the cancer in the subject. This response involves stronger T cell activation and penetration into the cancer. Anti-Cox2 siRNA reduces the inflammatory response of the subject to cancer and / or reduces the formation of exhausted T cells (exhausted T-cell) or regulatory T cells around the cancer.

[0057] The therapeutic drug combinations described herein can also be used to antigenically start (priming) T cells in a subject to recognize and kill cancer cells, and can be used to promote T cell-mediated immunity against cancer in a subject. The combination is administered to a subject in a therapeutically effective amount. The cancer is those described herein.

[0058] In a particular embodiment, the present invention relates to a method for treating liver cancer in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention or a therapeutic drug combination of the present invention. In one aspect of this embodiment, the liver cancer is primary liver cancer. In a particular aspect, the primary liver cancer is hepatocellular carcinoma or hepatoblastoma. In another aspect of this embodiment, the liver cancer is a cancer that has metastasized to the liver from another tissue in the subject. Such metastatic cancers include colon cancer and pancreatic cancer. In one aspect of this embodiment, the subject is a human.

[0059] In another particular embodiment, the present invention relates to a method for killing hepatocellular cancer cells in humans, comprising administering to a human a therapeutically effective amount of a pharmaceutical composition comprising an anti-TGF-beta 1 siRNA and an anti-Cox2 siRNA in a pharmaceutically acceptable carrier, the carrier comprising a branched histidine-lysine polymer, which forms nanoparticles with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA, wherein the anti-TGF-beta 1 siRNA comprises the following sequence: sense: 5'-cccaagggcuaccaugccaacuucu-3' (SEQ ID NO: 3); antisense: 5'-agaaguuggcaugguagcccuuggg-3' (SEQ ID NO: 4), and the anti-Cox2 siRNA comprises the following sequence: sense: 5'-ggucuggugccuggucugaugaugu-3' (SEQ ID NO: 19); antisense: 5'-acaucaucagaccaggcaccagacc-3' (SEQ ID NO: 20).

[0060] In another particular embodiment, the present invention relates to a method for killing hepatocellular cancer cells in humans, comprising administering to the human a therapeutically effective amount of an immune checkpoint inhibitor and a pharmaceutical composition, wherein the pharmaceutical composition comprises an anti-TGF-beta 1 siRNA and an anti-Cox2 siRNA in a pharmaceutically acceptable carrier, wherein the carrier comprises a branched histidine-lysine polymer, which forms nanoparticles with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA, wherein the anti-TGF-beta 1 siRNA comprises the following sequence: sense: 5'-cccaagggcuaccaugccaacuucu-3' (SEQ ID NO: 3); antisense: 5'-agaaguuggcaugguagcccuuggg-3' (SEQ ID NO: 4), and the anti-Cox2 siRNA comprises the following sequence: sense: 5'-ggucuggugccuggucugaugaugu-3' (SEQ ID NO: 19); antisense: 5'-acaucaucagaccaggcaccagacc-3' (SEQ ID NO:20), and wherein the checkpoint inhibitor includes a monoclonal antibody capable of binding to and blocking the interaction between PD1 and PDL1. Such monoclonal antibodies include Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab and Ipilimumab.

[0061] In a further particular embodiment, the present invention relates to a combination of therapeutic drugs, comprising an immune checkpoint inhibitor and a pharmaceutical composition, wherein the pharmaceutical composition comprises an anti-TGF-beta1 siRNA and an anti-Cox2 siRNA in a pharmaceutically acceptable carrier, wherein the immune checkpoint inhibitor comprises a monoclonal antibody selected from Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab and Ipilimumab, and the anti-TGF-beta1 siRNA comprises the following sequence: sense: 5'-cccaagggcuaccaugccaacuucu-3' (SEQ ID NO: 3); antisense: 5'-agaaguuggcaugguagcccuuggg-3' (SEQ ID NO: 4), and the anti-Cox2 siRNA comprises the following sequence: sense: 5'-ggucuggugccuggucugaugaugu-3' (SEQ ID NO:19); antisense: 5'-acaucaucagaccaggcaccagacc-3' (SEQ ID NO:20), and the pharmaceutically acceptable carrier comprises a branched histidine-lysine polymer, which forms nanoparticles with anti-TGF-beta 1 siRNA and anti-Cox2 siRNA. In one aspect of this embodiment, the branched histidine-lysine polymer has the formula (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO:17) or KHHHKHHHKHHHHKHHHK (SEQ ID NO:18), X=C(O)NH2, K=lysine, H=histidine, N=asparagine.

