SMC COMBINATION THERAPY FOR THE TREATMENT OF CANCER

BACKGROUND OF THE INVENTION

The death of cells by apoptosis (or programmed cell death), and other cell death pathways, is regulated by various cellular mechanisms. Inhibitor of apoptosis (lAP) proteins, such as X-linked lAP (XIAP) or cellular lAP proteins 1 and 2 (clAP1 and 2), are regulators of programmed cell death, including (but not limited to) apoptosis pathways, e.g., in cancer cells. Other forms of cell death could include, but are not limited to, necroptosis, necrosis, pyroptosis, and immunogenic cell death. In addition, these lAPs regulate various cell signaling pathways through their ubiquitin E3 ligase activity, which may or may not be related to cell survival. Another regulator of apoptosis is the polypeptide Smac. Smac is a proapoptotic protein released from mitochondria in conjunction with cell death. Smac can bind to the lAPs, antagonizing their function. Smac mimetic compounds (SMCs) are non-endogenous proapoptotic compounds capable of carrying out one or more of the functions or activities of endogenous Smac.

The prototypical XIAP protein directly inhibits key initiator and executioner caspase proteins within apoptosis cascades. XIAP can thereby thwart the completion of apoptotic programs. Cellular lAP proteins 1 and 2 are E3 ubiquitin ligases that regulate apoptotic signaling pathways engaged by immune cytokines. The dual loss of clAP1 and 2 can cause TN Fa, TRAIL, and/or IL-1 β to become toxic to, e.g., the majority of cancer cells. SMCs may inhibit XIAP, clAP1 , clAP2, or other lAPs, and/or contribute to other proapoptotic mechanisms.

Treatment of cancer by the administration of SMCs has been proposed. However, SMCs alone may be insufficient to treat certain cancers. There exists a need for methods of treating cancer that improve the efficacy of SMC treatment in one or more types of cancer.

SUMMARY OF THE INVENTION

The present invention includes compositions and methods for the treatment of cancer by the administration of an SMC and an immunostimulatory, or immunomodulatory, agent. SMCs and agents are described herein, including, without limitation, the SMCs of Table 1 and the agents of Table 2, Table 3, and Table 4.

One aspect of the present invention is a composition including an SMC from Table 1 , and one or more (e.g., two, three, four, five, or more) agents, wherein each agent is independently an immune checkpoint inhibitor (ICI) or is an agent from Table 2 or angent from Table 3 or is a STING agonist. In some embodiments, the ICI is an ICI from Table 4, .The SMC and the agent(s) are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In some embodiments, the two, three, or four agents are from different categories (i.e., one agent is an ICI, one agent is from Table 2, one agent is from Table 3, and/or one agent is a STING agonist).

Another aspect of the present invention is a method for treating a patient diagnosed with cancer, the method including administering to the patient an SMC from Table 1 and one or more (e.g., two, three, four, five, or more) agents, wherein each agent is independently an ICI or is an agent from Table 2 or angent from Table 3 or is a STING agonist. In some embodiments, the ICI is an ICI from Table 4, such that the SMC and the agent are administered. In some embodiments, the two, three, or four agents are from different categories (i.e., one agent is an ICI, one agent is from Table 2, one agent is from Table 3, and/or one agent is a STING agonist), simultaneously or within 28 days of each other in amounts that together are sufficient to treat the cancer.

In some embodiments, the SMC and the agent(s) are administered within 14 days of each other, within 10 days of each other, within 5 days of each other, within 24 hours of each other, within 6 hours of each other, or simultaneously.

In particular embodiments, the SMC is a monovalent SMC, such as LCL161 , SM-122, GDC- 01 52/RG741 9, GDC-091 7/CUDC-427, or SM-406/AT-406/Debio1 143. In other embodiments, the SMC is a bivalent SMC, such as AEG40826/HGS1 049, OICR720, TL3271 1 /Birinapant, SM-1387/APG-1387, or SM-1 64.

In particular embodiments, one of the agents is a TLR agonist from Table 2. I n certain embodiments, the agent is a lipopolysaccharide, peptidoglycan, or lipopeptide. In other embodiments, the agent is a CpG oligodeoxynucleotide, such as CpG-ODN 221 6. In still other embodiments, the agent is imiquimod or poly(l :C).

In particular embodiments, one of the agents is a virus from Table 3. In certain embodiments, the agent is a vesicular stomatitis virus (VSV), such as VSV-M51 R, VSV-MA51 , VSV-I FN , or VSV-I FN -NIS. In other embodiments, the agent is an adenovirus, maraba vesiculovirus, reovirus, rhabdovirus, or vaccinia virus, or a variant thereof. In some embodiments, the agent is a Talimogene laherparepvec, a variant herpes simplex virus.

In particular embodiments, one of the agents is an ICI. In certain embodiments, the agent is Ipilimumab, Tremelimumab, Pembrolizumab, Nivolumab, Pidilizumab, AM P-224, AM P-51 4, AUN P 1 2, PDR001 , BGB-A31 7, REGN281 0, Avelumab, BMS-935559, Atezolizumab, Durvalumab, BMS-986016, LAG525, IMP321 , MBG453, Lirilumab, or MGA271 .

In some embodiments, a composition or method of the present invention includes a plurality of immunostimulatory or immunomodulatory agents, including but not limited to interferons, and/or a plurality of SMCs.

In some embodiments, a composition or method of the present invention includes one or more interferon agents, such as an interferon type 1 agent, an interferon type 2 agent, and/or an interferon type 3 agent.

In any method of the present invention, the cancer can be a cancer that is refractory to treatment by an SMC in the absence of an immunostimulatory or immunomodulatory agent. In any method of the present invention, the treatment can further include administration of a therapeutic agent including an interferon.

In any method of the present invention, the cancer can be a cancer that is selected from adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, cancer of the head and neck, hepatocellular carcinoma, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplasia syndrome, multiple myeloma, oral cavity cancer, ovarian cancer, paediatric cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, cancer of the respiratory system, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and cancer of the urinary system.

The invention further includes a composition including an SMC from Table 1 and one or more (e.g., two, three, four, or more) agents described above. One of the agents may include a killed virus, an inactivated virus, or a viral vaccine, such that the SMC and the agent are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In particular embodiments, the said agent is a NRRP or a rabies vaccine. In other embodiments, the invention includes a composition including an SMC from Table 1 , a first agent that primes an immune response, and a second agent that boosts the immune response, such that the SMC and the agents are provided in amounts that together are sufficient to treat cancer when administered to a patient in need thereof. In certain embodiments, one or both of the first agent and the second agent is an oncolytic virus vaccine. In other particular embodiments, the first agent is an adenovirus carrying a tumor antigen and the second agent is a vesiculovirus, such as a Maraba-MG1 carrying the same tumor antigen as the adenovirus or a Maraba-MG1 that does not carry a tumor antigen.

"Neighboring" cell means a cell sufficiently proximal to a reference cell to directly or indirectly receive an immune, inflammatory, or proapoptotic signal from the reference cell.

"Potentiating apoptosis or cell death" means to increase the likelihood that one or more cells will apoptose or die. A treatment may potentiate cell death by increasing the likelihood that one or more treated cells will apoptose, and/or by increasing the likelihood that one or more cells neighboring a treated cell will apoptose or die.

"Endogenous Smac activity" means one or more biological functions of Smac that result in the potentiation of apoptosis, including at least the inhibition of clAP1 and clAP2. It is not required that the biological function occur or be possible in all cells under all conditions, only that Smac is capable of the biological function in some cells under certain naturally occurring in vivo conditions.

"Smac mimetic compound" or "SMC" means a composition of one or more components, e.g., a small molecule, compound, polypeptide, protein, or any complex thereof, capable of inhibiting clAP1 and/or inhibiting clAP2. Smac mimetic compounds include the compounds listed in Table 1 .

To "induce an apoptotic program" means to cause a change in the proteins or protein profiles of one or more cells such that the amount, availability, or activity of one or more proteins capable of participating in an lAP-mediated apoptotic pathway is increased, or such that one or more proteins capable of participating in an lAP-mediated apoptotic pathway are primed for participation in the activity of such a pathway. Inducing an apoptotic program does not require the initiation of cell death per se:

induction of a program of apoptosis in a manner that does not result in cell death may synergize with treatment with an SMC that potentiates apoptosis, leading to cell death.

"Agent" means a composition of one or more components cumulatively capable of inducing an apoptotic or inflammatory program in one or more cells of a subject, and cell death downstream of this program being inhibited by at least clAP1 and clAP2. An agent may be, e.g. , a TLR agonist (e.g., a compound listed in Table 2), a virus (e.g., a virus listed in Table 3), such as an oncolytic virus, or an immune checkpoint inhibitor (e.g., one listed in Table 4). "Treating cancer" means to induce the death of one or more cancer cells in a subject, or to provoke an immune response which could lead to tumor regression and block tumor spread (metastasis). Treating cancer may completely or partially abolish some or all of the signs and symptoms of cancer in a subject, decrease the severity of one or more symptoms of cancer in a subject, lessen the progression of one or more symptoms of cancer in a subject, or mediate the progression or severity of one or more subsequently developed symptoms.

