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Definitions

• the invention relates to certain pharmaceutical molecules and compositions and their use to treat cancer and, in particular, to the use of small interfering RNA (siRNA) molecules that inhibit TGF-beta 1 and Cox2, alone or in combination with immune checkpoint inhibitors, to treat cancer.
• siRNA small interfering RNA
• RNA interference is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene containing the homologous sequence.
• dsRNA double-stranded RNA
• Dicer small interfering RNA
• dsRNA small interfering RNA
• nt nucleotides
• RISC RNA-induced- silencing-complex
• siRNA One strand of siRNA remains associated with RISC to guide the complex towards a cognate RNA that has a sequence complementary to the guider ss-siRNA in RISC.
• This siRNA-directed endonuclease digests the RNA, resulting in truncation and inactivation of the targeted RNA.
• Recent studies have revealed the utility of chemically synthesized 21-27-nt siRNAs that exhibit RNAi effects in mammalian cells and have demonstrated that the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function. More detailed characteristics of RISC, siRNA molecules, and RNAi have been described in the scientific literature.
• RNAi in down-regulation of mammalian cell gene expression has been shown successfully in the laboratory by utilizing either chemically synthesized siRNAs or endogenously expressed siRNA.
• the endogenous siRNA is first expressed as small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector), and then processed by Dicer to become functional siRNAs.
• siRNAs can be delivered to the cells where the silencing must occur by transfection of these cells.
• One way to get siRNA delivery to these cells is to use a nanoparticle that can carry the siRNAs and allow uptake of the siRNAs across the external cell membrane, gaining access to the cytoplasm. Release of siRNAs into the cytoplasm allows these moieties to interact with the RISC complex, where the antisense strand is separated from the sense strand, the sense strand is degraded, and the antisense strand is used by the RISC complex to surveil the mRNAs within the cell for a sequence with complementarity to the antisense sequence.
• siRNA provides the template responsible for translation of the mRNA sequence into a viable protein needed by the cell. Cleavage of the mRNA reduces the ability of the cell to produce the peptide or protein encoded by the mRNA.
• Silencing of the genes by siRNA can exhibit a prolonged effect and is dependent on the turnover and balance between the rate of synthesis, the quantity of mRNA, and the rate of degradation of the mRNA and/or the protein itself. SiRNAs have been shown to have a potent silencing effect on the gene targeted, and this can further result in a prolonged decrease (days to weeks) of the protein product.
• TGF-beta 1 levels have been implicated in inhibiting the penetration of T- cells into the regions in proximity to tumors [1-3].
• TGF transforming growth factor b
• TGF -blocking antibody with anti-PD-Ll antibodies reduced TGF signaling in stromal cells, and this, in turn, allowed T-cell penetration into the center of the tumors - provoking anti-tumor immunity and tumor regression in this model [3].
• AF647 fluorecent- labeled siRNA was formulated with HKP to form a nanoparticle.
• the nanoparticles were administered in mice through IV injection (tail vein administration).
• the animals were euthanized, livers excised and dissected and dissociated cells were subjected to flow cytometry with labeled antibodies able to discriminate the different cell types shown.
• Kupffer cells KC
• Dendritic Cells DC
• Liver Sinusoidal Endothelial cells LSEC
• Hepatocytes Ly6c High (inflammatory monocytes)
• Ly6c Low PolyMorphoNuclear leucocyte cells (PMN)
• Lymphocytes and Stellate Cells This figure shows that hepatocytes, Kupffer cells and LSEC cells initially take up STP707 by lh. The stellate cell population within the liver is only 1.4% of the cell population. While the flow cytometry showed some evidence of uptake, the signal was too low, so we validated uptake in primary human stellate cells and showed excellent uptake and gene silencing within these cells.
• luciferin substrate administered to anesthetized animals, the amount of tumor was assessed by measuring the light efflux from each animal (measured using a digital imaging system). Animals were assigned to a cohort based on the normalization of tumor across all test groups. This figure shows the initial values produced at assignment to the groups - showing uniformity of tumor size between cohorts prior to commencing treatments.