[0062] definition

[0063] Liver cancer is any primary cancer in the liver, i.e., a cancer that starts in the liver; or any secondary cancer in the liver, i.e., a cancer that metastasizes to the liver from another tissue in the mammal's body. An example of a primary liver cancer is hepatocellular carcinoma. An example of a secondary liver cancer is colon cancer.

[0064] Cancer is any malignant tumor.

[0065] A malignant tumor is a mass of growths of cells.

[0066] Treating or treatment is killing some or all cancer cells, reducing the size of a cancer, inhibiting the growth of a cancer, or reducing the growth rate of a cancer in a subject.

[0067] Anti-TGF-beta 1 siRNA is a siRNA molecule that reduces or prevents the expression of the gene encoding the synthesis of TGF-beta 1 protein in mammalian cells.

[0068] Anti-Cox2 siRNA is a siRNA molecule that reduces or prevents the expression of the gene encoding the synthesis of Cox2 protein in mammalian cells.

[0069] siRNA molecules are duplex oligonucleotides, which are short double-stranded polynucleotides that interfere with the expression of genes in the cell after being introduced into the cell. For example, they target and bind to the complementary nucleotide sequence in the single-stranded target RNA molecule. The siRNA molecules are chemically synthesized or otherwise constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Patent Nos. 5, 898,031, 6,107,094, 6,506,559, 7,056,704 and European Patent Nos. 1214945 and 1230375, which are incorporated herein by reference as a whole. According to the practice in the art, when the siRNA molecule is identified by a specific nucleotide sequence, the sequence refers to the sense strand of the duplex molecule. The one or more ribonucleotides contained in the molecule can be chemically modified by techniques known in the art. In addition to modifying at the level of one or more individual nucleotides, the main chain of the oligonucleotide can also be modified. Other modifications include coupling of small molecules (such as sugar molecules), amino acids, peptides, cholesterol and other macromolecules to siRNA molecules.

[0070] Branched histidine-lysine polymers are peptides composed of histidine and lysine amino acids. By synthesizing multiple amino acids from a common lysine core, the peptide is composed of 4 arms or side chains. Such polymers are described in U.S. Patent Nos. 7,070,807B2 (authorized on July 4, 2006), 7,163,695B2 (authorized on January 6, 2007), and 7,772,201B2 (authorized on August 10, 2010), which are incorporated herein by reference in their entirety.

[0071] Immune checkpoint inhibitors are drugs that block certain proteins produced by some types of immune system cells, such as T cells and some cancer cells. These checkpoint proteins help keep immune responses in check and can prevent T cells from killing cancer cells. When these checkpoint proteins are blocked, the immune system's "brakes" are released and T cells are better able to kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2.

[0072] Cancer vicinity refers to tissues or cells that are close to or surround a tumor or a group of tumor cells.

[0073] Enhanced anti-tumor efficacy means providing a greater reduction in the growth rate of tumor cells, providing a greater effect of killing tumor cells and / or reducing tumor mass, and ultimately producing a better therapeutic effect by extending the life span of subjects with tumors. This effect may be mediated by a direct effect on the tumor cells themselves or by an enhancement of T cell activity or by a mechanism by which, after initial treatment, T cells are better able to approach tumor cells and / or are activated to promote a stronger immune response against the tumor, with or without an increased ability to recognize tumor cells.

[0074] The following examples illustrate certain aspects of the invention and should not be construed as limiting the scope thereof.

[0075] Example

[0076] Experimental Results

[0077] STP707 consists of 2 siRNAs (targeting TGF-beta 1 and Cox2 genes) protected by polypeptide delivery nanoparticles composed of branched polypeptide HKP (histidine lysine polymer). STP707 is described in U.S. Patent 9,642,873B2 (May 9, 2017) and U.S. Reissue Patent RE46,873E (May 29, 2018), the disclosures of which are incorporated herein by reference in their entirety.