"Prodrug" means a therapeutic agent that is prepared in an inactive form that may be converted to an active form within the body of a subject, e.g. within the cells of a subject, by the action of one or more enzymes, chemicals, or conditions present within the subject.

By a "low dosage" or "low concentration" is meant at least 5% less (e.g., at least 1 0%, 20%, 50%,

80%, 90%, or even 95%) than the lowest standard recommended dosage or lowest standard recommended concentration of a particular compound formulated for a given route of administration for treatment of any human disease or condition.

By a "high dosage" is meant at least 5% (e.g., at least 1 0%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition.

"Immune checkpoint inhibitor" means a cancer treatment drug that prevents immune cells from being turned off by cancer cells by antagonistically blocking respective receptors or binding their ligands thus re-establishing the immune system's capacity to attack a tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-1 F are a set of graphs and images showing that SMC synergizes with oncolytic rhabdoviruses to induce cancer cell death. All panels of FIG. 1 are representative of data from at least three independent experiments using biological replicates (n=3). FIG. 1 A is a pair of graphs showing the results of Alamar blue viability assays of cells treated with LCL1 61 and increasing MOIs of VSVA51 . Error bars, mean ± s.d. FIG. 1 B is a set of micrographs of cells treated with LCL161 and 0.1 MOI of VSVA51 -GFP. FIG. 1 C is a pair of graphs showing viability (Alamar Blue) of cells infected with VSVA51 (0.1 MOI) in the presence of increasing concentrations of LCL1 61 . Error bars, mean ± s.d. FIG. 1 D is a pair of graphs showing data from cells that were infected with VSVA51 for 24 hours. Cell culture supernatant was exposed to virus-inactivating UV light and then media was applied to new cells for viability assays (Alamar Blue) in the presence of LCL1 61 . Error bars, mean ± s.d. FIG. 1 E is a graph showing the viability of cells co-treated with LCL1 61 and non-spreading virus VSVA51 AG (0.1 MOI). Error bars, mean ± s.d. FIG. 1 F is a graph and a pair of images relating to cells that were overlaid with agarose media containing LCL1 61 , inoculated with VSVA51 - GFP in the middle of the well, and infectivity measured by fluorescence and cytotoxicity was assessed by crystal violet staining (images were superimposed; non-superimposed images are in FIG. 1 1 ). Error bars, mean ± s.d.

FIGS. 2A-2E are a set of graphs and images showing that SMC treatment does not alter the cancer cell response to oncolytic virus (OV) infection. All panels of FIG. 2 are representative of data from at least three independent experiments using biological replicates. FIG. 2A is a pair of graphs showing data from cells that were pretreated with LCL1 61 and infected with the indicated MOI of VSVA51 . Virus titer was assessed by a standard plaque assay. FIG. 2B is a pair of graphs and a set of micrographs captured over time from cells that were treated with LCL1 61 and VSVA51 -GFP. The graphs plot the number of GFP signals over time. Error bars, mean ± s.d. n=12. FIG. 2C, is pair of graphs showing data from an experiment in which cell culture supernatants from LCL161 and VSVA51 treated cells were processed for the presence of I FN by ELISA. Error bars, mean ± s.d. n=3. FIG. 2D is a pair of graphs showing data from an experiment in which cells were treated with LCL1 61 and VSVA51 for 20 hours and processed for RT-qPCR to measure interferon stimulated gene (ISG) expression. Error bars, mean ± s.d. n=3. FIG. 2E is a pair of images showing immunoblots for STAT1 pathway activation performed on cells that were pretreated with LCL161 and subsequently stimulated with I FN .

FIGS. 3A-3H are a set of graphs showing that SMC treatment of OV-infected cancer cells leads to type 1 interferons (type 1 I FN) and nuclear-factor kappa B (NF-Kb)-dependent production of proinflammatory cytokines. All panels of FIG. 3 are representative of data from at least three independent experiments using biological replicates (n=3). FIG. 3A is a graph showing Alamar blue viability assay of cells transfected with combinations of nontargeting (NT), TNF-R1 and DR5 siRNA and subsequently treated with LCL1 61 and VSVA51 (0.1 MOI) or I FNp. Error bars, mean ± s.d. FIG. 3B is a graph showing the viability of cells transfected with NT or I FNAR1 siRNA and subsequently treated with LCL1 61 and

VSVA51 AG. Error bars, mean ± s.d. FIG. 3C is a graph showing data from an experiment in which cells were pretreated with LCL1 61 , infected with 0.5 MOI of VSVA51 , and cytokine gene expression was measured by RT-qPCR. Error bars, mean ± s.d. FIG. 3D is a chart showing data collected from an experiment in which cytokine ELISAs were performed on cells transfected with NT or I FNAR1 siRNA and subsequently treated with LCL161 and 0.1 MOI of VSVA51 . Error bars, mean ± s.d. FIG. 3E is a graph showing the viability of cells co-treated with LCL161 and cytokines. Error bars, mean ± s.d. FIG. 3F is a graph showing data from an experiment in which cells were pretreated with LCL1 61 , stimulated with 250 U/mL (~20 pg/mL) I FN and cytokine m RNA levels were determined by RT-qPCR. Error bars, mean ± s.d. FIG. 3G is a pair of graphs showing the results of cytokine ELISAs conducted on cells treated with LCL1 61 and 0.1 MOI of VSVA51 . FIG. 3H is a graph showing the result of cytokine ELISAs performed on cells expressing ΙΚΚβ-DN and treated with LCL1 61 and VSVA51 or I FN . Error bars, mean ± s.d.

FIGS. 4A-4G are a set of graphs and images showing that combinatorial SMC and OV treatment is efficacious in vivo and is dependent on cytokine signaling. FIG. 4A is a pair of graphs showing data from an experiment in which EMT6-Fluc tumors were treated with 50 mg/kg LCL1 61 (p.o.) and, 5x1 0 ⁸ PFU VSVA51 (i.v.). The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m. n=5 per group. Log-rank with Holm- Sidak multiple comparison: **, p < 0.01 ; ***, p < 0.001 . Representative data from two independent experiments are shown. FIG. 4B is a series of representative I VIS images that were acquired from the experiment of FIG. 4A. FIGS. 4C and 4D are sets of immunofluorescence images of infection and apoptosis in 24 hour treated tumors using a- VSV or a-c-caspase-3 antibodies. FIG. 4E is an image showing an immunoblot in which protein lysates of tumors from the corresponding treated mice were immunoblotted with the indicated antibodies. FIG. 4F is a pair of graphs showing data from an experiment in which mice bearing EMT6-Fluc tumors were injected with neutralizing TNFa or isotype matched antibodies, and subsequently treated with 50 mg/kg LCL1 61 (p.o.) and 5x1 0 ⁸ PFU VSVA51 (i.v.). The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m. Vehicle a-TNFa, n=5; SMC a-TNFa, n= 5; vehicle+VSVA51 , n=5; a-TNFa, n=5; SMC+VSVA51 a-TN Fa, n=7; SMC+VSVA51 a-lgG, n=7. Log-rank with Holm-Sidak multiple comparison: ***, p < 0.001 . FIG. 4G is a set of representative I VIS images that were acquired from the experiment of FIG. 4F.

FIGS. 5A-5E are a series of graphs and images showing that small molecule immune stimulators enhance SMC therapy in murine cancer models. FIG. 5A is a graph showing the results of Alamar blue viability assays of EMT6 cells which were co-cultured with splenocytes in a transwell system, and for which the segregated splenocytes were treated with LCL1 61 and the indicated TLR agonists. Error bars, mean ± s.d. Representative data from at least three independent experiments using biological replicates (n=3) is shown. FIG. 5B is a pair of graphs showing the results of an experiment in which established EMT6-Fluc tumors were treated with SMC (50 mg/kg LCL1 61 , p.o.) and poly(l :C) (1 5 ug i.t. or 2.5 mg/kg i.p.). The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Vehicle, vehicle+poly(l :C) i.p., n=4; remainder groups, n=5.. Error bars, mean ± s.e.m. Log-rank with Holm-Sidak multiple comparison: **, p < 0.01 ; ***, p < 0.001 . FIG. 5C is a series of representative I VIS images that were acquired from the experiment of FIG. 5B. FIG. 5D is a pair of graphs showing the results of an experiment in which EMT6-Fluc tumors were treated with LCL161 or combinations of 200 μg (i.t.) and/or 2.5 mg/kg (i.p.) CpG ODN 221 6. The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Vehicle, n=5; SMC, n= 5; vehicle+CpG i.p., n=5; SMC+CpG i.p., n=7; vehicle+CpG i.t., n=5; SMC+CpG i.t., n=8; vehicle+CpG i.p.+i.t., n=5; SMC+CpG i.p.+i.t., n=8. Error bars, mean ± s.e.m. Log-rank with Holm-Sidak multiple comparison: *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 . FIG. 5E is a series of representative I VIS images that were acquired from the experiment of FIG. 5D.

