Together with natural killer cells and dendritic cells (DCs), cytotoxic T lymphocytes (CTLs) can orchestrate potent anti-tumor immune responses involving both adaptive and innate effector mechanisms. Nonetheless, tumor cells are often able to evade this immune surveillance by commandeering immune checkpoint inhibitory pathways that are hardwired into the immune system to retain self-tolerance and modulate the duration and amplitude of physiological immune responses in order to minimize potential collateral tissue damage.

The PD-1 immune checkpoint pathway is one such example of an immune checkpoint that has emerged as a critical mediator of immunosuppression in the local tumor microenvironment. The inliibitory co-receptor Programmed Death 1 (PD-1; also known as CD279), a member of the extended CD28/CTLA-4 family of T cell regulators, is expressed on immune cells, such as T, B and NIC cells, whereas its ligand, the Programmed Cell Death Ligand 1 (PD-L1 , also known as CD274 or B7-H1) is a cell surface glycoprotein expressed on the surface of tumor cells of solid tumors as well as on human tumor associated antigen presenting cells (APCs), e.g., dendritic cells and macrophages. The interaction of PD-L 1 ligand on tumor cells with the PD-1 receptor on immune cells delivers an inliibitory signal to T lymphocytes that ultimately leads to T cell anergy and immune evasion.

The development of immune checkpoint inhibitors that prevent the activation of the PD-L 1/PD- 1 immune checkpoint pathway has resulted in unprecedented and prolonged disease control in about 20-30% of cancer patients with melanoma, non-small cell lung cancer, renal cancer, or head/neck cancer (reviewed by Lipson et ah , Scmin. Oncol. (2015) 42(4): 587-600; Zou et at, Sci. Transl. Med. (2016) vol. 8, issue 328, pp. 328rv4). However, it remains unclear why the remaining 70-80% of cancer patients fail to respond to anti-PD-1 or anti-PD-Ll antibodies or why most patients with colorectal cancer, pancreatic cancer and other non- responsive tumor types are resistant to immune checkpoint inhibitors. Hence, there is a need in the art for treatment modalities targeting cancer ceils that are resistant or have acquired resistance to immune checkpoint therapies.

The present disclosure is based on the discovery that aberrant KRAS signaling is at least in part responsible for the activation of PD-L 1 gene expression in tumor ceils and for the subsequent suppression of tumor cell-specific T cell toxicity. The present disclosure provides compositions and methods that can prevent tumors from evading immune surveillance through Lhe aberrant activation of the PD-Ll/PD-1 immune checkpoint pathway in T cells, in certain embodiments, inhibition of abeiTant KRAS signaling in tumor cells sensitizes tumor cells to immune checkpoint inhibitors. In certain embodiments, the disclosure further provides methods for enhancing the therapeutic efficacy of existing anticancer treatment using KRAS signaling modulators.

In one aspect, a composition comprising an effective amount of a modulator of KRAS signaling is disclosed, wherein the modulator of KRAS signaling is effective at enhancing the sensitivity of a tumor cell to tumor cell-specific T cell cytotoxicity. In certain embodiments, the tumor cell is in a subject, in certain embodiments, the modulator of KRAS signaling enhances the efficacy of a therapeutic agent at treating a KRAS associated disease, e.g. cancer.

In certain embodiments, the modulator of KRAS signaling can be, for example, an inhibitor of aberrant KRAS signaling. In certain embodiments, the abeiTant KRAS signaling is induced by a modified KRAS, e.g., an oncogenic KRAS, expressed in tumor cells. In certai n, embodiments, the aberrant KR AS signaling comprises the KRAS induced activation of at least one member of the RAS/R AF/ME /'ERK/FR A~ 1 signal transduction pathway, in certain embodiments, the abeiTant KRAS signaling can result irs the KRAS induced activation of PD- L 1 gene expression in tumor cells. ITS certain embodiments, the aberrant. KRAS signal ing cars be induced by an effector of KRAS signaling, e.g. by a KRAS GEF.

In a second aspect, a composition is disclosed that comprises a combination of an effective amount of a modulator of KRAS signaling, and an effective amount of a therapeutic agent, wherein the modulator of KRAS signaling is effective at enhancing the sensitivity of a tumor cell to tumor cell-specific T cell cytotoxicity. In certain embodiments, the modulator of KRAS signaling can be an inhibitor ofKRAS signaling. In certain embodiments, the modulator of KRAS signaling can enhance the efficacy of the therapeutic agent at treating a KRAS associated disease, for example, cancer. In certain embodiments, the tumor cell is in a subj ect.

In a third aspect, a composition is disclosed that comprises a combination of an effective amount of an modulator of oncogenic KRAS signaling, and an effective amount of a therapeutic agent, wherein the modulator of oncogeni c KRAS signaling is effective at enhancing the sensitivity of a tumor cell to tumor cell-specific T cell cytotoxicity. In certain embodiments, the modulator of oncogenic KRAS signaling can be an inhibitor of oncogenic KRAS signaling. In certain embodiments, the modulator of oncogenic KRAS signaling can enhance die efficacy of the therapeutic agent at treating an oncogenic KR AS associated disease, in certain embodiments, the tumor cell is in a subject.

In certain embodiments, the therapeutic agent can be, for example, an anticancer therapeutic agent. In certain embodiments, the anticancer therapeutic agent can be, for example, an immunotherapeutic agent, such as an antigen-binding protein, or fragment thereof, e.g. an antibody that targets a cell surface antigen or extracellular growth factor. In certain embodiments, the anticancer therapeutic agent can be, for example, a small molecule inhibitor of a target protein required for the maintenance or progression of a cancer. In certain embodiments, the small molecule inhibitor can be a small molecule proteasome inhibitor, a small molecule tyrosine kinase inhibitor, a small molecule cyclin-dependent kinase inhibitor, a small molecule inliibitor of a transcription factor or s small molecule inhibitor of an immune checkpoint molecule. In certain embodiments, the anticancer therapeutic agent can be, for example, an RNA interfering agent that silences the expression of a target gene required for the maintenance or progression of a cancer. In certain embodiments, the therapeutic agent can be, for example, an epigenetic inhibitor, e.g. an HDAC inhibitor. In certain embodiments, the anticancer therapeutic agent can be, for example, an anti-estrogen or an anti-androgen therapeutic agent. In. certain embodiments, the anticancer therapeutic agent can be, for example, a chemotherapeutic agent arid/or radiotherapy. In certain embodiments, the anticancer therapeutic agent can be, for example, a cancer vaccine. In certain embodiments, the anticancer therapeutic agent can be, for example, an RNA interfering agent of an immune checkpoint molecule, e.g. PD-L1.

In a fourth aspect, a composition is disclosed that comprises a combination of an effective amount of a modulator of KRAS signaling, and an effective amount of an immune checkpoint inliibitor, wherein the modulator of KRAS signaling is effective at enhancing the sensitivity of a tumor cell to the immune checkpoint inliibitor. In certain embodiments, the tumor cells can be resistant or have acquired resistance to the immune checkpoint inliibitor. In certain embodiments, the modulator of KRAS signaling sensitizes a tumor cell to tumor cell - specific T cell cj' otoxicity. In certain embodiments, the modulator of KRAS signaling enhances the efficacy of the immune checkpoint inhibitor at treating a KRAS associated disease, e.g., cancer. In certain embodiments, the tumor cell is in a subject.

In a fifth aspect, a method for enhancing an immune response against a tumor is disclosed comprising administering an effective amount of a modulator of KRAS signaling and an effective amount of an immune checkpoint inhibitor to the subject with the cancer, wherein the administration of the modulator of KRAS signaling and the immune checkpoint inhibitor is effective at enhancing the sensitivity of the tumor cell s to the immune checkpoint inhibitor. In certain embodiments, the tumor cell is in a subject. In certain embodiments, the modulator of KRAS signaling, can act in synergy with the immune checkpoint inhibitor to enhance an immune response against a tumor.

In certain embodiments, the immune checkpoint inhibitor is effective at blocking the interaction of programmed cell death protein 1 (PD-1) receptor with programmed cell death 1 ligand 1 (RD-L1). In certain embodiments, the immune checkpoint inhibitor can be, for example, ipilimumab, tremelimumab, atezolizumab, nivolumab, pembrolizumab, JS001, REGN2810, SHR- 121Q, MEDI0680, FDR001, BGB-A317, TSR-042, PF-06801591, Ningbo Cancer Hosp. anti-PD-I CAR, Medimmune anti-PD- l , s anti-PD- i. UCB anti-PD-l or 948. gl, Dana- arber anti-PD-l, STI-1110, Suzhou Stainwei Biotech anti-PD-l, Haixi pembroiizumab biosimilar, Livzon anti-PD-l, MabQuest anti-PD- \, Singapore ASTR anti-PD- 1, Sutro anti-PD-l, Rinat anti-PD-l, Biocad anti-PD- l, Enumeral anti-PD-l or ENLM 388D4, Kadmon anti-PD- l, BMS-936559, avelumab and/or durvalumab or any combination thereof.

In certain embodiments, the mmune checkpoint inhibitor is effective at inhibiting an endogenous immune checkpoint protein or fragment thereof chosen from, for example, PD-1, PD-L1, PD-L2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7~ H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3. CD 137, CD137L, OX40, OX40L, CD27, CD70, CD4G, CD40L, T.IM3, GAL?, ADORA, CD276, VTCN1, IDOL KIR3DL1, KAVCR2, VISTA, and/or CD244 or any combination thereof.

In certain embodiments, the tumor cells express a modified KRAS, e.g. an oncogenic KRAS. In certain embodiments, the expressed oncogenic KRAS comprises a mutation of at least one amino acid residue of the amino acid sequence of SEQ ID No. :980. In certain embodiments, the mutation can be an activating mutation of KRAS. In certain embodiments, the oncogenic KRAS comprises an activating mutation of amino acid residues G12, G13, S17, P34 and/or Q61 of SEQ ID No.; 980. In certain embodiments, the oncogenic KRAS comprises a mutation chosen from G12C, G12S, G 12R, G12F, G12L, G12N, G12A, G12D, GOV, G13C, G13S, G1.3D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and/or Q61H.

In certain embodi ments, the modulator of KRAS signaling inhibits aberrant KRAS signaling. In certain embodiments, the aberrant KRAS signaling comprises signaling by an oncogenic KRAS expressed in the tumor cells. In certain embodiments, the aberrant KRAS signaling comprises the KRAS induced activation of at least one effector of the RAS/ RAF MEK'' ERK ^' FRA-1 signal transduction pathway in tumor cells. In certain embodiments, the aberrant KRAS signaling comprises the KRAS induced activation of PD-L1 gene expression in tumor ceils. In certain embodiments, the modulator of KRAS signaling is ineffective at reducing KRAS induced signaling activity in the absence of oncogenic KRAS gene expression.

. In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of KRAS mRNA in the tumor cells by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90% by at least about. 93% or by at least about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of KRAS mRNA in the tumor cells by at least 95%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of KRAS mRNA in the tumor cells by at least. 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of KRAS mRNA in the tumor cells from about 10% to about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of KRAS induced PD-L1 gene expression in the tumor cells by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%. by at least about 60%, by at least about 70%, or by at least about 75% by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95% or by at least about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at mhibiting the level of KRAS induced PD-L1 gene expression in the tumor cells by at least about 80%, in certain embodiments, the modulator of KR AS signaling can be effective at inhibiting the level of KRAS induced PD-L1 gene expression in the tumor cells from about 10% to about 99%

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of FRA-1 gene expression in the tumor cells by at least about. 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least, about 60% by at least about 70%, or by at least about 75% by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95% or by at least about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of FRA-1 gene expression in the tumor cells by at least about 80%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the level of FRA-1 gene expression in the tumor cells from about 10% to about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the KRAS induced activation of at least one effector molecule of the RAS/ RAF/ MEK/ ER ' FRA- 1 signal transduction pathway in tumor cells.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the KRAS induced activation of at least one effector molecule of the RAS/ RAF/ MEK/ ERK/ FRA- 1 signal transduction pathway in tumor by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, or by at least about 75% by at least about 80% by at least about 85%, by at least about 90%, by at least about 95% or by at least about 99%.

In certain embodiments, the modulator of KRAS signaling cart be effective at inhibiting the KRAS induced activation of at least one effector molecule of the RAS/ RAF/ MEK/ ERK/ FRA- 1 signal tra nsduction pathway in tumor from about 10% to about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the KRAS-induced phosphorylation of RAF, MEK. or ERK in tumor cells.

Trt certain embodiments, the modulator of KRAS signaling cart be effective at inhibiting the KRAS-induced phosphorylation of RAF, MEK or ERK in tumor cells by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, or by at least about 75% by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95% or by at least, about 99%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting the KRAS-induced phosphorylation of RAF, MEK or ERK in tumor cells from about 10% to about 99%.

In certain embodiments, the modulator of KRA.S signaling can be effective at inhibiting the level of both KRAS and PD-L1 gene expression in tumor cells by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, or by at least about 75% by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95% or by at least about 99%.

In certain embodimsnts, the modulator of KRAS signaling can be effective at inhibiting both KRAS and PD-L1 gene expression in tumor cel ls from about 10% to about 5)9%.

In certain embodiments, the modulator of KRAS signaling can be effective at inhibiting KRAS, RAF. IvIEK, ERK, FRA-1 and PD-L1 signaling activity in tumor cells from about 10% to about 99%.

In certain embodiments, the modulator of KRAS signaling comprises an RNA interfering agent. In certain embodiments, the RNA interfering agent targets the expression of one or more effectors of the RAS/ RAF MEK7 ERK' FRA-1 signal transduction pathway.

In certain embodiments, the modulator of KRAS signaling comprises an inhibitor of GTP bound KRAS activity. In certain embodiments, the modulator of KRAS signaling comprises an inhibitor of a KRAS GEF. in certain embod ments, the modulator of KRAS signaling comprises an activator of KRAS bound GTP hydrolysis, e.g. KRAS GAP activity. In certain embodiments, the modulator of KRAS signaling comprises an inhibitor of oncogenic KRAS. In certain embodiments, the modulator of KRAS signaling comprises a KRAS-specifie RNA interfering agent, e.g. a KRAS-specific asymmetric interfering RNA (referred to herein as KRAS aiRNA or aiKRAS). In certain embodiments, the modulator of KRAS signaling comprises an oncogenic KRAS-specific RNA interfering agent, e.g. an oncogenic KRAS- specific asymmetric interfering RNA. In certain embodiments, the modulator of KRAS si gnaling comprises an RNA interfering agent that targets both wild type and oncogenic KRAS, e.g. a KRAS-specific asymmetric interferin RNA.

In certain embodiments, the tumor can be, for example, a tumor caused by pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, or non-small-cell lung cancer (NSCLC).

In certain embodiments, the cancer can be a metastatic cancer, a cancer that is refractory to chemotherapy, a cancer that is refractory to radiotherapy and/or a cancer that has relapsed. In certain embodiments, the cancer can be resistant to an immunotherapeutic agent, e.g. an immune checkpoint inhibitor.

In a sixth aspect, the disclosure provides a modulator of oncogenic KRAS signaling. In Certain embodiments, the oncogenic KRAS signaling comprises aberrant KRAS signaling.

In certain embodiments, the modulator of KRAS signaling is effective at inhibiting oncogenic KRAS signaling.

In certain embodiments, the modulator of KRAS signaling is ineffective at reducing

KRAS induced signaling activity in the absence of oncogenic KRAS gene expression.

In certain embodiments, the modulator of KRAS signaling comprises an RNA interfering agent, for example, an asymmetric interfering RNA (aiRNA).

In certain embodiments, the asymmetric interfering RNA comprises a sense strand sequence that is at least 50% identical to a sequence chosen from SEQ ID NOs: 320-637.

In certain embodiments, the asymmetric interfering RNA comprises a sense strand sequence chosen from SEQ ID NOs: 320-637.

In certain embodiments, the asymmetric interfering RNA comprises an antisense strand sequence that is at least 50% identical to a sequence chosen from SEQ ID NOs: 638-955.

In certain embodiments, the asymmetric interfering RNA comprises an antisense strand sequence chosen from SEQ ID NOs: 638-955.

In a seventh aspect, the disclosure provides a composition comprising an effective amount of a modulator of oncogenic KRAS signaling.

In an eighth aspect, the disclosure provides a method for changing the efficacy or/and safety of a therapeutic agent comprising administering an effective amount of a modulator of KRAS signaling. In certain embodiments, the modulator of KRAS signaling can act in synergy with the therapeutic agent to enhance the efficacy and/or safety of the therapeutic agent at treating cancer.

In a ninth aspect, the disclosure provides a method for changing the efficacy or/and safety of a therapeutic agent comprising administering an effective amount of an asymmetric interfering RNA (aiRNA). In certain embodiments, the asymmetric interfering RNA (aiRNA) can act in synergy with the therapeutic agent to enhance the efficacy and/or safety of the therapeutic agent at treating cancer.

In a tenth aspect, the disclosure provides a method for changing the efficacy or/and safety of a therapeutic agent comprising administering an effective amount of an asymmetric

S interfering RNA (aiRN A) comprising a sense strand sequence chosen from SEQ ID.NOs: 320- 637.

In an eleventh aspect, the disclosure provides a method for changing the efficacy or/and safety of a therapeutic agent comprising administering an effective amount of an asymmetric interfering RNA (aiRN A) comprising an antisense strand sequence chosen from SEQ ID NOs: 638-955.

In certain embodiments, the efficacy of the therapeutic agent is enhanced,

In certain embodiments, the safety of the therapeutic agent is enhanced,

In certain embodiments, the therapeutic agent is an immune checkpoint inhibitor. In certain embodiments, the therapeutic agent is chosen, for example, from ipilimumab, trerneiimumab, atezolizumab, nivolumab, pembrolizumab, JS001, REGN2810, SHR-1210, MEDIQ68Q, PDR001, BGB-A317, TSR-042, PF-06801591, Ningbo Cancer Hosp. anti-PD- 1 CAR, edimmune anti-PD-1 ₅ Isis anti-PD-1, UCB anti-PD-l or 948,gl, Dana-Farber anti-PD- 1 , STT-1 1 10, Suzhou Stainwei Biotech anti-PD- 1 , Haixi pembrolizumab btosimilar, Livzon anti-PD- 1, MabQuest anti-PD-1, Singapore ASTR. anti-PD-1, Sutro anti-PD- 1, Rinat anti-PD- 1 , Biocad anti-PD-1, Enumeral anti-PD-1 or ENXJM 388D4, Kadmon anti-PD-1, BMS-936559 _; , avelumab and/or durvalumab or any combination thereof.

In certain embodiments, the therapeutic agent can be effective at inhibiting an endogenous immune checkpoint protein or fragment thereof chosen from, for example, PD-1 , PD-L1, PD-L2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7- H3, B7-H4, BTLA HVEM, KIR, TCR, LAG3, CD 137, CD137L, OX40, OX40L, CD27, CD70, CD40, CD40L, ΤΪΜ3, GAL9, ADORA CD276, VTCN1, IDOl, KJR3DL1, HAVCR2, VISTA and/or CD244 or any combination thereof.

Other features and advantages of the present disclosure are apparent from the additional descriptions provided herein including the different examples. Based on the present disclosure the skilled artisan may identify and employ other components and methodologies useful for practicing the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. ί A shows the nucleotide sequences within the KRAS gene that are targeted as well as the sequences of the sense and antisense strands of the scrambled aiControi and exemplar)'. interfering RNAs aiKRAS#l, aiKRAS#2, aiKRAS#3, aiKRAS#4, aiPD-Ll, aiFra-l#l, and aiFra-l#2.

FIG. IB depicts an exemplary nucleotide sequence of the human KRAS proto- oncogene, transcript variant B mRNA (NCBI Reference Sequence: NM 004985.4) along with the encoded amino acid sequence of KRAS B (SEQ ID NO.: 981). The locations of the most common activating missense mutations within the KRAS amino acid sequence are highlighted in grey. Nucleotide sequences targeted by the exemplar)' interfering RNAs aiR As#l-4 within SEQ ID NO. : 980 are in bold and underlined.

FIG, 2 shows an exemplary embodiment of aiKRAS silencing of KRAS and PD-L1 expression in KRAS ¹ ^ cells and ^' KRAS ^r cells, FIG. 2A and FIG, 2B show an exemplary embodiment of MDA-MB-231 cells treated with aiKRAS# l, aiKRAS#2, aiKRAS#3 and aiKRAS#4 (at InM and O. lnM). PD-L1 expression and KRAS silencing were confirmed by Western Blot (FIG. 2A) and quantitative real-time PGR (FIG. 2B). Data with mean and error bars represent mean of standard error (SEM). FIG. 2C shows an exemplary embodiment of KRAS silencing of PD-L1 expression in MDA-MB-231 cells at 0, 8, 24, 32 and 48 hours post- transfection. FIG. 2D shows an exemplary embodiment of aiKRAS silencing of KRAS and PD-L1 expression in KRAS mutant cells (H 58, H460 and H2009) and KRAS wild-type cells (RKO, TCCSIJP) transfected with aiKRAS# l (at InM and O. lnM). The expression of KRAS and PD-L1 was confirmed by Western Blot and quantitative real-time qPCR (data not. shown). FIG. 2E shows an exemplary embodiment of the down-regulation of PD-L1 expression resulting from aiRNA mediated silencing of KRAS gene expression in various KRAS ^MT cell lines.

Fig. 3 shows an exemplary embodiment of aiKRAS silencing of ERK phosphorylation in KRAS mutant (KRAS ¹ * ¹ ) cells. KR AS ^MT MDA-MB-231 cells and KRAS ^WT RKO cells were transfected with aiKRAS#l at InM for 48 hours. FIG, 3A shows an exemplary embodiment of KRAS ^MT MDA-MB-231 and KRAS ¹ RKO cell lysates applied to a phosphokinase array (R&D systems, USA). FIG. 3B shows an exemplary embodiment of phosphorylated ERK1/2 being inhibited only in KRAS*" MDA-MB-231.

FIG. 4 shows an exemplary embodiment of the effect of aiKRAS silencing on RAS MEK1 2 ERK1 2 signaling pathway in KRAS ^MT and K AS^ cells. KRAS*" ^" MDA- MB-231 cells and KRAS ^' * ^1' RKO ceils were treated with aiKRAS# 1 at InM. After a 48h transfection period, the eel! lysate was applied to a Western Blot, to confirm the amount of total or phosphorylaled MEK and ERK. FIG. 4A shows an exemplary embodiment of KRAS silencing inhibiting MEK/ERK pathway in KRAS ^MT MDA-MB-231 cells but not in KRAS ^WT RKO cells. FIG. 4B shows an exemplary embodiment of inhibition of PD-L 1 gens expression and ERK1/2 phosphorylation in MDA-MB-231 cells treated with the MEK inhibitor, U0126. PD-L1 expression and total and phosphorylated ERK were analyzed by Western Blot. FIGs. 4C and 4D show an exemplary embodiment of the failure of KRAS silencing to inhibit STATS signaling and nuclear localization of RELA RELB in KRAS** ^1" MDA-MB-231 cells treated with aiKRAS# l at lnM. After a 48h transfection period, the amount of nuclear localization of NF-KB (RELA and RELB) (FIG. 4C) or the amount of whole and phosphorylated STATS (FIG. 4D) was determined by Western Blot.

FIG. 5 shows an exemplary embodiment of KRAS silencing inhibiting the phosphorylation, the accumulation of ERA- 1 protein as well as the transcriptional activity of FRA- 1 gene. KRAS mutant MDA-MB-231, H358, and H460 cells were treated with asKRAS# l at lnM for 48h. FIG. 5A shows an exemplaiy embodiment of a Western blot of the cell lysate and the detection of total or phosphorylated FRA-1. FIG. SB shows an exemplary embodiment of KRAS mutant MDA-MB-231 cells and KRAS wild type RKO cells treated with either control aiRNA or aiKRAS 1. Nuclear extracts (5ug well) were then incubated in 96- well plates coated with an immobilized oligonucleotide containing the TRE (TPA response element) sequence that is required for AP- 1 binding. Competition with TRE oligonucleotides was performed to confirm DNA binding specificity. FIG. SB shows an exemplary embodiment of an analysis of the complexes formed in the RAS mutant or KRAS wild type cells by ELISA assay in the presence of FRA-l-specific antibodies. FIG, 5C shows an exemplary embodiment of a ChlP-qPCR analysis of the PD-L1 enhancer. ChlP was conducted in aiControl or aiKRAS treated KRAS mutant MDA-MB-231 cells and KRAS wild type RKO cells. The predicted PD- LI enhancer sequence was enriched in immunoprecipitated chromatin using an anti-FRA-1 antibody and an anti-cJUN antibody in KRAS mutant cells (Fig. SC Left) but not in KRAS wild type cells (Fig.SD Right), or in chromatin incubated with the negative control IP (normal rabbit IgG). Data with mean and error bars represent mean of standard error (SEM).

FIG. 6 shows an exemplary embodiment of the effects of FRA-1 on PD-L1 expression in KRAS mutant cells. KRAS mutant MDA-MB-231 and H460 cells were treated with control aiRNA, aiKRAS, and two different FRA-1 aiRNAs (aiFra-1 # 1, and aiFra-1 #2). PD-L1 and FRA- 1 expression were confirmed using Western Blot (Fig. 6A) and qPCR (Fig. 6B). Data with mean and error bars represent mean of standard error (SEM). FIG. 7 shows an exemplary embodiment of cytotoxic T cell activity against KRAS mutant cancer cells after aiKRAS silencing of PD-L1 expression, FIG. 7A shows an exemplary embodiment of cytotoxic T lymphocyte (CTL) activity against KRAS mntant MDA-MB-23.1 cells transfectecl with. aiKRAS. CMV-specific CTLs were expanded to culture human PBMC with HLA-A*02:01 CMV pp65 peptide. aiRNA-transfected Luc-MDA-MB-231 cells were incubated with or without CMV peptide. Peptide loaded or non-loaded MDA-MB-231 cells were plated into 96-well plates (2000 cells/well). CMV-specific CD 8+ T cells were subsequently added to 96-well plates with Effector : Target (E/T) ratio of 50: 1 and incubated for 24 hours. Live Luc~MDA-MB-231 cells were measured for intracellular luciferase activity with D-Luciferin K.+ salt. The percent, lysis was then calculated as: (Luminescence of CMV peptide pulsed Luc-MDA-MB-23.1/ Luminescence of CMV peptide un~pulsed Luc-MDA-MB- 231) x 100. Anti-PD-LI antibody (lOpg/mL) was used as a positive control, Data with mean and error bars represent mean of standard error (SEM). Data were subjected to one- way ANOVA with Dimnett's multiple comparison of means test. Statistical significance is displayed as p-value ** pO.01 and *** pO.001. FIG. 7B shows an exemplary embodiment of PD-L1 cell surface expression on MDA-MB-231 cells after aiKRAS and aiPD-Ll transfection. An exemplary embodiment of the calculated geometric mean of fluorescence intensity (MFI) measured by flo cytometry is depicted at the bottom.

FIG. 8A shows an exemplary embodiment of some of the canonical KRAS signaling pathways. In certain embodiments, growth factor binding to cell-surface receptors results in activated receptor complexes, which contain adaptors such as SHC (SH2-containing protein), GRB2 (growth-factor-receptor bound protein 2) and GAB (GRB2-associated binding) proteins. These proteins can recruit SHP2 and SOS1, a guanine nucleotide exchange f ctors (RAS GEF) protein that can increase RAS-guanosine triphosphate (RAS-GTP) levels by catalyzing nucleotide exchange on RAS. In contrast, the GTPase activating protein (GAP) neurofibromin (NF1) can bind to RAS-GTP and accelerate the conversion of RAS-GTP to inactive RAS- GDP (guanosine diphosphate), which can terminate signaling. The BRAF-mitogen-activated and extracellular-signal regulated kinase-kinase (MEKV-extraeel ar signal-regulated kinase (ERK) cascade can determine key cellular processes including cell proliferation. RAS can also activate the phosphatidylinositol 3-kinase (PI3K) - ^■ 3-phosphoinositide-dependent protein kinase 1 (PDK1)- AKT pathway that can determine cellular survival. RALGDS, RALGDS-like gene (RGL), RGL2 and TIAM1 can be exchange factors of RAL and RAC, respectively. Among the effectors of RAL is phospholipase D (PLD) an enzyme that, can regulate vesicle trafficking, RAC can regulate actin dynamics and, therefore, the cytoskelelon. RAS can also bind and activate the enzyme phospholipase Cepsilon (PLCep ^' silon), the hydrolytic products of which can regulate calcium signaling and the protein kinase C (PKC) family. FIG, 8B shows a proposed signaling mechanism along the KRAS/ME ERK Fra- 1 PD-L 1 axis, hi KRAS mutant cancer cells, mutant KRAS can activate ME ERK kinase. Stabilized phosphorylated FRA-1 can bind to the PD-L1 enhancer region where it can activate and maintain FD-L1 gene expression in tumor cells.

The methods and techniques of the present application are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present disclosure unless otherwise indicated. See, e.g., M.R. Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed, ₅ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), Ausubel et al, Current Protocols, John Wiley & Sons, Inc. (2000-2016), Antibodies: A Laboratory Manual, 2nd edition, edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2014), and RNA: A Laboratory Manual by Rio et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N. Y. (2011), all of which are incorporated herein by reference.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly miderstood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present disclosure, including definitions, will control. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. Tire materials, methods, and examples are illustrative only and not intended to be limiting.

As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

The phrase "and/or," as iised herein in the disclosure and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non- limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the disclosure and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combi nations of elements in the list of elements. This definition also allows mat elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-l imiting example, "at least one of A and B" (or, equivalent!}', "at least one of A or B." or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with mo A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a vari nce of 20%, 10%, 5%, or 1%, in certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 1.0%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 5%. In certain embodiments, the 'term "about" is used to modify a numerical value above and below the stated value by a variance of 1%,

When a range of values is listed herein, it is intended to encompass each value and subrange within that range. For example, "1-5 mg" or "1 to 5 mg" is intended to encompass 1 mg, 2 mg, 3 mg, 4 mg, 5 rng, 1-2 mg, 1-3 mg, 1-4 mg, 1-5 mg, 2-3 mg, 2-4 mg, 2-5 mg, 3-4 mg. 3- 5 mg, and 4-5 mg.