• Body weights of the animals were monitored after each treatment and the body weights averaged across all animals in the cohort. The data was plotted as % of initial bodyweight prior to dosing.
• Sorafenib alone (red squares) induced a slight change in body weights.
• all other treatment schemes (STP707 alone or +anti-PDLl) were well tolerated and no significant body weight loss was observed in the treatment arms.
• TABL Tumor Associated BioLuminescence
• luciferase luciferin
• Control (vehicle) treated animals showed a rapid growth of tumors as determined by a larger light signal.
• Sorafenib and anti-PDLl mAb treatments appeared to show a static effect on tumor growth over the dosing phase.
• STP707 alone (40ug per injection or ⁇ 2mgs/kg) or with the Anti-PDLl mAb showed a dramatic reduction in tumor cells after 5-6 doses. Tumors were not visible in these treatment arms.
• Fig 5. Control (vehicle treated) animals showed a dramatic effect of uncontrolled tumor growth on viability of the animals. 50% of the untreated animals died or were euthanized as a result of increased tumor burden during the dosing phase of the experiment. 1 animal was euthanized after 37 days on Sorafenib. No animals died on any of the other treatment arms.
• Fig 6. Data shows that control samples (PNP loaded with Non-Silencing (NS) siRNA) showed a dramatic increase in tumor cell growth as measured by the Flux reading from outside the animal using an I VIS imaging system. PDL1 antibody showed a weak inhibitory effect on tumor growth.
• NS Non-Silencing
• STP707 alone showed an even greater inhibition of tumor growth than the PDL1 Ab and, in the presence of the anti-PDLl mAb, STP707 abolished the tumor completely after 6 doses - suggesting some additivity of effect with the antibody.
• Fig 7. STP707 was administered at 3 doses at lmg/kg in animals with a syngeneic orthotopic HCC tumor and then livers were excised, sectioned and stained (H&E) to show tumor location and size. Note the dramatic reduction in tumor size when STP707 was administered for this short period of time. In the regions shown by the white boxes, the amount of CD4+ and CD8+ T-cells were quantitated by staining and counting the stained spots. These regions are expanded on the right of each figure and it can clearly be seen that STP707 treatment produced a dramatic increase in the number of CD4+ and CD8+ T-cells present within the liver-tumor margin - suggesting that STP707 treatment allows greater T-cell penetration into the tumor.
• Fig 8. Using images similar to those shown in Fig 7, T-cells were quantitated in various segments measured away from the tumor margin - either in towards the tumor, or out towards the liver. Within each segment (50um thick), the number of T-cells were
• the invention relates to the use of small interfering RNA (siRNA) molecules that inhibit TGF-beta 1 and Cox2 in a subject, alone or in combination with immune checkpoint inhibitors, to treat cancer in the subject.
• shRNA small interfering RNA
• the term "subject" refers to any mammal, including humans.
• the subject may be a laboratory animal, such as a rodent, ferret, or non-human primate.
• the subject is a human.
• the invention is directed 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.
• anti-TGF-beta 1 siRNA is administered intravenously to the subject.
• anti-TGF-beta 1 siRNA is administered into a tumor in the subject.
• anti- TGF-beta 1 siRNA is administered in proximity to the tumor or administered systemically in a vehicle that allows delivery to the tumor.
• the invention is directed to a method of treating a cancer in a subject by administering to the subject a therapeutically effective amount of an anti-TGF- beta 1 siRNA.
• anti-TGF-beta 1 siRNA is administered intravenously to the subject.
• anti-TGF-beta 1 siRNA is administered into a tumor in the subject.
• anti- TGF-beta 1 siRNA is administered in proximity to the tumor or administered systemically in a vehicle that allows delivery to the tumor.
• the cancer are any cancer that afflicts a subject.
• Such cancers include liver, colon, pancreatic, lung, and bladder cancer.
• the liver cancer can be a primary liver cancer or a cancer that has metastasized to the liver from another tissue.
• Primary liver cancers include hepatocellular carcinoma and hepatoblastoma.