[0078] By using branched peptide nanoparticles composed of histidine and lysine amino acids (HKP), we have demonstrated that intravenous administration results in a higher efficiency of nanoparticle uptake by cells within the liver. By measuring the uptake of fluorescently labeled siRNA in the nanoparticles using flow cytometry, we have demonstrated that delivery to specific cell types within the liver is possible, including astrocytes, LSEC cells, hepatocytes, and Kupffer cells ( Figure 1 ). So this suggests that we can get very good delivery efficiency of siRNA to these cells within the liver, thereby silencing the target of interest within these cells.

[0079] We examined STP707 as monotherapy and in combination with an anti-PD-L1 monoclonal antibody (mAb) (anti-PD-L1 mAb 10F.9G2 clone, from BioXcell, West Lebanon, NH03784) in an orthotopic murine hepatocellular carcinoma model using a bioluminescent variant of the Hepa 1-6 cell line to allow monitoring of tumor burden over time.

[0080] We used an orthotopic implantation model in which HCC liver cancer tumor cells (Hepa 1-6 cells) were surgically implanted into their source organ (liver). Mouse liver cancer cell lines were modified to express luciferase (Hepa 1-6-Lux). Then, after adding substrate to the animals, the extent of tumor growth in the livers of these animals could be monitored using a luminescence detection system. This allows tumor growth to be measured in a non-invasive manner that is harmless to the animals. We monitored the growth rate of tumors using this method. We examined the tumor growth rate of no treatment (control group), the gold standard for human hepatocellular carcinoma treatment (sorafenib, a kinase inhibitor; 50 mg / kg QD administration), and a mouse anti-PDL1 antibody (5 mg / kg BIW administration) that has been validated to inhibit tumor growth in animals with orthotopic Hepa1-6 tumors.

[0081] We also compared the effect of siRNA (STP707) shown to inhibit TGF-beta and Cox2 delivered using HKP peptide nanoparticles (administered BIW intravenously at a dose of 40ug or 20ug per injection). We further analyzed the effect of STP707 when administered with anti-PDL1 antibody.

[0082] We examined the efficacy of STP707 in the treatment of HCC using a syngeneic orthotopic murine hepatocellular carcinoma model using a bioluminescent variant of the Hepa 1-6 cell line to allow monitoring of tumor burden over time.

[0083] Experiments were performed using the C57BL / 6J mouse strain from Charles River labs.

[0084] Vehicle (HKP + non-silencing siRNA; control) or STP707 were administered intravenously.

[0085] Eight animals were randomly assigned to each treatment group. The treatment groups were as follows:

[0086] 1. Carrier only

[0087] 2. Sorafenib (50 mg / kg) po, QD

[0088] 3. Anti-PD-L1 (5 mg / kg) ip, BIW

[0089] 4. Anti-PD-L1 (5 mg / kg) ip, BIW + 20ug STP707 / BIW intravenous injection

[0090] 5. Anti-PD-L1 (5 mg / kg) ip, BIW+40ug STP707 / BIW intravenous injection

[0091] 6.40ug STP707 / BIW intravenous injection alone

[0092] The animals were randomized into groups based on weight at the beginning of the experiment. Figure 2 As shown, randomization provided very similar body weight distribution among the selected animals within each group.

[0093] The body weights of the animals were measured daily during the dosing phase of the efficacy study. The mean body weights of each group were plotted ( Figure 3 Sorafenib alone induced mild changes in body weight. However, all other treatment regimens (STP707 alone or + anti-PDL1) were well tolerated and no significant body weight loss was observed in the treatment groups.

[0094] Tumor growth was monitored by bioluminescent imaging and quantified by tumor-associated bioluminescence (TABL). TABL was plotted on each study day and is shown in Figure 4 .

[0095] After completion of the scheduled dosing period, tumor outgrowth was monitored in Groups 2 to 6. No tumor regrowth was observed until the last day of the study (Day 50), indicating that the treatment was highly effective in inhibiting tumor growth and preventing regrowth, indicating a significant effect on tumor viability.