As used herein, a "polynucleotide" refers to a polymeric chain containing two or more nucleotides. "Polynucleotides" includes primers, oligonucleotides, nucleic acid strands, etc. A polynucleotide ma contain standard or non-standard nucleotides. Typically, a polynucleotide contains a 5' phosphate at one terminus ("5' terminus") and a 3' hydroxy! group at the other terminus ("3* terminus) of the chain. The most 5' nucleotide of a polynucleotide may be referred to herein as the "5 -term.inal nucleotide" of the polynucleotide. The most 3' nucleotide of a polynucleotide may be referred to herein as the "3 '-terminal nucleotide" of the polynucleotide, The term "subject" generally refers to an organism to which a compound or pharmaceutical composition described herein can be administered. A subject can be an animal or animal cell, including a mammal or mammalian cell (e.g., a human or human cell). The term also refers to an organism, which inchjdes a cell or a donor or recipient of such cell. In various embodiments, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, fish, nematode, and insects, which is to be the recipient of compound or pharmaceutical composition described herein. Under some circumstances, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

The terms "administer,"" "administering," or "administration" are used herein in their broadest sense. These terms refer to any method of introducing to a subject a compound or pharmaceutical composition described herein and can include, for example, introducing a compound systemically, locally, or in situ to the subject. Thus, a compound of the present disclosure produced in a subject from a composition (whether or not it includes the compound) is encompassed by these terms. When these terms are used in connection with the term "systemic" or "systemically," they generally refer to in vivo systemic absorption or accumulation of the compound or composition in the blood stream and its distribution throughout the entire body. In certain embodiments, the terms "administer," "administering." or "administration" can refer to, for example, delivering one or more recombinant vectors to a tumor cell, wherein the vector expresses an RNA interfering agent as defined herein. In certain embodiments, the tumor cell is in a subject.

The terms "combination," "combinatorial," or "combination treatment," as used herein, mean the administration of at least two different agents (e.g., at least one compound chosen from modulators of KRAS signaling and/or at least one compound chosen from therapeutic agents, and, optionally, one or more additional agents) to treat a disorder, condition, or symptom, e.g., a cancer condition. Such combination treatment may involve the administration of one agent before, during, and/ or after the administration of a second agent. The first agent and the second agent can be administered concurrently, separately, or sequentially in separate pharmaceutical compositions. The first agent and the second agent may be administered by the same or different routes of administration, In certain embodiments, a treatment combination comprises a therapeutically effective amount of at least one compound chosen from modulators of KRAS signaling and a therapeutically effective amount of at least, one compound chosen from therapeutic agents, e.g. immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitor can be. for example, an inhibitor of PD-L1.

For example, the at least one compound chosen from modulators of KRAS signaling and at least one compound chosen from therapeutic agents can have different mechanisms of action. In certain embodiments, a combination treatment improves the prophylactic or therapeutic effect of the at least one compound chosen from modulators of KRAS signaling and the at least one compound chosen from therapeutic agents by functioning together to have an additive, synergistic, or enhanced effect. In certain embodiments, a combination treatment of the present disclosure reduces adverse side effects associated with the at least one compound chosen from modulators of KRAS signaling and the at least one compound chosen from therapeutic agents. The administration of the at least one compound chosen from modulators of KRAS signaling and the at least, one compound chosen from therapeutic agents may be separated in time by up to several weeks, but more commonly within 48 hours, and most commonly within 24 hours.

As used herein, a "therapeutic agent" that may administered with a modulator of KRAS signaling can be an anticancer therapeutic agent, i.e. an agent that may be administered in vivo to treat cancer. In certain embodiments, the anticancer therapeutic agent can be a small molecule, a peptide, a modified peptide, a peptidomimetic, an antibody, an antibody fragment, a recombinant antibody, a recombinant antigen-binding protein, an aptamer, a nucleic acid or RNA interfering agent,

In certain embodiments, the anticancer therapeutic agent can be, for example, a chemotherapeutic agent, i.e. a chemical compound useful in the treatment of cancer. Exemplary classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, mitotic inhibitors, spindle poison plant alkaloids, cytotoxic /antitumor antibiotics, topisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), anti-progesterones, estrogen receptor down-regulators ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, anti- androgens, aron atase inliibitors, EGFR inliibitors, angiogenesis inhibitors, VEGF inhibitors, and inhibitors of the translation and/or transcription of genes implicated in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the treatment methods disclosed herein include cytostatic and/or cytotoxic agents.

In certain embodiments, the therapeutic agent can be, for example, a biotherapeutic agent, such as an antibody or recombinant antigen-binding protein, in certain embodiments, the antibody or recombinant antigen-bind protest! can block ligand / receptor signaling its any biological pathway that supports tumor maintenance and/or growth or suppresses the antitumor immune response. In certain embodiments, the therapeutic agent can be, for example, a targeted therapeutic agent, such as a small molecule drug, hi certain embodiments, the therapeutic agent can include radiation and/or surgery.

In certain embodiments, the therapeutic agent can be, for example, a small molecule such as a small molecule kinase inhibitor (SMKI), SM Is that can be combined with a modulator of KRAS signaling are disclosed, for example, in Wu et al Drug Discovery Today (2016) volume 21, issue 1, pp. 5-10, which is incorporated herein by reference.

Exemplary SMKIs are depicted in TABLE 1 below (dates indicate year when FDA approved).

(¾a

The terms "effective amount" and "therapeutically effective amount" refer to the amount of a compound or pharmaceutical composition described herein that is capable of invoking, for example, one or more of the following effects: (1) inhibition, to some extent, of cancer or tumor growth, including a decrease or cessation in the progression of cancer; (2) reduction in the number of cancer or tumor cells; (3) reduction in tumor size; (4) inhibition, e. g., a decrease or a cessation, of cancer or tumor cell infiltration into peripheral organs; (5) inliibition, e.g., a decrease or a cessation, of metastasis; (6) enhancement of anti-tumor immune response, which may, but is not required to, result in the regression or rejection of the tumor, or (7) relief, to some extent, of one or more symptoms associated with the cancer or tumor. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), the subject and disease condition being treated, e.g., the sex, weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which cars readily be determined by one of ordinary skill m the art, e.g., a board-certified oncologist. A 'therapeutically effective amount" is an amount of a compound where any toxic or detrimental effects resulting from the administration of the compound are outweighed by the therapeutically beneficial effects.

The terms "progress," "progressed," and "progression" as used herein refer to at least one of the following: (1) a response of progressive disease to prior therapy (e.g., chemotherapy and/or immune checkpoint therapy); (2) the appearance of one or more new lesions after treatment with prior therapy (e.g.. chemotherapy and/or immune checkpoint therapy); and (3) at least a 5% (e.g., 10%, 20%) increase in the sum of diameters of target lesions, taking as a reference the smallest sum on study.

The term "sensitize" means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy. In certain embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the cancer therapy. In certain embodiments, an increased sensitivity or a reduced sensitivity to a therapeutic treatment can be measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa et ah Cancer Res 1982; 42: 2159-2164) or cell death assays (Weisenthal et al Cancer Res 1 84; 94: 161. - 173; Weisenthal et al Cancer Treat Rep 1985; 69: 615-632; Weisenthal et al Drug Resistance in Leukemia and Lymphoma. Langhome, P A: Harweod Academic Publishers, 1993: 41 -432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animals by measuring the tumor size reduction over a period of time, for example, 6 months for humans and 4-6 weeks for mice. A composition or a method sensitizes cancer cells or tumor cells to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is about 25% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more, compared to treatment sensitivity or resistance in the absence of such composition or method. In certain embodiments, the increase in treatment sensitivity or the reduction in resistance is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fo!d, about 20-fold or more compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

The terra "synergy," "synergistic," "synergisticaily," or "enhanced" as used herein refers to an effect of interaction or combination of two or more components to produce a combined effect greater than the sum of their separate effects (or "additive effects"). A synergistic effect may be attained when the compounds are: (.1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes. A synergistic anticancer effect denotes an anticancer effect which is greater than the predicted purely additive effects of the individual compounds of the combination administered separately.

Terms such as "treating" or "treatment" or "to treat" as used herein refer to (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder _. or/and (2) prophylactic or preventative measures that prevent or slow the development and/or progression of a targeted pathologic condition or disorder, Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented, A subject is successfully "treated" according to the methods of the present disclosure if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis: inhibition or an absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduced morbidity and mortality; and improvement in quality of life. In certain embodiments, "treating cancer," "treatment of cancer," or an equivalent thereof, mean preventing recurrence of cancer after surgical removal or other anticancer therapies,

As used herein, a "modulator" refers to a compound or combination of compounds that is capable of modulating KRAS signaling activity, including but not limited to, oncogenic KRAS signaling or otherwise aberrant KRA8 signaling activity,

In certain embodiments, a "modulator" can refer to a compound or combination of compounds that is capable of modulating the expression a target gene required for KRAS signaling.

In certain embodiments, a "modulator" can refer to a compound or combination of compounds thai are capable of modulating the expression of KRAS.

In certain embodiments, a "modulator" can refer to a compound or combination of compounds that, are capable of modulating tine expression of one or more of KRAS, RAF, MEK, ERK and FRA- 1.

As used herein, "modulating" and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating KRAS signaling activity, e.g., by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, 99% or 100%, compared to KRAS signaling activity in the absence of a modulator.

In certain embodiments, a "modulator" can refer to an inhibitor of a cellular activity, e.g, an inhibitor of oncogenic KRAS signaling. In certain embodiments, a "modulator" can refer to an activator of a cellular activity, e.g. RAS GAP induced ^' hydrolysis of GTP bound to KRAS.

As used herein, the terms "inhibiting", "to inhibit" and their grammatical equivalents, when used in the contest of a bioactivity, refer to a down-regulation of the bioactivity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. When used in the context of an organism (including a cell), the terms refer to a down-regulation of a bioactivity of the organism, which may reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule, in particular embodiments, inhibition may refer to a reduction, e.g., of about 10%, of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of the targeted activity. When used in the context of a disorder or disease, the terms refer to success at preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition or disorder.

The term "cancer" h a subject refers to the presence of cells possessing characteristics typical of cancer-causing ceils, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain morphological features. Often, cancer cells will he in the form of a tumor or mass, hut such cells may exist alone within a subject or may circulate in the blood stream as independent cells, such as leukemic or lymphoma cells.

Examples of cancer as used herein include, but are not limited to lung cancer, pancreatic cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, breast cancer, uterine cancer, ovarian cancer, peritoneal cancer, colon cancer, tnicrosatellite instability-high metastatic colorectal cancer, microsatellite stable metastatic colorectal cancer, colorectal cancer with mismatch-repair deficiency, colorectal cancer without mismatch-repair deficiency, small bowel adenocarcinoma, rectal cancer, colorectal adenocarcinoma, cancer of the anal region, stomach cancer, gastric cancer, gastrointestinal cancer, gastric adenocarcinoma, adrenocorticoid carcinoma, genitourinary cancer, gynecologic cancer, uterine cancer, uterine sarcoma, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, cervical cancer, Hodgkm's Disease, esophageal cancer, gastroesophageal junction cancer, gastroesophageal adenocarcinoma, chondrosarcoma, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, E wing's sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, testicular cancer, cancer of the ureter, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, kidney cancer, renal cell carcinoma, endometrial cancer, chronic or acute leukemia, multiple myeloma, lymphocytic lymphomas, neoplasms of the central nervous system (CNS), spinal axis tumors, brain cancer, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. Some of the exemplified cancers are included in general terms and are included in this term. For example, urological cancer, a general term, includes bladder cancer, prostate cancer, kidney cancer, testicular cancer, and the like; and hepatobiliary cancer, another general term, includes liver cancers (itself a general term that includes hepatocellular carcinoma or cholangiocarcmoma), gallbladder cancer, biliary cancer, or pancreatic cancer. Both urological cancer and hepatobiliary cancer are contemplated by the present disclosure and included in the term "cancer."

Also included within the term "cancer" is the term "solid tumor" or "advanced solid tumor." A "solid tumor" refers to those conditions, such as cancer, that form an" abnormal tumor mass, such as sarcomas, carcinomas, and lymphomas. Examples of solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, th omas, fibrous tumors, metastatic colorectal cancer (mCRC), and the like, in certain embodiments, the solid tumor disease is an adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like.

In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is colorectal adenocarcinoma. In certain embodiments, the cancer is small bowel adenocarcinoma. In certain embodiments, the cancer is hepatocellular carcinoma. In certain embodiments, the cancer is head and neck cancer. In certain embodiments, the cancer is renal cell carcinoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is uterine sarcoma. In certain embodiments, the cancer is esophageal cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is a gastric cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is cholangiocarcinoma. In certain embodiments, each of the cancers is unresectable, advanced, refractory, recurrent, or metastatic. In certain embodiments, the cancer is resistant or has acquired resistance to an anticancer therapeutic agent, e.g. treatment with an immune checkpoint inhibitor.

in certain embodiments, the efficacy of a compound or a combination of compounds is tested in a xenograft cancer model in which cells isolated from a solid tumor are injected into a host animal, e.g. an immunocompromised host, to establish solid tumors. In certain embodiments, the cells isolated from a solid tumor comprise cancer stem cells. The host animal can be a model organism such as nematode, fruit fly, zebrafish; preferably a laboratory mammal such as a mouse (nude mouse, SCID mouse, or NOD/'SCID mouse, Beige /SCED Mouse), rat, rabbit, or primate. The severely immunodeficient NOD-SCID mice ma be chosen as recipients to maximize the participation of injected cells.

Solely to provide certain background and not to limit the scope of this disclosure, a KRAS gene (also called C-K-RAS; CFC2; K-RAS2A; K-RAS2B; K-RAS4A; K-RAS4B; KI- RAS; KRASl ; KRAS2; NS; NS3; RASK2) encodes tire human cellular homoiog of a transforming gene isolated from the Kirsten rat sarcoma virus. KRAS is a member of the mammalian RAS gene family that encode a group of closely related 21 kDa GDP/GTP-binding proteins that can act as intracellular signal transducers. Alternative splicing of the Human KRAS pre-mRNA generates transcript, variants "A" and "B" encoding two isoforms that differ in the C-teifninal region. Human transcript variant "B" having the exemplary nucleotide sequence of SEQ ID NO. : 980 (see FIG. IB; Accession No. : NM_004985), the prevalent KRAS tsoform, comprises five exons but lacks exon 4a which the longer transcript, variant "A" includes.

As used herein, KRAS protein refers to a polypeptide having at least about 40%, e.g., about 80%, identity to the amino acid sequence provided at Genbank Accession No. AAB41942 or ABY87538.

In certain embodiments, the KRAS protein can refer to a polypeptide comprising at least 10 contiguous amino acids of the amino acid sequence of SEQ ID NO. : 981.

In certain embodiments, the KRAS protein refers to the KRAS isoform B having the exemplary amino acid sequence of SEQ ID NO. : 981. 1 Si SKSiW S JM3SV»¾CS&ldi QS.XQ¾HF¥B STOSFTXEDSX KKQWVXOQftF ΟΪΟ¾Ι¾»ΧΆ.ΐ¾

SI QESXSfiKHBQ TMRTSS3 I.C &XKSS&SF SDXHKXRSQI SR KBS¾»VF WV& ^CDL

121 ^SRSWBtKQA Q02o&&S3feX& F2S5SAKTRQ gSS $^«LV SSl&KHKEK S^SIF^S

181 « ¾ 0fS ^' CSEQ a SO, j SSI)

Solely to provide certain background and not to limit the scope of this disclosure, the N-terminal portion (residues 1- 165) of KRAS comprises a highly conserved G domain which is also found in H-RAS and N-RAS isoforms. RAS proteins can diverge substantially at the C- terminal end, which is known as the hypervariabl e region. This region can contain residues that specify post-translational protein modifications that are essential for targeting RAS proteins to the cytosolic leaflet of cellular membranes. All RAS proteins are famesylated at a terminal CAAX motif, in which C is cysteine, A is usually an aliphatic amino acid, and X is any amino acid. KRAS4A is additionally modified by one or two palmitic acids just upstream of the CAAX motif. The addition of the hydrophobic farnesyl moiety is complemented by the hydrophobic palmitates (the so-called "second signal") to firmly anchor KRAS4A to the membrane. By contrast, KRAS4B, the predominant splice variant, contains an alternative second signal that is composed of a polybasic stretch of lysine residues. In this case, membrane anchoring is mediated by the electropositive lysines that form ionic bonds to the predominantly electronegative lipid head groups of the inner leaflet of the plasma membrane.

In certain embodiments, the term "KRAS" encompasses wild type KRAS 4 A and/or KRAS4B. In certain embodiments, the term "KRAS" encompasses both wild type and modified forms of KRAS 4 A. and/or KRAS4B.

In certain embodiments, modified forms of KRAS include, but are not limited to, KRAS proteins having one or more activating mutations, for example, missense mutations at positions G12, G13 and/or Q61. in certain embodiments, KRAS encompasses KRAS proteins having one or more alterations in the post-translational modifications of KRAS, including, but not limited to, acetylation, methylation, lipidation, palmitoylation, prenyl ati on, and S- nitrosylation.

Tethered to the inner leaflet of the plasma membrane, KRAS can act as a binary molecular switch at the apex of a signaling hub where it can control the transmission of signals from cell surface receptors to intracellular effectors by cycling between a GDP -bound inactive and a transient GTP-bound active slate. In its active GTP-bound form, KRAS can activate downstream effectors that control cellular processes in the cytoplasm (actin organization, endocytosis) or modify the activity of nuclear transcription factors that, regulate gene expression important for cell cycle progression, differentiation, or survival.

The KRAS molecular switch can function by responding to upstream signals by activating a class of proteins known as guanine nucleotide exchange factors (RAS GEFs) that can stimulate the dissociation of GDP from the RAS protein. For example, SOS I, a RAS GEF in the MAPK/ER pathway, can be recruited by the adaptor protein GRB2 in response to epidermal growth factor receptor (EGFR) activation. The binding of SOS 1 (Son of Sevenless 1 ) to GBR2 can localize it to the plasma membra ne, where it can activate membrane bound RAS. Activation of KRAS by release of GDP then can allow RAS to bind free GTP which can trigger a conformational change in the protein's structure that can facilitate the transient transduction of extracellular signals from the active GTP-bound RAS to downstream signaling pathways. The inactive GDP bound KRAS can then be restored by GTPase acting proteins (GAPs), like pl2QGAP., that enhance GTP hydrolysis by the otherwise slow intrinsic GTPase activity of RAS proteins.

Once activated, RAS-GTP can preferentially bind to and activate downstream RAS- binding-domain (RBD) or RAS-association (RA)-domain-containing effectors. It can be estimated that there are at least 11 distinct RAS effector families, each of which can activate a distinct protei n signaling cascade. Exemplary downstream effector pathways that respond to the KRAS activation are depicted in FIG. 8A and summarized below.

One exemplary downstream effector of activated GTP-bound KRAS is the RAS-RAF- MAP-MEK-ERK kinase cascade which can be an essential, shared element of mitogenic signaling involving tyrosine kinase receptors that leads to a wide range of cellular responses, including growth, differentiation, inflammation, and apoptosis, GTP-bound R AS can recruit RAF serine/threonine kinases (A-RAF, B-RAF and C-RAF- 1), The interaction of activated GTP-bound KRAS with RAF can initiate the RAF→ MEK→ ERK kinase cascade. Activated RAF, a MAPK (Mitogen-activated protein) kinase-kinase, can phosphorylate the MAPK kinase proteins EK1 and MEK2 (also known as MAP2K1 and MAP2K2), which in turn can phosphorylate the praline-directed kinases ERK1 and 2 (extracellular signal-regulated kinase; also known as MAPK3 and MAPK1). Activated ERK1/2 (p42/44 MAP kinase) can then phosphorylate many substrates, including kinases that are important for control of translation (e.g., p90RSK) and transcription factors thai control genes involved in cell cycling (e.g., ELK1, FOS, MYC, FRA-1)

Another exemplar}' downstream effector of activated GTP-bound KRAS is the RA8- phosphoinositide 3 -kinase (PI3K)-AKT signaling pathway involved in cell survival and proliferation. PBKs are heterodimeric lipid kinases composed of a regulatory subunit (p85) and a catalytic subunit (pi 10). GTP-bound RAS bound to the pi 10 (o, β, γ, and δ) catalytic subunits of class I PBK can trigger the synthesis of the secondary messenger, phophatidylinositol-3,4 _s 5-triphosphate (PIP3), PIPS can then be free to engage the pleckstrin homology (PH) domain of AKT/PKB (Protein kinase B), thereby stimulating its Ser/Thr kinase activity and the phosphorylation of a host of other proteins involved in cell growth, cell cycle entry, and cell survival. AKT-raediated phosphorylation can inhibit some proteins that promote programmed cell death (BAD, FoxO), and stimulates others (MDM2) that, promote cell survival.

Many additional RAS effectors have now been identified. These include RAIN, an endomembrane receptor for RAS, NOREL a pro-apoptotic tumor suppressor, and AF-6, a mediator of membrane-cytoskeleton interactions. RAS can also engage in cross- talk with other GTPase signaling pathways involved in regulating acttn reorganization and/ or endocytic trafficking. This can occur through interactions of RAS with RAL guanine nucleotide dissociation stimulator for the RA8-!ike (RAL) small GlPases, RALA and RALE, a GEF that facilitates GDP/GTP exchange with RAS (RALGDS). RAS can also interact with GEFS like RINl, for RAB5, and TIAM.1 (tumor invasion and metastasis inducing protein 1), for RAC. In addition, accumulating evidence indicates signaling specificity may also be dictated by differential localization of KRAS isoforms in discrete plasma membrane microdomains or distinct intracellular membrane compartments (e.g., endosomes, Golgi), where the activated GTPase may encounter a unique sets of effectors,

KRAS can be essential for mammalian embryonic development. KRAS -deficient mice can die of anemia and defective fetal liver erythropoiesis after only about 12-14 days of gestation. Germline mutations that affect components of the RAS-RAF— MEK-ERK pathway can cause several developmental disorders, including Noonan Syndrome (NS3), Costello Syndrome and Cardio-Facio-Cutaneous (CFC2) syndrome. The developmental disorders associated with RAS pathway mutations may share phenotypic features that include facial abnormalities, heart defects, impaired growth and development, and. in some instances, a predisposition to specific cancers.

KRAS is also a proto-oncogene, A proto-oncogene can become oncogenic by increased KRAS expression or the acquisition of an "activating" mutation, i.e. a mutation that leads to constitutive aberrant activation of KRAS signaling. Indeed, activating somatic KRAS mutations have been detected in—30% of all human cancers. Amongst those cancers. KRAS cart be found in a predominantly mutated form in pancreatic ductal adeno-carcinoma (71 %), colorectal cancer (35%), non-small cell lung adenocarcinoma (19%), and endometrial cancer (17%) (see TABLE 2 below; collated from the Catalogue of Somatic Mutations in Cancer (COSMIC) database). Activating KRAS gene point mutations can also be present in other cancers, including but not limited to, biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid lexikemia and breast cancer.

TABLE 2

Mutant KRAS, as used herein, refers to somatic or germlme KRAS4 A and/or K AS4B mutations including, but not limited to, point mutations, nonsense substitutions, missense substitutions, synonymous substitutions, in frame insertions, frameshift insertions and/or deletions, KRAS missense gain-of -function activating mutations can be found predominantly at one of three mutational hotspots: G12 (89%), G13 (9%), and Q61 (1%). In certain embodiments, these mutations disrupt intrinsic as well as GAP-mediated GTP hydrolysis. In certain embodiments, the disruption results in an accumulation of constitutive! y active GTP- bound RAS in cancer cells.

In certain embodiments, a mutant KRAS can refer to aberrant post-translational modification of KRAS. In certain embodiments, the aberrant post-translational modification of KRAS includes, but not limited to. phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation (C-terminal glycosyl phosphatidylinositol (GPI) anchor, N-tercninal myriatoylation, S-myristoylation, S-prenylation) and/or proteolysis.

In certain embodiments, a mutant KRAS can refer to aberrant splicing of KRAS mRNAs.

TABLE 3 below provides an exemplary list of KRAS4B mutations and their reported association with human cancers and developmental disorders. Oncogenic KRAS mutations associated with cancer include, without limitation, KRAS ^G52D , KRAS ^GI2V , KRAS ^GUD , KRAS ^G12C , KRAS ^Q61R , KRAS¾ ^S1L , KRAS¾ ^6IK , KRAS ^{Gi 2R} , and KRAS ^G12C . The skilled artisan will understand that a KRAS gene comprising a different KRAS mutation than one of those above and/or combinations of the above and/or other KRAS mutations that lead to constitutive activation of KRAS signaling, is also an oncogenic KRAS encompassed by the present disclosure, A comprehensive list of KRAS mutations present in human cancer is available online from UniProt Consortium, EMBL.

TABLE 3

EXEMPLARY KRAS MUTATIONS ¹

¹ reproduced from OMIM ^® (Online endeliait Inheritance in Man ^® ), Johns Hopkins University:

In certain embodiments, "KRAS signaling" can refer to wild ty e KRAS4A and/or KRAS4B GTPase activity, GTP/GDP binding activity or any signaling activity induced by ■GTP-bound KRAS4A and or KRAS4B, including, but not limited to, the RAS-RAF- AP- MEK-ER , the RAS-PDKs-AKT and RAS-RalGDS signal transduction pathways as summarized in. part above (e.g., see FIG, 8 A).

In certain embodiments, "KRAS signaling" encompasses any form of aberrant KRAS signaling. For example, in certain embodiments, aberrant KRAS signaling can occur as a result of sig aling by an oncogenic KRAS protein having one or more activating mutations, including, but not limited to, missense mutations at positions G12, GI3 and/or Q61.

In certain embodiments, the aberrant KRAS signaling refers to the activity of a hyperactive wild type GTP bound KRAS as a result of changes in GDP--GTP regulation, loss of GAPs or persistent receptor tyrosine kinase-mediated activation of GEFs. Thus, in certain embodiments, aberrant KRAS signaling can be caused by the aberrant, activation of effector molecules downstream of KR AS, including, but not limited to, A-RAF. B-RAF, C-RAF, MEK, ERK and/or F.RA-L In certain embodiments, aberrant KRAS signaling, e.g. oncogenic KRAS signaling, can occur in the presence of wild type KRAS. in certain embodiments, aberrant KRAS signaling, e.g. oncogenic KRAS signaling, can occur as a result of the overexpression of KRAS, e.g. via gene ainpliileaiion. In certain embodiments, KRAS signaling comprises hyperactive KRAS signaling initiated as a result of the inactivation of a tumor suppressor that, when inactivated, provides an alternative mechanism of activating RAS, In certain embodiments, the hyperactive KRAS signaling results from the inactivation of a tumor suppressor such as a RAS GAP, Exemplary RAS GAPs include, but are not limited to, RASAl, RASA2, RAS A3, RASA4, RASALl, NFl , DAB2IP, RASAL2, RASAL3, SynGAPl, IQGAP1, IQGAP2 and IQGAP3.

The aberrant signaling by mutant or otherwise modified KRAS has been implicated in developmental disorders and virtually all aspects of the malignant phenotype of the cancer cell, including cellular proliferation, transformation, invasion and metastasis. However, despite the importance of oncogenic KRAS in the etiology of human cancers, efforts to develop small molecule drugs targeting, for example, oncogenic KRAS over the past three decades have been largely unsuccessful.

For example, as reported by Stephen et ah, Cancer Cell (2014) vol, 25, 3 272-281, efforts to block RAS associated cancers by preventing RAS farnesylation, once thought to be an essential posttranslational modification for RAS activity, were thwarted by the unexpected presence of a backup system (geranylgeranvitransferase) that restored activity of KRAS and NRAS after famesyltransferase treatment. Likewise, efforts to block one of RAS' major downstream effectors, RAF kinase, ran into the unexpected discovery that, in RAS-transformed cells, RAF inhibitors activate the pathway rather than inhibit it. MAP kinase-kinase (MEK) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors have not yet shown significant clinical activity in RAS associated cancers, for reasons relating to feedback loops and poor therapeutic windows as well as lack of specificity. This failure has led some to dismiss KRAS as an "undruggable" target. Even strategies employing siR s to target mutant KRAS remain challenging primarily because of off target silencing of genes unrelated to KRAS or the induction of a robust interferon response.

In one aspect, the present disclosure reports on an approach for specifically inhibiting aberrant KRAS signaling in tumor cells without the known caveats associated with RNA interference.

As shown in Examples 2 and 3, oncogenic KRAS signaling in KRAS associated cancer cell lines can induce aberrant RAF MEK ERK/F A-1 signaling that can stimulate the constitutive high level expression of the immune checkpoint, PD-L1. The interaction of PD-L1 expressed on the surface of tumor cells with PD-1 receptor on T cells ca trigger the activation of the PD-Ll/PD- 1 immune checkpoint pathway in T cells which can lead to the suppression of tumor cell-specific T eel! cytotoxicity, m certain embodiments _; , the targeted inhibition of K AS signaling, e.g., by RAS-specific asymmetric interfering RNAs, can down-regulate PD-L1 gene expression and restore the sensitivity of cancer ceils expressing an oncogenic 5 KRAS to killing by antigen-specific cytotoxic T cells (see Example 4). in certain embodiments, tire inhibition of oncogenic KRAS signaling in cancer cells can inhibit MEK/ERK-dependenl phosphorylation, in certain embodiments, the inhibition of oncogenic KRAS signaling in cancer cells can inhibit the accumulation of FRA.-1 protein, In certain embodiments, FRA-1 protein is a transcription factor required for the activation of the AP-1 responsive enhancer 10 within the first intron of the PD-L1 gene, Inhibition of KRAS signaling may therefore provide a novel approach to sensitizing cancer cells resistant to immunotherapies, such as immune checkpoint therapies, as well as improving the efficacy of known anticancer therapeutics.