FIG. 6 is a graph showing the responsiveness of a panel of cancer and normal cells to the combinatorial treatment of SMC and OV. The indicated cancer cell lines (n=28) and non-cancer human cells (primary human skeletal muscle (HSkM) and human fibroblasts (GM38)) were treated with LCL161 and increasing VSVA51 for 48 hours. The dose required to yield 50% viable cells in the presence in SMC versus vehicle was determined using nonlinear regression and plotted as a log EC50 shift toward increasing sensitivity. Representative data from at least two independent experiments using biological replicates (n=3) are shown.

FIG. 7 is pair of graphs showing that SMC and OV co-treatment is highly synergistic in cancer cells. The graphs show Alamar blue viability of cells treated with serial dilutions of a fixed ratio combination mixture of VSVA51 and LCL1 61 (PFU: μΜ LCL1 61 ). Combination indexes (CI) were calculated using Calcusyn. Plots represent the algebraic estimate of the CI in function of the fraction of cells affected (Fa). Error bars, mean ± s.e.m. Representative data from three independent experiments using biological replicates (n=3) is shown.

FIG. 8 is a pair of graphs showing that monovalent and bivalent SMCs synergize with OVs to cause cancer cell death. The graphs show the result of Alamar blue viability assay of cells treated with 5 μΜ monovalent SMCs (LCL161 , SM-122) or 0.1 μΜ bivalent SMCs (AEG40730, OICR720, SM-1 64) and VSVA51 at differing MOIs. Error bars, mean ± s.d. Representative data from three independent experiments using biological replicates (n=3) is shown.

FIGS. 9A and 9B are a set of images and graphs showing that SMC-mediated cancer cell death is potentiated by oncolytic viruses. FIG. 9A is a series of images showing the results of a virus spreading assay of cells that were overlaid with 0.7% agarose in the presence of vehicle or LCL1 61 and 500 PFU of the indicated viruses were dispensed in to the middle of the well. Cytotoxicity was assessed by crystal violet staining. Arrow denotes extension of the cell death zone from the origin of OV infection. FIG. 9B is a set of graphs showing the Alamar blue viability of cells treated with LCL1 61 and increasing MOIs of VSVA51 or Maraba-MG1 . Error bars, mean ± s.d. Representative data from two independent experiments using biological replicates (n=3) is shown.

FIGS. 10A and 10B are a set of graphs and images showing that clAP1 , clAP2 and XIAP cooperatively protect cancer cells from OV-induced cell death. FIG. 10A shows Alamar blue viability of cells transfected with nontargeting (NT) siRNA or siRNA targeting clAP1 , clAP2 or XIAP, and subsequently treated with LCL161 and 0.1 MOI VSVA51 for 48 hours. Error bars, mean ± s.d.

Representative data from three independent experiments using biological replicates (n=3) is shown. FIG. 10B is a representative siRNA efficacy immunoblots for the experiment of FIG. 10A.

FIG. 1 1 is a set of images used for superimposed images depicted in FIG. 1 G. Cells were overlaid with agarose media containing LCL161 , inoculated with VSVA51 -GFP in the middle of the well, and infectivity measured by fluorescence and cytotoxicity was denoted by crystal violet (CV) staining. Note: the bars represent the same size.

FIGS. 12A and 12B are a set of images and a graph showing that SMC treatment does not affect OV distribution or replication in vivo. FIG. 12A is a set of images showing images from an experiment in which EMT6-bearing mice were treated with 50 mg/kg LCL1 61 (p.o.) and 5x1 0 ⁸ PFU firefly luciferase tagged VSVA51 (VSVA51 -Flue) via i.v. injection. Virus distribution and replication was imaged at 24 and 48 hours using the I VIS. Outline denotes region of tumors. Representative data from two independent experiments are shown. Arrow indicates spleen infected with VSVA51 -Flue. FIG. 1 2B is a graph showing data from an experiment in which tumors and tissues at 48 hour post-infection were homogenized and virus titrations were performed for each group. Error bars, mean ± s.e.m.

FIGS. 13A and 13B are images showing verification of siRNA-mediated knockdown of non- targeting (NT), TN FR1 , DR5 and IFNAR1 by immunoblotting. FIG. 1 3A is an immunoblot showing knockdown in samples from the experiment of FIG. 3A. FIG. 13B is an immunoblot showing knockdown in samples from the experiment of FIG. 3B.

FIGS. 14A-14G are images and graphs showing that SMC synergizes with OVs to induce caspase-8- and RI P-1 -dependent apoptosis in cancer cells. All panels of FIG. 14 show representative data from three independent experiments using biological replicates. FIG. 14A is a pair of images of immunoblots in which immunoblotting for caspase and PARP activation was conducted on cells pretreated with LCL1 61 and subsequently treated with 1 MOI of VSVA51 . FIG. 1 4B is a series of images showing micrographs of caspase activation that were acquired with cells that were co-treated with LCL1 61 and VSVA51 in the presence of the caspase-3/7 substrate DEVD-488. FIG. 14C is a graph in which the proportion of DEVD-488-positive cells from the experiment of FIG. 14B was plotted (n=1 2). Error bars, mean ± s.d. FIG. 14D is a series of images from an experiment in which apoptosis was assessed by micrographs of translocated phosphatidyl serine (Annexin V-CF594) and loss of plasma membrane integrity (YOYO-1 ) in cells treated with LCL1 61 and VSVA51 . FIG. 14E is a graph in which the proportion of Annexin V-CF594-positive and YOYO-1 -negative apoptotic cells from the experiment of FIG. 14D was plotted (n=9). Error bars, mean ± s.d. FIG. 14F is a pair of graphs showing alamar blue viability of cells transfected with nontargeting (NT) siRNA or siRNA targeting caspase-8 or RI P1 , and subsequently treated with LCL161 and 0.1 MOI of VSVA51 (n=3). Error bars, mean ± s.d. FIG. 14G, is an image of an immunoblot showing representative siRNA efficacy for the experiment of FIG. 14F.

FIGS. 15A and 15B are a set of graphs showing that expression of TNFa transgene from OVs potentiates SMC-mediated cancer cell death further. FIG. 1 5A is a pair of graphs showing Alamar blue viability assay of cells co-treated with 5 μΜ SMC and increasing MOIs of VSVA51 -GFP or VSVA51 -TNFa for 24 hours. Error bars, mean ± s.d. FIG. 1 5B is a graph showing representative EC50 shifts from the experiment of FIG. 1 5A. The dose required to yield 50% viable cells in the presence in SMC versus vehicle was determined using nonlinear regression and plotted as EC50 shift. Representative data from three independent experiments using biological replicates (n=3).

FIG. 16 is a set of images showing that oncolytic virus infection leads to enhanced TNFa expression upon SMC treatment. EMT6 cells were co-treated with 5 μΜ SMC and 0.1 MOI VSVA51 -GFP for 24 hours, and cells were processed for the presence of intracellular TNFa via flow cytometry. Images show representative data from four independent experiments.

FIGS. 17A-1 7C are a pair of graphs and an image showing that TNFa signaling is required for type I I FN induced synergy with SMC treatment. All panels of FIG. 1 7 show representative data from at least three independent experiments using biological replicates (n=3). FIG. 1 7A is a graph showing the results of an Alamar blue viability assay of EMT6 cells transfected with nontargeting (NT) or TN F-R1 siRNA and subsequently treated with LCL1 61 and VSVA51 (0.1 MOI) or l FN . Error bars, mean ± s.d. FIG. 17B is a representative siRNA efficacy blot from the experiment of FIG. 1 7A. FIG. 17C is a graph showing the viability of EMT6 cells that were pretreated with TNFa neutralizing antibodies and subsequently treated with 5 μΜ SMC and VSVA51 or I FN .

FIGS. 18A and 18B are a schematic of OV-induced type I IFN and SMC synergy in bystander cancer cell death. FIG. 1 8A is a schematic showing that virus infection in refractory cancer cells leads to the production of Type 1 I FN, which subsequently induces expression of I FN stimulated genes, such as TRAIL. Type 1 I FN stimulation also leads to the N F-KB-dependent production of TNFa. IAP antagonism by SMC treatment leads to upregulation of TNFa and TRAIL expression and apoptosis of neighboring tumor cells. FIG. 18B is a schematic showing that infection of a single tumor cell results in the activation of innate antiviral Type 1 I FN pathway, leading to the secretion of Type 1 I FNs onto neighboring cells. The neighboring cells also produce the proinflammatory cytokines TNFa and TRAIL. The singly infected cell undergoes oncolysis and the remainder of the tumor mass remains intact. On the other hand, neighboring cells undergo bystander cell death due upon SMC treatment as a result of the SMC-mediated upregulation of TNFa/TRAIL and promotion of apoptosis upon proinflammatory cytokine activation.