[0096] Survival analysis of all mice in the study (using a humane surrogate endpoint) further underscored the role of the combination therapy ( Figure 5 ). The endpoint of humane termination was defined as when the tumor reached the maximum allowed size or when the tumor showed adverse clinical symptoms. All treatment regimens produced statistically significant improvements in survival as demonstrated by the Log-rank (Mantel-Cox) test (p=0.0001) and the Gehan-Breslow-Wilcoxon test (p=0.0002). No differences in survival were observed between treatment groups.

[0097] To confirm the results obtained above, we repeated the study using a lower dose of STP707 (1 mg / kg) ( Figure 6 ).

[0098] The data obtained supported this observation: STP707 showed a single-agent effect against the tumor - a reduction in growth relative to the control (untreated) cohort. The STP707 group showed better efficacy than the antibody group alone (PDL1).

[0099] In this study, STP707's single-agent activity was superior to that of the anti-PDL1 antibody alone. However, combining STP707 with the antibody treatment reduced tumors to undetectable levels after four doses.

[0100] Since there were no obvious tumors in the STP707-treated groups, we repeated the study but only used 1 mg / kg of STP707 for 3 doses. After the third dose, the animals were euthanized, and the livers were removed and sectioned and stained for CD4+ and CD8+ T cells using immunohistochemistry.

[0101] Even in these reduced-dose studies, we saw a large difference in overall tumor size between untreated (control) and STP707-treated samples. In untreated animals, tumors were almost exactly the size of the liver, whereas in STP707 samples, tumors were significantly smaller ( Figure 7 ).

[0102] Furthermore, IHC staining of CD4+ and CD8+ T cells showed a substantial increase in these T cells infiltrating the tumors of STP707-treated samples ( Figure 7 and Figure 8 ).

[0103] Image analysis was performed to quantify CD4+ and CD8+ T cells at the edge between the tumor and the liver, as indicated by the colored lines in the tumor samples. These lines were drawn at a distance of 50um from the edge of the tumor - either inwards towards the tumor or outwards away from the tumor but towards the liver. Image analysis was performed to count all CD8+ T cells in each 50um segment, and the data were plotted as Figure 8 shown.

[0104] Treatment with STP707 together with anti-PDL1 Ab showed a 2-fold increase in CD8+ T cells 500um inward toward the tumor. It also showed an increase in CD8+ T cells within the liver close to the tumor - suggesting that a reduction in the TGF-beta "wall" surrounding the tumor may induce T cell recruitment.

[0105] in conclusion

[0106] The primary objective of this study was to determine the tolerability and efficacy of STP707 (HKP peptide nanoparticles containing siRNA targeting TGF-beta 1 and Cox2) as monotherapy and in combination with anti-PD-L1 in an orthotopic murine hepatocellular carcinoma model using a bioluminescent variant of the Hepa 1-6 cell line.

[0107] All treatment regimens were well tolerated at the doses and formulations tested; no adverse clinical symptoms or weight loss were noted in any treatment group.

[0108] All treatment regimens produced statistically significant reductions in tumor growth compared to control. However, STP707 monotherapy (2 mg / kg) resulted in reduced tumor growth, which was also observed in combination with anti-PDL1 Ab. Lowering the dose of STP707 to 1 mg / kg showed that STP707 alone was less effective, but the additivity with anti-PDL1 Ab was more evident at this time.

[0109] The combination of anti-PD-L1 5 mg / kg and STP705 (20 ug (1 mg / kg) per injection) appeared to be more effective than STP705 monotherapy, which was more effective than anti-PD-L1 5 mg / kg.

[0110] Our results show that STP707 enhances the effects of anti-PDL1 antibodies - it also leads to a dramatic reduction in tumor viability, with no regrowth of tumor cells even after more than 2 weeks of cessation of dosing. The fact that we show that this formulation given intravenously is delivered to the liver suggests that we could treat tumors present in the liver with this regimen. This would include any tumor that occurs naturally in the liver (hepatoblastoma or hepatocellular carcinoma (HCC)) or that metastasizes to the liver (e.g., colon cancer).