In certain embodiments, the modulator of ICR AS signaling may comprise, for example, an inhibitor that reduces or prevents KRAS-mediated cell signaling in tumor cells, in certain I S embodiments, the tumor cell expresses an oncogenic KRAS, in certain embodiments, the inhibitor can directly target both wild type and/or oncogenic KR AS by inhibiting the expression of KRAS in tumor cells. In certain embodiments, the inhibitor of KRAS can be, for example, an RNA interfering agent.

An "RNA interfering agent," as used herein, is defined as any agent that can inhibit the 0 expression of a target gene by RNA. interference (RNAi).

Solely to provide a background and not to limit the scope of this disclosure, "RNA interference (RNAi)" is an evoiutionally conserved process whereby the expression or introduction of an RNA comprising a sequence that is identical to or highly similar to a target gene sequence can result in the sequence-specific degradation or specific post-transcriptional 5 gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene, thereby iiimbiting target gene expression, in nature, RNAi is initiated by the dsRNA-specific endonuclease, Dicer, a member of RNase III rihonuclease family. Dicer cleaves long, double- stranded RNA (dsRNA), pre-microRN A (miRNA), and short hairpin RNA (shRNA) into short double-stranded RNA fragments called small interfering RNAs (siRNA) of about 20-25 0 nucleotides in length, usually with a two-base overhang on the 3' end. Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component, argonauts, is an endonuclease capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.

RNAi can be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, having a guide RNA thai targets a specific expressed gene sequence. Examples of such RNA interfering agents include, but are not limited to, small non-coding RNAs such as antisense oligonucleotides, shRNAs (e.g. as disclosed in U.S. Patent No. 7,750, 144, which is incorporated by reference herein in its entirety for any purpose), siRNAs (e.g. as disclosed in U. S. Patent No, 7,056,704 and 9,260,470, which are incorporated by reference herein in their entireties for any purpose), a microRNA or a mature microRNA molecule or a pre-microRNA molecule or a primary microRNA molecule, or a variant thereof, (e.g. as disclosed in U. S. Patent No. 8,609,831, which, is incorporated by reference herein in its entirety for any purpose), gapraers (e.g. as disclosed in U. S. Patent No. 6,107,094, which is incorporated by reference herein in its entirety for any purpose), IncRNA (e.g. as disclosed in the International Publication No. WO2012018881, which is incoiporated by reference herein in its entirety for any purpose), a piRNA (piwiR A) molecule (e.g. as disclosed in the International Publication No. WO2008109142, which is incorporated by reference herein in its entirety for any purpose), a triplex oligonucleotide (e.g. as disclosed in U.S. Patent. No. 5,693,773, which is incorporated by reference herein in its entirety for any purpose), or ribozym.es (e.g. as disclosed in U.S. Patent No. 5,225,347, which is incorporated by reference herein in its entirety for any purpose).

Exemplary chemical modifications of RNA interfering agents are disclosed in Dar et al siRNAmod: A database of experimentally validated chemically modified siRNAs. Sci. Rep. (2016) 6, 20031.

The present disclosure provides compositions comprising a class of short double stranded RNA. interfering agents, called asymmetrical interfering RNAs (aiRNA), that can induce potent gene silencing in mammalian cells, aiRNA. is described, for example, in PCX Publications WO 2009/029688 and WO 2009/029690, the contents of which are hereby incorporated by reference in their entireties for any purpose. In one aspect, this class of RNAi- inducers is characterized in the length asymmetry of the two RNA strands. This structural design can not only be functionally potent in effecting gene silencing but offer several advantages over the current state-of-art siRNAs, Among the advantages, aiRNA can have RNA duplex structure of much shorter length than the other siRNA, which should reduce the cost of synthesis and abrogate/reduce the length-dependent triggering of nonspecific interferon- like responses. In addition, the asymmetry of the aiRNA structure abrogates and/or otherwise reduces the sense-strand ^' mediated off-target effects. aiRNA is therefore, in certain embodiments, more efficacious, more potent, with a more rapid-onset, and more durable ai inducing gene silencing than any of the other RNA interfering agents.

In certain embodiments, aiRNAs disclosed herein each comprises a first strand with a length from 18-23 nucleotides (nt) and a second strand with & length from 12-17 nucleotides. In certain embodiments, the second strand is substantially complementary to the first strand. In certain embodiments, the second strand forms a double-stranded region with the first strand, in certain embodiments, the first strand has a 3 -overhang from 1-9 nucleotides. In certain embodiments, the first strand has a 5 -overhang from 0-8 nucleotides. In certain embodiments, the aiRNA is capable of effecting at silencing KRAS signaling in a eukaryotic cell.

In certain embodiments, the first strand is 18, 19, 20, 22, or 23 nucleotides long.

In certain embodiments, the second strand is 12, 13, 14, 15, 16, or 17 nucleotides long. In certain embodiments, the 3 -overhang is greater than 0 nucleotides in length. In certain embodiments, the first strand comprises a sequence being substantially complementary to a target KRAS mRNA sequence. In certain embodiments, the first strand comprises a sequence being at least 70 percent complementary to a target mRNA sequence.

In certain embodiments, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the firs strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20-100 nt longer than the second strand. In a further embodiment, the first, strand is 2-12 rit longer than the second strand. In an even further embodiment, the first strand is 3-10 nt longer than the second strand.

In certain embodiments, the first strand, or the long strand, has a length of 5-100 nt, or preferably 10-30 or 12-30 nt, or more preferably 15-28 nt. In one embodiment, the first strand is 21 nucleotides in length. In some embodiments, the second strand, or the short strand, has a length of 3-30 nt, or preferably 3-29 nt or 10-26 nt, or more preferably 12-26 nt. In some embodiments, the second strand has a length of .15 nucleotides.

In certain embodiments, the double-stranded region has a length of 3-98 base pairs (bp).

In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-19 bp, The length of the double- stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.

In certain embodiments, aiRNAs disclosed herein comprise a duplex RNA comprising a first strand, wherein the 5 '-terminal and 3 '-terminal nucleotides of the first strand are from 16 to 21 nucleotides apart, and a second strand, wherein the 5 '-terminal and 3 '-terminal nucleotides of the second strand are from 10 to 17 nucleotides apart, wherein the 5 '-terminal nucleotide of the second strand is complementary to a nucleotide of the first strand other than its 3 '-terminal nucleotide, wherein the 3 '-terminal nucleotide of the second strand is complementary to a nucleotide of the first, strand,

In certain embodiments,the penultimate nucleotide from the 3 '-terminal nucleotide of the first strand is not dT.

In certain embodiments, the duplex RNA is more effective at silencing an expressed nucleotide sequence of a target gene than a corresponding 21-mer siRNA duplex targeting the same expressed nucleotide sequence of the target gene.

In certain embodiments, the second strand is from Ito 9 nucleotides shorter than the first strand. In certain embodiments, the 5 '-terminal and 3 '-terminal nucleotides of the first strand are 19 nucleotides apart. In certain embodiments, the 5 '-terminal and 3 '-terminal nucleotides of the second strand are 13 nucleotides apart, in certain embodiments, the 5'- terminal and 3 '-terminal nucleotides of the first strand are 19 nucleotides apart and the 5'- terminal and 3 '-terminal nucleotides of the second strand are 13 nucleotides apart.

In certain embodiments, the 3 '-terminal nucleotide of the second strand is complementary to a nucleotide of the first strand that is within 3 nucleotides from the 5'- terminal nucleotide of the first strand, in certain embodiments, the 3' -terminal nucleotide of the second strand i s complementary to the 5 '-terminal nucleotide of the first strand.

In certain embodiments, the S'-terminal nucleotide of the second strand is complementary to a nucleotide of the first strand that is 1-4 nucleotides from the S'-terminal nucleotide of the first strand. In certain embodiments, the 5 '-terminal nucleotide of the second strand is complementary to a nucleotide of the first strand that is 1-2 nucleotides from the 3'- terminal nucleotide of the first, strand. In certain embodiments, a least one nucleotide of the sequence of 5' overhang is selected from the group consisting of A, U, and dT.

In certain embodiments, the GC content of the double stranded region is 20%-70%.

In certain embodiments, the first strand has a length from 19-22 nucleotides.

In certain embodiments, the first strand has a length of 21 nucleotides. In certain embodiments, the second strand has a length of 14-16 nucleotides.

In certain embodiments, the first strand has a lengtli of 21 nucleotides, and the second strand has a lengtli of 15 nucleotides. In certain embodiments, the first strand has a 3'-overhang of 2-4 nucleotides. In certain embodiments, the first strand has a 3 '-overhang of 3 nucleotides. In certain embodiments, the first strand has a 5'-overhang of 3 nucleotides,

In certain embodiments, a duplex RNA molecule of the present disclosure, e.g. aiRNA, can contain at least one modified nucleotide or its analogue. In certain embodiments, the at least one modified nucleotide or its analogue can be a sugar-, backbone-, and/or base- modified ribonucleotide. In certain embodiments, the backbone-modified ribonucl eoti de can have a modification in a phosphodiester linkage with another ribonucleotide. In certain embodiments, the phosphodiester linkage is modified to include at least one of a nitrogen or a sulphur heteroatom. In another embodiment, the modified nucleotide or its analogue can be a backbone- modified ribonucleotide containing a phosphothioate group. In certain embodiments, the at least one modified nucleotide or its analogue is an unusual base or a modified base. In certain embodiments, the at least one modified nucleotide or its analogue comprises inosine, or a tritylated base. In certain embodiments, the modified nucleotide or its analogue is a sugar- modified ribonucleotide, wherein the 2 -OH group is replaced by a group selected from H, OR, R, halo, SH, SR. NH2, NHR, NR2, or CN, wherein each R is independently Ci-Ce alkyl, alkenyl or aikynyl, and halo is F, CI, Br, or I.

In certain embodiments, the first strand comprises at least one deoxynueleotide. In certain embodiments, the at least one deoxynueleotide is in one or more regions chosen from 3 '-overhang, 5 ~overhang, or double-stranded region. In another embodiment, the second strand comprises at least one deoxynueleotide.

In certain embodiments, a modulator of KRAS signaling comprises, for example, an aiRNA In certain embodiments, a modulator of KRAS signaling comprises a RAS-re!ated aiRNA In certain embodiments, a modulator of KRAS signaling comprises a KRAS-specific aiRNA.

In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more KRAS-specific aiRNAs that target one or more KRAS activating mutations as defined herein. In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more KRAS-specific aiRNAs that target KRAS transcripts having one or more KRAS activating mutations at amino acid residues G12, G13, S 17, P34, and/or Q61.

In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more KRAS-specific aiRNAs that target both wild type and oncogenic KRAS RNA sequences.

Wild type KRAS-specific aiRNAs are described, for example, in the PCT International Application WO2015139044, the content of which is hereby incorporated by reference in its entirety for any purpose.

Exemplasy KRAS aiRNA molecules, comprising a sense strand sequence, an arstisense strand sequence or a combination of a sense strand sequence and antisense strand sequence are also shown in TABLE 4.

TABLE 4

Antisense aiRNA Sense Strand Sense Strand Antisense Strand

Strand

IX) HO: Sequence SEQ ID NO: Sequence

SEQ ID NO:

1 C GUI J AU AGCG U AUO 320 AAUAAUAAGCUAUAACUGGCC 638

2 CCU AGUAGGAAAi JAA 321 AAUUUAUUUCCUACUAGGACC ^: ; 639

3 AG A CXX; AGU AU G AAA. 322 AAAUUU CAU ACU GGGUCIJ GGG 64 0

4 GUGCCAAGA.CAUimA 323 AAAUU AAU GUCUU GG GAG AC G 641

CUCUuClATCCAUAUU 324 AAUAAU U GGAAGAAGAQUCC 642

6 AAU GGAAACU. AUG. AU 325 AAU AUAAU AGUT JGCC'AUUGCC 643

7 GUUGAUUACUUCUUA .326 AAAUAAGAAGUAAU CAACUGC 644

8 CUUAGCAAGAAGIAJA 327 AAAUAA JUaJUGCTJAAGUCC 645

9 CAGCAGAAUCUGUAA 323 : AAUUGACAGAGIJGGGCUGA.GC 545

1 0 CUUGCCACGGCGAiJU 329 AAuAAuAGCAGUGGAAAGGAG 547

1 1 GGU GU GAAA.CAAA.U U 330 AAUAAuuuGLJuUCACACCAAC 643 ^™ "

1 2 TJA C GCU AAUU C AG A 331 AAUUCUGAAUUAGCUGUATJCG 64 9

13 CGAAUUCAGAAUCAU 33.2 AAAAU GAUU CU GAAUUAGCU G 63 0

1 AAUUCAGAAUCAIJUU 333 AAAAAAUGAGXJaJGAAUGAGC _.... 65 _. 1

1 AUAUG AUG CAACAAU 334 AAUAUUaJUGGAiJCAiiAlJUCG 652

_.. .... _.. 1 6 GGU AACAGUAAGACA 335 AAAUGUAUUACuXXiUACOAGG 65 3

17 CAGG ACGG A GCAAGA 336 A&UUC XJGCUAAGUCCUGAGC 65 4

1 8 UGUGCCAGCUCUAUA 337 AAUU AUAGAGCU GGGACAGAG 655 aiRNA Antlsense

Sense Strand Sense Strand Antisen.se Strand

ID NO: Strand

Sequence SEQ ID NO: Sequence

SEQ ID NO:

1 9 CU AUAUUU ACAU GCU 538 AAUAGCAUGUAAAU AU AGCCC 65 6

20 GUCUCUUGGAU AUG C 339 AAAGAAUAUCCAAGAGACAGG 6 7

21 GGGCU GU CU UU GU GU 340 AAU AGACAAAGAAAGCCCUC C 65 8

GAUAUCUCCAG G AAG 341 AAACUUCAUGGAGAU ^' AUCCAC 65 3

23 CAUUAUAGAGAACAA 342 AAUlATGUUCUCUAUAAUGGUG 660

34 CUGUAAUAUCUUACU 343 AAUAGUAAGAUAUJACAGACC 661

25 U GACGAUACAGCUAA 344 AAA UUA GCU6UAUCGUCAAGG 662

26 GAUGCUUUGAAC ^' A.UC 345 AAAGA UGUUCAAAGCAUCAGC 663

27 CCCUGA.UGAAUGUAA. 3 46 AAUUUACAUUCAUCAGGGAIJG 664

28 vj AUUUGCCAllAAAUA 347 AA.UUAUUUAUGGCAAAUACAC 665

29 ■ &0?v &CUAAA.U C 348 AAU GAUUGAGU AUU AUU U AU G 666

30 AAUCAUUUGAAGAUA 3 49 AAAUAGCUUGAAAGGAUUU G 667 1 GAUAGU CACCAUGAIJ 35 0 AAU AUAAU GGU GAAI AUCT A J G 663

W32 UUUAACAAAAGAUX!U 35 1 AACAAATJCUUUUGUUAAACCA 669

S33 CCUAAU UAUACAU 352 AACUAUGU AU AU AU' JAG GACA 670

W34. AAAAGAAACUGAAU 353 AAGUAUUCAGUUUCUUUUUCA 67

^{" "} 35 ^" AGCACAAUCUGUAAA. 35 4 AAAUUUACAGAUUGUGCUGAG 672

_... 36 CUUUCAUAGUAUAAC 355 AAA.GUllAUAGiJA.UGAAA.GAGC 673

37 CUAGUGUGG JCUGUA 3 6 A AUUA GAGA CCACACUAGCA. C 67

38 GU GUGGUCUGU AAUA 357 AAAUAUUACAGACCACACUAG 675

39 GACGUAGAUUGU UC .35 8 AAU GAUACAAU AUACGU CUGC 676

40 CCCAAGUAGGCAUIJC 359 AAAGAAUGC ; ACUGGGGAAC 677 1 GGAAUAUGAUCCAAC 360 AAUGJUGGAUCAUAUUCGUCC 678 2 GCAAGUAGT TAAUUG A 361 A UCA UU CUACi Π 5GC( Jt JC 673

43 UCCUGAUGAUGAUUC 362 AAAGAAUCAUCAUCA GGAA.G C 680

44 GACCUCAAGUGAUUC 363 AAUGAAUCACUUGAGGUCAGG 681. 45 UCCCUA.CCUGCCACA 3 64 AAAUGGGGAAGGUAGGGAGGC «82 6 ^■ AUUUCCUUUUCACAU 365 AAAUGUGAA AGG AAAU GGC 633

W 7 GlJAtXAUUGUAUCAlJG 366 AAAAAU GAUACAAU AUACGU C 684

W4 S CA KJUCCUUUGCACA 367 A A AU Gl ) GAAAAGGAAAU G GGC 685

W49 GGAGAAUG CUAGAAA ^{" "} 368 AAAUUU CUAGAAUU CUGCCGC 686 0 COC G U GGCA U C 36 AAGAA!J GCCU AOJ !J GGGAAC 687

5.1 GGAAUAUGAUCCAAC 370 AAUGUUGGAUCAUAUUCGUCC 688

52 ^' GCAAGUAGUAAUUGA 371 AAAUCAAJUACUACIA/GC AJG 689

5 3 UCCUGAUGAUGAUUC 372 AA GAAUCAUCAUCAGGAAGC 6 0

^{" "} 5 4 CUGUACUACUCCUAA 373 AAA.GUAGGA.GUAGU ACAGUU C ^{' " "} 691

55 GACCUCAAGUGAUUC 3 '74 A.A1JGAAUCACUUGA.GGUCAGG 692

5 6 CUU AGGUAGUGCGAG 375 AAACIJAGCACUACCUAAGGAC 693

57 U CAGACUGCUCUUU G 376 AAU GAAAG GCAGU CUGACAC 694

58 UGCUCUUUCAUAGUA :. 377 AAAUACUAU GAAAGAGCAGJ C 695

5 9 G AUG AAUG UAAA GU U 378 AAU AACUUU AC AUT J C A J CAGG 696

60 GUGUGAUCCAUAUUU 37 AAUAAAUAUGGAUCAGACUUG 697

; 6 ^: · CAGGAAAGAUGCUAA. 380 AAUUUACCAUCUUUGCUCAUC 6 8

6 · GAGGUGAAGUUUAUA _. 331 699

63 GGGUGUUAAGACUU 382 AAUAAGUCUUAACACCCUACC 700

6 GGCAUCAGGUCCU.AU 383 AAUAUAGGACAUGAUGCCUAG 701 Antisense alRNA Sense Strand Sense Strand Antisense Strand

Strand ID NO ; Sequence SEQ ID NO: Sequence

SEQ ID NO;

I I S; ACAGGAAGCAA.GIJAG 40 0 AAACUACUUGCUUCCUSUAGG ^" 748

1 1 3 CAGGAA CAAGU AGU 431 AAU CUACUUGCUUCCUGUAG 74 9

1 1 4 GAAGCAAGU AGU AAU 432 AAAJ^UUACUACUUGCIJUCCUG 75 0

1 1 5 A G G G ϋ AAU U G AU G G * 4 33 AAU CCAUCAAUU GU ACOJUG C 7 1

1 .1 6 GUAAUUGAUGGAGAA 434 A AG GGCGCCAU CAAO ^' U A CGA C 752

1 1 7 GGAGAAACCUGUCUC 435 AAAGAGACAGGOiO!GCuCCAGG 753

1. 1 8 AAACCUGUCUCUUGG 436 AAU CCAAGAGA.CAGGUUU CU C 75 4

1 1 9 ACCUGGC07CUGGGAU 437 AAUA CCAA.GAGA.CA.GGUUUC 75 ^: 5 ^"

1 20 UCGiuGGOOUAUUCUCG 438 AAU CGA.GAAUA1 J CCAAGAGA.C 75 6

1 21 ^■ 1UG GA GAGU CU ^' CGAC 4 30 AAUGUCGAGAAUAUCGAAGAG 757

1 22 GGAUAUUCUCGACAC 4 40 AAU GUGUCGAGAAU AO!CCAAG 758

1 23 UAGGCGGGAGACAGC 441 AAU GGU GU GUCGAGAAU AOiCC 75 9

1 24 GA CA.CA.GCAGGU C A A 442 AACOnJGACaJGCUGUGUCGAG 760

125 GGUCAAGAGGAGUAC 443 AAU GO? A J C C J CO" J G AC C- GG C 761 26 GAGGAGUACAGUGCA 444 AAUGGCACUSJAaiCCUCGTJG 762 ^'

127 GAGUACAGUGCAAuG 4 45 AAGCA GGGGA CU GO? GG CCGC 763

128 UGAGGGACCAGUAC 4 46 AAUGUACUGGI ^■ GCCUCAUUG 764

1 29 CCAGOiA.GA.GGA.GGAC 447 AAAGU C G CAU GUACU GGU C C 765

130 AGGGCGGGCUUUGUG 4 48 AAAC ACAAAGAAAGCCCU GCC 766

1 31 GGCUUUC7UUUGUGUA 4 49 AAAUACACAAAGAAAGCCaJC 767

1 32 CUUU CU UU GGG ^: J AU U 45 0 'AAAAAUACACAAAGAAAGCCC 768

1 33 UGUGOOAUUUGCCAO!A 451 AAO!UAO) GGCAAAUACAGAAAG 769

1 34 GGGGCCAU AAAUAA 402 AAAUU AUUU AU GGGAAAUAC 7 ^' 70

1 35 AAUCATJOAJGAAGAU.A 45 2 AAAUAUCUUCAAAUGAUUUAG 77 !

136 G AUAOAJCA CCAIRIAU 45 4 AAIJAUAAUGGUGAA JAUGIAJC 772

137 UUAGAGAGAAC.AAAU 455 AAAGOUUUGUUCUCUAUAAUGG 773

1 36 UAGAGAGAACAAAUU 45 6 AAU AAUUU GUU CUGU AU AA J G 77

.1 39 AA.CAA.AUU AAAAGAG 4.57 AAACllCIAlUUAAUUUGUIj ^' CUC 775

1 0 CAAAUUAAAAGAGiJG 4 5 8 AAUAACacaOiJUAAOAATGGGC 776

1 41 UUAAGGACUGUGAAG 45 9 A AO! CG G GA G AGO? CC07UAA.CU C ^' 777

1 2 A AG G A C fj C !J G AA GAD 460 AACAUCGGCAGAGGCCUGAAC 773

1 43 CG ^' CGGAAGAGGU CC 461 ^:' AAA GGGAGA UC ^' U UCA GA GGCC '7 '70

1 44 UCUGAAGAuG!uACCU 4 62 AAU AGGUACAUCIAJ CAGAGU C 730

1 5 A A GA.GGUACGU AU GG 4 63 AAACCAUAGGUACAUCUUCAG 781

1 4 6 AUGGGCCUAGU GGA 4 64 AAUUCCUACUAGGACCAU AGG 762

1 7 U GGU CCU AGO!AGGAA 4 65 783

1 8 CU AGU AGO AAAOAAA 466 AAAOATUAOOTJCCUAC'JAGGAC 784

1 49 GGAAAUA-A-AUGUGiiU 467 AAAAGCACAiAJGAUGGCCUAC 735

1 50 U JAGAACAGuAGAC 463 AAUGGCUACGGOIGCGAGAAGG 766

15 1 GAACAGOJAGACACAA. 469 AAUGGGGGUCUACUGGUCUAG 787

152 CAGGA GACACAAAAC 470 A A U GUUUU GUGU GGA.CG GUUC 766

153 AACAGGCUCAGGACU 471 A AAAGU CCU GA GCCU JIR A J G 783

1 54 GCUCAGGACGUAGCA 472 AAUUGCUAAGU CCU GAG GGU G 700

1 5 5 GCAGGA'G iU AGCAAG ''57 AAU GUU GCUAAGUCCUGAGCC 7

1 5 6 GiACuUAGCAAGAAGG : 474 AAAACUUCUUGC AAGUCCOG 792

AGCAAGAAGGGAuGG 475 AAUCCAUAACUO'CUUGCOOOAG 793 Antlsense aiftra Sense Strand Sense Strand Antisense Strand

Strand ID NO: Sequence SEQ ID NO: Sequence

SEQ ID NO:

158 AGAAGUUAUGGAAUU 476 AAGAAUU CCAU AACGUCIJI!G C 79

159 G AGAUGGAADU C CUU 477 AAAAA G GAA UU C CA GA ACGLJ C 79

160 AGGGAGIUUCCUUUUA 476 AAAUAAAAGGAAWCCAUAAC 796

16.1 AITUCCUUUUAUUGAA 479 AAGGG¾ «A&A-ASGAAGGGG 797

162 ACAliCAGCAAAGACA 430 AAUUGUCUUUGCUGAUGGUUC 793

.163 CAGCAA AG ACA A GA C 431 AAUGuCUuGUCUUUG JGAUG 799

164 AAGACAAGACAGGGU 482 AACACCCUGUCUUGUCUUUGC 800

165 AGACAAGACAGGGUG 483 AAACACCCU GU CUU GO CUT JU G 80.1

166 AAGACA.GGGUG lUSA 404 A AUCAACACCCUGUCUUGUC 002

16 GACAGGGUGIJOGAUG 485 AAUCAUCAACACCajffjCUUG 803

168 GGUGUUGAUGAUGCC 486 AAA G G G AU ^' GAG ^' GA A CA G GCU G 304

169 GGUGAUGAUGCCUA!C 487 A A GA GGCAGGAG GAA C CC 305

170 UUGAL!GAUGCCUUCU 483 AAUAGAAGGCAUCAUCAACAC 806

171 GAUGAUGCC rtJCnjAU 439 AAUAUAGAAGGCAU CAU CAAC 307

172 AUGC JGCUALJACAU 430 AAAAUGUAUAGAAGGCA JCAU 808

173 GCC ^' UuCLJAuACA AJA 49.1 AACUAAUGUAUAGAAGGCAG ^' C 809

174 ^: UUCtiAUACAUUAGUU 492 AAGAACUAAUGUAU AGAAGGC 8 0

1 S CaAUACAUU AGUU ^' CG 493 AAGCGAACUAAUGUAUAGAAG 811

176 - UACAUUAGUUCGAGA 494 AAUUCUCG AACUAAUGUAUAG 812

177 GAAAUUCGAAAACAU 495 ^" 813

178 AAAUUCGAAAACA.U ^' A. 496 AAGilAUGUUUUCGRAUUUCUC 81

179 AAACAUAAAGAAAAG 497 AAIACUUUUCUUUAUGUUUUCG 815

180 AAC U GAAAAGA 498 AAAUCUUUUCUUUAUGUUUI C 816

181 AAAGAAAAGAUGAGC 499 AAUGCUCAUCUUGUCUUU ^' AUG 817

;! 82 AAGAGGGUAAAAAGA 500 AAUUCT AiGXTACCAUCU ^' GUGC ^' 618

183 A GAUGGUAAAAAG A 501 AACGGCGGGGilACCAGCGUGG 813 _.

164 ^" ; ^' 502 ^" AAUUWCUUCUUUUUACCAUC 820

185 AAAAAGAAGAAAAAG ■503 AAUCUlilJUUCUUCUUUUUACC 321

186 AAAAGAAGAAAAAGA 50 AAIAJCIAAAJUCUUCUUUUUAC 822

187 GAAAAAGAAGUCAAA 505 AACUUU GACGU CUUUUU CUT j G 823

188 AAAGAAGU CAAAGAC 506 AAU GU CUUU GACUU CUUUGUC 824

.189 GGCAAAGACAAAGU G 507 AAACACU ^' U UGG CU U U GACUU C 825

.1 0 AAAGACAAAGUGUGU 508 AAUACACACGUiJGO'CUUUGAC 826

191 AGACAAAGU GU G LJAA 509 AAAl;UA.CACACUUUGUCUl)lTG 827 2 A A AGU G UG ^' U A A i. · UA U 510 _.. A A GAG AAUUACACACUUU GIJ C _. 323

1.93 AGGGGGUAAGGAUGU 5 1 AAU AC AUAAT JU ACAC ACUUU G 829

U ^' U TGT ACU ^' UUUUUCU 512 AAAAGAAAAAAGUACAAAUUa 830

195 ClAJUGUUCUUAAGGC 13 AAUGCCITGAAGAAAAAAGUAC 831

.196 UlTUl JCUUAAGGC AU A 51 AAGUAUGCC171AAGAAAAAAG 332

197 AAGGCAUACUAGUAC 15 AAG GG ACU AGU AUGCCUU ^' AAG 833

198 ^"" ACU AG AC A GG G U 5 6 A AT J A C GA C U UGU A CUAGUAU G 834

199 GUACAAGG GG ^' U AAUU 517 AAAAAGuAGCACA:gA7UAC:UAG 835

200 CAAGUGGUAAUUI JU 518 AAGAAAAAUUACCACUUGUAC 836

201 GU AAUUUU U G-G A CAU 519 AAAAGGU.ACAAAAAUUACCAC 837

202 AuTIUUUGUACAUUAC 520 AAU GU AAU GUACAAAAAUUAC 838

203 C UU C ACU AAUU A 521 AAAU AAUU GAGUGU AU UA C 839 Antisense

Sense S and Sense Steand Sntisease Strand

Strand

ID NO : Sequence SEQ ID HO: Sequence

SEQ ID NO:

296 UU O" AJCUU A AG AA A. 61 4 AAUUUUCUUAAGAAACAAAAG 932

29" AAGAAAACAAACUCU AAAAGAGUUUGUUGUCUUAAG 933

29 S AAGAAAaJCUUUUUU 61 AAG A A A A A A GAG UU UGU UUUC 934

299 U G AAGU GAAAAAGUU 617 AAAA ACiAJS. T u U CA.CU 1? GA UUG 936

300 GUGAAAAAGOnjIAJAG 61 8 A» ^" υ GUA&AACUUUUUCACUU C 936

301 UG ACACU GGUUAAA 61 9 AAAUUUAACCAGUGUUAAGAG 937

302 AACACO'CGUGAAAIJU 620 AAUAAUUUAACCAGO' GGGAAG S3 S

303 AAAOPOAACAUUGCAG 62.1 AAUAUGCAAUGirUAAirUUAAC 939

304 UAAACACUUUUCAAG 622 AAAO. JUGA A A A G UG UiATA UGG 940

3 OS ^{" "'} rj CGU uUUGAIlAAAUU 6.23 A A A A At AAiAUCA A A A GG A UUG 94 1

306 A CUUAGGUDQj A G AU 624 AAGAUCUAGAACCUAAGUCAC ^" 942

30? UUAGGA a CU GAUUOS ^' 625 AAAAAAUCAGAGUCCUAAAAG 943

309 CACUUACUAUCCAiJu 626 AAAAAU GGAUAGUAAGG G AG G 944 ^"

309 UUACUAOTCCAiJLrUCU 627 AAAAGAAAUGGAUAGU AAGU G 4 ¾

3 i 0 ACL' AOi ^' CCAGUUCUUC 626 AAU GAAGAAAU GGAG AGG AAG 946

3 ; I GCCAOrUGCUUCAUGU ⁶² .A AAAACAG ^' GAAGAAAUGGAUAG 947

^" 31 2 ^" UUUGUGCAUGUUAAA 630 AAU O!U O!AACAG G A A GA A A UGG 948

313 GUCAUCU C A A ACG CU 631 ^' Λ ·ν· ··-· ··· : . :· ··· :··.· . - . GGGG 949 .

31 GL.iC.AAAa; CGI J GU" 632 AAAAACUAAGAGUUUGAGAUG 95 0 ■

3 1. 5 AAACUCUIJAGUUUUU 633 AAAA A CUAAGAGUUU GAG 5 1

31 6 U GU AAG GU A UA U U C C 63 AAUGGAAGAUAAAUU C UAG 952

3 7 AAGQAUACAca Aaa 63,9 AAAAAUAAGUGUAUCajUAUG 953

31 8 CAAUCliGUAAAUUUU 636 AAAAAAAUUuACAGAOrUGUGC 95 4

3 19 UGUUACACCAUCUUC 637 A UGAAGAUGGUGGAACAUAG 95 5

In certain embodimenis, the RNA duplex molecule (aiRNA) comprises a sense strand sequence selected from the group consisting of SEQ ED NOs: 320-637. In certain embodiments, the RNA duplex molecule (aiRNA) comprises an antisense strand sequence selected from the gxoup consisting of SEQ ID NOs: 638-955, In certain embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence selected from the group consisting of SEQ ID NOs: 320-637 and its corresponding complimentary antisense strand sequence as depicted in TABLE 4 above.