FIGS. 19A and 19B are a graph and a blot showing that SMC treatment causes minimal transient weight loss and leads to downregulation of clAP1 /2. FIG. 1 9A is graph showing weights from LCL1 61 treated mice female BALB/c mice (50 mg/kg LCL161 , p.o.) that were recorded after a single treatment (day 0). n=5 per group. Error bars, mean ± s.e.m. FIG. 19B is a blot of samples from an experiment in which EMT6-tumor bearing mice were treated with 50 mg/kg LCL161 (p.o.). Tumors were harvested at the indicated time for western blotting using the indicated antibodies.

FIGS. 20A-20C are a set of graphs showing that SMC treatment induces transient weight loss in a syngeneic mouse model of cancer. FIGS. 20A-20C are graphs showing measurements of mouse weights upon SMC and oncolytic VSV (FIG. 20A), poly(l :C) (FIG. 20B), or CpG (FIG. 20C) co-treatment in tumor-bearing animals from the experiments depicted in FIGS. 4A, 5B, and 5D, respectively. Error bars, mean ± s.e.m.

FIGS. 21 A-21 D are a series of graphs showing that VSVA51 -induced cell death in HT-29 cell is potentiated by SMC treatment in vitro and in vivo. FIG. 21 A is a graph showing data from an experiment in which cells were infected with VSVA51 , the cell culture supernatant was exposed to UV light for 1 hour and was applied to new cells at the indicated dose in the presence of LCL1 61 . Viability was ascertained by Alamar blue. Error bars, mean ± s.d. FIG. 21 B is a graph showing Alamar blue viability of cells co- treated with LCL1 61 and a non-spreading virus VSVA51 AG (0.1 MOI). Error bars, mean ± s.d. FIGS. 21 A and 21 B show representative data from three independent experiments using biological replicates (n=3). FIG. 21 C is a pair of graphs showing data from an experiment in which CD-1 nude mice with established HT-29 tumors were treated with 50 mg/kg LCL1 61 (p.o.) and 1 x1 0 ⁸ PFU VSVA51 (i.t.). Vehicle, n=5; VSVA51 , n=6; SMC, n=6; VSVA51 +SMC, n=7. The left panel depicts tumor growth relative to day 0 post-treatment. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m. Log-rank with Holm-Sidak multiple comparison: ***, p < 0.001 . FIG. 21 D is a graph showing measurement of mouse weights upon SMC and OV co-treatment in tumor-bearing animals. Error bars, mean ± s.e.m.

FIG. 22 is a blot showing that type I IFN signaling is required for SMC and OV synergy in vivo. EMT6 tumor bearing mice were treated with vehicle or 50 mg/kg LCL1 61 for 4 hours, and subsequently treated with neutralizing I FNAR1 or isotype antibodies for 20 hours. Subsequently, animals were treated with PBS or VSVA51 for 18 hours. Tumors were processed for Western blotting with the indicated antibodies.

FIGS. 23A and 23B are a pair of graphs showing that oncolytic infection of innate immune cells leads to cancer cell death in the presence of SMCs. FIG. 23A is a graph showing data from an experiment in which immune subpopulations were sorted from splenocytes (CD1 1 b+ F4/80+:

macrophage; CD1 1 b+ Gr1 +: neutrophil; CD1 1 b- CD49b+: NK cell; CD1 1 b- CD49b-: T and B cells) and were infected with 1 MOI of VSVA51 for 24 hours. Cell culture supernatants were applied to SMC-treated ETM6 cells for 24 hours and EMT6 viability was assessed by Alamar Blue. Error bars, mean ± s.d. FIG. 23B is a chart showing data from an experiment in which bone marrow derived macrophages were infected with VSVA51 and the supernatant was applied to EMT6 cells in the presence of 5 μΜ SMC, and viability was measured by Alamar blue. Error bars, mean ± s.d.

FIGS. 24A-24H are a series of images of full-length immunoblots. Immunoblots of FIGS. 24A- 24H pertain to (a) FIG. 2E, (b) FIG. 4E, (c) FIG. 1 0B, (d) FIG. 13, (e) FIG. 14A, (f) FIG. 14G, (g) FIG. 1 9, and (h) FIG. 17, respectively.

FIGS. 25A and 25B are a set of graphs showing that non-replicating rhabdovirus-derived particles (NRRPs) synergize with SMCs to cause cancer cell death. FIG. 25A is a set of graphs showing data from an experiment in which EMT6, DBT, and CT-2A cancer cells were co-treated with the SMC LCL1 61 (SMC; EMT6: 5 μΜ, DBT and CT-2A: 1 5 μΜ) and different numbers of NRRPs for 48 hr (EMT6) or 72 hr (DBT, CT-2A), and cell viability was assessed by Alamar Blue. FIG. 25B is a pair of graphs showing data from an experiment in which unfractionated mouse splenocytes were incubated with 1 particle per cell of NRRP or 250 μΜ CpG ODN 221 6 for 24 hr. Subsequently, the supernatant was applied to EMT6 cells in a dose-response fashion, and 5 μΜ LCL1 61 was added. EMT6 viability was assessed 48 hr post- treatment by Alamar blue.

FIGS. 26A and 26B are a graph and a set of image showing that vaccines synergize with SMCs to cause cancer cell death. FIG. 26A is a graph showing data from an experiment in which EMT6 cells were treated with vehicle or 5 μΜ LCL1 61 (SMC) and 1 000 CFU/mL BCG or 1 ng/mL TN Fa for 48 hr, and viability was assessed by Alamar blue. FIG. 26B is a set of representative I VI S images depicting survival of mice bearing mammary fat pad tumors (EMT6-Fluc) that were treated twice with vehicle or 50 mg/kg LCL1 61 (SMC) and PBS intratumorally (i.t.), BCG (1 x1 0 ⁵ CFU) i.t, or BCG (1 x1 0 ⁵ CFU) intraperitoneal^ (i.p.) and subjected to live tumor bioluminescence imaging by I VIS CCD camera at various time points. Scale: p/sec/cm2/sr.

FIGS. 27A and 27B are a pair of graphs and a set of images showing that SMCs synergize with type I I FN to cause mammary tumor regression. FIG. 27A is a pair of graphs showing data from an experiment in which mice were injected with EMT6-Fluc tumors in the mammary fat pad and were treated at eight days post-implantation with combinations of vehicle or 50 mg/kg LCL161 (SMC) orally and bovine serum albumin (BSA), 1 g IFNa intraperitoneally (i.p.), or 2 μg IFNa intratumorally (i.t.). The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m. FIG. 27B is a series of representative I VIS images from the experiment described in FIG. 27A. Scale: p/sec/cm2/sr.

FIGS. 28A-28C are graphs showing that VSV-I FN or VSV synergizes with SMCs to cause cancer cell death. FIG. 28A shows data from an experiment in which EMT6 cells were co-treated with vehicle or 5 μΜ LCL1 61 (SMC) and differing multiplicity of infection (MOI) of VSVA51 -GFP, VSV-IFN , or VSV-NIS-I FN . Cell viability was assessed 48 hr post-treatment by Alamar blue. FIG. 28B are a pair of graphs where EMT6 mammary tumor bearing mice were treated twice with vehicle or 50 mg/kg LCL1 61 (SMC) orally and PBS or 1 x1 08 PFU of VSV-I FN -NIS intratumourally. FIG. 28C are a pair of graphs where EMT6 mammary tumor bearing mice were treated twice with vehicle or 50 mg/kg LCL1 61 orally and 1 x1 08 PFU of VSV intratumourally.

FIG. 29 is a graph showing that non-viral and viral triggers induce robust expression of TN Fa in vivo. Mice were treated with 50 mg of poly(l :C) intraperitoneally or with intravenous injections of 5x10 ⁸ PFU VSVA51 , VSV-ml FNp, or Maraba-MG1 . At the indicated times, serum was isolated and processed for ELISA to quantify the levels of TNFa.

FIGS. 30A-30C are a set of graphs and images showing that virally-expressed proinflammatory cytokines synergizes with SMCs to induce mammary tumor regression. FIG. 30A is a pair of graphs showing data from an experiment in which mice were injected with EMT6-Fluc tumors in the mammary fat pad, and were treated at seven days post-implantation with combinations of vehicle or 50 mg/kg LCL161 (SMC) orally and PBS, 1 x1 0 ⁸ PFU VSVA51 -memTNFa (i.v.), or 1 x1 0 ⁸ PFU VSVA51 -solTNFa (i.v.). The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m. FIG. 30B is a set of representative bioluminescent I VI S images that were acquired from the experiment described in FIG. 30A. Scale: p/sec/cm2/sr. FIG. 30C is a pair of graphs showing data from an experiment in which mice were injected with CT-26 tumors subcutaneously and were treated 1 0 days post-implantation with combinations of vehicle or 50 mg/kg LCL161 orally and either PBS or 1 x1 0 PFU VSVA51 -solTNFa intratumorally. The left panel depicts tumor growth. The right panel represents the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± s.e.m.