[0111] Furthermore, in previous studies, we have demonstrated that this product is able to reduce gene expression of these 2 gene targets when it is administered by injection, such as intradermally. This suggests that when this product is administered near the tumor by injection, we will be able to obtain the same therapeutic benefit of promoting an immune response to the tumor. This could be used for skin cancers, such as non-melanoma skin cancers or melanoma tumors in the skin, or for tumors in other organs, where the material could be injected close to the tumor site to promote the same effect.

[0112] References

[0113] [1] Liu et al., Cancer Cell Int (2015) 15:106-112 "Cyclooxygenase-2promotestumor growth and suppresses tumor immunity"

[0114] [2]Zelenay et al., Cell (2015) 162:1257–1270 “Cyclooxygenase-Dependent TumorGrowth through Evasion of Immunity”

[0115] [3] Mariathasan et al., Nature (2018) 554; 544-548 "TGFβattenuates tumourresponse to PD-L1 blockade by contributing to exclusion of T cells"

[0116] All publications identified herein, including issued patents and published patent applications, and all database entries identified by URL addresses or accession numbers, are incorporated herein by reference in their entirety.

[0117] Although the present invention has been described in conjunction with certain embodiments thereof and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to alternative embodiments and that certain details described herein may be varied without departing from the basic principles of the invention.

Claims

1. Use of a combination of anti-TGF-beta 1 siRNA and anti-COX2 siRNA in the preparation of a medicament for treating cancer, wherein the combination is administered together with an immune checkpoint inhibitor, wherein the combination is administered intravenously or administered into or near a malignant tumor of the cancer, wherein the cancer is selected from liver cancer, colon cancer, pancreatic cancer, lung cancer and bladder cancer, wherein the anti-TGF-beta 1 siRNA is selected from SEQ ID NO: 1-16, wherein the anti-COX2 siRNA is selected from SEQ ID NO: 19-34, and wherein the immune checkpoint inhibitor comprises a monoclonal antibody or a small molecule that blocks the interaction between cell surface receptors and ligands of these receptors, wherein the receptors are selected from PD-1, PD-L1, CTLA4, Lag3 and Tim3.

2. The use according to claim 1, wherein the cancer is liver cancer.

3. The use according to claim 1, wherein the liver cancer is selected from hepatocellular carcinoma, hepatoblastoma and cancer metastasized to the liver from another tissue. 4 . The use according to claim 1 , wherein the anti-TGF-beta 1 siRNA has the following sequence: sense: 5′-cccaagggcuaccaugccaacuucu-3′ (SEQ ID NO: 3); antisense: 5′-agaaguuggcaugguagcccuuggg-3′ (SEQ ID NO: 4).

5. The use according to claim 1, wherein the anti-Cox2 siRNA has the following sequence: sense: 5'-ggucuggugccuggucugaugaugu-3' (SEQ ID NO: 19); antisense: 5'-acaucaucagaccaggcaccagacc-3' (SEQ ID NO: 20).

6. The use according to claim 1 or 2, wherein the anti-TGF-beta 1 siRNA and anti-Cox2 siRNA are administered in a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier comprising a branched histidine-lysine polymer, wherein the branched histidine-lysine polymer has the formula (R)K(R)-K(R)-(R)K(X), wherein R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 17) or KHHHKHHHKHHHHKHHHK (SEQ ID NO: 18), X=C(O)NH2, K=lysine, H=histidine, and wherein the branched histidine-lysine polymer forms nanoparticles with the anti-TGF-beta 1 siRNA and anti-Cox2 siRNA.

7. The use according to claim 1, wherein the monoclonal antibody is a monoclonal antibody against PD1 or PD-L1.

8. The use according to claim 7, wherein the monoclonal antibody is selected from atezolizumab, durvalumab, nivolumab, pembrolizumab and ipilimumab.

9. The use according to claim 1, wherein the small molecule blocks the binding between PD1 and PD-L1.

10. The use according to claim 1, wherein the small molecule is BMS202.

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