In certain embodiments, a KRAS RNA duplex molecule (aiRNA) comprises a sense strand sequence that is at least e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 320-637, In certain embodiments, the RNA duplex molecule (aiRNA) comprises an antisense strand sequence that is at least, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% _* or more identical to a sequence selected from the group consisting of SEQ ID NOs: 638-955. In certain embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence thai is at least, e.g., 50%, 60%, 70%. 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 320-637 and a substantially complimentary antisense strand sequence that independently is at least, e.g., 50%, 60%, 70%, 75%, 80% 85%, 90%, 95% or more identical to the corresponding antisense strand as depleted in TABLE 4 above.

In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more aiRNAs that target one or more effectors of the RAS/RAF/MEK/ERK/FRA signal transduction pathway. In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more aiRNAs that target wildtype or mutant RAS GEFs. In certain embodiments,, a modulator of KRAS signaling comprises, for example, one or more aiRNAs that target wildtype or mutant A-RAF, B-RAF and/or C-RAF/RAF-I. In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more aiRNAs that target wildtype or mutant MEK 1/2. In certain embodiments, a modulator of KRAS signaling comprises, for example, one or more aiRNAs that target wildtype or mutant ERK 1/2. In certain embodiments, a. modulator of KRAS signaling comprises, for example, one or more aiRNAs that target wildtype or mutant. FRA-1.

The present disclosure also provides a method of preparing a duplex RNA molecule of the disclosure. In certain embodiments, the method comprises synthesizing the first strand and the second strand. In certain embodiments, the method comprises combining the synthesized strands under annealing conditions, in certain embodiments, the duplex RNA molecule is capable of effecting KRAS silencing. In certain embodiments, the RNA strands are chemically synthesized, or biologically synthesized. In certain embodiments, the first strand and the second strand are synthesized separately or simultaneously. In certain embodiments, the method comprises introducing at least one modified nucleotide or its analogue into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step.

In certain embodiments, the present disclosure provides an expression vector for the generation of the duplex RNA in vivo. In certain embodiments, the vector comprises a nucleic acid or nucleic acids encoding the duplex RNA molecule operably linked to at least one expression-control sequence, e.g. a U6 snRNA promoter or an inducible promoter, in certain embodiments, the vector comprises a first nucleic acid encoding the first strand operably linked to a first expression- control sequence. In certain embodiments, the vector comprises a second nucleic acid encoding the second strand operably linked to a second expression-control sequence, in another embodiment, the vector is a viral, eukaryotic, or bacterial expression vector. The present disclosure also provides a cell comprising the expression vector. In another embodiment, the cell comprises a duplex aiRNA molecule, e. g. a KRAS-specific aiRNA

The present disclosure further provides a method of modulating KRAS signaling. In certain embodiments, the method comprises administering an. asymmetrical duplex RNA. molecule of the disclosure, in certain embodiments, the method comprises administering an asymmetrical duplex RNA molecule of the disclosure in an amount effective to silence KRAS expression or otherwise reduce KRAS expression. ID certain embodiments, the method is in a cell or an organism. In certain embodiments, the method comprises contacting said cell or organism with an asymmetrical duplex RNA molecule of the disclosure, for example, under conditions wherein selective KRAS gene silencing can occur, in certain embodiments, the method comprises mediating a selective KRAS gene silencing affected by the duplex RNA molecul e towards KR AS or nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA. In certain embodiments, said contacting step comprises introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective KRAS silencing can occur. In certain embodiments, the introducing step is chosen from transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical administration, or regional administration.

In certain embodiments, the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is chosen from a pharmaceutical carrier, a positive- charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid.

In certain embodiments, a modulator of KRAS signaling may comprise one or more inhibitors that target the KRAS activation of the RAF/MEK ERK ^' FR A- 1 signaling pathway. In. certain embodiments, the method comprises administering an asymmetrical duplex RNA molecule of the disclosure in an amount effective in reducing (e.g., silencing) the KRAS activation of the RAF/MEK/ERK FRA- 1 signaling pathway.

In certain embodiments, a modulator of KRAS signaling may comprise one or more inhibitors that target the oncogenic KRAS activation of the RAF MEK/ERK/FRA~ 1 signaling pathway. In certain embodiments, the method comprises administering an asymmetrical duplex RNA molecule of the disclosure in an amount effective in reducing (e.g., silencing) the oncogenic KRAS activation of the RAF/MEK/ERK/F A- 1 signaling pathway.

In certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of RAF activity (e.g. A-RAF, B-RAF, RAF1). in certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of mitogen-activated kinase activity (e.g., MEKl, MEK2), an inhibitor of ER activity (e.g., ERKl, ERK2). In certain embodiments, a modulator of KRAS signaling may comprise inhibitor of FRA-1 activity or any combination thereof. The skilled artisan will understand, however, that other downstream targets may also be suitable targets for an inhibitor of KRAS signaling, e.g. oncogenic KRAS signaling, and such targets are also encompassed by the present disclosure.

In certain embodiments, a modulator of KRAS signaling may comprise, for example, an RNA. interfering agent that inhibits one or more downstream effectors of KRAS signaling.

In certain embodiments, a modulator of KRAS signaling m )' comprise an inhibitor of A-RAF, B-RAF, and/or C-RAF, for example, an A-RAF, B-RAF, and/or G-RAF RNA interfering agent (e.g., see WO2009143372, the content of which is hereby incorporated by reference in its entirety for any purpose).

In certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of MEK activity, for example, a MEK RNA interfering agent (e.g., see published Patent Application No. 2009/0239936, the content of which is hereby incorporated by reference in its entirety for any purpose). .

In certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of ERK activity, for example, a ERK RNA interfering agent (e.g., see published Patent Application No. 2009/0239936, the content of which is hereby incorporated by reference in its entirety for any purpose).

in certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of

FRA-1 activity, for example, a FRA-1 RNA interfering agent (e.g. see U.S. Patent No. 6,124,133, the content of which is hereby incorporated by reference in its entirety for any purpose).

In certain embodiments, a modulator of KRAS signaling may comprise an antigen- binding protein or fragment thereof (e.g. a recombinant antigen-binding protein) that targets RAS protein, e.g. an oncogenic KRAS (e.g. see U.S. Patent Nos. 4,820,631; 5,084,380, 4,898,932; 5,081,230; 5, 1 12,737; 5,028,527, the contents of which, are hereby incorporated by reference in their entireties for any purpose), RAF protein (e. g. See WO2014047973, the content of which is hereby incorporated by reference in its entirety for any purpose), MEK protein, ERIC protein and/or FRA- 1 protein.

In certain embodiments, a modulator of KRAS signaling may comprise a small molecule inhibitor of KRAS signaling (see, for example, WO2016123378A1, the content of which is hereby incorporated by reference in its entirety for any purpose and TABLE 5 below).

TABLE 5

Exem lary inhibitors of the RAS/RAF MEK/ERK signal transduction pathway

■779,450 Raf leukemia Precl inical Merck modified from Chappefl si at Oncoiargel. (2011) 2:135-64, INHIBITOR ! TARGET(S) CANCER EXAMINED CLINICAL COMPANY j

TRIALS

Gw5074 i Rat i (c-Raf) mel noma, glioblastoma Preclinical Glaxo SiTiithKline j j Raf Preclinical GlaxoSmitbKlinel j MEKT, colorectal, NSCLC, 1¾¾ e i Pfizer j j MKK5 pancreatic, kidney. (disconti nued)

PD0325901 j MEK I /2 breast, colon, SCLC, Phase I, Π Pfizer ] melanoma (disconti nued)

xl5 I 8 ΐ MEK Phase I Exelixis 1 selumetinib j MEK melanoma, HCC, Phase I,. II Astra j (AZD6244, pancreatic, colon, king, : Zeneca/Array 1 ARRY- 142886) breast 1 BioPharma 1 advanced tumors Ardea Bayer 1

(BAY $69766) j (MAPK/ERK

1 kinase 1 )

PD098059 j MEKI/2 advanced hematological j Preclinical Parke- j

and advanced solid j Davis/Pfizer j cancers ■

u0126 j MEKI/2 advanced . ί Preclinical DisPont j hematological and Pharmaceuticals I advanced solid cancers j

sl-327 j MEKI /2 not evaluated for use in Preclinical ] D Pont i j cancer treatment j Pharmaceuticals !

In certain embodiments, a modulator of KRAS signaling may comprise a covalent inhibitor of KRAS G12C (see for example, WO2014152588 and WO2016049524, the contents of which are hereby incorporated by reference herein in their entireties for any purpose). in certain embodiments, a modulator of KRAS signaling may comprise an inhibitor of prenyl transferase activity, in certain embodiments, a modulator of KRAS signaling comprises one or more agents each independently chosen from inhibitors of farnysyltransferase or geranylgeranyl transferase I (e.g. , see U.S. Patent No. 5,965,539, the content of which is hereby- incorporated by reference in its entirety for any purpose).

In certain embodiments, a modulator of KRAS signaling may comprise, for example, a small molecule RAF inhibitor. In certain embodiments, a small molecule RAF inhibitor can inhibit the RAF--MEK---ERK pathway signal ing in cells expressing the BRAFV600E oncogene. Exemplary BRAF inhibitors include, but are not limited to, vemurafenib (RG7204 or PLX4032) which has been approved by the FDA for treatment of melanoma., dabrafenib which has been approved by FDA for treatment of cancers associated with a mutated version of the gene BRAF, GDC-Q879, PLX-4720 (Plexxikon Roche) (R72Q4), Sorafenib Tosylate, dabrafenib and/or LGX818 or any combination thereof.

In certain embodiments, a modulator of KRAS signaling may comprise, for example, a small molecule MEK inhibitor. In certain embodiments, a MEK inhibitor may comprise, for example, an ATP-competitive MEK inhibitor, a non- ATP competitive MEK inhibitor, and/or an ATP- unc o mpet i t i ve MEK inliibiior.

Exemplary MEK inhibitors include, but are not limited to, trametinib (GSK1120212; JTP 74057), for treatment of BRAF-mutated melanoma and possible combination with BRAF inhibitor dabrafenib (GSK2118436) to treat BRAF-mutated melanoma; selumetmib (ARRY- . 142886, AZD6244), for non-small cell lung cancer (NSCLC); binimeti ib (MEK162, ARRY- 162, ARR Y -43 162), for biliary tract cancer and melanoma; PD-3259Q1, for breast cancer, colon cancer, and melanoma; Cobimetinib (GDC-0973; XLS 18); AS703026 (pimasertib), CI- 1040, FD035901, RG7304, AZD6244, GSK1 120212, GDC-0973, XL-518, ARRY-162, ARRY-300, PD184161, RO4927350, CIP- 1374, TAK-733, CH4987655, and/or RDEA1 19, or any combination thereof. In certain embodiments, the modulator of KRAS signaling may comprise, for example, RG7304, a small molecule MEK inhibitor with a structure based on a coumarin skeleton. It selectively inhibited RAFl (C-RAF), B-RAF, mutant B-RAF (V600E), and MEKl In in vitro studies and showed a strung and broad spectrum of antitumor activities both in vitro in various tumor cell lines and in vivo in mouse xenograft models.

In certain embodiments, a modulator of KRAS signaling may comprise, for example, a non-ATP-competitivc small- molecule MEK inhibitor (e.g. PD 098059, U0126, PD 184352 and its derivatives) or a biological inhibitor (e.g. anthrax lethal toxin and Yersinia outer protein J). In certain embodiments, a modulator of KRAS signaling may comprise, for example, a pyrrole derivative of MEK kinase. In certain embodiments, a modulator of KRAS signaling may comprise, for example, a 4-anilino-3-cyano-6,7-dialkoxyqumoline, including 4-anilino-3- cyano-6,7-dialkoxyquinolines (e.g., see WO2013059320, the content of which is hereby incorporated by reference in its entirety for any purpose).

In certain embodiments, a modulator of KRA S signaling may comprise, for example, a small molecule ERIC inhibitor. Exemplary ERK inhibitors include, but are not limited to, SCH772984 which is an ATP-cotnpetitive ER l and ERK2 inhibitor, MK-8353/SCH900353, a clinical grade analogue of SCH772984, which is currently being tested in Phase I clinical trials, BVD-523 (Biomed Valley Discoveries) and RG7842 (GDCQ994; Genentech/Roche) which is a selective inhibitor of ERK 1/2. Phase I clinical trials evaluating RG7842 as a single agent or in combination with cobirnetinib in solid tumors are ongoing. Studies conducted with SCH772984 indicate that the small molecule can inhibit cellular proliferation and cause apoptosis selectively in tumor cell lines that carry RAS or BRAF mutations, and also induce significant tumor regressions in mice with BRAF- or RAS-mutant xenografts. SCH772984 also demonstrated inhibition activity in cells that were resistant to either BRAF or MEK inhibitors and in cells that became resistant to the dual combination of these inhibitors.

In certain embodiments, a modulator of KRAS signaling may comprise, for example, a small molecule inhibitor with a thienyl benzenes ulfonate scaffold that can inhibit ERK 1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif (e.g. see U.S. Patent No. 9, 115,122, the content of which is incorporated by reference herein in its entirety for any purpose). In certain embodiments, a modulator of KRAS signaling may comprise, for example, an inhibitor of ERK2-mediated phosphorylation of c-FOS, e.g. SF-3-026 and its analogues. In certain embodiments, the modulator of KRAS signaling may comprise, for example, an inhibitor of AP-.1 promoter activity in cells with constitutively active ERK 1/2, e.g. SF-3-030 and its analogues. Exemplary small molecule inhibitors of ERKl/2-regulated proteins include, but are not limited to the compounds depicted in TABLE 6.

TABLE 6

In certain embodiments, a modulator of RAS signaling may comprise, for example, an antigen-bi ding protein or fragment thereof (e.g. a recombinant antigen-binding protein) that targets RAS protein, In certain embodiments, the RAS protein is chosen from an oncogenic KR AS (e.g., see U.S. Patent Nos. 4,820,631; 5,084,380, 4,898,932; 5,081,230; 5, 112,737; 5,028,527, the contents of which are hereby irseoiporaied by reference in their entireties for any purpose), RAF protein (e.g., see WO2014047973, the content, of which is hereby incorporated by reference in its entirety for any purpose), MEK protein, ERK. protein and/or FRA-1 protein.

In certain embodiments, a modulator of KRAS signaling may comprise, for example, a peptide that binds to and inhibits the activity of RAS protein. In certain embodiments, the RAS protein is chosen from RAF protein, MEK protein, ERK protein and/or FRA-1 protein, in certain embodiments, the peptide can be, for example, a chimeric peptide comprising a cell penetrating peptide, e.g. pro-apoptotic RAS and/or RAF peptides (see e.g. WO2015001045, the content of which is hereby incorporated by reference in its entirely for any purpose).

The present disclosure further provides compositions comprising a modulator of KRAS signaling. In certain embodiments, the composition is for the treatment of a KRAS-assoeiated disorder or disease, e.g. cancer. In certain embodiments;, the composition comprises a therapeutic agent .

In certain embodiments, the therapeutic agent can be, for example, a chemotherapeutic agent, a targeted agent, or an immunotherapeutic agent. In certain embodiments, a modulator of signaling may be combined, lor example, with an immunotherapeutic agent, e.g., any agent that can induce, enhance, or suppress an immune response in a subject,

Jn certain embodiments, an immunotherapeutic agent comprises an antibody or a recombinant antigen-binding protein or fragment thereof, in certain embodiments, a recombinant antigen-binding protein, or fragment thereof, can be, for example, monospecific, bispecific or multi-specific and monovalent or bivalent recombinant antigen-binding protein. In certain embodiments, an antigen-binding protein, or .fragment thereof, can be an asymmetric bispecific antibody, an asymmetric bispecific IgG4, a CrossMab binding protein, a DAI ⁷ (dual action Fab antibody; two-in-one), a DM (dual action Fab antibody; four-in-one), a DutaMab, a DT-IgG, a knobs~½~holes binding protein, a Charge pair binding protein, a Fab-arm exchange binding protein, a SEEDbody, a Triomab (Triomab quadroma bispecific or removab bispecific), a LUZ-Y, a Fcab, a λ-body, an iMab (innovative multimer), an Orthogonal Fab, a DVD-Ig binding protein, an IgG(H)-scFv, an scFv-(H)IgG, an IgG(L)-scFv, an scFv-(L)IgG, an IgG(L, H)~Fv, an IgG(H)-V, a V(H)-IgG, an IgG(L)-V, a V(L)-IgG ₅ a KIH IgG-scFab, a 2scFv-IgG, an IgG-2scFv, an scFv4-Ig, a Zybody, a DVI-IgG (four-in-one), a nanobody (or VHH). a bispecific tandem nanobody. a bispecific trivalent tandem nanobody, a nanobody- HSA, a BiTE (bispecific T-cell engager) binding protein, a Diabody, a DART (dual affinity retargeting) binding protein, a TandAb (tetravalent bispecific tandem antibody), an scDiabody, an scDiabody-CH3, a Diabody-CH3, a Triple Body, a Miniantibody, a Minibody, a TriBi. minibody, an scFv-CFB ΚΓΗ, a Fab-scFv, an scFv-CH-CL-scFv, a F(ab')2, a F(ab')2 scFv2, an scFv-KIH, a Fab-scFv-Fc, a Tetravalent HCAb, an scDiabody-Fc, a Diabody-Fc, a Tandem scFv-Fc, a Fabsc, a bsFc-1/2, a CODV-Ig (cross-over dual variable immunoglobulin), a hiclonies antibody, an Inlrabody, a Dock and Lock binding protein, an ImmTAC, an HSAbody, an scDiabody- HS A, a Tandem scFv-Τοχΐη, an IgG-IgG binding protein, a Cov-X-Body, and/or an scFvl-PEG-scFv2. 1.

In certain embodiments, an immunotherapeutic agent specifically binds to a specific cytokine, cytokine receptor, co-stimulatory molecule, co-inhibitory molecule, or immunomodulatory receptor that modulates the immune system. In certain embodiments, an immunotherapeutic agent .specifically binds to a component of a regulatory T cell, myeloid suppressor cell, or dendritic cell. In certain embodiments, an immunolherapeuti c agent can be a cytokine, for example, an interferon (IFN), interleukin, or the like. Specifically, an immuno therapeutic agent can be interferon (IFNa or ΙΡΝβ), type 2 (IFNy), or type III (IFNX). An immunothsrapeuiic agent, can also be interleukin- 1 (JL-1), interieukin- 1 a (IL- la), interleukin- 1β (IL-Ιβ), interleukin-2 (IL- 2), interleukin-3 (11,-3), interleukin-4 (1L-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 10 (IL-10), interleukin- .1 1 (EL- 11), interieukin- 12 (IL-12), interleukin- 13 (IL-13), or interleukin- 18 (EL- 18), or the like.

In certain embodiments, a modulator of KRAS signaling described herein may be combined, for example, with an immunotherapeutic agent. In certain embodiments, the immunotherapeutic agent targets and/or binds a cancer or tumor cell marker or component. Exemplary cancer or tumor cell markers or components include , but are not limited to, are not limited to, epidermal growth factor receptor (EGFR; EGFR ; ErbB-1; HERE); ErbB-2 (HER2/neu); ErbB-3/HER3; EAB-4/HER4; EGFR ligand family; insulin-like growth factor receptor (IGFR) family; IGF-binding proteins (IGFBPs) IGFR ligand family (IGF-IR); platelet derived growth factor receptor (PDGFR) family; PDGFR ligand family; fibroblast growth factor receptor (FGFR) family: FGFR ligand family; vascular endothelial growth factor receptor (VEGFR) family; VEGF family; HGF receptor family; TRK receptor family; ephrin (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family; angiopoietin 1; 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; diseoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor alpha (TGF- a); TGF-a receptor; Transforming growth factor-beta (TGF-β); TGF-β receptor; interieukin β receptor alpha2 chain (EL13Ralpha2); interleukin- 6 (IL-6): IL- 6 receptor; interleukin-4: IL-4 receptor; Cytokine receptors; Class Ϊ (hematopoietiii family) and Class II (interferon,'! L- 10 family) receptors; tumor necrosis factor (TNF) family; TNF-a; tumor necrosis factor (TNF) receptor superfamily (T ^' NTRSF); death receptor family; TRAIL- receptor; cancer-testis (CT) antigens; lineage-specific antigens; differentiation antigens; alpha-actinin-4 ARTCl ; breakpoint cluster region- Abelson (Bcr-Ahl) fusion products: B- RAF; caspase-5 (CASP-5); caspase-8 (CASP-8); beta-catenin (CTNNBl); cell division cycle 27 (CDC27); cyclin- dependent kinase 4 (CDK4); CDK 2A; CO A- 1; dek-can fusion protein: EFTUD-2; Elongation factor 2 (ELF2); Ets variant gene 6/aeute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein; fibronectin (FN); GPNMB; low density lipid receptor/GDP-L fxicose: beta-D- Galactose 2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein; HLA-A2; MLA-A11 ; heal shock protein 70-2 mutated (HSP7G-2M); KIAA0205; MART2; melanoma ubiquitous mutated 1; 2; 3 (MUM- 1; 2; 3); prostatic acid phosphatase (PAP); neo-PAP; Myosin class 1; NFYC; OGT OS~9; pml-RAR alpha fusion protein; PRDX5; PTPRK; K-RAS (KRAS2); N- RAS (NRAJS); HRAS; RBAF600; SIRT12; SNRPD1; SYT-SSX1 or -SSX2 fusion protein; triosephosphate isomers se; BAGE; BAGE- 1; BAGE-2; 3; 4; 5; GAGE- 1; 2; 3; 4; 5; 6; 7; 8; GnT-V (aberrant N-aeetyl glucosaminyl transferase V: MGAT5); HERV-K MEL; KK-LC;

M-H -I; LAGE; LAGE-1; CTL-recognizsd antigen on melanoma (CAMEL); MAGE-A1 (MAGE-1) MAGE-A2; MAGE- A3; MAGE-A4; MAGE- AS; MAGE-A6; MAGE-A8; MAGE-A9; MAGE- A 10; MAGE- Al J ; MAGE-A12; MAGE- 3; MAGE-B !; MAGE-B2; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2 mucin 1 (MUC1); MART-l Melan-A (MLA A); gplOO; gplOO/Pmel 17; tyrosinase (TYR); TRP-1; HAGE; NA-88; NY-ESO-1; N Y-ES O-l L AGE-2: SAGE; Sp!7; SSX- 1; 2; 3; 4; TRP2-1NT2; carcinoembryonic antigen (CEA); Kallikrein 4; mammaglobin-A; OA1; prostate specific antigen (PSA); prostate specific membrane antigen; TRP-1/; TRP-2 adipophilin; interferon inducible protein absent in melanoma 2 (AJM-2); BiNG-4; CPSF; cycHn D ; epithelial cell adhesion molecule (Ep-CAM); EpbA3; fibroblast growth factor- 5 (FGF-5); glycoprotein 250 (gp250 intestinal carboxyl esterase (iCE); alpha-fetopiotein (AFP); M-CSF; mdm-2; MUCi; p53 (TP53); PBF; FRAME; PSMA RAGE-1; RNF43; RU2AS; SOX10; STEAPl; survmn (BIROS); human telomerase reverse transcriptase (hTERT); telomerase; Wilms' tumor gene (WT1); SYCP1; BRDT; SPANX; XAGE; ADAM2; PAGE-5; LIP1; CTAGE- 1 ; CSAGE; MMA1 ; CAGE; BORIS; HOM-TES-85; AF15q!4; HCA66I; LDHC; MORC; SGY-1; SPOl l; TPX1; Y-SAR-35; FTHI.J7; NXF2 TDRD l : TEX 1 ; FATE; TPTE; immunoglobul n k!iotypes; Bence-Jones protein; estrogen receptors (ER); androgen receptors ( AR); CD40; CD30; CD20; CD19; CD33; CD4; CD25; CD3; cancer antigen 72-4 (CA 72-4); cancer antigen 15-3 (CA 15-3); cancer antigen 27-29 (CA 27-29); cancer antigen 125 (CA 125); cancer antigen 19-9 (CA 19-9); beta- human chorionic gonadotropin; 1-2 microglobulin; squamous cell carcinoma antigen; neuron- specific enoiase; heat shock protein gp96; GM2; sargramostim; CTLA-4; 707 alanine proline (707- AP); adenocarcinoma antigen recognized by T cells 4 (ART-4); carcinoembryonic antigen peptide- 1 (CAP- 1); calcium- activated chloride channel- 2 (CLCA2); cyclophilin B (Cyp-B); and/or human signet ring tumor-2 (HST-2).

In certain embodiments, a modulator of RAS signaling described herein may be combined, for example, with one or more immunotherapeutie antibodies, each independently chosen from trastuzumab (antj- HER2/neu antibody); pertuzumab (anti-HER2 inAb); celuximab (chimeric monoclonal antibody to epidermal growth factor receptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); za!utumumab (anti- EGFR mAb); necitumumab (anti- EGFR mAb); MDX-210 (humanized anti-HER-2 bispecifie antibody); MDX-210 (humanized anti-HER-2 bispecifie antibody); MDX-447 (humanized anti-EGF receptor bispecifie antibody); rituximab (chimeric murine/human- anti-CD20 mAb); obinutuzumab (anti-CD20 mAb); ofalumumab (anti-CD20 mAb); tosilumumab-1131 (anti- CD20 mAb); ibritumomab tiuxetan (anti-CD20 mAb): bevacizumab (anti-VEGF mAb); ramucirumab (anti-VEGFR2 mAb); ranibizuinab (anti-VEGF mAb); aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgGl Fc); AMG386 (angiopoietin-1 and -2 binding peptide fused to IgGl Fc); dalotuzumab (anti-IGF-lR mAb); gemtazumab ozogainicin (anti- CD33 mAb); alemtuzumab (anti-Campath-l/CD52 mAb); brentuximab vedotin (anti-CD30 mAb): catumaxomab (bispecifie mAb that targets epithelial cell adhesion molecule and CD3); naptumomab (anti-5T4 mAb); girentuximab (anti-Carbonic anhydrase ix); farletuzumab (anti- folate receptor); Panorex™ ( 17-lA) (murine monoclonal antibody); Panorex (@.(17- 1A)) (chimeric murine monoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope) (with. BCG); oncolym (Lym-1 monoclonal antibody); SMART M195 Ah, humanized 13' 1 LYM-1 (Oncolym); Ovarex (B43. 13, anti-idiotypic mouse mAb); 3622W94 mAb that binds to EGP40 (17-lA) pancarcinoma antigen on adenocarcinomas; Zenapax (SMART Anti-Tac (IL- 2 receptor); SMART Ml 95 Ab, humanized Ab, humanized); ovoMAb~G2 (pancarcinoma specific Ab); TNT (chimeric mAb to histone antigens); TNT (chimeric mAb to hi stone antigens); Gliomab-H (Monoclonal s— Humanized Abs); GN1-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); or MDX-260 (bispecifie, targets GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab), and/or ImmuRAIT-CEA.