• Metastasized cancers include colon and pancreatic cancer.
• the anti-TGF-beta 1 siRNA molecules include the sequences identified in Table 1.
• the anti-TGF-beta 1 siRNA comprises the following sequences: sense: 5'- cccaagggcuaccaugccaacuucu-3'; antisense: 5'-agaaguuggcaugguagcccuuggg-3'.
• the anti-TGF-beta 1 siRNA is administered to the subject in a pharmaceutically acceptable carrier.
• Such carriers include branched histidine-lysine polymers.
• Such polymers form a nanoparticle with the anti-TGF-beta 1 siRNA.
• the nanoparticle can be administered intravenously or intratumorally to the subject.
• the invention is directed 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 the therapeutically effective amount of the anti-TGF-beta 1 siRNA.
• the administration of the immune checkpoint inhibitor with the anti-TGF beta 1 siRNA increases the efficacy of the anti-TGF beta 1 siRNA.
• the invention is directed to a method of treating a cancer in a subject by administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor with the therapeutically effective amount of the anti-TGF-beta 1 siRNA.
• the administration of the immune checkpoint inhibitor with the anti-TGF beta 1 siRNA increases the efficacy of the anti-TGF beta 1 siRNA.
• the immune checkpoint inhibitor and the anti-TGF-beta 1 siRNA are administered intravenously to the subject, into a tumor in the subject in proximity to the tumor, or systemically in a vehicle that allows delivery to the tumor.
• the immune checkpoint inhibitor is a monoclonal antibody that blocks the interaction between receptors, such as PD-1, PD-L1, CTLA4, Lag3, and Tim3, and ligands for those receptors on mammalian cells, such as human cells.
• the monoclonal antibody is a monoclonal antibody to PD1 or PDL1.
• monoclonal antibodies examples include Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab, and Ipilimumab.
• the immune checkpoint inhibitor is a small molecule that blocks the interaction between receptors, such as PD-1, PD-L1, CTLA4, Lag3, and Tim3, and ligands for those receptors on mammalian cells, such as human cells.
• the small molecule blocks binding between PD1 and PDL1.
• BMS202 and similar ligands are examples of such small molecules.
• the invention is directed 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 with the therapeutically effective amount anti-TGF-beta 1 siRNA.
• the combination is administered intravenously to the subject.
• the combination is administered into a tumor in a subject.
• the combination is administered in proximity to the tumor or administered systemically in a vehicle that allows delivery to the tumor.
• the invention is directed to a method of treating a cancer in a subject by administering to the subject a therapeutically effective amount of an anti-Cox-2 siRNA with the therapeutically effective amount anti-TGF-beta 1 siRNA.
• the combination is administered intravenously to the subject.
• the combination is administered into a tumor in a subject.
• the combination is administered in proximity to the tumor or administered systemically in a vehicle that allows delivery to the tumor.
• anti-Cox2 siRNA molecules include the sequences identified in Table 2.
• Table 2 Anti-Cox2 siRNA Sequences hmCX-25-1: sense 5'-r(GGUCUGGUGCCUGGUCUGAUGAUGU)-3'
• the anti-Cox2 siRNA comprises the following sequences: sense: 5'- ggucuggugccuggucugaugaugu-3'; antisense: 5'-acaucaucagaccaggcaccagacc-3'.
• the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA are administered in a pharmaceutically acceptable carrier.
• Such carriers include branched histidine-lysine polymers.
• Such polymers form a nanoparticle with the anti- TGF-beta 1 siRNA and the anti-Cox2 siRNA.
• the nanoparticle can be administered intravenously or intratumorally to the subject.
• the cancer (and the 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, colon, pancreatic, lung, and bladder cancer.
• the liver cancer can be a primary liver cancer or a cancer that has metastasized to the liver from another tissue.
• Primary liver cancers include hepatocellular carcinoma and hepatoblastoma.
• Metastasized cancers include colon and pancreatic cancer.
• the invention is directed 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 to which this method is directed are any cancer that afflicts a subject, including those described above.