FIGS. 31 A and 31 B are a set of images showing that SMC treatment leads to down-regulation of clAPI /2 protein in vivo in an orthotopic, syngeneic mouse model of glioblastoma. FIG. 31 A is an image showing an immunoblot from an experiment in which CT-2A cells were implanted intracranially and treated with 50 mg/kg orally of LCL161 (SMC) and tumors were excised at the indicated time points and processed for western blotting using antibodies against clAP1 /2, XI AP, and β-tubulin. FIG. 31 B is an image showing an immunoblot from an experiment in which CT-2A cells were implanted intracranially and treated with 1 0 uL of 1 00 μΜ LCL1 61 intratumorally and tumors were excised at the indicated time points and processed for western blotting using antibodies against clAP1 /2, XIAP, and β-tubulin.

FIGS. 32A-32E are a set of graphs and images showing that a transient proinflammatory response in the brain synergizes with SMCs to cause glioblastoma cell death. FIG. 32A is a graph showing data from an experiment in which an ELISA was conducted to determine the levels of soluble TNFa from 300 mg of crude brain protein extract that was derived from mice injected intraperitoneally (i.p.) with PBS or 50 mg poly(l :C) for 12 or 24 h. Brain protein extracts were obtained by mechanical homogenization in saline solution. FIG. 32B is a graph showing data from Alamar blue viability assays of mouse glioblastoma cells (CT-2A, K1 580) that were treated with 70 mg of crude brain homogenates and 5 μΜ LCL1 61 (SMC) in culture for 48 h. Brain homogenates were obtained from mice that were treated for 1 2 h with i.p. injections of poly(l :C), or intravenous injections of 5x1 0 ⁸ PFU VSVA51 or VSV-ml FNp. FIG. 32C represents the Kaplan-Meier curve depicting survival of mice that received three intracranial treatments of 50 mg poly(l:C). Treatments were on days 0, 3, and 7. FIG. 32D represents the Kaplan- Meier curve depicting survival of mice bearing CT-2A intracranial tumors that received combinations of SMC, VSVA51 or poly(l :C). Mice received combinations of three treatments of vehicle, three treatments of 75 mg/kg LCL1 61 (oral), three treatments of 5x10 ⁸ PFU VSVA51 (i.v.), or two treatments of 50 mg poly(l :C) (intracranial, i.e.). Mice were treated on day 7, 1 0, and 14 post tumor cell implantation with the different conditions, except for the poly(l :C) treated group that received i.e. injections on day 7 and 1 5. Numbers in brackets denote number of mice per group. FIG. 32E is a series of representative MRI images of mouse skulls from the experiments depicted in FIG. 32D, which shows an animal at endpoint and a representative mouse of the indicated groups at 50 days post-implantation. Dashed line denotes the brain tumor.

FIG. 33 is a graph showing that SMCs synergize with type I IFN to eradicate brain tumors. The graph represents the Kaplan-Meier curve depicting survival of mice bearing CT-2A that received intracranial injections of vehicle or 1 00 μΜ LCL161 (SMC) with PBS or 1 I FNa at 7 days post- implantation.

FIG. 34 is an overview of the NF-κΒ signalling pathway. Upon ligand engagement with a TN F family receptor, either the classical or alternative pathway will be activated depending on the activity of clAPI /2. In classical NF-κΒ activation, RI P1 receives K63 ubiquitin linkages from clAP1 /2 to form a signalling complex, which allows phosphorylation of the inhibitor of κΒ (ΙκΒ) following activation of the ΙκΒ- inase (IKK). Phosphorylated ΙκΒ is degraded, freeing the p50/p65 heterodimer. The alternative pathway is kept inactive by clAP1 /2 K48 linked ubiquitination of NF-κΒ inducing kinase (NIK). When NIK is stable, it allows phosphorylation of IKK and downstream p100, resulting in processing of p100 to p52. The pathway culminates with N F-κΒ heterodimers translocating to the nucleus to act as transcription factors to regulate expression of target genes.

FIGS. 35A-35C describe the process of combining SMC with monoclonal antibodies against PD-1 delayed disease progression and prolonged survival in a murine MM model. FIG. 35A shows images of mice bearing MPC-1 1 Flue cells that were treated with 250 g of ICI and 50mg/kg three times/week for two weeks. Mice are treated with SMC and monoclonal antibodies against either PD-1 or CTLA-4. Mice treated with the combination of anti-PD-1 and SMC showed almost no tumour burden as determined by S bioluminescence images of the cancer burden on the days post cell implantation. FIG. 35B shows the treatment regimen with anti-PD-1 , anti-CTLA-4 and SMC. FIG. 35C is a graph showing the number of days mice survived post implantation of M PC-1 1 Flue cells as indicated in a Kaplan-Meier curve

FIGS. 36A-36C are a series of graphs demonstrating that innate immune stimulants synergize with SMC to cause MM cell death. FIG. 36A is a series of bar graphs showing the viability of human cell lines U266, MM1 R, and MM 1 S that were treated with 1 ΙΙ/μΙ_ I FNa, IFNp, and I FNy in the presence of either vehicle or 5 μΜ SMC. Viability was determined by trypan blue exclusion after 24 hours. FIG. 36B and FIG. 36C are graphs showing the viability of the murine MM cell line M PC-1 1 that was treated with 5 μΜ SMC and various multiplicity of infections (MOI) VSVA51 and VSVm l FN respectively. Viability was assessed after 24 hours with Alamar blue.

FIGS. 37A-37C show IFN and SMC synergize to delay MM disease progression in mice. Mice bearing M PC-1 1 Flue cells were treated with 1 μg of recombinant I FNa and 50 mg/kg SMC 3 times. FIG. 37A is a series of I VIS bioluminescence images of cancer burden taken at the indicated days post MM cell implantation. FIG. 37B is a Kaplan-Meier curve showing survival times. FIG. 37C is a schematic showing thetreatment regimen.

FIGS. 38A-38C indicate that oncolytic virus can delay MM disease progression and increase survival. FIG. 38A is I VIS bioluminescence images taken at indicated days post implantation of mice bearing M PC-1 1 Flue cells that were treated 4 times with 5x10 ⁸ pfu VSVA51 and 50 mg/kg SMC. FIG.

38B is a Kaplan-Meier curve showing survival times. FIG. 38C shows the treatment regimen.

FIGS. 39A-39C show glucocorticoid receptor ligands synergize with SMC to sensitize resistant cell lines to SMC-mediated cell death. FIG. 39A is a schematic showing protein was extracted from

MM1 R and MM1 S cell for western blotting, equal amounts of protein were used. FIGS. 39B and 39C are graphs showing that cells were treated with 5 μΜ SMC, 1 0 μΜ Dex and 1 0 μΜ RU486 for the indicated times and dead cells were determined as YOYO-1 positive, a cell impermeable DNA binding dye, and normalized to confluency of the cells within the well.

FIGS. 40A-40C show SMC increases NF-κΒ signalling and causes apoptosis. Human MM cell lines MM 1 R and MM1 S were treated with 5 μΜ SMC then collected after 1 , 1 6 or 48 hours. FIG. 40A shows western blots for various components of N F-κΒ pathway. FIGS. 40B and 40C are quantification of bands from FIG. 40A, expressed as ratios of p-p65 to p65 and p52:p1 00 respectively, that were normalized to an untreated control.

FIG. 41 shows SMC and I FN combination treatment increases N F-κΒ activity to cause apoptosis. Human cell lines U266, MM 1 R and MM1 S and murine cell line M PC-1 1 and a Flue tagged subline were treated with 5 μΜ SMC and 1 ΙΙ/μΙ_ I FN for 1 or 1 6 hr. Cell pellets were harvested and lysates were loaded equally for western blotting. FIGS. 42A-42C shows an oncolytic virus combined with SMC activates NF-κΒ signalling leading to apoptosis in murine MM cells. MPC-1 1 cells were treated with VSVA51 or VSVmlFN for 1 , 1 2, or 24 hours. FIG. 42A is a western blot showing cell pellets were harvested and lysates were loaded equally for western blotting. FIGS. 42B and 42C are protein levels quantified from the bands in FIG. 42A and expressed as ratios of phospho-p65 to p65, or p52 to p1 00 respectively.

FIG. 43 show PD-L1 and PD-L2 expression are increased in human MM cell lines after treatment with I FN . Expression of PD-L1 and PD-L2 mRNA are increased at 6, 1 2 and 24 hours posts I FN or IFN and SMC treatment relative to a no-treatment control.