In certain embodiments, an i mmun otherapeut ic agent can be a cell, for example, an immune cell. For example, an immune cell, particularly one that is specific to a tumor, can be activated, cultured, and administered to a patient. The immune cell can be a natural killer cell, lymphokine-aciivated killer cell, cytotoxic T-cell, dendritic cell, or a tumor infiltrating lymphocyte (TIL). As used herein, tumor infiltrating lymphocytes (or TILs) refers to white blood cells ((i.e., T cells, B cells, N cells, macrophages) that have left the bloodstream and migrated into a tumor. An analysis of patients with metastatic gastrointestinal cancers suggest CD4 ⁺ and CDS ⁺ T cells within the TIL population are able to recognize neo-epi topes derived from somatic mutations expressed by the patient's tumor. In certain embodiments, an imr inotherapeutic agent can be, for example, sipuleucel-T (Provenge).

In certain embodiments, a modulator of KRAS signaling as disclosed herein may be combined with an inhibitor of an immune checkpoint.

Immune checkpoint proteins can regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses including cytotoxic T cell activity.

Non-limiting examples of immune checkpoint proteins include cytotoxic T- lymphocyte-associated antigen (CTLA, for example, CTLA4) and its ligands CD 80 and CDS 6; programmed cell death protein (PD, for example, PD-1) and its ligands PD-Ll and PDL2; indoleamine-pyrrole 2,3-dioxygenase-l (IDOl); T cell membrane protein (TIM, for example, TIM3); adenosine A2a receptor (A2aR); lymphocyte activation gene (LAG, for example, LAGS); killer immunoglobulin receptor (KIR); or the like.

As used herein, the term "immune checkpoint inhibitor" refers to a molecule that can completely or partially reduce, inhibit, interfere with, or modulate one or more immune checkpoint proteins that regulate T~cell activation or function. For example, the term "immune checkpoint inhibitor" can refer to a molecule that can interfere or/and prevent the interaction of PD- 1 with its !igand, either PD-Ll or PD-L2.

In certain. embodiments, the immune checkpoint inhibitor can target CTLA4. In certain embodiments, the immune checkpoint inhibitor can target PD-1. In certain embodiments, the immune checkpoint inhibitor can target PD-Ll. In certain embodiments, the immune checkpoint inhibitor can target PD-L2. In certain exnbodiments, the immune checkpoint inhibitor can target LAG 3. in certain embodiments, the immune checkpoint inhibitor can target B7-H3. In certain embodiments, the immune checkpoint inhibitor can target B7-H4. In certain embodiments, the i mmune checkpoint inhibitor can target TIMS.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule that, competes with an antibody or other antigen-binding protein, or fragment thereof, for binding to an immune checkpoint molecule.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule that competes with an anti-PD- 1 antibody, e.g., nivolumab, pembrolizumab, pidilizumab, BMS 936559, or atezolizumab as disclosed herein, for binding to PD- 1. In certain embodiments, the immune checkpoint inhibitor can be a small molecule that competes with an anti-PD-Ll antibody, e.g., atezolizumab, avelumab, or durvahimab as disclosed herein, for binding to PD-L1.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule that competes with an anti-PD-L2 antibody, e.g., rHIgM12B7 or Dana-Farber patent anti-PD-L2 as disclosed herein, for binding to PD-L2.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule that competes with an anti-CTL4 antibody, e.g., ipilimumab, tremelimumab or AGEN1884 as disclosed herein, for binding to CTL4.

In certain embodiments, the immune checkpoint inhibitor can be a small molecule that competes with an anti- VISTA antibody, e.g., the Janssen patent anti-VISTA antibody, Igenica patent anti-C10orf54 antibody . or the Amplimmune patent anti-B7-H5 antibody as disclosed herein, for binding to the immune checkpoint protein, VIST

In certain embodiments, the immune checkpoint inhibitor can be a small molecxile that rescues the inhibition of cell proliferation by recombinant PD-L1 in a mouse splenocyte assay by about 10% to about 95% (the mouse splenocyte assay is described in detail in, for example, WO2016142833).

In certain embodiments, the immune checkpoint inhibitor can be, for example, a 1,2,4- oxadiazole and thiadiazole compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. In certain embodiments, the immune checkpoint inhibitor can suppress or inhibit the programmed cell death 1 (PD-1) signaling pathway in T cells.

In certain embodiments, the immune checkpoint inhibitor can be, for example, a compound of formula (I)

(I)

or a pharmaceutically acceptable salt or a stereoisomer thereof; wherein,

is an optional double bond; X is O or S;

Ri and R2 independently are a side chain of an amino acid or hydrogen. Ci-C6)alkyl, (C2-Ce)alkenyl, (C2-Ce)alkynyl or cycloalkyl; wherein (Ci-Cs)a!kyL (Cs-CeJaJkenyl, (C2- Ce)alkynyl and cycloalkyl are optionally substituted by one or more substituents selected from amino, alkylamino, acylamino, carboxylic acid, earboxylate, oarboxylic acid ester, thiocarboxylate, thioaeid, ~CONR?Rs, hydroxy, cycloalkyl, (cyc!oalkyl)alkyl, aryl, arylalkyl .heterocyclyl, (heterocyciyl)alkyl, heteroaryl, (heteroaryl)alkyl, guanidino, -SH and -S(alkyl); optionally wherein cycloalkyl, aryl. heterocyclyl and heteroaryl are further substituted by one or more substituents such as hydroxy, alkoxy, halo, arnino, nitro, cyano or alky! and optionally wherein two or three carbon atoms of the (Ci-Cfi)alkyl _? (C2-C6)alkenyl or (C2-G>)alkynyl form part, of a 3-7-membsred carbocyclic or heterocyclic ring (such as a cyclobutyl. or oxirane ring);

R3 is hydrogen, -CG-[Aaal] _m , [Aaal.] _m , [Aaal] _m -CO-[Aa.a l] _m , -S(0) _P -[Aaal] _m , - CONR-Rs, -CORe, -SQiRc, (Ci-Cejalkyl, (C2-Gs)alkenyl or (Cs-C^alkynyl; wherein (Ci- C6)alkyL (C2-C6)alkenyl and (C2-C6)alkynyl are optionally substituted by one or more substituents selected from amino, alkylamino, acylamino, -COO-alkyl, carboxylic acid, carboxylate, thiocarboxylate, thioaeid, -CONRTRS, hydroxy, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl, (cycloalkyl)alkyl, (heterocyclyl)alkyl, (neteroaryl)alkyl, guanidino, - SH and -S(alkyl): optionally wherein cycloalkyl, aryl, heterocyclyl and heteroaiyl are further substituted by one or more substituents such as hydroxy, alkoxy, halo, amino, nitro, cyano or alkyL optionally wherein two or three carbon atoms of the (C: -Gs)alkyl, (CVCf alkeny) or (C2- Ce)alkynyl form part of a 3-7-membered carbocyclic or heterocyclic ring (such as a cyclobutyl or oxirane ring);

R4 and R5 independently are hydrogen or absent;

Re is hydrogen, alkyl, alkenyl, alkynyi, aralkyl, aryl, heteroaralkyl, heteroaiyl, cycloalkyl, (cycloalkyl )alkyl, amino, aminoalkyl, hydroxyalkyl, alkoxyalkyl, acyl, [Aaa2]n, - CO[Aaa2]n, [Aaa2]n-CO-[Aaa2] _n or -S(0) _P -[Aaa2]n;

R.7 and Rs independently are hydrogen, (Ci-C<>)alkyl, (C2-Ce)alkenyl, (C2-C¾)alkynyL aryl or heterocyclyl; wherein (Ci-Cs)alkyi, (C2-C6)alkenyl, (C2-C6 ^' )alkynyl, aryl and heterocyclyl are optionally substituted by one or more substituents selected from halogen, hydroxy!, amino, nitro, cyano, cycloalkyl, heterocyclyl. heteroaryl, aryl, guanidino, (ey oalkyl)alkyl, (heterocyclyl)alkyl and (heteroaryl)alkyl; optionally wherein two or thrse carbon atoms of the (Ci-C6)a1kyl, (C2-C6)alkenyl or (C2-Ce)alkynyl form part of a 3-7- membered carbocyclic or heterocyclic ring (such as a cyclobutyl or oxirane ring);

alternatively R? and Rs together with the nitrogen to which they are attached form an optionally substituted 3-7-membered ring containing 0-2 additional heteroatoms independently selected from N, O and S in any stable combination; wherein the optional substituent at each occurrence is selected from hydroxyl, -COOH, -CQO-aikyl, amide, halo, amino, nitro and eyano;

[Aaal] and [Aaa2], independently for each occurrence, represents an amino acid residue; wherein a C-terminai carboxyj. group of amino acid residue is a free C-terminal carboxyl group (-COOH) or a modified C-terminal carboxyl group and an -terminal amino giOup of amino acid residue is a free N-terminus (-NHz) or a modified N-terminal amino group;

R ₃ is hydrogen or alkyl. alkenyl. aikynyl, acyl, aralkyL aryl, heteroaralkyl, heteroaryl, cycloalkvl, (cycloalkyi )alkyl. aminoalkyl, hydroxy alkyl or alkoxyalkyl;

Rb is hydrogen., alkyl, alkenyl, aikynyl, acyl, aralkyl, aryl, heteroaralkyl, heteroaryl, cycloalkvl, (cycloalkyl)alkyl, aminoalkyl, hydroxyalkyl or alkoxyalkyl; or Rb and R ₂ , together with the atoms to which they are attached, may form pyrrolidine or piperidine optionally substituted with one or more groups independently selected from hydroxy, halo, amino, eyano and alkyl;

R is (Ci-Ce)alkyl, cycloalkvl, aryl, heterocyclcyl or heteroaryl; wherein the said (Ci- C(5)alkyi, cycloalkyi aryl, heterocyclcyl or heteroaryl is optionally substituted by one or more substituents selected from carboxylic acid, hydroxy, alkyl, alkoxy, amino, alkylamino, acylamino, carboxylic ester, cycloalkyi, heterocyclyL heteroaryl, (cycloalkyl)alkyL (helerocyclyl)alkyl or (heieroaryl)aikyl;

m and n independently are integers selected from 1 to 3;

p is an integer selected from 1 to 2;

with a proviso that Ri is not a side chain of Ser or Thr, when R2 is a side chain of Asp, Asa, Glu or Gin, R3 is hydrogen, -CO-Ser or -CO-Thr, Re is hydrogen, alkyl or acyl and Ra and Rfi are hydrogen.

Compositions and methods of using and making the compounds of Formula (I) are disclosed in WO2016142833 (e.g. Compound Nos. 1- 124 of WO2016 42833), the content of which is hereby incorporated by reference herein in its entirety. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of t! modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of a compound of Formula (I). In certain embodiments, the method is effective at. sensitizing tumor cells to a compound of Formula (I). In certain embodiments, the method changes (e.g., enhances) the efficacy of a compound of Formula (I). In certain embodiments, the tumor cells are resistant to treatment with a compound of Formula (I) alone.

In certain embodiments, the compound of Formula (I) can be, for example. Compound No. 7 ving the structure of:

In certain embodiments, the compound of Formula (I) can be, for example, Compound No. 14 o

In certain embodiments, the compound of Formula (I) can be, for example, Compound No, 60 of WO2016142833 havin the structure of:

In certain embodiments, the compound of Formula (I) can be, for example, Compound No, 75 of WO2016142833, havin the structure of:

In certain embodiments, the immune checkpoint inhibitor can be, for example, a 3~ substituted 1,3,4-oxadiazo!e and thiadiazole compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the immune checkpoint inhibitor can be, for example, a compound of formula (II):

or a pharmaceutically acceptable salt thereof or a stereoisomer thereof;

wherein,

X is O or S

each dotted line [ ] independently represents an optional bond;

Ri is hydrogen or -CO-Aaa;

Aaa represents an amino acid residue;

R.2 is side chain of an amino acid, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, heteroaraikyi, aralkyl, heteroaryl or aryl, each optionally substituted by one or more substituents selected from carboxylate, carboxylic acid, carboxylic acid ester, thiocarboxylate, thio acid, amido, amino, heterocyclyl, hydroxy! ,· cycloalkyl, aryl, aryl-COOH, heteroaryl, guamdino, amidino, -NH, -N(alkyl), -SH and -S( alkyl), optionally wherein two or three carbon atoms of the alkyl, alkenyl or alkynyl form part, of a 3-7-memhered carbocyclic or heterocyclic ring which is optionally substituted with 1 to 4 substituents, each independently selected from alkyl, a!koxy, carboxylic acid, carboxylate and hydroxyl; Rs is aryl, heteroaryl, heterocyclyl or cycioalkyl;, wherein the said aryl, heteroaryl, heterocyclyl or cycioalkyl is optionally substituted by 1 to 4 occurrences of R¾

^' Ra, independently for each occurrence, is alkyl, alkoxy, halo, hydroxy!, amino, - C(0)OH, aralkyl, aryl, a!koxy, heteroaralkyl, heteroaryl, cycioalkyl., (cyeloalkyl)alkyl, hydroxyalkyl, alkoxyalkyl or acyk or any two Ra groups attached to the same carbon atom together represent an oxo ( ^~ 0) or tJhioxo ( ^:;; S);

each of R4 and Rs independently is hydrogen or absent; and

R0 is hydrogen or alkyl.

Compositions and methods of using and making the compounds of Formula (II are disclosed in WO2016142894 (see, for example. Compound Nos, 1- 30 of WO2016142894) and WO2016142886 (see, for example, Compound Nos. 1-62 of WO2016142886), the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

Irs certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor compri sing administering an effective amount of a modulator of RAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of a compound of Formula (II). In certain embodiments, the method is effective at sensitizing tumor cells to a compound of Formula (II). In certain embodiments, the method changes (e.g., enlianc.es) the efficacy of a compound of Formula (Π). In certain embodiments, the tumor cells are resistant to treatment with a compound of Formula (II) alone.

In certain embodiments, the compound of Formula (II) can be, for example, Compound No. 11 of WO2016142894, having the structure of:

In certain embodiments, the compound of Fornmla (II) can be, for example. Compound No. 13 of WO2016142894 having the structure of:

In certain embodiments, the compound of Formula (II) can be, for example, Compound No. 32 of O2016142886, having the structure of:

In certain embodiments, the compound of Formula (II) can be, for example, Compound No. 43 of WO2016142886, havin the structure of:

In certain embodiments, the immune checkpoin inhibitor can be, for example, a 1,3,4- oxadiazols and thiadiazole compound or a stereoisomer thereof or a pharmaceutically acceptable salt thereof that can suppress or inhibit the programmed cell death 1 (PD- 1) signaling pathway.

In certain embodiments, the immune checkpoint inhibitor can be, for example, a compound of formula (III):

or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; wherein, each dotted line [- - - - ] independently represents an optional bond; X is O or S;

Ri and R2 independently are a side chain of an amino acid or hydrogen. (Ci-Ce)alkyl, (C2-C6)alkenyl, (C ₂ -Cs)aikynyl, heterocycloalkyl or cycloalkyl; wherein (Ci-Cs)alkyl _s (C2- Ce)alkenyl, (C2-Cs)alkynyl, heterocycloalkyl and cycloalkyl are optionally substituted by one or more substituents selected from amino, alkylamino, acylamino, carboxylic acid, carboxylate, carboxylic acid ester, thiocarboxylate, thioacid, -CONR7R8, hydroxy, cycloalkyl, (cycloalkyl)alkyL aryl, heterocyclyl, (heteroeycly )alkyL heteroaryl, (hefceroaryi)alkyL guanidino, -SH. and -S(alkyl); optionally wherein cycloalkyl, aryl, heterocyclyl and heteroaryl are further substituted by one or more substituents such as hydroxy, alkoxy, halo, amino, nitro, cyano or alkyl and optionally wherein two or three carbon atoms of the (Ci -GOalk l, (Ci- C6)alkenyl or (C2-C6)alkynyl form part of a 3-7-membered carbocyciic or heterocyclic ring (such as a cyelobutyl or oxirane ring);

R3 is hydrogen, -CO-[Aaa l]m, [Aaaljm, [Aaall]m-CO-[Aaal]m, -8(0 )p-[ Aaaljm, - CONR7R8, -CORc, -SChRc, (Ci-C«)alkyL (C ₂ -C<s)alkenyl or (C2-Gs)alkynyl; wherein (Ci- Ce)alkyl, (C2-C6)alkenyl and (C2~Ce)aJkynyl are optionally substituted by one or more substituents selected from amino, alkylamino, acylamino, -COO-alkyl, carboxylic acid, carboxylate, thiocarboxylate, thioacid. - CO R7R8, hydroxy, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl, (cycloalkyl)alkyl. (heterocyelyl)alkyl, (heteroaryl)alkyl, guanidino, - SH and -S(alkyl); optionally wherein cycloalkyl, aryl, heterocyclyl and heteroaryl are further substituted by one or more substituents such as hydroxy, alkoxy, halo, amino, nitro, cyano or alkyl, optionally wherein two or three carbon atoms of the (Ci~C¾)alkyl, (C2-Cs)aSkenyl or (C2- Ce)alkynyl form part of a 3-7-membered carbocyciic or heterocyclic ring (such as a cyelobutyl or oxirane ring);

R.4 and R5 independently are hydrogen or absent;

R<5 is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, ary!, heteroaralkyl, heteroaryl, cycloalkyl, (cycloalkyl )alkyi, amino, aminoalkyl, hydroxyalkyl, alkoxyalkyl, acyl, [Aaa2]n, - CO- 1 Aaa2Jn, [Aaa2]n-CO-[Aaa2jn or -S(0)p-[Aaa2] _n ;

R? and Rs independently are hydrogen, (Ci~Cs)alky1, (C2-C6)alkenyl, (C2-C6)alkynyl, aryl, cycloalkyl or heterocyclyl; wherein (Ci-Ce)alkyL (C2-C«)aikenyl and (C2-Ce)alkynyl, aryl and heterocyclyl axe optionally substituted by one or more substituents selected from halogen, hydroxy!, amino, nitro, cyano, cycloalkyl, heterocyclyl, heteroaryl, aryl, guanidino, (cycloalkyl)alkyi, (heterocyclyl)alkyl and (heteroaryl)aikyl; optionally wherein two or three carbon atoms of the (Ci-Cs)alkyl, (C2-Ce)alkenyl or (C2-Ce)alkynyl form part of a 3-7- membered carbocyclic or heterocyclic ring (such as a cyclobutyl or oxirane ring);

alternatively, R? and Rs together with the nitrogen to which they are attached form an optionally substituted 3-7-membered ring containing 0-2 additional heteroatoms independently selected from N, O and S in any stable combination; wherein the optional suhstituent at each occurrence is selected from hydroxyl, -COOH, -COOalkyl, amide, halo, amino, nitro and cya.no;

[Aaal] and [Aaa2], independently for each occurrence, represents an amino acid residue; wherein a C-terminal carboxyl group of amino acid residue is a free C-terminal carboxyl group (-COOH) or a modified C-terminal carboxyl group and an N-terminal amino group of amino acid residue is a free N-termimis (-NEb) or a modifi ed N-teiminal amino group;

Ra is hydrogen or alky , alkenyl, alkynyl., acyl, aralkyl, aryl, heteroaralkyl, heteroaryl, cycloalkyl, (cycloalkyl)alkyl, aminoalkyl, hydroxyalkyi or alkoxyalkyl; or Ra and R2, together with the atoms to which they are attached, form hetcrocycloatkyl ring optionally substituted with one or more groups independently selected from hydroxy!, halo, amino, cyano and alkyl;

Rb is hydrogen or alkyL alkenyl, alkynyl, acyl, aralkyl, aryl, heteroaralkyl, heteroaryl, cycloalkyl, (cycloalkyl)alkyL aminoalkyl, hydroxyalkyi or alkoxyalkyl;

Re is (Ci-C6)alkyi, cycloalkyl, aryl, heterocyclyl or heteroaryl; wherein the said (Ci- Cs)aikyL cycloalkyl, aryl, heterocyclyl or heteroaryl is optionally substituted by one or more substituents selected from carboxyiic acid, hydroxy, alkyl, alkoxy, amino, alkylamino, acylamino, carboxyiic ester, cycloalkyl, heterocyclyl, heteroaryl, (cycloalkyl)alkyL (heterocyclyl)alkyl or (heteroaryl)alkyl;

m and n independently are integers from 1 to 3; and

p is an integer selected from 1 to 2;

with a proviso that Ri is not. a side chain of Ser, Thr, Phe, Ala or Asn, when R-i is side chain of Ser, Ala, Glu, Gin, Asn or Asp, R3 is hydrogen, -CO-Ser, -CO-Thr or -CO- Asn and Ra, Rb and Re are hydrogen.

Compositions and methods of using and making the compounds of Formula (1) are disclosed in WO2016142852 (e.g. Compound Nos. 1-55 of WO2016142852), the content of which is hereby incorporated by reference herein in its entirety. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against, a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of a compound of Formula (III). In certain embodiments, the method is effective at sensitizing tumor cells to a compound of Formula (III). In certain embodiments, the method changes (e.g., enhances) the efficacy of a compound of Formula (III). In certain embodiments, the tumor cells are resistant to treatment with a compound of Formula (III) alone.

In certain embodiments, the compound of Formula (III) can be, for example, Compond No. 1 havin the structure of:

In certain embodiments, the immune checkpoint inhibitor can be, for example, a cyclic compound of formula (TV) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof that can suppress or inhibit the programmed cell death 1 (PD- 1) signaling pathway.

In certain embodiments, the immune checkpoint inhibitor can be, for example, a compound of formula IV):

or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; wherein. R, R

ft ^■ ,G

-C(0)-(CH2) _m -(X-CH-CH2) _n -NH- or -C(OMCH ₂ ) _m .(CH)-(CH2) _n -IMH-

L is

wherein the -C(O)- group marked with * is connected to the nitrogen bearing Rs in Formula (IV );

X is Cm, O, NH or S;

Rj , R2 and R$ independently are a side chain of an amino acid, hydrogen, (C_-C«)aikyl,

(Ca-G alkenyi or (C2-C6)alkynyl; wherein (Q -C6)alkyl, (C2-C6)alkenyi and ^' (C2-Cs)alkynyl are optionally substituted by one or more substituents selected from hydroxy, amino, amido, alkyiamino, acylamino, -(Ctt COOH, -(CH.)m-COO-alkyl, cycloalk l, heterocyclyl, aryl heleroaryL gtianidmo, (cycloalkyl)alkyl. (heterocyclyl)aikyL (heteroaryl)alkyl, -SH and -S- (alkyl); optionally wherein cycloalkyl, aryl, heterocyclyl and heteroaiyl are further substituted optionally by one or more substituents such as hydroxy, alkoxy, halo, amino, nitro, cyano or alkyl; optionally wherein two or three carbon atoms of the (Ci -C6)alkyL (C2-C6)aikenyl or (C2- Cs)alkynyl form part of a 3-7-membered carbocyclic or heterocyclic ring (such as a cyciobutyl or oxirane ring);

Ri', Ri Ri and R5 independently are hydrogen or alkyl;

or Ri and Ri', together with the carbon atom to which they are attached, may optionally form an optionally substituted cycloalkyl or heterocycloalkyl ring;

or Ri and R. together with the atoms to which they are attached, may optionally form a heterocyclic ring optionally substitated with one or more groups independently selected from amino, cyano, alkyl, halo and hydroxy;

or R2 and R?.', together with the carbon atom to which they are attached, may optionally form a optionally substituted cycloalkyl or heterocycloalkyl ring;

or R2 and R. together with the atoms to which they are attached, may optionally form a heterocyclic ring optionally substituted with one or more groups independently selected from amino, cyano, alkyl, halo and hydroxy;

RA and R4' independently are hydrogen or alkyl;

Ra and Ra' are each hydro •;gen; or together represent an oxo (-O) group;

Rb and Rb ' are each hydrogen; or together represent an oxo ( ^~ C)) group; Rc at each occurrence is independently hydrogen or lkyl;

Rd is amino or - H-C(0)"(CH ₂ )r~CH3

m is an integer from 0 to 3;

11, independently for each occurrence, is an integer from 2 to 20; r, is an integer from 0-20; and

with a proviso thai Rs is not a side chain of Ser, Asp, Ala, He, Phe, Trp, Lys, Glu and Thr, when Ri is a side chain of Ala, Ser, Thr or Leu, I½ is a side chain of Asp, Asn, Glu or Gin and R; and c are hydrogen.

Compositions and methods of using and making the compounds of Formula (I) are disclosed in WO2016142835 (e. g. Compound Nos, 1-20 of WO2016142835), the content of which is hereby incorporated by reference herein in its entirety,

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of RAS signaling, e.g., a KRAS aiRNA, as defined herein. In. certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount, of a modulator of KRAS signaling, e. g., a KRAS aiRNA, as defined herein, combined with an effective amount of a compound of Formula (TV). In certain embodiments, the method is effective at sensitizing tumor cells to a compound of Formula (XV). In certain embodiments, the method changes (e.g., enhances) the efficacy of a compound of Formula (TV). In ceitain embodiments, the tumor cells are resistant to treatment with a compound of Formula (IV) alone.

In certain embodiments, the compound of Formula (IV) can be, for example, the compound No. 12 havin the structure of;

In certain embodiments, the immune checkpoint inhibitor can be, for example, a peptidomimetie compound that inhibits the immunosuppressive signal induced by an immune checkpoint, e.g. PI , FD-L1, PD-L2, CTL-4 and/or VISTA. Non-limiting exemplary psptidomimetic compounds are disclosed in U.S. Patent Nos. 8,907,033; 9,044,442;

9,096,642; 9,233,940 and U.S. Patent Publication Nos. 2015/0087581 ; 2015/0125491 ;

2016/0113901 and 2016/0194295, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, CA-170 (previously AUPM 170), a first-in-class oral, small molecule antagonist that selectively targets PD-Ll, PD-L2 and V-domain ig suppressor of T cell activation (VISTA) immune checkpoints CA- 170 is currently being evaluated in a phase I trial for the treatment of advanced solid tumors or lymphomas (see Abstract 4861, AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA; developed by Cutis and Aurigene).

In certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, one or more of the Bristol-Myers Squibb (BMS) compounds based on the (2-methyl- 3-biphenylyl) methanol scaffold disclosed in WO2015034820, the content of which is hereby incorporated by reference herein in its entirety.

In certain embodiments, he small molecule immune checkpoint inhibitors can be, for example, compounds 8, 37, 202 and 242 of WO2015034820 (designated herein as BMS-8, BMS-37, BMS-202 and BMS-242) and having the structure of:

B S-8. B S-37. BMS-202 and BMS-242 bind directly to PD-Li and not PD-1 and effectively dissociate a preformed PD-1/PD-L1 complex in vitro. NMR studies indicate these molecules block PD-1/PD-L1 interaction by inducing PD-LI dimerization through PD-1 interacting surface.

In certai embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA. as defined herein, combined with an effective amount of BMS-202. in certain embodiments, the method is effective at sensitizing tumor cells to BMS-202. In certain embodiments, the method changes (e.g., enhances) the efficacy of BMS-202. In certain embodiments, the tumor cells are resistant to treatment with BMS-202 alone.

in certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, an immune checkpoint-specific peptide aptamer.

In certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, an immune checkpoint-specific affimer.

Affimers are peptide aptamers that are engineered into a modified human protease inhibitor Stefin A scaffold (see, for example. U.S. Patent No. 9,447,170, the content of which is hereby incorporated by reference herein in its entirety for any purpose).

In certain embodiments, the small molecule immune checkpoint inliibitor may comprise an amino acid sequence having at least 80% identity to the Stefin A scaffold polypeptide sequence of SEQ ID NO. : 987. .

MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAG

TNYYIKVRAGDNKY HLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF

(SEQ ID NO. : 987)

In certain embodiments, the small molecule immune checkpoint inhibitor nsay comprise an amino acid sequence having at least 80% identity to the modified Stefin A scaffold polypeptide sequence of SEQ ID NO, : 988.

MIP WGL SE AKP ATPEIQEI VDKVKP QLEEKTNET YGKLE AV Q YKTQ VD AG TNYYIKVRAGDNKYMHLKVFNGPPGQNEDLVLTGYQVDK KDDELTGF (SEQ ID NO. : 988)

In certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, a PD-Ll-specific affimer (e.g. PDL1-141 Or PDL1- 179; see, for example, Avacta Life Sciences poster entitled "Generation and Formatting of Mfmier® Biotherapeutics for the Inhibition of the PD-Ll/PD-1 Pathway" 14 ^th Annual Discovery on Target, September 19-22, 2016, Boston, MA).

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount, of a PD-L1 -specific affimer. In certain embodiments, he method is effective at sensitizing tumor cells to PD-Ll-specific affimer. hi certain embodiments, the method changes (e.g., enhances) the efficacy of PD-Ll- specific affimer. In certain embodiments, the tumor cells are resistant to treatment with PD- Ll-specific affimer alone. In certain embodiments, the PD-Ll-specific affimer can comprise PDL1- 141 and/or PDL1-179.

In certain embodiments, the small molecule immune checkpoint inhibitor can be, for example, a bromodomain and extraterminal domain (BET) inhibitor e.g.. JQ 1 (also known as TEN-01Q in clinical trials NCT02308761 and NCT01987362). In certain embodiments, a bromodomain and extraterminal domain (BET) inhibitor can be an inhibitor of BRD4.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor compri sing administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In. certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of a BET inhibitor, in certain embodiments, the method is effective at sensitizing tumor cells to BET inhibitor. In certain embodiments, the method changes (e.g., enhances) the efficacy of BET inhibitor, hi certain embodiments, the tumor cells are resistant to treatment with BET inhibitor alone. In certain embodiments, the BET inhibitor can comprise an inhibitor of BRD4, e.g. JQ1. In certain embodiments, the immune checkpoint inhibitor is a monoclonal or polyclonal antibody directed at PD-1 , PD-1 . 1 , PDL2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7-H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3, CD137, CD .1371., OX40, OX40L, CD27, CD70, CD40, CD40L, TJM3 (i.e„ HAVcr2), GAL9, and/or A2aR.