• the administration of an immune checkpoint inhibitor with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA increases the efficacy of either one of the siRNA's alone.
• the invention is directed to a method of treating a 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 to which this method is directed are any cancer that afflicts a subject, including those described above.
• the administration of an immune checkpoint inhibitor with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA increases the efficacy of either one of the siRNA's alone.
• the immune checkpoint inhibitor, the anti-TGF-beta 1 siRNA, and the anti-Cox2 siRNA are administered intravenously to the subject, into a tumor in the subject in proximity to the tumor, or systemically in a vehicle that allows delivery to the tumor.
• the immune checkpoint inhibitor administered with the combination of the siRNA molecules is a monoclonal antibody or a small molecule as described above. It can be administered before, after, or concurrently with the combination of the siRNA molecules.
• the invention is directed to certain pharmaceutical compositions.
• the composition comprises an anti-TGF-beta 1 siRNA as described herein in a pharmaceutically acceptable carrier as described herein.
• this pharmaceutical composition is used in connection with an immune checkpoint inhibitor as described herein.
• this embodiment of the invention is directed 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.
• the invention is directed to a combination of therapeutic drugs comprising an immune checkpoint inhibitor and a pharmaceutical composition comprising an anti-TGF-beta 1 siRNA and an anti-Cox2 siRNA and a
• the therapeutic drug combination described herein is also useful for enhancing the anti-tumor efficacy of an immune checkpoint inhibitor in a subject with a cancer.
• a therapeutically effective amount of a pharmaceutical composition comprising an anti-TGF- beta 1 siRNA and an anti-Cox2 siRNA is administered to the subject along with a
• the anti-TGF-beta 1 siRNA decreases the subject's inflammatory response to the cancer and allows better penetration of T-cells and other immune cells into the tumor. It also creates a stronger immune response to the cancer in the subject than the immune response created by the checkpoint inhibitor alone. This response involves greater T-cell activation and penetration into the cancer.
• the anti-Cox2 siRNA decreases the subject's inflammatory response to the cancer and/or decreases the formation of exhausted T-cells or regulatory T-cells around the cancer.
• the therapeutic drug combination described herein is also useful for antigenically priming T cells to recognize and kill cancer cells in a subject and for promoting T-cell- mediated immunity against a cancer in a subject.
• a therapeutically effective amount of the combination is administered to the subject.
• the cancers are those described herein.
• the invention is directed to a method of treating a liver cancer in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention or a therapeutic drug combination of the invention.
• the liver cancer is a primary liver cancer.
• the primary liver cancer is a hepatocellular carcinoma or a
• the liver cancer is a cancer that has metastasized to the liver from another tissue in the subject's body.
• metastasized cancers include colon cancer and pancreatic cancer.
• the subject is a human.
• the invention is directed to a method of killing hepatocellular carcinoma cells in a human comprising administering to the 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 comprising a branched histidine-lysine polymer that forms a nanoparticle with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA, wherein the anti-TGF-beta 1 siRNA comprises the sequences: sense: 5'-cccaagggcuaccaugccaacuucu-3'; antisense: 5'-agaaguuggcaugguagcccuuggg-3' and the anti-Cox2 siRNA comprises the sequences: sense: 5'-ggucuggugccuggucugaugaugu- 3'; antisense: 5'-acaucaucagaccaggcaccagacc-3'.
• the invention is directed to a method of killing hepatocellular carcinoma cells in a human comprising administering to the human a therapeutically effective amount of an immune checkpoint inhibitor and a pharmaceutical composition comprising an anti-TGFbeta 1 siRNA and an anti-Cox2 siRNA in a
• the pharmaceutically acceptable carrier comprising a branched histidine-lysine polymer that forms a nanoparticle with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA
• the anti-TGF-beta 1 siRNA comprises the sequences: sense: 5'-cccaagggcuaccaugccaacuucu-3'
• the anti-Cox2 siRNA comprises the sequences: sense: 5'-ggucuggugccuggucugaugaugu-3'
• the checkpoint inhibitor comprises a monoclonal antibody able to bind to and block interactions between PD1 and PDL1.