FIGS. 44A-44D are graphs showing that the combination of SMCs and immunomodulatory agents leads to cancer cell death that also involves CD8+ T cells. FIGS. 44A and 44B are graphs showing data from an experiment in which double treated cured mice were re-injected with EMT6 cells in the mammary fatpad (180 days from the initial post-implantation date) or reinjected with CT-2A cells intracranially (1 90 days from the initial post-implantation date). FIG. 44C is a graph showing data from an experiment in which CT-2A glioma or EMT6 breast cancer cells were trypsinized, surface stained with conjugated isotype control IgG or anti-PD-L1 and processed for flow cytometry. FIG. 44D is a graph showing data from an experiment in which CD8+ T-cells were enriched from splenocytes (from nal ^' ve mice or mice previously cured of EMT6 tumours) using a CD8 T-cell positive magnetic selection kit, and subjected to ELISpot assays for the detection of I FNy and Granzyme B. CD8+ T-cells were co-cultured with media or cancer cells (12:1 ratio of cancer cells to CD8+ T-cells) and 1 0 mg of control IgG or anti- PD-1 for 48 hr. Three mice were used as independent biological replicates (were previously cured of EMT6 tumors). 4T1 cells serve as a negative control as 4T1 and EMT6 cells carry the same major histocompatibility antigens.

FIGS. 45A-45D are graphs showing that SMCs synergize with immune checkpoint inhibitors in orthotopic mouse models of cancer. FIG. 45A is graph showing data in which EMT6 mammary tumor bearing mice were treated once with PBS or 1 x1 08 PFU VSVD51 intratumorally, and five days later, the mice were treated with combinations of vehicle or 50 mg/kg LCL1 61 (SMC) orally and 250 mg of anti-PD- intraperitoneally (i.p.). FIGS. 45B and 45C are graphs showing data in which mice bearing intracranial CT-2A or GL261 tumors were treated four times with vehicle or 75 mg/kg LCL161 (oral) and 250 mg (i.p.) of control IgG, anti-PD-1 or anti-CTLA-4. FIG. 45D is a graph showing data in which athymic CD-1 nude mice bearing CT-2A intracranial tumors were treated with 75 mg/kg LCL1 61 (oral) and 250 mg (i.p.) anti- PD-1 .

FIGS. 46A-46C are graphs showing that SMCs induces the death of glioblastoma cells in the presence of cytokines or oncolytic viruses. Alamar blue viability assay of human (M059K, SNB75, U1 1 8) and mouse (CT-2A, GL261 ) glioblastoma cells treated with vehicle or 5 μΜ LCL1 61 (SMC) and 0.1 ng mL-1 of TNF-a or 0.01 MOI of VSVA51 for 48 h (FIG. 46A). Error bars, mean, s.d. n = 4. The indicated primary mouse NF1 -/+p53-/+ lines were treated with vehicle or 5 μΜ LCL1 61 (SMC) and 0.01 % BSA, 1 ng mL-1 TNF-a or the indicated MOI of a nonspreading version of VSVA51 (VSVA51 AG) for 48 h, and viability was assessed by Alamar blue (FIG. 46B). Error bars, mean, s.d. n = 4. Alamar blue viability assays of human brain tumor initiating cells (BTICs) treated with vehicle or 5 μΜ LCL1 61 and 0.001 MOI of VSVA51 or Maraba-MG1 for 48 h (FIG. 46C). Error bars, mean, s.d. n = 3. FIGS. 46A and 46B show representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. Significance is reported if p < 0.0001 (*).

FIG. 47 is a graph showing that SMCs potently synergize with TN F-a to induce the death of glioblastoma cells. Viability of mouse glioblastoma CT-2A cells to the treatment of 0.01 % BSA or 0.1 ng mL-1 TNF-a and vehicle or 5 μΜ of the indicated monomeric or dimeric for 48 h. Viability was assessed by Alamar blue. Error bars, mean, s.d. n = 4. Representative data from two independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. Significance is reported if p < 0.0001 (*).

FIGS. 48A and 48B is a series of graphs and an image showing that resistance to SMC-based combinations in glioblastoma cells is circumvented with downregulation of cFLI P. Primary mouse NF1 - /+p53-/+ (K5001 ) or human (SF539) glioblastoma cells or human nontransformed cells (GM38) were transfected with nontargeting (NT) or cFLI P siRNA for 48 h and subsequently treated for 48 h with vehicle or 5 μΜ LCL1 61 (SMC) and BSA, 0.1 ng mL-1 TNF-a or the indicated MOI of a nonspreading version of VSVA51 (VSVA51 AG; FIG. 48A). Viability was determined by Alamar blue. Error bars, mean, s.d. n = 4. Representative data from three independent experiments using biological replicates. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. Significance is reported if p < 0.0001 (*). Efficacy of NT siRNA or siRNA targeting cFLI P from the experiment in (FIG. 48B).

FIGS. 49A and 49B are images showing establishment of a mouse syngeneic orthotopic model of glioblastoma. Shown are MRI (FIG. 49A) and gross (FIG. 49B) images of a C57BL/6 mouse injected intracranially with PBS or 5x10 ⁴ CT-2A cells and sacrificed at 35 days post-implantation. Scale bar, 2 mm. Ruler is in cm with mm divisions.

FIGS. 50A and 50B are graphs showing that SMCs synergize with innate immunostimulants for the treatment of glioblastoma. Scale bar, 2 mm. Alamar blue viability assay of CT-2A cells treated with vehicle or 5 μΜ LCL1 61 and 0.01 % BSA or 1 μg mL-1 I FN-aB/D. Error bars, mean, s.d. n = 4 (FIG. 50A). Mice bearing 7 d old intracranial CT-2A tumors were treated with combinations of 75 mg kg-1 LCL1 61 (oral) and BSA or 1 μg of I FN-a B/D (i.p. ; FIG. 50B). FIG. 50B shows data representing the Kaplan- Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison: **, p < 0.01 ; ***, p < 0.001 . Numbers in parentheses represent number of mice per group.

FIG. 51 is an image showing that SMC treatment does not induce the downregulation of the lAPs in brain tissue from non-tumor bearing mice. Mice were treated with 75 mg kg ^"1 of LCL1 61 (SMC) for the indicated time, and tissues were processed for Western Blotting using the indicated antibodies, n = 2 for each timepoint.

FIGS. 52A-52C are graphs showing that SMC-based combination treatment results in long-term immunological anti-tumor memory. CT-2A cells were treated for 24 h with vehicle or 5 μΜ LCL1 61 (SMC) and 0.01 % BSA, 1 ng mL-1 TNF-a, 250 U mL-1 I FN-β or 0.1 MOI of VSVA51 , and viable cells (Zombie Green negative) were analyzed by flow cytometry using the indicated antibodies (FIG. 52A).

Representative data from at three independent experiments using biological replicates. Nal ^' ve mice or mice previously cured with SMC-based treatments of mammary fat pad EMT6 (mammary carcinoma, FIG. 52B) or intracranial CT-2A (glioblastoma, FIG. 52C) tumors were reinjected with EMT6 or mammary carcinoma 4T1 cells within the mammary fat pad or with CT-2A cells subcutaneously (s.c.) or intracranially (i.e.). Cells were implanted at 1 80 days initial post-implantation. Data represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison (compared to method of implantation): *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 . Numbers in parentheses represent number of mice per group.

FIG. 53 is a graph showing that SMC treatment does not abrogate expression of checkpoint inhibitor molecules or M HC l/l I proteins. SNB75 cells were treated for 24 h with vehicle or 5 μΜ LCL1 61 (SMC) and 1 ng mL-1 TNF-a, 250 U mL-1 IFN-β or 0.1 MOI of VSVA51 , and viable cells (Zombie Green negative) were processed for flow cytometry using the indicated antibodies. Representative data from three independent experiments.

FIGS. 54A-54G are graphs showing that SMCs synergize with antibodies targeting immune checkpoints mouse models of glioblastoma. Splenic CD8+ T-cells were enriched from naive mice or mice previously cured of CT-2A tumors, and subjected to ELISpot assays for the detection of I FN-γ and GrzB. Cancer cells (CT- 2A, LLC) were cocultured with CD8+ cells (25:1 ratio) and 10 μg mL-1 of control IgG or a-PD-1 for 48 h. n = 4 of mice per group (FIG. 54A). Significance was compared to naive CD8+ T-cell co- incubated with CT-2A cells as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05; *, p < 0.01 ; ***, p < 0.001 . Mice bearing intracranial CT-2A tumors were treated with 75 mg/kg LCL161 orally (SMC) on post-implantation d 14, 1 6, 21 and 23 (FIG. 54B). Viable cells from tumor masses were analyzed by flow cytometry for the detection of CD45 (BV605), CD3 (APC-Cy7), CD8 (PE) and PD-1 (BV421 ). Statistical significance for each pair was assessed by a t-test. *, p < 0.05; **, p < 0.01 (FIG. 54C). Viable tumor cells from the experiment in (were analyzed by flow cytometry using the antibodies CD45 (PE) and PD-L1 (BV421 ; FIG. 54C). n = 6 of mice per group. FMO, fluorescence minus one. Statistical significance was assessed by a t-test. Mice bearing intracranial CT-2A (FIGS. 54D, 54F, and 54G) or GL261 (FIG. 54E) tumors were treated at the indicated times with combinations of vehicle, 75 mg kg-1 LCL161 orally (FIGS. 54D, 54E, and 54G) or vehicle or 30 mg kg-1 Birinapant intraperitoneally (i.p. ; FIG. 54F) and 250 Mg of IgG, a-PD-1 or a-CTLA4 (i.p.) or both combined (FIG. 54G). Data represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison: *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 . Numbers in parentheses represent number of mice per group. In FIGS. 54A- 54C, crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. FIG. 54D shows representative data from two independent experiments.