Exemplaiy PD- 1 immune checkpomt inhibitors that may be combined with a modulator of KRAS signaling, as disclosed herein, include, but are not limited, to:

Nivolumab

Synonym: Anti-PD- 1 human monoclonal antibody MDX-1106

Description: A fully human IgG4 antibody blocking the programmed cell death- 1 receptor (Medarex/Gno Pharmaceuticals Bristol-Myers Squibb) gamma 1 heavy chain ( 1-440) [Homo sapiens VH (IGHV3 -33*01 (91.80%) - (IGHD) - IGHJ4*01) [8.8.6] (1- 113) - IGHG4*01 hinge S 10>P (221) (1 14-440)], (127-214') - disulfide with kappa light chain ( F-214') [Homo sapiens V- KAPPA (IGKV3- 11*01 (98,90%) - IGKJ1 *01) [6.3.9] (l '-107') - IGKC*01 (108'-214')]; (219-219":222- 222") - bisdisulfi.de dimer

US brand name: Opdivo™ (developed by Bristol-Myers Squibb, Medarex

(Bristol-Myers Squibb) and Ono Pharmaceuticals)

167 clinical trials

2015 FDA Approved - Lung cancer (non- small cell)

2012 Start of Phase 3 - Lung cancer (non- small ceil)

2 11 Start of Phase 2 - Renal cell carcinoma

2006 Start of Phase 1 - Lung cancer (non-small cell)

Conditions: B-cel! malignancies. Lung cancer (non-small cell), Melanoma,

Renal Cell Carcinoma

Code name: BMS-936558, MDX-1 106, ONO-4538, 5C4

CAS Registry number: 946414-94-4

Nivolumab is a fully human immunoglobulin (Ig) G4 monoclonal antibody directed against the negative immuno-regulatory human cell surface receptor programmed death- 1 (PD- 1, PCD-1). Nivolumab can bind to and block the activation of PD- 1 by its ligands programmed cell death ligand 1 (PD-L1), overexpressed on certain cancer cells, and programmed cell death ligand 2 (PD-L2). which is primarily expressed on APCs. This can result in the activation of T-cells and cell-mediated immune responses against tumor cells or pathogens. Compositions and methods of using and making nivolumab are disclosed, for example, in U.S. Patent Nos. 9,387,247; 8,779, 105; 8,779,105; 8, 168, 179; and 8,008,449, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a RAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of nivolumab. In certain embodiments, the method is effective at sensitizing tumor cells to nivolomab. 3 certain embodiments, the method changes (e.g., enhances) the efficacy of nivolomab. In certain embodiments, the tumor cells are resistant, to treatment with nivolomab alone,

Pembrolizumab

Synonym: anti-PD- 1 monoclonal anti body MK -3475

Description: humanized mouse monoclonal (228~L-proime(H10->P)) gamma 4 heavy chain (134-218)-disulfide with humanized mouse monoclonal kappa light chain dimer (226~226":229-229")-bisdisulfide

immunoglobulin G4, anti -(human programmed cell death 1 )

US brand name: Keytroda™ (developed by Merck and Schering-Plough);

261 clinical trials

2014 FDA Approved - Solid tumors

2013 Start of Phase 3 - Melanoma

Code name: M -3475, SCH 900475, lambrolizumab

Conditions: Melanoma, solid tumors

CAS Registry- number: 1374853-91-4

Pembrolizumab is a humanized monoclonal immunoglobulin (Ig) G4 antibody directed against human cell surface receptor PD-1 (programmed death- 1 or programmed cell death- 1). Upon administration, pembrolizumab can bind to PD-1, an inhibitory signaling receptor expressed on the surface of activated T ceils, and block the binding to and activation of PD-1 by its ligands, which can result in the activation of T-cell-mediated immune responses against tumor ceils. Compositions and methods of using and making pembrolizumab are disclosed, for example, in U.S. Patent Nos. 8,354,509; 8,900,587; 8,952, 136; and 9,220,776, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, combined with an effective amount of pembrolizumab. In certain embodiments, the method is effective at. sensitizing tumor cells to pembrolizumab. In certain embodiments, the method changes (e.g., enhances) the efficacy of pembrolizumab. In certain embodiments, the tumor cells are resistant to treatment with pembrolizumab alone.

JS001

Synonym: anti-PD-1 humanized antibody (developed by Shanghai .Tunshi

Biosciences Inc. and Junmeng Biosciences Co., Ltd.)

Description: Immunoglobulin G4, anti-(human programmed cell. death protein 1)

(human monoclonal JS001 y4-chain), disulfide with human

monoclonal JS001 κ-chain, dimer

Status: ^" 2016 Start of Phase I clinical trials (NCT02836834, NCT02838823

NCT02836795 & NCT02857166).

Conditions: Breast cancer, solid tumors

CAS Registry number: 1924598-82-2

Compositions and methods of using and making JS001 are disclosed, for example, in International Patent Application No. PCT/CN2014/072574, the content of which is hereby incorporated by reference herein in its entirety for any purpose

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor in a subject comprising admin stering to the subject an effective amount of a modulator of KRAS signaling, e.g.. a KRAS aiRNA ^as defined herein, combined with an effective amount of JS001. In certain embodiments, the method is effective at sensitizing tumor cells lo JS001. in certain embodiments., the method changes (e.g., enhances) the efficacy of JS00 L In certain embodiments, the tumor cells are resistant to treatment with JS001 alone.

REGN2810

Synonym: anti-PD-1 human monoclonal antibody (developed by Regeneron

Pharmaceuticals, Inc. and Sanofi-Aventis)

Description: Immunoglobulin G4, anti-(human programmed cell death protein 1)

(human monoclonal REGN2810 heavy chain), disulfide with human monoclonal REGN2810 κ-chain, dimer

Status: 2015-2016 Start of Phase I/II clinical trials for treatment of advanced

cutaneous squamous cell carcinoma (NCT02760498, NCT02651662,

NCT02520245 & NCT02383212; see also J Clin Oncol 34, 2016

(s ppl; abstr 3024)

Conditions: Advanced malignancies CAS Registry number: 1801342-60-8

REGN2810 is a human monoclonal antibody directed against the negative immunoregulatory human cell surface receptor programmed cell death 1 (PD-I) protein. Upon administration, anti-PD-1 monoclonal antibody REGN2810 can bind to PD-1, inhibit its binding to the PD-1 ligand programmed cell death- 1 iigand 1 (PD-L1), and prevent the activation of its downstream signaling pathways.

Compositions and methods of using and making REGN2810 are disclosed, for example, in the published U.S. Patent Application No. 2015/0203579, the content of which is hereby incorporated by reference herein in its entirety for any purpose

In certain embodiments, the present disclosure provides a method ibr enhancing an immune response against a tumor compri sing administering an effective amount of a modulator of KRAS signaling, e.g.. a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immun response against, a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of REGN2810. In certain embodiments, the method is effective at sensitizing tumor cells to REGN2810. In certain embodiments, the method changes (e.g., enhances) the efficacy of REGN2810. In certain embodiments, the tumor cells are resistant to treatment with REGN2810 a!one.

SHR-1210

Synonym: Immunoglobulin G4, itnti-(celi surface receptor) Qxas&avi-Mus muscuhis

monoclonal SHR-1210 heavy chain), disulfide with human-A/tis musculus monoclonal SHR-1210 light chain, dimer (developed by Incyte Corporation. Jiangsu Hengrui Medicine Co,, Ltd and Shanghai Hengrai Pharmaceutical Company, Ltd.)

Description: Immunoglobulin G4~kappa, anti-[Homo sapiens PDCD1 (programmed cell death 1, PD- L CD279)j, humanized monoclonal antibody;

gamma4 heavy chain (1-443) [humanized VH (Homo sapiens IGHV3- 7*01 (90.80%) -(IGHD) -IGHJ4*01) [8.8,9] (1-1 16) -IGHG4*01 (CHI (117-214), hinge S 10>P (224) (215-226), CH2 (227-336), CH3 (337- 441), CHS (442- 443)) ( 117-443)], (130-214') disulfide with kappa light chain (Γ-214 ⁵ ) [humanized V-KAPPA (Homo sapiens IGKV1- 39*01 (87.40%) -IGKJ 1 *01) [6.3.9] (Γ-107') -Homo sapiens

IGKC*01, Km3 (108'~214')]; dimer (222-222":225 225")-bisdisulfide

Status: 2015-2016 Start of Phase I clinical trials for treatment of advanced melanoma and Advanced Solid Tumors (NCT02492789,

NCT02721589, NCT02738489 & NCT02742935).

Solid tumors (e.g., breast cancer, lung cancer)

Code name 1NCSHR-1210; camrelizumab

CAS Registry Number: 1798286-48-2 (camrelizumab); 1923896-09-6 (SHR-1210)

SHR- 1210 is a monoclonal antibody directed against the negative immunoregulatory human cell surface receptor programmed death- 1 (PD-1). Upon administration, anii-PD-1 monoclonal antibody SHR-1210 can bind to and block the binding of PD- 1 to its iigands programmed cell death ligand 1 (PD-L1), overexpressed on certain cancer cells, and programmed cell death ligand 2 (PD-L2), which is primarily expressed on antigen presenting cells (APCs). Compositions and methods of using and making SHR-1210 are disclosed, for example, in international Patent. Application No. PCT/CN2014/091090, the content of which is hereby incorporated by reference herein in its entirety for any purpose

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effeciive amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of SHR- 1210. In certain embodiments, the method is effective at sensitizing tumor cells to SHR-1210. hi certain embodiments, the method changes (e.g., enhances) the efficacy of SHR-1210. In certain embodiments, the tumor cells are resistant to treatment with SHR-1210 alone.

MEDJ0680

Synonym: anti-PD- 1 human G4 monoclonal antibody (developed by

Amplimmune Inc. &- edimmune)

Description: Immunoglobulin G4, anti -(human programmed cell death 1 ligand protein PDCD1) (human-M s m sc l s monoclonal MEDI0680 γ4- chain), disulfide with human- iis musculus monoclonal MEDI0680 κ- chain, dimer

Status 2014 NCT02271945 (Phase I/Phase II)

2014 NCT02118337 (Phas I)

2013 NCT0201 804 (Phase i)

Code name: AMP-514, MEDI0680

Conditions: Advanced malignancies, aggressive B cell lymphomas

CAS Registry Number: 1607465-69-9

MED 10680 is a humanized immunoglobulin (Ig) G4 monoclonal antibody directed against the negative immunoregulatory human eel! surface receptor programmed cell death 1 (PD-1. Upon administration, anti-PD- l monoclonal antibody MEDI0680 can bind to and inhibit PD-1 and its downstream signaling pathways.

Compositions and methods of using and making MED10680 are disclosed, for example, in U.S. Patent No. 9,205, 148 and the published U.S. Patent Application No. 2016/0130348, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, In. certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of MED10680. In certain embodiments, the method is effective at sensitizing tumor cells to MED 10680. In certain embodiments, the method changes (e.g., enhances) the efficacy of MED10680. In certain embodiments, the tumor cells are resistant to treatment with ED10680 alone.

PDR001

Synonym: humanized anti-PD-1 IgG4 antibody (developed by Novartis)

Status 201 5-201.6 Phase Ι/Π clinical trials (NCT02807844. NCT02460224,

NCT02404441, see also J Clin Oncol 34, 2016 (suppl; abstr 3060)

Conditions: Advanced malignancies, solid tumors

CAS Registry Number: 1859072-53-9

PDR.001 is a fully humanized monoclonal antibody that binds to PD-1 with high affinity and inhibits the biological activity of PD-1. Upon administration, anti-PD-1 monoclonal antibody PDR001 can bind to PD-1 expressed on activated T-cells and block the interaction with its ligands, programmed cell death 1 ligand 1 (PD-Ll, PD-lLl) and PD-1 ligand 2 (PD- L2. PD-1L2).

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of PDR001. In certain embodiments, the method is effective at sensitizing tumor cells to PDR001. In certain embodiments, the method changes (e.g., enhances) the efficacy of PDR001. In certain embodiments, the tumor cells are resistant to treatment with PDR001 alone. BGB-317

Synonym: humanized anti-PD-1 monoclonal antibody

(developed by BeiGene Ltd.)

Status 2015-2016 Phase I clinical trials (NCT02660034, NCT02407990; see also J Clin Oncol 34, 2016 (suppl; abstr 3066)

Code name; BeiGene patent anti-PD-1

Conditions: Advanced solid tumors

CAS Registry- Number: 1863119-16-7

BGB-317 is a monoclonal antibody directed against the negative immunoregulatory human cell surface receptor programmed cell death 1 (PD-1). Upon administration, anti-PD-1 monoclonal antibody BGB-3 17 can bind to PD-1 and inhibit the binding of PD- 1 to the PD- 1 ligands programmed cell death- 1 ligand 1 (PD-L1), and PD- 1 Hgatid 2 (PD-L2).

Compositions and methods of using and making BGB-317 are disclosed, for example, in U.S. Patent Nos. 9,217,034 and 8,735,553, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present, disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, combined with an effective amount of BGB-317. In certain embodiments, the method is effective at sensitizing tumor cells to BGB-317. In certain embodiments, the method changes (e.g., enhances) the efficacy of BGB-317. In certain embodiments, the tumor cells are resistant to treatment with BGB-317 alone. TSR'042

Synonym: humanized anti-PD-1 monoclonal antibody

(developed by AnaptysBio and Tesaro, Inc.)

Status 2016 Phase I clinical trial (NCT02715284)

Code name: ANB01 1, TSR-042

Conditions: Advanced solid tumors

CAS Registry Number: 1923896-12-1 TSR-042 is a humanized monoclonal antibody directed against the negative immunoregulatory human cell surface receptor programmed cell death 1 (PD-1 ; programmed death-1), with potential smmune checkpoint inhibitory d antineoplastic activities, Upon administration, anti-PD- 1 monoclonal antibody TSR-042 can bind to and inhibit PD-1 and its downstream signaling pathways.

Compositions and methods of using and making TSR-042 are disclosed, for example, in U.S. published Patent Application Patent No. 2016/0075783, the content of which is hereby incorporated by reference herein in its entirety for any purpose

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprisiiig administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of TSR-042. In certain embodiments, the method is effective at sensitizing tumor cells to TSR-042. In certain embodiments, the method changes (e.g., enhances) the efficacy of TSR-042. In certain embodiments, the tumor cells are resistant to treatment with TSR-042 alone.

Anti-PD- 1 monoclonal antibody

Status: 2016 NCT02573259 Phase 1 (developed by Pfizer & Merck)

Conditions: Melanoma, Head and Neck Cancer (SCHNC), Ovarian, Sarcoma,

Hodgkin Lymphoma

PF-06801591 is an inhibitor of the human inhibitory receptor programmed cell death 1 (PD-1; PDCDl ), with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, anti-PD- 1 checkpoint inhibitor PF-06801591 can target and bind to PD- 1 and blocks the interaction between PD-1 and its ligands, PD- 1 ligand 1 (PD-L1) and PD-1 ligand 2 (PD-L2).

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of PF-06801591. in certain embodiments, the method is effective at sensitizing tumor cells to PF-06801591. in certain embodiments, the method changes (e.g.. enhances) the efficacy of PF-06801591. hi certain embodiments, the tumor ceils are resistant to treatment with PF-06801591 alone.

Ningbo Cancer Hasp. (NCH) anti-PD-1 CAR

Synonym: Anti-PD- 1 Chimeric antigen receptor

Status: 2016 NCT02873390 Phase MI

(developed by Ningbo Cancer Hospital, China)

Condition ^' s: EGFR Family Member Positive Advanced Malignancies

Code name: HfirinCAR-PD- 1 cells (PD-1 Antibody Expressing CAR-T Cells)

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA. as defined herein, In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of NCH anti-PD- 1 CAR. in certain embodiments, the method is effective at sensitizing tumor cells to NCH anti-PD- 1 CAR. In certain embodiments, the method changes (e.g.. enhances) the efficacy of NCH anti-PD- 1 CAR. In certain embodiments, the tumor cells are resistant to treatment with NCH anti-PD-1 CAR alone.

Pidilizumah

Synonyms: CT-011, K-3475, hBat-1, MDV9300

Status: NCT02077959 Phase I H: Lenalidomide and pidilizumab are currently being e valuated for the treatment of patients with relapsed or refractory multiple myeloma (developed by CureTech Ltd. Medivation Inc., Teva Pharmaceutical Industries)

Description: Immunoglobulin Gl , anti-( receptor PD- 1 (Programmed Death 1))

(human-mouse monoclonal CT-011 γΐ-chain), disulfide with human- mouse monoclonal CT-011 κ-chain, dirtier CAS Registry Number: 1036730-42-3

Compositions and methods of using and making pidilizumab are disclosed, for example, in U. S. Patent Nos. US7332582; US8686119; US8747847; US9309308 and US9416175, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of pidilizumab. In certain embodiments, the method is effective at sensitizing tumor cells to pidilizumab. In certain embodiments, the method changes (e.g., enhances) the efficacy of pidilizumab. In certain embodiments, the tumor cells are resistant to treatment with pidilizumab alone.

TABLE 7 provides non-limiting examples of pre-clinical inhibitors of the PD~ 1 immune checkpoint reported in the recited "Related Patent Documents, '' each one of which is hereby incorporated by reference herein in its entirety for any purpose

TABLE 7

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, hi certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of a preclinical inhibitor of PDl shown in TABLE 7. In certain embodiments, the method is effective at sensitizing tumor cells to the preclinical inhibitor of PDl, In certain embodiments, tiie metliod changes (e.g.. enhances) the efficacy of the preclinical inhibitor of PDl. In certain embodiments, the tumor cells are resistant to treatment with the preclinical inhibitor of PDl alone.

Exemplary PD-L1 immune checkpoint inhibitors that may be combined with a modulator of KRAS signaling, as disclosed herein, include, but are not limited, to:

Ateza!izumab

Synonym: Anti-PD-LI monoclonal antibody MPDL3280A

Description: Immunoglobulin Gl, anti -(human CD antigen CD274) (human

monoclonal MPDL3280A heavy chain), disulfide with human monoclonal MPDL3280A κ-chain, dimer

US brand name; Tecentriq™ (developed by Genentech/Hoffmann-La Roche) FDA approved for the treatment of bladder cancer

Code name: MPDL3280A, RG7446, R05541267, YW243.55.S70

CAS Registry Number: 1380723-44-3

Atezolizumab is a human, Fc optimized, monoclonal antibody directed against the protein ligand PD-Ll (programmed cell death- 1 ligand 1), with potential immune checkpoint inhibitory and antineoplastic activities. PD-Ll is overexpressed on many human cancer cell types and on various tumor-infiltrating immune cells, Atezolizumab can bind to PD-Ll, blocking its binding to and activation of its receptor programmed death 1 (PD-1) expressed on activated T-eells, which may enhance the T-cell-mediated immune response to neoplasms and reverse T-cell inactivation. In addition, by binding to PD-Ll, atezolizumab can also prevent binding of this ligand to B7. 1 expressed on activated T cells, which further enhances the T- cell-mediated immune response. The Fc region of atezolizumab is modified in such a. way that it does not induce either antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

Compositions and methods of using and making atezolizumab are disclosed, for example, in U.S. Patent No. 8,217.149, the content of which is hereby incorporated by reference herein in its entirety for any purpose

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g.. a KRAS aiRNA. as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of atezolizumab. In certain embodiments, the method is effective at sensitizing tumor cells to atezolizumab. In certain embodiments, the method changes (e.g., enhances) the efficacy of atezolizumab. In certain embodiments, the tumor cells are resistant to treatment with atezolizumab alone.

Avelumab

Synonym: Anti-PD-Ll monoclonal antibody

Description: Immunoglobulin G l, anti-(human CD antigen CD274) (human

monoclonal MSB0010718C heavy chain), disulfide with human monoclonal MSB0010718C light chain, aimer Code .name: MSBOG 10718C, A09-246-2 (developed by Merck Serono and Pfizer)

CAS Registry Number: 1537032-82-8

Avelumab is a human immunoglobulin Gl (IgGl) monoclonal antibody directed against the human immunosuppressive ligand programmed death-ligand 1 (PD-Ll) protein, with potential immune checkpoint inhibitory and antineoplastic activities. Upon administration, avelumab can bind to PD-Ll and prevent the interaction of PD-Ll with its receptor programmed cell death protein ί (PD- 1 ). This can inhibit the activation of PD-1 and its downstream signaling pathways. This may restore immune function through the activation of cytotoxic T-lymphocytes (CTLs) targeted to PD-Ll-overexpressing tumor cells. In addition, avelumab can induce an antibody-dependent cellular cytotoxic (ADCC) response against PD- Ll -expressing tumor cells.

Compositions and methods of using and making avelumab are disclosed, for example, in the published International Patent Publication Nos. WO2013079174 and WO2016137985, the contents of which are hereby incorporated by reference herein in their enti eties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of avelumab. In certain embodiments, the method is effective at sensitizing tumor cells to avelumab. In certain embodiments, the method changes (e. g., enhances) the efficacy of avelumab. In certain embodiments, the tumor cells are resistant to treatment with avelumab alone. Durvalumab

Code name: MEDI-4736, MEDI4736

(Developed by Medimmune/AstraZeneca/Celgene)

Description: Immunoglobulin GL anti-(human protein B7-H1) (human monoclonal

MEDI4736 heavy chain), disulfide with human monoclonal

MEDI4736 kappa-chain, dimer

CAS Registry Number: 1428935-60-7 Durvalumab is an Fc optimized monoclonal antibody directed against programmed cell death- 1 ligand 1 (PD-Ll; B7 homolog 1; B7H1), with potential immune checkpoint inhibitory and antineoplastic activities. Upon intravenous administration, durvalumab can bind to PD-Ll, thereby blocking its binding to and activation of its receptor programmed death 1 (PD-1) expressed on activated T-cells. This may reverse T-cell inactivation and activate the immune system to exert a cytotoxic T-lymphocyte (CTL) response against PD-Ll-expressing tumor cells. The Fc region of durvalumab is modified in such a way that it does not induce eitlier antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

Compositions and methods of making and using durvalumab are disclosed, for example, in U.S. Patent No. 8,779, 108 and the published U.S. Patent Application No. 2016/0222120, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, combined with an effective amount of durvalumab. In certain embodiments, the method is effective at sensitizing tumor cells to durvalumab. In certain embodiments, the method changes (e.g., enhances) the efficacy of durvalumab. In certain embodiments, the tumor ceils are resistant to treatment with durvalumab alone.

AMP-224

Code name: B7-DC Ig

(Developed by Medimmune/Amplimmune/ AstraZeneca)

Description: AMP-224 is a recombinant B7-DC- Fc-fusion protein composed of the extracellular domain of the PD- l ligand programmed cell death ligand 2 (PD-L2, B7-DC) and the Fc region of human immunoglobulin (Ig) Gl

CAS Registry Number: 1422184-00-6 AMP-224 is a recombinant B7-DC Fc-fusion protein that binds to PD-1. In a mouse model of colon cancer, an optimized treatment regimen with AMP-224 eradicated established tumors of the animals and conferred long-term protective anti-cancer immunity,

Compositions and methods of making and using AMP-224 are disclosed, for example, in the International Publication Nos. WO2010027827 and WO2011066342, the contents of which are hereby incorporated by reference herein in their entireties for any purpose,

In certain embodiments, the present, disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of AMP-224. In certain embodiments, the method is effective at sensitizing tumor cells to AMP-224. In certain embodiments, the method changes (e.g., enhances) the efficacy of AMP-224. In certain embodiments, the tumor cells are resistant to treatment with AMP-224.

MDX-1105

Code name: MDX- 1 105, BMS-936559, 12A4

(Developed by Medarex, (Bristol-Myers Squibb)

anti-PD-Ll antibody

CAS Registry Number: 1422185-22-5

MDX- 1105 is an antt-PD-L 1 antibody that is in Phase Ι/Π clini cal trials for the treatment of advanced cancers, including hematologic malignancies, melanoma, renal cell carcinoma and solid tumors (Brahmer et ah N. Engl. J. Med. (2012) 366:2455-65).

Compositions and methods of making and using AMP-224 are disclosed, for example, in U.S. Patent Nos. 7,943,743; 8,383,796; 9, 102,725; 9,212,224 and 9,273,135, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiinents, the present disclosure provides a method for enhancing an immune response against a tumor comprismg administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprismg administering a effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of MDX-1105. In certain embodiments, the method is effective at sensitizing tumor cells to MDX-1105. In certain embodiments, the method changes (e.g., enhances) the efficacy of MDX-1105. In certain embodiments, the tumor cells are resistant to treatment with MDX-1105, LY3300054

LY3300054 is an anti-PD-Ll antibody that has entered Phase I clinical trial in June 2016 for the treatment of advanced refractory solid tiunors (Clinical Trial ID; NCT02791334)

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of K AS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of LY3300054. In certain embodiments, the method is effective at sensitizing tumor cells to LY3300054. In certain embodiments, the method changes (e.g., enhances) the efficacy of LY33G0054. In certain embodiments, the tumor cells are resistant to treatment with LY3300054.

KN035

Alphamab is an anti-PD-Ll heavy chain antibody selected from a large camel naive phage displ y Nanobody library.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of Alphamab. In certain embodiments, the method is effective at sensitizing tumor cells to Alphamab. In certain embodiments, the method changes (e.g., enhances) the efficacy of Alphamab. In certain embodiments, the tumor cells are resistant to treatment with Alphamab.

TABLE 7 provides non-limiting examples of pie-clinical inhibitors of the PD-L1 immune checkpoint reported in the recited "Related Patent. Documents," each one of which is hereby incorporated by reference herein in its entirety for any purpose : Preclinical inhibi ors of 04.1 j Com any

Scientific iterature iCom positions and methods of jusing and making Mayo patent janti- B7-H1 are disclosed in

Mayo patent a nti-B7-Hl Mayo |US8460927; USS981063, the

[contents of which are hereby

[incorporated by reference herein jin their entireties for any purpose iiCom positions a nd methods of iiusing and making Dana-Farber jjpatent a nti-PD- Ll are disclosed in

Checkpoint |US20160108123; WO2016061142;

Therapeutics |US20160137731; WO2015061668;

|US20150274835; WO2014055897;

Dana-Farber patent anti-PD-Ll

Dana-Farber |W02014022758; US20150197571;

|U520130291136; US8552154; Novartis |W02010036959; US20H0271358,

Ijthe contents of which are hereby Incorporated by reference herein [ in their entireties for any purpose

Compositions a nd methods of I using and making STI-1014 are I disclosed in US9175082, the j iSTI-1014 Sorrento

content of which is hereby incorporated by reference herein j in its entirety for any purpose j

Com ositions a d mei jads of jj using and makin ftegerserofs j patent a nt¼£ _. &»U are disclosed if? I

Regeneron patent anti-PD-Ll Regeneron US20150203380; WO201 Sil OS, j

¾e contents-; of whic h are f r e ^' By j linear era ^' e ^: b;y reference erein I jin their entireties for any ur ose J

Compositions a nd methods of I using and making Kadmon patent j anti- PD-L1 are disclosed in j

Kadmon patent anti-PD-Ll Kadmon WO2015109124, the content of j ^;

which is hereby incorporated by Ϊ preference herein in its entirety for j jany purpose i

Merck patent anti-PD-Ll / TG F Compositions and ^' methods of j

Ij erck Serono

beta R! i using and making Merck patent I [ Re a ed Pa ent Documents/

: Preclinical Inhibitors of PO-ti mi Scie t fic Literature

lanti- PD-Ll / TG F beta Rl l are [[disclosed in WO2015118175, the [[content of which is hereby

[[incorporated by reference herein [[in its entirety for any purpose

[[Com positions a nd methods of [[using and making Merck patent anti- PD-Ll are disclosed in

[Merck patent anti-PD-Ll Merck & Co WO2014100079, the content of

[[which is hereby incorporated by Ireference herein in its entirety for [[any purpose

[Compositions a nd methods of [using and making Spring

Bioscience patent a nti- PD-Ll a re

Spring Bioscience patent anti- [Genentech disclosed in WO2015181342; PD-Ll [Spring Bioscience WO2016OO7235, the contents of which are hereby incorporated by reference herein in their entireties for any purpose

Compositions a nd methods of using and making R-Pha m patent anti- PD-Ll are disclosed in

R-Pharm patent a nti-PD- Ll R-Pharm WO2015195163, the content of which is hereby incorporated by reference herein in its entirety for any purpose

Compositions a nd methods of using and making BeiGene patent anti- PD-Ll are disclosed in

BeiGene patent anti- PD-Ll BeiGene WO2016000619, the content of which is hereby incorporated by reference herein in its entirety for any purpose

[Com positions a nd methods of [using and making B60-55 are [disclosed in Lua n et ai. Int.

[imm unopharmacol. (2016) 31:248

B60-55 DingFu Biota rget

[256, the content of which is

[hereby incorporated by reference [herein in its entirety for any

[purpose

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signal ing, e. g., a KRAS aiRNA, as defined herein, combined with an effective amount of a preclinical inhibitor of PD- Ll shown in TABLE 7. In certain embodiments, the method is effective at sensitizing tumor cells to the preclinical inhibitor of PD-L l . In certain embodiments, the method changes (e. g., enhances) the efficacy of the preclinical inhibitor of PD-L l . In certain embodiments, the tumor cells are resistant to treatment with the preclinical inhibitor of PD-L l alone. Exemplary PD-L2 immune checkpoint inhibitors that may be combined with a modulator of KRAS signaling, as disclosed herein, include, but are not limited, to: rIiIgM12B7

Status: Currently being evaluated in a Phase I clinical trial NCT00658892 for the treatment of Stage IV Melanoma.

Synonym: Mayo patent anti-PD-L2

A recombinan form of the monoclonal IgM antibody Ml 2 isolated from a Waldenstrom macroglobulinaemia patient (rHTgM12) with potential immunomodulating activity, B7-DC cross-linking antibody rHIgM12B7 binds and crosslinks the B7 co-stimulatory family member B7-DC (PD-L2) on dendritic cells (DCs), antigen presenting cells (APCs) that play a crucial role in the human immune response.