• Such monoclonal antibodies include Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab, and Ipilimumab.
• the invention is directed to a combination of therapeutic drugs comprising an immune checkpoint inhibitor and a pharmaceutical composition comprising an anti-TGF-beta 1 siRNA and an anti-Cox 2 siRNA in a
• the checkpoint inhibitor comprises a monoclonal antibody selected from the group consisting of Atezoluzimab, Durvalumab, Nivolumab, Pembrolizumab, and Ipilimumab
• the anti-TGF-beta 1 siRNA comprises the sequences: sense: 5'-cccaagggcuaccaugccaacuucu-3'; antisense: 5'- agaaguuggcaugguagcccuuggg-3'
• the anti-Cox2 siRNA comprises the sequences: sense: 5'- ggucuggugccuggucugaugaugu-3'; antisense: 5'-acaucaucagaccaggcaccagacc-3'
• the pharmaceutically acceptable carrier comprises a branched histidine-lysine polymer that forms a nanoparticle with the anti-TGF-beta 1 siRNA and the anti-Cox2 siRNA.
• Liver cancer is any primary cancer within the liver, i.e., one that starts in the liver; or any secondary cancer within 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 a colon cancer.
• a cancer is any malignant tumor.
• a malignant tumor is a mass of neoplastic cells.
• Treating/treatment is killing some or all of the cancer cells, reducing the size of the cancer, inhibiting the growth of the cancer, or reducing the growth rate of the cancer in a subject.
• Anti-TGF-beta 1 siRNA is an siRNA molecule that reduces or prevents the expression of the gene in a mammalian cell that codes for the synthesis of TGF-beta 1 protein.
• Anti-Cox2 siRNA is an siRNA molecule that reduces or prevents the expression of the gene in a mammalian cell that codes for the synthesis of Cox2 protein.
• siRNA molecule is a duplex oligonucleotide, that is a short, double-stranded polynucleotide, that interferes with the expression of a gene in a cell, after the molecule is introduced into the cell. For example, it targets and binds to a complementary nucleotide sequence in a single stranded target RNA molecule.
• SiRNA molecules are chemically synthesized or otherwise constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5, 898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties.
• siRNA molecule By convention in the field, when an siRNA molecule is identified by a particular nucleotide sequence, the sequence refers to the sense strand of the duplex molecule.
• One or more of the ribonucleotides comprising the molecule can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide can be modified. Additional modifications include the use of small molecules (e.g. sugar molecules), amino acids, peptides, cholesterol, and other large molecules for conjugation onto the siRNA molecule.
• a branched histidine-lysine polymer is a peptide consisting of histidine and lysine amino acids. By synthesizing multiple amino acids from a common lysine core, the peptide is composed of 4 arms or branches.
• Such polymers are described in US Patent Numbers 7,070,807 B2, issued July 4, 2006, 7,163,695 B2, issued January 6, 2007, and 7,772,201 B2, issued August 10, 2010, which are incorporated herein by reference in their entireties.
• An immune checkpoint inhibitor is a drug that blocks certain proteins made 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 keep T cells from killing the cancer cells. When these checkpoint proteins are blocked, the "brakes" on the immune system are released, and T cells are able to kill cancer cells better. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7-2.
• In proximity to the cancer means in tissue or cells close to or surrounding a tumor or series of tumor cells.
• Enhancing the antitumor efficacy means providing a greater reduction in growth rate of the tumor cells, greater effect in killing the tumor cells and/or reducing tumor mass and eventually producing a better therapeutic effect by prolonging life of the subject with the tumor.
• Such effects may be mediated by a direct action on the tumor cells themselves or an augmentation of the activity of the T-cells or a mechanism by which the T-cells are afforded better access to the tumor cells and/or are activated to promote a stronger immune reaction against the tumor with or without an increase in the ability to recognize tumor cells even after the initial treatment.