FIG. 55 is a series of graphs showing that SMC treatment leads to the upregulation of PD-1 in CD8 T-cells. Mice bearing intracranial CT-2A tumors were treated with 75 mg kg-1 LCL1 61 orally (SMC) on post-implantation days 14, 1 6, 21 , and 23. Viable cells from CT-2A tumors were processed for flow cytometry using the antibodies CD45 (BV605), CD3 (APC-Cy7), CD8 (PE), and PD-1 (BV421 ).

FIGS. 56A and 56B are graphs showing that SMCs synergize with immune checkpoint inhibitors for the treatment of a mouse model of multiple myeloma. M PC-1 1 cells were treated with vehicle or 5 μΜ LCL1 61 (SMC) and 0.1 ng mL-1 TNF-a, 250 U mL-1 IFN-a, or 250 U mL-1 I FN-β (FIG. 56A). Viability was determined by Alamar blue at 48 h post-treatment. Error bars, mean, s.d. n= 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. Significance is reported if p < 0.0001 (***). Representative data from three independent experiments using biological replicates. M PC-1 1 cells were dissociated and processed for flow cytometry with PE-Cy7-conjugated isotype IgG or PD-L1 (FIG. 56B).

FIGS. 57A-57C are graphs showing that the combination of SMCs with antibodies targeting immune checkpoint inhibitors in a mouse model of mammary cancer. Viability assay of EMT6 cells treated with vehicle or 5 μΜ LCL1 61 (SMC) and 0.1 ng mL-1 TNF-a, 250 U mL-1 IFN-β or 0.1 MOI of VSVA51 for 48 h (FIG. 57A). Error bars, mean, s.d. n = 4. Statistical significance was compared to vehicle and BSA treatment using ANOVA using Dunnett's multiple comparison test. Significance is reported if p < 0.0001 (***). Representative data from three independent experiments using biological replicates. EMT6 cells were dissociated and processed for flow cytometry with PE-Cy7-conjugated isotype IgG or PD-L1 (FIG. 57B). Representative data from three independent experiments. Mice bearing ~1 00 mm3 EMT6-Fluc tumors were treated at the indicated post-implantation times with PBS or 5x1 08 PFU of VSVA51 intratumorally, and then with vehicle or 50 mg/kg LCL1 61 (SMC) orally and 250 μg of IgG or a-PD-1 intraperitoneally (FIG. 57C). The left panel depicts tumor growth. Error bars, mean, s.e,m. Right panel represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm- Sidak multiple comparison: *, p < 0.05; **, p < 0.01 . Numbers in parentheses represent number of mice per group.

FIGS. 58A and 58B are graphs showing that the inclusion of SMCs increases the immune response in the presence of glioblastoma cells. The expression of the indicated factors was detected by ELISA from cell culture supernatants of CT-2A cells that were co-incubated for 48 h with splenocytes derived from naive mice or mice previously cured with intracranial CT-2A tumors by SMC and anti-PD-1 cotreatment (1 :20 ratio of CT-2A cells to splenocytes; FIG. 58A). Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Statistical significance was compared to naive CD8+ T-cell as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05; ** p < 0.01 ; ***, p < 0.001 . The indicated cytokines were determined by ELISA from CT-2A cells that were cocultured with splenocytes derived from naive or cured mice and treated with vehicle or 5 μΜ LCL1 61 (SMC) for 48 h (FIG. 58B). Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Statistical significance was compared to vehicle and IgG treated T-cells as assessed by ANOVA with Dunnett's multiple comparison test. **p < 0.01 ; ***, p < 0.001 .

FIGS. 59A-59E are images and graphs showing that CD8+ T-cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma. The expression of the indicated immune factors was detected by ELISA from cell culture supernatants of CT-2A cells that were co-incubated for 48 h with splenocytes derived from naive mice or mice previously cured of intracranial CT-2A tumors by SMC and anti-PD-1 cotreatment (1 :20 ratio of CT-2A cells to splenocytes; FIG. 59A). Data is plotted as heat maps using normalized scaling. Box and whisker plots of the data are shown in FIG. 58A. Quantification of the indicated factor was determined by ELISA from CT-2A cells that were cocultured with splenocytes derived from naive or cured mice (1 :20 ratio) and treated with vehicle or 5 μΜ LCL1 61 (SMC) for 48 h (FIG. 59B). Splenocytes from naive or cured mice were cocultured with mKate2 tagged CT-2A cells (CT-2A-mKate2) in the presence of 20 μg mL-1 control IgG or anti-PD1 and 5 μΜ of the indicated SMC (FIG. 59C). Enumeration of CT-2A-mKate2 cells was performed using the Incucyte Zoom. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Significance was compared to na ^' ive splenocytes as assessed by ANOVA with Dunnett's multiple comparison test. Significance is reported as * when < 0.0001 . n = 6 for na ^' ive mice and n = 6 for cured mice. Scale bar, 1 00 μηι. C57BL/6 mice harboring intracranial CT-2A tumors were treated at the indicated date with combinations of either IgG (i.p.) and vehicle (oral) or a-PD-1 (i.p) and 75 mg kg-1 LCL161 (oral) and i.p. administration of either IgG, a-CD4 or a-CD8 (all antibodies were 250 μg; FIG. 59D). CD-1 nude mice bearing intracranial CT-2A tumors were treated at the indicated times with combinations of vehicle or 75 mg kg-1 LCL161 orally and PBS or 250 μg of IgG or a-PD-1 intraperitoneally (i.p. ; FIG. 59E). Data represents the Kaplan-Meier curve depicting mouse survival. Log-rank with Holm-Sidak multiple comparison: *, p < 0.05; **, p < 0.01 . Numbers in parentheses represent number of mice per group.

FIG. 60 is a series of graphs showing that combinatorial SMC and immune checkpoint inhibitor treatment leads to the increased systemic presence of proinflammatory cytokines. Serum from mice was processed for multiplex ELISA for the quantitation of the indicated proteins. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05. n = 6 for each treatment group.

FIGS. 61 A-61 G are graphs showing that SMC and immune checkpoint inhibitor treatment in mouse models of glioblastoma leads to changes in immune effector cell infiltration. Mice bearing intracranial CT-2A tumors were treated at the indicated times with vehicle or 75 mg kg-1 LCL1 61 orally (SMC) and 250 IgG or anti-PD-1 intraperitoneally (FIG. 61 A). Mice were sacrificed on d 27 post- implantation. Viable T-cells isolated from tumors were processed for flow cytometry using the following antibodies: CD45 (PE-Cy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605) and PD-1 (BV421 ; FIGS. 61 B-61 E). Viable cells from the experiment in (a) were processed for flow cytometry using the following antibodies: CD45 (BV605), CD1 1 b (APC-Cy7), Gr1 (BV786), F4/80 (PE) and CD3 (APC; FIGS. 61 F and 61 G). All panels: Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts minmax range of the values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05; **, p < 0.01 . n = 6 for each treatment group.

FIGS. 62A-62G are graphs and images showing that SMC and immune checkpoint inhibitor combination induces a proinflammatory cytokine response and efficacy is dependent on type I I FN signaling. Viable cells from brain tumors were isolated and processed for flow cytometry using the following antibodies: CD45 (BV605), CD3 (APC-Cy7), Cd4 (PE-Cy7), CD8 (BV786/0), I FN-γ (BV421 ), TNF-a (PE) and GrzB (AF647; FIGS. 62A-62D). Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values.

Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05. n = 6 for each treatment group. Serum from mice was processed for multiplex ELISA for the quantitation of the indicated proteins (FIG. 62E). Data is plotted as heat maps using normalized scaling, n = 6 for each treatment group. Mice were treated, and intracranial CT-2A tumors were processed for quantitation of 1 76 cytokine and chemokine genes by RT-qPCR (FIG. 62F). Shown are normalized heat maps of two major groups identified by hierarchical clustering, n = 4 for each treatment group. Mice bearing intracranial CT-2A tumors were treated at the indicated postimplantation day with vehicle or 75 mg kg-1 LCL1 61 (oral) or intraperitoneal^ with the relevant isotype IgG control or 2.5 mg a-I FNAR1 , 350 μg a-I FN-γ or 250 μg a-PD-1 (FIG. 62G). Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05. Numbers in brackets denote the size of the treatment groups.