Compositions and methods of using and making rHIgM12B7 are disclosed in the published U.S. Patent Application No. 2016/0122431 and U.S. Patent No. 9,255, 147, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, in certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e. g., a KRAS aiRNA, as defined herein, combined with an effective amount of rHIgM12B7. In certain embodiments, the method is effective at sensitizing tumor cells to rHIgM12B7. In certain embodiments, the method changes (e.g., enhances) the efficacy of rHIgMI2B7. In certain embodiments, the tumor cells are resistant to treatment with rHIgM12B7 alone Dana-Farber patent anti-PD-L2 (preclinical)

Compositions and methods of using and making the Dana-Farber patent anti-PD-L2 are disclosed in the published U.S. Patent Application Nos. 2011/0271358; 2013/0291136; 2015/0197571 and 2016/0137731, the contents of which are hereby incorporated by reference herein in their entireties for any purpose. Exemplary CTLA-4 immune checkpoint inhibitors thai may be combined with a modulator of KRAS signaling, as disclosed herein, include, but are not limited, to:

Ipilimumab

Synonym: anti-cytotoxic T-lymphocyte-associated antigen-4 monoclonal

antibody; Monoclonal antibody CTLA-4 (MOAB CTLA-4)

US brand name: Yervoy™ (developed by Bristol-Myers Squibb)

FDA. approved for the treatment of melanoma

Code name: BMS-734016, MDX-010, MDX101 , MDX-CTLA-4, 10D1

Description: immunoglobulin GL anti-(human CTLA-4 (antigen)) (human γ1~

chain), disulfide with human -chain, dimer

CAS Registr Number: 477202-00-9

Ipilimumab is a recombinant human immunoglobulin (Ig) Gl monoclonal antibody directed against the human T-cell receptor cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), with immune checkpoint inhibitory and antineoplast c activities. Ipilimumab ca bind to CTLA4 expressed on T-cells and inhibit the CTLA4~mediated downregulation of T- cell activation. This can lead to a cytotoxic T- lymphocyte (CTL)-mediated immune response against cancer cells.

Compositions and methods of making and using i ilimumab are disclosed, for example, in the U. S. Patent Nos. 9,358,289; 9,320,81 1; 8,784,815; 9,062, 111; 8,685,394; 8,475,790; 8,119,129; 8,449,886; 8, 110, 194; 6,984,720 and the International Publication Nos. WO2015134603, WO2014066532, WO2013142796, WO2013019620, WO2012027536, WO2011045704, WO201 101 1027, WO2010042433, WO2009100140, WO2007067959, WO2007056539, WO2003086459, WO2001014424, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

hi certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amotsnt of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of ipilimumab. In certain embodiments, the method is effective at sensitizing tumor cells to ipilimumab. In certain embodiments, the method changes (e.g., enhances) the efficacy of ipjiimumab. In certain embodiments, the tumor cells are resistant to treatment with ipilimumab alone.

Tremelimumah

Synonym: anti-CTLA4 human monoclonal antibody CP-675,206; ticilimumab Description: inimuno lobulin G2, anti -(human CTLA-4 (antigen) (human

monoclonal CP-675206 clone 11.2. 1 heavy chain) disulfide with human monoclonal CP-675206 clone 11.2.1 light chain, dimer

Code name: CP-675; CP-675, 206 (developed by AstraZeneca)

CAS Registry Number: 4772.02-00-9

Tremelimumah is a human immunoglobulin (Ig) G2 monoclonal antibody directed against the human T-cell receptor protein cytotoxic T-lymphocyte-associated protein 4 (CTLA4), with potential immune checkpoint inhibitory and antineoplastic activities. Tremelimumah ca bind to CTLA4 on activated T-iymphocytes and block the binding of the antigen-presenting cell ligands B7-1 (CD80) and B7-2 (CD86) to CTLA4, resulting in inhibition of CTLA4-mediated downregulation of T-cell activation. This can promote the interaction of B7-1 and B7-2 with another T-cell surface receptor protein CD28, and result in a B7-CD28-mediated T-cell activation that is unopposed by CTLA4-mediated inhibition.

Compositions and methods of making and using tremelimumah are disclosed, for example, in U. S. Patent Nos. 8,883,984; 8,491,895; 8,685,394; 7,824,679; 8, 143,379; 7,411,057; 7, 132,281; 7, 109,003; 6,682,736 and the International Publication Nos. WO2016030455, WO2015173267, WO2011045704, O2007U3648, WO2006101691, WO2006101692, WO2006096491, WO2006048749, WO2005092380, the contents of which are e eb}' incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, combined with an effective amount of tremelimumah. in certain embodiments, the method is effective at sensitizing tumor ceils to tremelimumah. hi certain embodiments, the method changes (e.g., enhances) the efficacy of tremelimumab. In certain embodiments, the tumor cells are resistant to treatment with tremelimumab alone.

AGEN1884

AGEN-1884 developed by Agenus is an anti-CTLA~4 antibody that is Phase I clinical trials for the treatment of solid tumors (Clinical Trial ID: NCT02694822).

In certain embodiments, the present disclosure providss a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e. g. ₅ a KRAS aiRNA, as defined herein, combined with an effective amount of AGEN-1884. In certain embodiments, the method is effective at sensitizing tumor cells to AGEN-1884. in certain embodiments, the method changes (e.g., enhances) the efficacy of AGEN-1884. In certain embodiments, the tumor cells are resistant to treatment with AGEN-1884 alone.

TABLE 8 provides non-limiting examples of prc-clinical inhibitors of the CTLA-4 immune checkpoint reported in the recited "Related Patent Documents," each one of which is hereby incorporated by reference herein in its entirety for any purpose

2017/063116

Exemplary VISTA immune checkpoint inhibitors thai may be combined with a modulator of KRAS signaling, as disclosed herein, include, but are not limited, to: Janssen patent anti-VISTA antibody (preclinical)

Compositions and methods of making and using Janssen patent, anti-VISTA antibody are disclosed, for example, in WO2015097536, the content of which is hereby incorporated by reference herein in its entirety.

in certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embod ments, the present disclosure provides a method foi" enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of Janssen patent anti-VISTA a tibody. In certain embodiments, the method is effective at sensitizing tumor cells to Janssen patent anti-VISTA antibody. In certain embodiments, the method changes (e.g., enhances) the efficacy of Janssen patent anti-VISTA antibody. In certain embodiments, the tumor cells are resistant to treatment with Janssen patent anti-VISTA . ntibody alone.

Igenica patent anii-ClOorf5 ^' 4 antibody

Compositions and methods of making and using igenica patent anti-C10orf54 antibody are disclosed, for example, in O20.14197849 and WO2016094837, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein. In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of Igenica patent anii-C lOorf antibody. In certain embodiments, the method is effective at sensitizing tumor cells to Igenica patent anti-C10orf54. In. certain embodiments, the method changes (e.g., enhances) the efficacy of Igenica patent anti-C10orf54 antibody. In certain embodiments, the tumor cells are resistant, to treatment with Igenica patent anti-ClQorf54 antibody alone.

Amplimmune patent anti-B7-H5 antibody

Compositions and methods of making and using Amplimmune patent anti-B7-H5 antibody are disclosed, for example, in WO2014190356 and the published U.S. Patent Application No. 20.16/0096891, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, hi certain embodiments, the present disclosure provides a method for enhancing an immune response against a tumor comprising administering an effective amount of a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, combined with an effective amount of Igenica patent Amplimmune patent anti-B7-H5 antibody. In certain embodiments, the method is effective at sensitizing tumor cells ^' to Ampl immune patent anti-B7-H5 antibody, m certain embodiments, the method changes (e.g., enhances) the efficacy of Amplimmune patent anti-B7-H5 antibody. In certain embodiments, the tumor cells are resistant to treatment with Amplimmune patent anti-B7-H5 antibody alone.

In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a co-administered therapeutic agent.

In certain embodiments, the change in the efficacy of a therapeutic agent, as a result of the co- dministration of a modulator of KRAS signaling as defined herein, can be evaluated in subcutaneous tumor animal models at endpoints such as the percent test/control (%T/C) tumor weights calculated on each day that tumors are measured, tumor growth delay, net log cell kill, median days to a defined tumor weight or to a specified number of tumor doublings, and tumor regression. In certain embodiments, the lowest calculated %T/C seen over time can be defined as the optimal %T/C because it defines the greatest level of activity seen with the therapeutic agent. The rate and duration of partial and complete tumor regressions can also be considered to be clinically relevant endpoints.

For example, a T/C = 0% means no tumor growth. A T/C = 100% means no antitumor activity, i.e., the treated and control tumors grew equally. A T/C equal to or less than 42% is considered significant antitumor activity by the Drag Evaluation Branch of the Division of Cancer Treatment (NCI). A T/C value < 10% is considered to indicate highly significant antitumor activity, and is the level used by NCI to justify a clinical trial if toxicity, formulation, and certain other requirements are met (termed DN- 2 level activity). In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KR AS aiRNA, wherein the modulator of KRAS signaling changes (e.g.. enhances) the efficacy of radiotherapy, In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, can be combined with radiotherapy. In certain embodiments, the method is effective at enhancing the efficacy of the radiotherapy.

In certain embodiments, the present disclosure provides a method of treating cancer comprising admiriistering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of an anti-angiogenesis agent. In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with an anti-angiogenesis agent. In certain embodiments, the method is effective at enhancing the efficacy of the anti-angiogenesis agent.

Non-limiting examples of anti-angiogenesis agents include, for example, MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (inatrix- etalloproteinase 9) inhibitors, and COX- 11 (eyelooxygenase 11.) inhibitors, rapamycin, temsirolimus (CCI-779). everoiiraus (RADOOl), sorafenib, sxinitinib, and bevacizumab. COX-Π inhibitors include but are not limited to, Celebrex™ (alecoxib), valdecoxib, and rofecoxib.

Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172, WO 96/27583, WO 98/07697, WO 98/035 16, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, PCX International Application No. PCT IB98/01113, U. S. Patent Nos. 5,863,949 and 5,861,510, all of which are incorporated by reference herein in their entireties for any purpose. Examples of MMP inhibitors include, but are not limited to, AG-3340, RO 32-3555, and RS 13-0830.

In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a chemotherapeutic agent. I certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent includes, but not limited to, antimetabolites, antibiotics, alkylating agents, plant alkaloids, and/or hormonal agents. In certain embodiments, the method is effective at enhancing the efficacy of the chemotherapeutic agent. Non-limiting examples of ehemotherapeulie agents (e.g., suitable for use in compositions and methods described herein) include, but are not limited to:

.* alkaloids, including isiicrotubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (Taxol), and docetaxel (taxotsre), etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxins (e.g., etopossde ^' (VP- 16), and teniposide (VM-26), etc.), and agents that target topoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.);

« covalent DN A- binding agents (alkylatmg agents), inchiding nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosphami.de, and busulfan (Myleran). etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g., dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin, etc.);

* noncovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubic n (daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g., anthracycline analogues, such as mitoxantrone, etc.), bleomycins (Blenoxane), etc., and piicamycin (mithramycin), etc.;

* antimetabolites, including antifolaies (e.g., methotrexate, Folex, and Mexate, etc.), purine antimetabolites (e.g., 6-mercaptopurme (6-MP, Purinethol), 6-thioguanine (6~ TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2- chlorodeoxyadenosine (CdA), and 2'-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g., fiuoropyrimidinea (e.g., S-fluorouracil (Adracil), 5- iluorodeox uridine (FdUrd) (floxuridine)) etc.), and cylosine arabinosides (e.g., Cytosar (ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase, and hydroxyurea, etc.;

« hormones, including glucocorticoids, antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g., fmtamide, etc.), and aromatase inhibitors (e.g., anastrozole (Arimidex), etc.);

* platinum compounds (e.g., cisplatin and carboplatin, etc.);

In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of RAS signaling changes (e.g., enhances) the efficacy of a cancer sternness inhibitor. In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer sternness inhibitor.

As used herein, the term "cancer sternness inhibitor" means a molecule that can target, reduce, inhibit, interfere with, or modulate at least one of a plurality of pathways involved in cancer sternness or the expression (e.g.. the production of a functional product, e.g., a protein) of at least one of a plurality of cancer sternness genes,

In certain embodiments, a cancer sternness inhibitor can be, for example, a small molecule that selectively binds a protein encoded by a cancer sternness gene. In certain embodiments, a cancer sternness inhibitor is a biologic, e.g., a recombinant binding protein or peptide (e.g. APTSTAT3) or nucleic acid (e.g. STAT3 aiRNA see U.S. Patent No, 9,328,345, the content of which is incorporated by reference herein in its entirety for any purpose), or conjugate thereof. In certain embodiments, a cancel- sternness inhibitor is a cell. In certain embodiments, a cancer sternness inhibitor is a STAT3 inhibitor that binds to and inhibits a biological activity of STAT3, In certain embodiments, STAT3 refers to mammalian STATS, In certain embodiments. STATS refers to the human "Signal Transducer and Activator of Transcription 3" having a canonical 770 amino acid sequence (Accession No: P40763-1 ; MP 644805. 1). STATS, as used herein, includes pSTAT3 or pSTATS dimer.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, can be combined with, for example, a cancer sternness inhibitor. In certain embodiments, a cancer sternness inhibitor can be, for example, a compound chosen from 2-(l- hydroxyethyl)-naphtho[2,3~b]fui'an-4,9-dione, 2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9- dione, 2-aeelyl-7-fluoro-naphlho[2,3-b]furan-4,9-dione ₅ 2-acetylnaphtho[2.3-b]furan-4,9- dionc, or 2-ethyI-naphtho[2,3-b]furan-4,9-dione, prodrugs thereof, derivatives thereof. pharmaceutically acceptable salts of any of the foregoing, and solvates of any of the foregoing.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer sternness gene inhibitor chosen from compounds having formula A, shown below,

(A) or prodrugs, derivatives, pharmaceutically acceptable salts or solvates thereof.

The compound having formula A may also be known as 2- cetylnaphtho[2,3-b]furan- 4,9-dione, napabucasin, or BBI608 and include tautomers thereof. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a K AS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of 2-ac«tylnaphtrio[2 _: ,3~b]fura3i--4,9-'dione.

Non-limiting examples of prodrugs of compounds having formula A include, for example, the phosphoric esters and phosphoric dicstcrs described in U.S. pre-grant Publication No. 2012/0252763 as compound numbers 4011 and 4012 and also suitable compounds described in U.S. Patent No. 9, 150,530. Non-limiting examples of derivatives of compounds having formula A include, for example, the derivatives disclosed in U.S. Patent No. 8,977,803. The disclosures of U.S. pre-grant Publication No. 2012/0252763 and U.S. Patent Nos. 9, 150,530 and 8,977,803 are incorporated by reference herein in their entireties for any purpose.

' Suitable methods of preparing 2~acetylnaphtho[2,3~h]fui¾n-4,9-dione, including its crystalline forms and additional cancer sternness inhibitors, are described in the co-owned PCT applications published as WO 2009/036099, WO 2009/036101, WO 2011/116398, WO 2011/116399, and WO 2014/169078; the contents of each of these applications are incorporated by reference herein by reference in their entireties for any purpose.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, atovaquone, a hydroxy naphthoquinone having the structure of

or prodrugs, derivatives, pharmaceutically acceptable salts or solvates thereof.

In certain embodiments, atovaquone can downregulate cell-surface expression of glycoprotein 130 (gpl30), which is required for the activation of the cancer sternness gene, STAT3 (see, for example, WO 2015050844 Al, the content of which is hereby incorporated by reference in its entirety for any purpose).

in certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, can be combined with an RNA interfering agent that targets a cancer sternness gene, e.g. STATS aiRNA U.S. Patent No. 9,328,345 provides exemplary asymmetric interfering RNA duplexes (aiRNA) and uses thereof to silence STATS expression and treat cancer. The disclosure of US, Patent No, 9.328,345 is hereby incorporated by reference herein in its entirety for any purpose In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a STATS aiRNA.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with 2-acetylnaphtho[2,3-b]furan-4,9-dione thai targets the cancer sternness gene, e.g. STATS. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA. in combination with 2-acetylnaphtho[2,3-b3furan-4,9-dione wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of 2-acetymaphtho[2,3- b]furan-4,9-dione.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with atovaquone that downregulates cell-surface expression of glycoprotein 130 (gpl30), which is required for the activation of the cancer sternness gene, STATS. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS' signaling, e.g., a KRAS aiRNA wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of atovaquone.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA as defined herein, can be combined with, for example, an inhibitor of a cancer stem cell pathway kinase (CSCPK) such as for example, STK33, MELK, AXL, p70S6K, and PDGFRo. In certain embodiments, an inhibitor of a cancer stem cell pathway kinase (CSCPK) can be, for example, an inhibitor, and derivatives thereof, that inhibit STK33, disclosed in U.S. Patent No. 9, 187,434, which is hereby enclosed herein by its entirety for any purpose

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, an aiRNA that targets one or more cancer stem cell pathway kinases (CSCPK). In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a CSCPK.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA that targets STK33, In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets STK33.

in certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA that targets MELK. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets MELK.

in certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA that targets AXL. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets AXL.

in certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA thai targets p70S6K. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets p70S6K.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA that targets PDGFR. In certain embodiments, the present disclosure provides a method of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets PDGFR.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase aiRNA that, targets NANOG, In certain embodiments, the present disclosure provides a metliod of treating cancer comprising administering a modulator of KRAS signaling, e.g., a KRAS aiRNA, wherein the modulator of KRAS signaling changes (e.g., enhances) the efficacy of a cancer stem cell pathway kinase aiRNA that targets NANOG.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase inhibitor including, but not limited to, compounds, or derivatives thereof, disclosed in U.S. Patent No. 8,299, 106 and PCT Patent Application Publication No. WO2014160401. The disclosures of U.S. Patent No. 8,299, 106 and PCT Patent Application Publication No. WO2014160401 are incorporated by reference herein by reference in their entireties for any purpose.

In certain embodiments, a modulator of KRAS signaling, e.g., a KRAS aiRNA, as defined herein, can be combined with, for example, a cancer stem cell pathway kinase inhibitor chosen from compounds having formula B, shown below,

(B)

or prodrugs, derivatives, pharmaceutically acceptable salts or solvates thereof. The present disclosure reports on the surprising discovery that a treatment combination of at least one modulator of K AS signaling and a therapeutic agent have a greater effect in inhibiting cancer cells than the added effects of both the at least one modulator of KRAS signaling and the at least one therapeutic agent alone,

The present disclosure reports on the surprising discovery that a treatment combination of at least one modulator of KRAS signaling and an immune checkpoint inhibitor have a greater effect, in inhibiting cancer cells than the added effects of both the at least one modulator of KRAS signaling and. the at least one immune ^' checkpoint inhibitor alone.

The present disclosure reports on the surprising discovery that a treatment combination of at least one modulator of KRAS signaling and an immunotlierapeutic agent have a greater effect in inhibiting cancer cells than the added effects of ^' both the at least one modulator of KRAS signaling and the at least, one immunotlierapeutic agent alone.

The present disclosure reports on the surprising discovery that a treatment combination of at least one modulator of KRAS signaling and at least one cancer sternness inhibitor have a greater effect in inhibiting cancer cells than the added effects of both the at least one modulator of KRAS signaling and the at least one cancer sternness inhibitor alone.

The present disclosure reports on the surprising discovery that, a treatment combination of at least one modulator of KRAS signaling and at least one cancer stem cell pathway kinase inhibitor have a greater effect in inhibiting cancer cells than the added effects of both the at least one modulator of KRAS signaling arid the at least one cancer stem cell pathway kinase inhibitor alone.

The compositions disclosed herein may be in the form of a pharmaceutical composition. The present disclosure further provides for a pharmaceutical composition comprising an RNA interfering agent, e.g. an aiRNA. The pharmaceutical comprises (as an active agent) at least one RNA interfering agent, e.g., an asymmetrical duplex RNA molecule. In certain embodiments, the pharmaceutical compositions may comprise a modulator of KRAS signaling, e.g. a KRAS aiRNA, and at least one immunotlierapeutic agent. In certain embodiments, the pharmaceutical compositions may comprise a modulator of KRAS signaling, e.g. a KRAS aiRNA, and at least one immune checkpoint inhibitor. In certain embodiments, the pharmaceutical compositions may comprise a modulator of KRAS signaling, e.g. a KRAS aiRNA, and at least one cancer sternness inhibitor, In certain embodiments, the pharmaceutical compositions may comprise a modulator of KRAS signaling, e.g. a KRAS aiRNA, and at least one cancer stem cell pathway kinase (CSCPK) inhibitor.

in certain embodiments, the pharmaceutical compositions may comprise a modulator of KRAS signaling, e.g. a KRAS aiRNA, and one or more compounds and at least one pharmaceutically acceptable carrier, where the one or mors compounds are capable of being converted into the at least one therapeutic agent, for example, a cancer sternness inhibitor in a subject, a cancer stem cell pathway kinase (CSCPK) in a subject or an immunotherapeutic agent, in a subject (i.e., a prodrug).

In some embodiments, the pharmaceutical composition comprises one or mors carriers selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanopartid , a nanoemulsion, a lipid, and a lipoid, in some embodiments, the composition may comprise more than RNA interfering agent species for therapeutic applications. In certain embodiments, the RNA interfeiing agent species may target different genes required for cancer initiation and progression chosen from, for example, a modulator of KRAS signaling as disclosed herein, in combination with RNA interfering agent(s) that target one or more cancer sternness genes and/or cancer stem cell kinase as disclosed herein.

The pharmaceutical compositions and formulations of the present disclosure can be the same or similar to the pharmaceutical compositions and formulations developed for siRNA, miRNA, and antisense RNA, except for the RNA ingredient. For example, the siRNA, mi R A, and antisense RN A in the pharmaceutical compositions and formulations can be replaced by the aiRNA molecules of the present disclosure. The pharmaceutical compositions and formulations can also be further modified to accommodate the RNA interfering agent, e.g. aiRNA, of the present disclosure.

A "pharmaceutically acceptable salt" or "salt" of an RNA interfering agent, e.g. aiRNA, is a product of the disclosed RNA interfering agent that contains an ionic bond, and is typically produced by reacting the disclosed RNA interfering agent with either an acid or a base, suitable for administering to a subject. Pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonat.es, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, Li, alkali earth metal salts such as Mg or Ca., or organic amine salts. A "pharmaceutical composition" is a formulation containing the disclosed RNA interfering agent, e.g. aiRNA, in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a .tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient, (e.g., a formulation of the disclosed duplex RNA molecule or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular ^' treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for the topical, or transdermal administration of a RNA interfering agent, e.g. aiRNA, of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active RNA interfering agent, e.g. aiRNA, is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

The present disclosure provides a method of treatment comprising adm nisteiing an effective amount of the pharmaceutical composition to a subject in need thereof. In some embodiments, the pharmaceutical composition is administered via a route selected from the group consisting of iv, sc, topical, po, and ip. In another embodiment, the effective amount is 1 ng to 1 g per day, 100 ng to 1 g per day. or 1 ug to 1 mg per day.

The present disclosure also provides pharmaceutical formulations comprising a RNA interfering agent, e.g. aiRNA, of the present disclosure in combination with at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA,, which is incorporated by reference herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active RNA interfering agent, e.g. aiRNA, use thereof in the compositions is contemplated. Supplementary active RNA interfering agents, e.g. aiRNAs, can also be incorporated into the compositions.

An RNA interfering agent, e.g. aiRNA, of the present disclosure is administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an efficacious level sufficient to achieve the desired therapeutic effect through inhibition of tumor growth, killing of tumor cells, treatment, or prevention of cell proliferative disorders, etc.) of a RNA interfering agent, e.g. aiRNA, of the present disclosure (as an active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the disclosure). These procedures may involve mixing, granulating, compressing, or dissolving the ingredients as appropriate to attain the desired preparation, h another embodiment, a therapeutically effective amount of a RNA interfering agent, e.g. aiRNA, of the present disclosure is administered in a suitable dosage form without standard pharmaceutical carriers or diluents.

Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like.- Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this disclosure.

The term "solvate" represents an aggregate that comprises one or more molecules of a compound of the present disclosure with one or more molecules of a solvent or solvents. Solvates of the compounds of the present disclosure include, for example, hydrates.

The pharmaceutical compositions containing active RNA interfering agent, e.g. aiRNA, of the present disclosure may he manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries which facilitate processing of the active duplex RNA molecules into preparations thai can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

An RNA interfering agent, e.g. aiRNA, or pharmaceutical composition of the disclosure can be administered to a subject in many of the well-known methods currently used for ehemoihsrapeutic treatment. For example, for treatment of cancers, a RNA interfering agent, e.g. aiRNA, of the present disclosure rnity be injected directly into tumors, injected into the blood stream or body cavities, taken orally, or applied through the skin with patches.

In certain embodiments, aiRNA nanopartieies may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form' ^' as used herein refers to a physically discrete unit of nanoparticle appropriate for the patient, to be treated. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physic ian within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of nanopartieies can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in. 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

Compositions suitable for parenteral administration may comprise at least one more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, baeteriosfats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

When in vivo administration of the polypeptides or antibodies described herein are used, normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of mammal body weight or more per day, preferably about 1 mg kg/day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212, the contents of which are hereby incorporated by reference herein in its entireties for any purpose. hi various embodiments, a composition described herein includes at least one compound and pharmaceutically acceptable salts and solvates thereof and one or more surfactants, in certain embodiments, the surfactant, is sodium lauryi sulfate (SLS), sodium dodecyl sulfate (SDS), or one or more polyoxylgiycerides. For example, the pol oxylglyeeride can be lauroyl polyoxylgiycerides (sometimes referred, to as Gelucke™) or iinoleoyl polyoxylgiycerides (sometimes referred to as Labrafil™). Examples of such compositions are disclosed in PCT Patent. Application No. PCT US2014/033566, the content of which is incorporated by reference herein in its entirety for any purpose.

The present disclosure provides further embodiments of suitable pharmaceutical formulations having selected particle size distribution and methods for identifying an optimum particle size distribution, suitable drug regimen, dosage and interval, suitable methods of preparing 2-acetylnaphtho[2,3-6]furan-4,9-dione including their crystalline forms, and further specific suitable cancer sternness inhibitors and kinase inhibitors as described in the co-owned PCT applications published as WO 2009/036099, WO 2009/036101 , WO 201 1/1 16398, WO 201 1/116399, WO 2014/169078, and WO 2009/033033, the contents of which are hereby incorporated by reference herein in their entireties for any purpose.

In certain embodiments, a kit is disclosed that comprises (1) at least one compound chosen from a modulator of KRAS signaling and (2) an immune checkpoint inhibitor, an immunotherapeutic, a chemotherapsutic agent, a cancer sternness inhibitor, a cancer stem cell pathway kinase inhibitor or prodrugs thereof, derivatives thereof, pharmaceutically acceptable salts of any of the foregoing, together with instructions for administration and/or use.

EXAMPLES

Examples are provided below to further illustrate different features of the present disclosure. The examples also illustrate useful methodology for practicing the disclosure. These examples do not limit the claimed disclosure.

EXAMPLE 1: KRAS SILENCING IS SUFFICIENT TO DEPLETE PD-L1

EXPRESSION IN KRAS MUTANT CELLS Asymmetric interfering RNA (aiRNA) silences target genes potently and specifically with minimal off-target effects. aiRNAs targeting KRAS were therefore used to determine the effects of KRAS depletion on PD-L1 expression in KRAS mutant MDA-MB-231 cells expressing endogenous PD-L1.

aiRNAs were first synthesized in DMT-on mode. Following completion of the synthesis, the solid support was suspended in 600 μΐ EtOH/MHkOH solution (prepared by mixing 1 volume of 200 proof ethanol wit 3 volumes of 28% NH4OH) and heated at 55 °C for 2 hours. After primary de-protection, EtOH/NiliOH was evaporated and the RNA oligo was dried to a pellet ΙΟΟμΙ of RNA de-protection solution (NMP/TEA.3HF (3:2)) was added and the solution was heated at 65 °C for 1.5 hours. The reaction was then quenched with 400 μΐ of 1.5 M ammonium bicarbonate. Purification was performed with Clarity® QSP Cartridges (Phenomenex, USA). The annealing of the resulting duplexes was confirmed on 15% PAGE gel. All sequences are shown in FIG. 1A. The location of each of the sequences targeted by aiKRAS is shown in FIG. IB.

The human breast adenocarcinoma ceil line, MDA-MB-231, that is heterozygous for the KRAS GOD mutation, was obtained from ATCC (ATCC® CRM-HTB-26™) and maintained in DMEM supplemented with 10% (vol/Vol) FBS (Gemini Bio Products, USA) and 1% penicillin/streptomycin (Life Technologies, USA).

MDA-MB-231 cells were then transfected with multiple aiRNAs targeting KRAS (aiKRAS# l-#4). Cells were seeded to 60mm plates (2 x 10 ⁵ cells/4 mL/well). aiRNA was transfected by Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's protocol. aiRNA and RNAiMAX were incubated for 20 mi in serum free ΟΡΊΊ-ΜΕΜ (Thermo Fisher, USA), then added to the cells along with culture medium, 48 hours after transfection, cells were harvested using Aecutase® cell detachment solution (Sigma Aldrich, USA). Scrambled aiRNA was used as a control. The amount of PD-L1, KRAS and actin protein was then determined by Western blot analysis.