• STP707 consists of 2 siRNAs (targeting TGF-beta 1 and Cox2 genes) protected by a polypeptide delivery nanoparticle consisting of the branched polypeptide HKP (Histidine Lysine Polymer). STP707 is described in US Patent 9,642,873 B2, dated May 9, 2017, and US Reissued Patent RE46,873 E, dated May 29, 2018, the disclosures of which are incorporated by reference herein in their entireties.
• branched polypeptide nanoparticle consisting of histidine and lysine amino acids (HKP)
• IV injection allows the nanoparticles to be taken up with higher efficiency by cells within the liver.
• flow cytometry to measure the uptake of fluorescent tagged siRNAs from the nanoparticles, we have demonstrated delivery to specific cell types within the liver, including the Stellate cells, LSEC cells, and the
• hepatocytes as well as the Kupffer cells (Fig 1). This would therefore suggest that we can get very good delivery efficiency of siRNAs to these cells within the liver and can therefore silence targets of interest within these cells.
• HCC liver cancer tumor cells Hepa 1-6 cells
• the mouse liver cancer cell line was modified to express luciferase (Hepa 1-6-Lux). Then, upon addition of the substrate to the animals, the degree of growth of the tumors in the livers of these animals could be monitored using a luminescence detection system. This allows measurement of the tumor growth in a non-invasive manner that is not harmful to the animals. We monitored the rate of growth of the tumors using this method.
• STP707 siRNAs shown to inhibit TGF-beta and Cox2 (STP707) delivered using the HKP peptide nanoparticles (administered IV BIW at a dose of 40ug or 20ug per injection).
• STP707 when administered along with the anti-PDLl antibody.
• STP707 for efficacy in treating HCC using a syngeneic, orthotopic murine hepatocellular carcinoma model using a bioluminescent variant of the Hepa 1-6 cell line to allow monitoring for tumor load over time.
• the treatment groups were as follows:
• Anti-PD-Ll (5mgs/Kg), i.p., BIW
• Anti-PD-Ll (5mgs/Kg), i.p., BIW + 20ugs STP707/injection iv BIW
• Anti-PD-Ll (5mgs/Kg), i.p., BIW + 40ugs STP707/injection iv BIW
• the animals were randomized at the outset of the experiment based on weight.
• the randomization provided very similar weight distributions in the selected animals within each group as shown in Fig 2.
• the bodyweights of the animals were measured daily during the dosing phase of the efficacy study. Mean body weights of each group were plotted ( Figure 3). Sorafenib alone induced a slight change in body weights. However, all other treatment schemes (STP707 alone or +anti-PDLl) were well tolerated and no significant body weight loss was observed in the treatment arms.
• TABL tumor associated bioluminescence
• tumor outgrowth was monitored for groups 2-6. No tumor regrowth was observed prior to the last day of the study (day 50), suggesting that the treatments were very efficacious at inhibiting tumor growth and preventing regrowth suggesting a pronounced effect on tumor viability.
• the data obtained supported the observation that STP707 shows single agent action against the tumor - diminishing growth relative to the control (untreated) cohort.
• the STP707 arm showed better efficacy than the antibody arm (PDL1) alone.
• STP707 shows single agent activity better than anti-PDLl antibody alone.
• combining STP707 with the antibody treatment reduced the tumor to undetectable levels after 4 doses.
• Image analysis was performed to quantitate the CD4+ and CD8+ T-cells at the margins between the tumor and the liver as shown by the colored lines in the tumor samples. These lines are drawn at 50um distances away from the tumor margin - either in towards the tumor or out, away from the tumor but towards the liver. Image analysis counted all CD8+ T-cells in each 50um segment and the data was plotted as shown in Figure 8.
• the primary objective of this study was to determine the tolerability and efficacy of STP707 (HKP polypeptide nanoparticles containing siRNAs against TGF betal and Cox2), as a monotherapy and in combination with anti-PD-Ll, in an orthotopic murine hepatocellular carcinoma model using a bioluminescent variant of the Hepa 1-6 cell line.
• nonmelanoma skin cancers or melanoma tumors in the skin or in tumors in other organs where material can be injected close to the tumor site to promote the same effect.

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