The cells were washed twice with ice cold PBS and lysed in lysis buffer [50 mM Hepes (pH 7.5), 1% Nonidet P-40, 150 mM NaCL 1 mM EDTA, and l x protease inhibitor mixture (EMD Millipore, USA)]. Soluble protein (20 μ ) was separated by SDS PAGE and transfen-ed to PVDF membranes and probed with commercially available antibodies specific for actin (Sigma Aldrich, USA), KRAS and PD-LI (Santa Craz Biotechnology, inc., USA). As shown in FIG. 2A, Iransfection of the KRAS aiRNAs resulted in a marked reduction in the amount of PD-L1 even at concentration of ΙΟΟρΜ (Fig. 1A),

The amount of KRAS and PD-Ll mRNA in the aiKRAS RNA. transfected cells was then determined by quantitative RT-PCR. Cells transfected with aiKRAS# l-#4 or scrambled control aiRNA, as described above, were seeded in six-well plates with culture media. At the appropriate time points, cells were harvested and RNA extracted using the SimplyRNA kit (Promega, USA.) according the manufacturer's protocol. Reverse transcription was performed on l|ig of RNA from each sample using the GoScript reverse transcription kit (Promega, USA). Real-time PGR was carried out using the RT2 q CR Primer Assays (SABiosciences, USA) except for KRAS. Primers of KRAS were synthesized by integrated DNA Technologies, US KRAS primer sequences were as follows: 5 '-CCTACT AGGACCAT AGGT ACATCT-3 ' (SEQ ID NO.: 982) and 5 ' -AGGGCTTTCTTTGTGT ATTTGC-3 ' (SEQ ID NO.: 983). Genes of interest were normalized to the expression of β-2-microglobulin (B2M).

As shown in FIG. 2B, the level of PD-Ll mRNA was also down -regulated by KRAS silencing with aiKRAS R s 1-4. These results suggested that. KRAS silencing down- regulated PD-Ll expression in a transcript on factor dependent manner. Time course experiments showed that aiRNA mediated KRAS silencing was induced very quickly arid reached maximum activity 8 hours post-transfection whereas PD-Ll was down-regulated only after 32 hours post-transfection (Fig. 2C) suggesting the presence of intermediate steps between KRAS silencing and the subsequent reduction in PD-Ll mRNA levels.

To investigate the dependency of PD-Ll downregulation on KRAS mutation status, three KRAS mutant cancer cell lines (H358, H460, and H2009) and two KRAS wild-type cancer cell lines (RKO and TCCSUP) were transfected with aiKRAS#l or aiControl (FIG. 2D). As observed earlier in MDA-MB-231 cells, PD-Ll expression was down-regulated with KRAS depletion in KRAS mutant cancer cells at both the protein and mRNA level (Fig. 2D and Fig. 2E). In contrast, PD-Ll expression protein expression in KRAS wild-type cancer cells was not altered, even though KRAS was potently silenced by aiRNA (Fig. 2D and 2E). Thus oncogenic KRAS mutant but not wild-type KRAS is essential to maintain PD-Ll expression in KRAS mutant cancer cells.

EXAMPLE 2: KRAS MAINTAINS PD-Ll EXPRESSION THROUGH

ENDOGENOUSLY ACTIVATED MEK/ER PATHWAY Mutant KRAS activates various down-stream signaling pathways including the RAF/MEK ERK pathway, the P13K/AKT pathway, and the RalGTP pathway. To determine the effect of KRAS depletion on downstream signaling ©vents, aiKRAS or aiControl transfected KRAS mutant MDA-MB-231 and KRAS wild-type RKO cells were washed twice with ice cold PBS and lysed in lysis buffer [50 mM Hepes (pH 7.5), 1% Nonidet P-40, 150 mM NaCi, 1 mM EDTA, and 1.x protease inhibitor mixture (EMD Miilipore, USA)]. Soluble protein was then probed using a Human Phospho-Kinase Antibody Array according to the manufacturer's instructions (R&D Systems; Catalog # ARY003B). Among the anti- phosphokinase antibodies spotted on the array, phosphorylated ERK 1/2 was most prominently down-regulated with KR AS depletion in KRAS mutant MDA-MB-231 cells but not in KRAS wild-type RKO cells (Fig. 3 A),

To confirm the effect, of KRAS silencing on the RAF/MEK ERK pathway, the phosphorylation state of MEK/ERK was determined by Western blot. Soluble protein (20 ug) from aiKRAS or aiControl transfected KRAS mutant MDA-MB-231 and KRAS wild-type RKO cell tysates was separated by SDS/PAGE and transferrsd to PVDF membranes and probed with actin (Sigma Aldrich, USA) KRAS (Santa Cruz Biotechnology, Inc., USA), Phospho-MEKl/2, MEK1/2, Phospho~p44/42 MARK (Erkl/2), p44/42 MAPK (Erkl/2) antibodies (Cell Signaling Technology, Inc., USA). As shown in FIG- 4A, KRAS silencing downregulated the phosphorylation of both MEK and ERK only in KRAS mutant MDA-MB- 231 cells but not in RKO cells.

To explore further the role of the RAF MEK/ERK pathway on PD-Ll expression, KRAS mutant MDA-MB-231 cells were treated with the MEK-specific inhibitor, U0126. Similar to aiKRAS, U0126 also down-regulated PD-Ll expression in MDA-MB-23 1 cells and inliibited ERK phosphorylation (Fig. 4B). These results indicate the ERK pathway is involved in KRAS dependent PD-Ll expression in KRAS mutant cancer cells.

EXAMPLE 3: KRAS MAINTAINS PD-Li EXPRESSION THROUGH

EJNDOGENOUSLY ACTIVATED API

ELISA based detection ofAP-1 transcript-fan factor activation

The down-regulation of PD-Ll mRNA levels by KRAS silencing suggested that PD- LI expression levels were maintained by a transcriptional factor regulated by mutant KRAS. Among the transcriptional factors regulated by RAS and ERK, one of the most well studied transcriptional factors is Activator protein 1 (AP- 1). The AP-! transcriptional factor is formed by either the homo-dimerization of a Jun family protein (eJun, JunB, or JunD) or by the more stable hetero-dimerization of a Jun family protein with a Fos family protein (cFOS, FGSB, FRA- 1, or FRA-2). In RAS activated cells, the accumulation of stabilized FRA- 1 was reported to be dependent on phosph rylation by ERK1/2.

To investigate the effect, of KRAS depletion on FRA-1 expression, ai-KRAS# l was transfected into KRAS mutant cancer cell lines (MDA-MB-231, H358, and H460) and a KRAS wild- type cancer ceil line (RKO). Immunoblotting analysis with a Phospho-FRA-1 and FRA- 1 antibody (Cell Signaling Technology, Inc., USA) showed thai KRAS depletion down- regulated the phosphorylated and total forma of FRA- 1 in MDA-MB-231, H358, and H460 cells harboring KRAS mutations but not in KRAS wild-type RKO cells (Fig.5 A). This result confirmed that FRA-1 is a KRAS dependent transcriptional factor as reported previously by Casalino et l, (2003).

The effect of KRAS silencing on DNA binding activity of FRA-1 was then quantified by ELISA using the Trans- AM AP-1 transcription factor assay (Active Motif, USA). Nuclear extract was prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA). 5p of nuclear extracts were incubated with coated immobilized oligonucleotides containing a 12-0-tetradecanoylphorbol-13-acetate (TP A) response element (TRE) with the 5'-TGA(C/G) TCA-3' (SEQ ID NO. : 984) sequence. To ensure the specificity of the assay, 20pM of consensus oligonucleotides were incubated in parallel as competition experiments.

As shown in FIG. 5B, KRAS silencing reduced the endogenous FRA-1 binding activity in KRAS mutant MDA-MB-231 cells but not in KRAS wild-type RKO cells. This result was consistent with the observed down-regulation of FRA-1 expression by KRAS silencing in KRAS mutant cells (Fig. 4A).

Intron 1 of the PD-L1 gene, located approxim tely 5kb downstream from the transcription start site, contains a conserved candidate enhancer element with putative API binding sites. To determine if FRA-1 could bind to the putative PD-L1 enhancer in a KRAS dependent manner, a chromatin immunoprecipitation (ChIP) quantified PGR assay was performed in the presence of anti-phospho-Fra- 1 or cJun antibody using SimpleChIP® Enzymatic Chromatin IP Kit (Cell signaling Technologies. USA) according to the manufacturer's protocol. aiRNA transfected MDA-MB-231 cells were cross-linked by treatment with 1% formaldehyde in culture medium for IGmin at room temperature. After washing with cold PBS, nuclear pellets were prepared by treating the cells with Buffers A and B, The nuclear pellets were then suspended in buffer B and treated with SDS buffer and micrococcal nuclease to digest chromatin for 20 min at 37°C. Cell suspensions were then sonicated to elute nuclear chromosomal content in preparation for immunoprecipitation. 2 ,g each of Phospho-FRA-1 and cJun were incubated with the lysate overnight at 4°C. Immunocomplexes were collected with magnetic protein A beads and eluted after extensive washing. Cross-linkage was reversed by heating the solution at 65°C with NaCl and proteinase K. DNA was purified and used as the template for quantitative PGR to amplif ' the region including the AP-1 binding site. The primer pairs used were 5'- GTC AC ATTTC AAGC AGG ATG ACT A-3 ' (SEQ ID NO. : 985) and 5'- GGAAGGGGAGAGAG TTGGATT-3 ' (SEQ ID NO. : 986). Quantitative PGR. was performed as described in Example 1.

As shown in FIG. 5C (left side), both FRA- 1 and cJUN were recruited to candidate PD~ LI enhancer sites in MDA-MB-231 cells. Binding of FRA-1 and cJUN to the PD-LI enhancer decreased with KRAS depletion. In contrast, as with PD-L1 gene expression. FRA-1 and cJUN binding to the PD-LI enhancer was not. altered by KRAS depletion in KRAS wild-type RKO cells (Fig. 5C, right side).

To determine the effect of FRA- 1 silencing on PD-LI expression, aiFra-l#L aiFra- l#2 (see FIG. 1 A) were transfected into KRAS mutant MDA-MB-231 and KRAS wild-type RKO cells, iramunoblotting and quantitative RT-PCR confirmed that transfection of aiFra-l#l or #2 resulted in depletion of FRA-1 protein and mR A (FIG. 6 A and 6B). Moreover, knockdown of FRA-1 also resulted in a marked down-regulation of PD-LI protein and mRNA expression (Fig. 6A and 6B).

STAT3 or NF-KB are also known to promote the expression of PD-LI .mRNA by binding directly to the PD-LI promoter region. However, unlike as with FRA- 1, KRAS silencing did not alter the expression of either STAT3 or NF-KB (see FIGs. 4C and 4D).

These results indicate that PD-LI expression was maintained in KRAS mutant cells via the RAS/ERK/Fra-1 signaling pathway (see FIG. 8B). EXAMPLE 4: KRAS SILENCING IS SUFFICIENT TO RESTORE SENSITIVITY OF KRAS MUTANT CANCER CELLS TO ΑΝΊΊ-PD-Ll OR TO CTL KILLING

The functional significance of these results on cancer immune evasion was assessed using luciferass expressing KRAS mutant MDA-MB-231 cells transiected with control aiRNA or ai RAS# l.

Luc-MDA-MB-231, a cell line stably expressing luciferase, was purchased .from Cell Biolabs, lac, USA Luc-MD A-MB-231 cells were grown in Dulb cco's modified Eagle's medium (D EM) supplemented with 10% inactivated fetal calf serum (FCS) and transfected with either control aiRNA or aiKRAS#l as described in Example 1.

Cell surface expression of PD-L1 in the control aiRNA or aiKRAS#l transfected Luc- MDA-MB-231 cells was determined by staining the cells with PE conjugated anti-CD274 (BD Biosciences, USA) in Stain Buffer (BD Biosciences, USA) on ice for 20 min and washed once with Sta n Buffer. CD274 positive population was then detected using flow cytometry (Attune Acoustic Focusing Cytometer, Life Technologies, USA) and analyzed with Flowjo software (Flow] o, LLC, LISA). Flow cytometry analysis confirmed that KRAS silencing down-regulated cell surface PD-L1 expression (Fig, 7B).

To determine if KRAS silencing altered T cell cytotoxicity, aiRNA-transfected MDA- MB-231 cells were either pulsed or not pulsed with CMV antigen peptide, and then co-cultured with CMV specific cytotoxic T lymphocytes (CTLs) expanded from HL A typed human PBMC from healthy donors,

HLA typed PBMCs from healthy donor were purchased from Cellular Technology, Ltd., USA PBMCs were diluted at 5 x 10 ⁶ cells/mL in culture medium (RPMI-1640 supplemented with 10% inactivated human serum and 50 μΜ 2-mercaptoethanol) and seeded into 24-well plates (5 x 10 ⁶ cells/mL/well). HLA-A*02:01 CMV pp65 peptide (NLVPMVATV) (IB A Lif ©sciences, Germany) was added to a final concentration of 5 μΜ on day 0 as well as 23 !U/mL IL-2 (R&D Systems, USA) and 5 ng/mL 3L-15 (R&D Systems, USA). The cultures were supplemented with fresh medium containing 10 μΜ HLA-A*02:01 CMV pp63 peptide, 50 lU/mL IL-2 and 10 ng/mL IL- 15 every 3-4 days. CD8 ⁺ T cells were isolated from PBMC using a CD8 ⁺ T cell Isolation Kit (Miltenyi Biotec, USA) as described by the manufacturer's protocol. The aiRNA transfected Luc-MDA-MB-23 1 cells were incubated with or without HLA- A*02:01 C V pp65 peptide (NL VPMV AT V) for 2 hours at 37°C in 5% CO2 and then washed twice with PBS, CMV peptide loaded or unloaded Luc-MDA-MB-23 1 cells were plated into 96-well plates (2000 cells/well). CMV specific CD8 ⁺ T cells were subsequently added to a 96- well plate with an Effector: Target (E/T) ratio of 50: 1 and incubated for 24 hours. Anti-Human PD-L 1 Ab at a final concentration of was used as a control (Clone: ΜΓΗ1, Functional grade purified, Affymetrix eBioseienee, USA). Live Luc-MDA-MB-23 ί cells were measured by intracellular luciferase activity with XenoLight D-Luciferin K ^v sait (PerkinElmer, USA) by GloMax® Discovery (Promega, USA). The percent Lysis was calculated with the formula: (Luminescence of CMV peptide pulsed Luc-MDA-MB-231/ Luminescence of CMV peptide un-puised Luc-MDA-MB-23 1)* 100.

As shown in FIG-. 7 A, the cytotoxic activity of antigen specific CTLs were significantly enhanced with aiKRAS transfection (pO.01) compared with scramble aiRNA transfected cells. To validate PD-L 1 dependency of the observed cytotoxic activity, aiRNA targeting PD- L I was compared to the activity of a commercially available functional grade anti-PD-L l antibody. Both treatments enhanced CTL killing activity against MDA-MB-231 cells (Fig. 7A) to a level that was comparable to the cytotoxic activity seen with KRAS depletion.

The results support the conclusion that KRAS activates immune evasion of cancer cells by maintenance of constitutive PD-L1 expression in the cancer cells. EXAMPLE S: , PREPARATION OF KRAS aiRNA NANOP ARTICLES (NPs) aiRNAs were synthesized in DMT-on mode as described in Example 1, For certain applications, selected nucleotides in the aiRNAs were modified with a 2' -OH methyl group.

Nanoparticles (NPs) loaded with aiRNA were prepared using the double-emulsion process described in WO2014123935, the content of which is hereby incorporated by reference herein in its entirety.

Specifically, in each of vials A and B, 5 mL of cyciohexane/Igepal CO-520 solution (71/29 v/v) were prepared from reagents respectively available from EMD and Sigma.

aiRNA was dispersed in Ix RNAse-free buffer to make the desired concentrat n (e.g., about 5 μ /'μΙ). A volume of aiRNA (e.g., about 50 p.g) was mixed with 100 uJL of 500 mM MgCk. Then, the MgCk-aiRNA solution was added drop-wise to the oil/surfactant solution in vial A to form a well-dispersed emulsion without reverse micro-emulsion. In vial B, 100 uL- of 25 mM NaaHPCH (pH=9) was added drop-wise to the oil/surfactant solution. The contents of vials A and B were then mixed and stirred for 30 minutes at room temperature. Afterwards, the contents were transferred into 10 centrifuge tubes ( 1.5 mL) and centrifuged for 30 minutes at 13,000g. The supernatant was discarded and the pellet was washed with absolute ethanol (1 mL-) twice. After the alcohol was removed, the resulting pellet was air-dried for 3-4 hours,

A polymer- based shell was coated onto the MgP nanoparticle cores already loaded with aiRNA Specifically, biodegradable polymers, PEG(5k)-Poly-L-Lysine ( lOU), Poly-L- Arginine (SOU) were coated onto the cores at a polymer ratio of 2.5 : 1 (PLL: PLR) & a complex ratio of 2.5: 1 (polymer: aiRNA).

Measurement for size Pdl determination of liposomes was performed. The average size of the nanoparticl.es was about 70 nm and the surface charge was about +25 mV. The nanoparticles exhibited good plasma stability, cellular uptake and endosomal escape.

EXAMPLE 6: INHIBITION OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF RADIATION THERAPY

The human breast adenocarcinoma cell line, MDA-MB-231 (ATCC® CRM-HTB- 26™) is cultured in DMEM supplemented with 10% (vol/vol) FBS (Gemini Bio Products, USA) and 1% penicillin/streptomycin (Life Technologies, USA). Approximately, 6 X 10 ⁶ cells /mouse are then inoculated subcutaneously into female athymic nude mice.

Xenografted mice are randomized into the following treatment groups: .

Group 1: Control aiRNA NP

Group 2: Radiotherapy + saline ^■

Group 3: aiKRAS NP + Control aiRNA NP

Group 4: aiKRAS NP + Radiotherapy

Irradiations are performed 7- 10 days after inoculation (when tumors were at least 100 mm ³ ) using a Pantak HF-320 320 kV x~ray unit, (Gulmay Medical, U.K.). The machine is operated at 300 kV, 9.2 rnA, with filtration fitted in the x-ray beam to give a radiation quality of 2.3 mm Cu half -value layer. Mice are positioned at a distance of 350 mm from the x-ray focus, where the dose rate is 0.80 Gy/min. The intravenous administration of nanoparticle (NP) preparations comprising either KRAS aiRNAs or an aiRN A control commences on day 1 of the fractionated radiation therapy cycle and is repeated every other day for 8 days (at a dose of 2.5 mg/kg/NP preparation). The treatment is well tolerated with minimal adverse side effects. The size of the tumors is measured every day. Experimental groups contain at least 5 mice/group and are representative of at least 2 independent experiments.

Low doses of local fractionated dose radiotherapy are delivered at about. 10 Gy in 5 fractions leads to increased tumor cell expression of PD-L1 with elevated expression evident 1, 3 and 5 days after the last dose of radiotherapy when compared to time-matched mice receiving the control aiRNA NP. Radiotherapy increases tumor cell PD-L1 expression which peaks at about 72 hours after the last dose of radiation and remains elevated as compared to non-treated control mice.

The radiotherapy-mediated local tumor control is improved tlirough the administration of aiKRAS#l-#4 nanoparticle formulation. However, a synergistic anti-tumor response with a T/C value < 10% is observed in mice that receive radiotherapy in combination with the aiKRASirl-#4 NP as compared to mice that received radiotherapy in combination with the scrambled control aiRNA formulation.

EXAMPLE 7: INHIBITION OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF A CHEMOTHERAPEUTIC

AGENT

Approximately, 6 X 1() ⁶ human breast adenocarcinoma cells (MDA-MB-231 (ATCC® CRM-HTB-26™)) /mouse are inoculated s beutaneously into female ath mic nude mice. Treatment starts when the tumor burden (mouse weight) reaches approximately 20 grams (~21 days after injection of cells).

Xenografted mice are randomized into the following treatment groups:

Group 1: Control aiRNA NP + saline

Group 2: Control aiRNA NP + paclitaxel

Group 3: aiKRAS# l-#4 NP ÷ saline

Group 4: aiKRAS#l-#4 NP + paclitaxel

Nanoparticle (NP) preparations comprising either aiKRAS#l-#4 or an aiRNA control are administered by intravenous injection to the xenografted mice of Groups L 3 and 4 starting on day 1. The aiR A injections are repeated every other day for 12 days (i.e., a dose of 2, 5 mg/kg/NP preparation on day L 3, 5, 8, and 10).

Paclitaxel (Taxol®) is administered to Groups 2 and 4 by intraperitoneal injection on day 2, 4, 6, 9 and 11 at a dose of 10 mg/kg preparation.

The treatment is well tolerated with miniinal adverse side effects, Xeno grafted animals are periodically weighed to assess the effect of treatment on tumor burden. Experimental groups contain 10 mice/group and are representative of at, least 2 independent experiments.

The intraperitoneal injection of paclitaxel alone or aiKRAS NP alone results in a delay in tumor progression. However, the administration of aiKRAS#l-#4 NP together with paclitaxel to the xenografted mice of Group 4 results in a synergistic anti-tumor response with a T/C value < 10% when compared to time- matched, mice receiving either the aiKRAS NP alone with saline or paclitaxel with control aiRNA NP.

EXAMPLE 8: INHIBITIO OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF AN IMMUNE CHECKPOINT RNA INTERFERING AGENT

Approximately, 6 X 10 ⁶ human breast adenocarcinoma cells (MDA-MB-231 (ATCC® CRM-HTB-26™)) /mouse are inoculated subcutaneously into female athymic nude mice. Treatment starts when the tumor burden (mouse weight) reaches approximately 20 grams (—21 days after injection of cells).

Xenografted mice are randomized into the following treatment groups:

Group 1: Control aiRNA NP

Group 2; aiPD-Ll NP + Control aiRNA NP

Group 31 aiKRAS NP + Control aiRNA NP

Group 4: aiKRAS NP + aiPD-Ll NP

Nanoparticle (NP) preparations comprising either PD-L1 aiRNA (e.g. comprising aiRNA sequences SEQ ID NOs. : 972 and 973), KRAS aiRNA, or an aiRNA control are administered to Groups 1-4 by intravenous injection starting on day 1. The aiRNA injections are then repeated every other day for 12 days, i.e., at a dose of 2.5 mg/kg/NP preparation on day I, 3, 5, 8, and 10). Xenografted animals are periodically weighed to assess the effect of treatment on tumor burden. The treatment is well tolerated with minimal adverse side effects. Experimental groups contain .10 mice/group and are representative of at least 2 independent experiments.

Intravenous injection of the aiKRAS NP with control aiRN A NP (Group 3) or aiPD-Ll NP with control aiRNA NP (Group 2) results in a delay of tumor progression. However, the administration of aiKRAS NP together wit PD-L1 aiRNA to the xenografted mice of Group 4 results in a synergistic anti-tumor response with a T/C value < 10% when compared to time- matchsd, mice receiving either the aiKRAS NP with control aiRNA NP (Group 3) or aiPD-Ll NP with control aiRNA NP (Group 2).

EXAMPLE 9: INHIBITION OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF A SMALL MOLECULE

IMMUNE CHECKPOINT INHIBITOR

Approximately, 6 X 10 ⁶ human breast adenocarcinoma ceils (MDA-MB-231 (ATCC® CR -HTB-26™)) /mouse are inoculated subcutaneously into female athymic nude mice. Treatment, starts when the tumor burden (mouse weight) reaches approximately 20 grams (~21 days after injection of cells).

The small molecule Immune checkpoint inhibitor used in this example is Compound No. 75 of WO2016142833, havin the structure of:

Compound Nos.75 rescues the PD-Ll induced inhibition of cell proliferation in a mouse splenocyte assay by 119% (see Table I of WO2016142833).

Xenografted mice are randomized into the following treatment groups:

Group 1: Control aiRNA NP + saline

Group 2: Control aiRNA NP + Compound No. 75

Group 3: aiKRAS NP† saline

Group 4: aiKRAS NP + Compound No, 75

Nanoparticle (NP) preparations comprising either KRAS aiRNA or an aiRNA control are administered to Groups 1-4 by intravenous injection starting on da)' 1. The aiRNA injections are then repeated every other day for 12 days, i.e., at a dose of 2.5 mg/kg/NP preparation on clay 1, 3, 5, 8, and 10).

Compound No. 75 is administered orally at a dail}' dose of 10 mg fcg for 12 days.

Xenografted animals are periodically weighed to assess the effect of treatment on tumor burden. The treatment is well tolerated with minimal adverse side effects. Experimental groups contain 10 mice/group and are representative of at least 2 independent experiments.

Administration of either aiKRAS NP with saline or Compound No. 75 with the aiRNA control NP results in a delay in tumor progression. However, the administration of aiKRAS NP together with Compound No. 75 to the xenografted mice of Group 4 resul ts in a synergistic anti-tumor response with a T/C value < 10% when compared to time-matched, mice receiving either the aiKRAS NP with saline or Compound No. 75 with the aiRNA control NP.

EXAMPLE 10: INHIBITION OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF AN ANTI-PD- LI ANTIBODY

Mice are inoculated subeutaneously with 0.5 10 ⁶ MC38.0va cells on Day 0. On Day 1 or on Day 14 mice (when tumors reach an average size of 250 mm ³ ), the xenografted mice are randomized into the following treatment groups ( 10 mice/group):

Group 1: Control Ig + Control aiRNA NP

Group 2: Atezolizumah + Control aiRNA NP

Group 3: Control Ig + aiKRAS NP

Group 4: atezolizumah + aiKRAS NP

Nanoparticle (NP) preparations comprising either KRAS aiRNA or an aiRNA control are administered to Groups 1-4 by intravenous injection starting on day 1. The aiRNA injections are then repeated every other day for 14 days, i.e., at a dose of 2.5 mg kg NP preparation on day 1, 3, 5, 8, 10 and 12),

Anti-PD-Ll (atezolizumah; Genentech/Hoffmann-La Roche) or control Ig is injected 3 x/week for 2 weeks at a dose of 10 mg/kg/NP preparation.

Xenografted animals are periodically weighed to assess the effect of treatment on tumor burden. The treatment is well tolerated with minimal adverse side effects. Experimental groups contain 10 mice/group and are representative of at least 2 independent experiments. Administration of alezolizumab and control aiRNA NP (Group 2) or control ig and aiKRAS NP (Group 3) results in a delay in tumor progression. However, the administration of aiKRAS NP together with atezolizumab to the xenografted mice of Group 4 results in a synergistic anti-tumor response with a T/C value < 10% when compared to time-matched, mice receiving either the atezolizumab and control aiRNA NP (Group 2) or control Ig and aiKRAS NP (Group 3).

EXAMPLE 11: INHIBITION OF KRAS SIGNALING ENHANCES THE

THERAPEUTIC EFFICACY OF A CANCER STEMMESS INHIBITOR

FaDu human head and neck cancer cells were inoculated subcutaneously into female athymic nude mice (6*106 cells/mouse) and allowed to form palpable tumors. When the tumors reached approximately 100 mm ³ , the xenografted mice are randomized into the following treatment groups (10 mice/group):

Group 1 : vehicle control + Control aiRNA NP

Group 2: BBI-608+ Control aiRNA NP

Group 3: vehicle control ^■ +· aiKRAS NP

Group 4: BBI608 + aiKRAS NP

Naiioparticle (NP) preparations comprising either KRAS aiRNA or an aiRNA control are administered to Groups 1-4 by intravenous injection starting on day 1. The aiRNA injections are theo repeated every other day for 14 days, i.e., at a dose of 2.5 mg/kg/NP preparation on day 1, 3, 5, 8, 10 and 12).

Animals are treated orally (po) with 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-di-one

(ΒΒΪ608) at 100 mg kg or vehicle control daily. BBI-608 was formulated at 10 mg/ml in 20% geSueire, Suitable methods of preparing 2-acetylnaphlho[2,3-b]furan-4,9-dione, mcluding its crystalline forms, are described in the co-owned PCT applications published as WO 2009/036099, WO 2009/036101, WO 2011/116398, WO 2011/116399, and WO 2014/169078. Xsnugrailsd animals are periodically weighed to assess the effect of treatment or! tumor burden. The treatment is well tolerated with minimal adverse side effects. Experimental groups contain 10 mice/group and are representative of at least 2 independent experiments.

Administration of BBI608 and control aiRNA NP (Group 2) or vehicle control and aiKRAS NP (Group 3) results in a delay in tumor progression. However, the administration of aiKRAS NP together with BBI-608 to the xenografted mice of Group 4 results in a synergistic anti-tumor response with a T/C value < 10% when compared to time-matched, mice receiving either the BBI608 and control aiRNA NP (Group 2) or vehicle control and aiKRAS NP (Group

3). EXAMPLE 12: KRAS SILENCING ENHANCES THE THERAPEUTIC

EFFICACY OF CANCER STEM CELL PATHWAY INHIBITOR

SW480 colon cancer cells are inoculated subcutaneously into male athyinic nude mice (8 x 10 ^s cells/mouse) and allowed to form palpable tumors. Once the tumors readied approximately 200 mm ³ , the xenograftsd mice are randomized into the following treatment groups (10 mice/group):

Group 1: vehicle control - Control aiRNA NP

Group 2: BBI503+ Control aiRNA NP

Group 3: vehicle control + aiKRAS NP

Group 4: ΒΒΪ503 + aiKRAS NP

Nanoparticle (NP) preparations comprising either KRAS aiRNA or an aiRNA control are administered to Groups 1-4 by intravenous injection starling on day 1. The aiRNA injections are then repeated every other day for 14 days, i.e., at a dose of 2.5 mg/kg NP preparation on day 1, 3, 5, 8. 10 and 12).

Animals are treated orally (po) at 5 mg/kg or vehicle control daily with the cancer stem cell kinase (CSCK) inhibitor (BBI503) having the structure of:

Xeno grafted animals are periodically weighed to assess the effect of treatment on tumor burden. The treatment is well tolerated with minimal adverse side effects. Experimental groups contain 10 mice/group and are representative of at least 2 independent experiments. Administration of BBI503 and control aiRNA NP (Group 2) or vehicle control and aiKRAS NP (Group 3) results in a delay in tumor progression. However, the administration of aiKRAS NP together with BBI-503 to the xenografted mice of Group 4 results in a synergistic anti-tumor response with a T/C value < 10% when compared to time-matched, mice receiving either the BBI503 and control aiRNA NP (Group 2) or vehicle control and aiKRAS NP (Group 3).