FIG. 63 is an image showing that proinflammatory cytokine and chemoattractant chemokine gene signatures are upregulated with SMC and immune checkpoint inhibitor combinatorial treatment.

Intracranial CT-2A tumors were processed for quantitation of 1 76 cytokine and chemokine genes by RT- qPCR. Shown are normalized heat maps of major groups identified by hierarchical clustering, n = 4 for each treatment group.

FIG. 64 is a series of graphs showing that SMCs enhance clonal expansion of CD8+ T-cells in the presence of glioblastoma target cells. Isolated splenic CD8+ T-cells derived from mice previously cured of CT-2A tumors were loaded with CFSE and co-incubated with CT-2A cells (10:1 ratio) for 96 h in the presence of vehicle or 5 μΜ LCL1 61 (SMC) or 20 μg mL-1 of control IgG or anti-PD1 . Viable cells were processed for flow cytometry. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p <0.05; **, p < 0.01 ; ***, p < 0.001 . n = 5 for each treatment group.

FIGS. 65A-65C are graphs and images showing that the proinflammatory cytokine TNF-a is required for T-cell mediated death of glioblastoma cells upon Smac mimetic and immune checkpoint inhibitor treatment. Isolated CD8 T-cells derived from the spleen and lymph nodes from mice previously cured of intracranial CT-2A tumors were cocultured with CT-2A cells in the presence of vehicle or 5 μΜ LCL1 61 and 20 μg mL-1 isotype-matched IgG or a-PD-1 for 24 h. Viable T-cells were processed for flow cytometry using the following antibodies: CD3 (APC-Cy7), CD8 (BV71 1 ), GrzB (AF647) and TNF-a (PE; FIG. 65A). Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 . n = 5 for each treatment group. CD8+ T-cells were cocultured with mKate2-tagged CT-2A cells (CT-2A-mKate2) for 72 h in the presence of vehicle or 5 μΜ LCL161 and 20 μg/mL of control IgG, a-PD-1 or a-TNF-a (FIG. 65B). Enumeration of mKate2-positive cells was acquired using the Incucyte Zoom software. Crosses depicts mean, solid horizontal line depicts median, box depicts 25th to 75th percentile, and whiskers depicts min-max range of the values. Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test, p < 0.01 ; ***, p < 0.001 . n = 5 for each treatment group. Scale bar, 1 00 μηι. Mice bearing intracranial CT-2A tumors were treated at the indicated post-implantation day with vehicle or 75 mg kg-1 LCL161 (oral) or intraperitoneally with the relevant isotype IgG control or 500 μg a-TNF-a or 250 μg a-PD-1 (FIG. 65C). Significance was compared to vehicle and IgG treated mice as assessed by ANOVA with Dunnett's multiple comparison test. **, p < 0.01 . Numbers in parentheses represent number of mice per group.

FIG. 66 is a schematic showing that SMCs are immunoregulatory drugs that act on tumor and immune cells to eradicate cancer through the innate and adaptive immune systems. Shown is a model depicting the single agent and combinatorial immunomodulatory effects of Smac mimetics based on our results. The effects of IAP antagonism on these immune or tumor cells are outlined below: (1 ) SMCs stimulates the production of cytokines and chemokines from various immune cells, such as macrophages or T-cells, which results in infiltration of immune cells within the tumor microenvironment. (2) SMC treatment decreases the immunosuppressive macrophage M2 population and concomitantly increases the pro-inflammatory M1 population. (3) SMCs deplete clAP1 and clAP2 to sensitize tumors to death by immune ligands, such as TNF-a or TRAIL1 . Tumor cell death is sensed by the immune system resulting in the priming of a cytotoxic T-cell (CTL) response. (4) SMCs stimulate the TNF/TNFR family member CD40L/CD40 signaling pathway on antigen-presenting cells (APCs) to promote the

differentiation and maturation of dendritic cells (DCs) and macrophages. APCs present tumor antigens to the immune system and further release cytotoxic inflammatory cytokines. (5) As a consequence of degrading clAP1 and clAP2 by SMC treatment, SMCs activate the alternative NF-κΒ pathway, removing the need for a TNF superfamily ligand (such as 4-1 BB) and therefore providing a T-cell costimulatory signal. (6) SMCs have been shown to increase CTL and natural killer cell mediated cell death.

Granzyme B-mediated cell death is blocked by the X-linked IAP, XIAP, and this block can be overcome by the mitochondrial release of Smac or by its drug mimic, SMC13-1 5.

FIG. 67 is a schematic showing that cooperative and complimentary mechanisms for synergy between SMCs and immune checkpoint inhibitors (ICI). (1 ) The presence of therapeutic recombinant antibodies that block the PD-1 /PD-L1 axis allows for signaling of the T-cell receptor (TCR) of a CD8+ T- cell with its associated antigen presented by the cancer cell through a major histocompatibility complex I (MHC-I) molecule. Concurrent depletion of the lAPs through SMC treatment can enhance T-cell activation, likely by providing a Tumor Necrosis Factor Receptor Superfamily (TNFRSF) co-stimulatory response (similar to 4-1 BB or OX40 activation), resulting in enhanced activation and expansion of tumor- specific CD8+ T-cells. As a result, Granzyme B (GrzB) and Perforin (Pfn) are secreted to kill target cells. (2) SMC-mediated antagonism of the casp-3 inhibitor, XIAP, can result in enhanced death of tumor cells by GrzB. (3) The depletion of clAP1 and clAP2 by SMCs leads to increased local production of TNF-a by T-cells in the tumor microenvironment, an effect that is likely mediated by activation of the alternative NF- KB pathway. (4) As a result of clAP1 / 2 loss, SMC-treated cancer cells are sensitized to cell death induction in the presence of proinflammatory cytokines, such as TNF-a.

FIGS. 68A-68D are images showing full-length Western blots.

DETAILED DESCRIPTION

The present invention includes methods and compositions for enhancing the efficacy of Smac mimetic compounds (SMCs) in the treatment of cancer. In particular, the present invention includes methods and compositions for combination therapies that include an SMC and a second agent that stimulates one or more cell death pathways that are inhibited by clAP1 and/or clAP2. The second agent may be, e.g., a TLR agonist a virus, such as an oncolytic virus, or an interferon or related agent.

The data provided herein demonstrates that treatment with an agent and an SMC results in tumor regression and durable cures in vivo (see, e.g., Example 1 ). These combination therapies were well tolerated by mice, with body weight returning to pre-treatment levels shortly after the cessation of therapy. Tested combination therapies were able to treat several treatment refractory, aggressive mouse models of cancer. One of skill in the art will recognize, based on the disclosure and data provided herein, that any one or more of a variety of SMCs and any one or more of a variety of agents, such as a TLR agonist, pathogen, or pathogen mimetic, may be combined in one or more embodiments of the present invention to potentiate apoptosis and treat cancer.

While other approaches to improve SMC therapy have been attempted, very rarely have complete responses been observed, particularly in aggressive immunocompetent model systems. Some embodiments of the present invention, including treatment of cancer with a pathogen mimetic, e.g., a pathogen mimetic having a mechanism of action partially dependent on TRAIL, can have certain advantages. First, this approach can evoke TN Fa-mediated apoptosis and necroptosis: given the plasticity and heterogeneity of some advanced cancers, treatments that simultaneously induce multiple distinct cell death mechanisms may have greater efficacy than those that do not. Second, pathogen mimetics can elicit an integrated innate immune response that includes layers of negative feedback. These feedback mechanisms may act to temper the cytokine response in a manner difficult to replicate using recombinant proteins, and thus act as a safeguard to this combination therapy strategy.

Multiple myeloma (MM) is an incurable cancer that is characterized by rapid expansion of plasma cells in the bone marrow. MM is the second most common haematological malignancy and has a median survival of only three to five years after diagnosis. The MM cells cause bone resorption leading to fractures and immune suppression as they populate the bone marrow compartment. MM cells can disseminate to other tissues to form plasmacytomas, and the disease can have an aggressive leukemic phase. Current therapies can prolong survival and mitigate symptoms, but they are no curative treatments. New therapies are desperately needed to combat treatment resistance and inevitable relapse.

The malignant cells are reliant on the bone marrow microenvironment in early stages of the disease, specifically TNFa and interleukin-6 (IL-6) from cells within the bone marrow microenvironment. As the disease progresses, the cells become independent of their environment, surviving on high autocrine production of TNFa. Throughout all stages the cells have high levels of N F-κΒ signalling that enhance their survival, in part due to common mutations in key components of the pathway Targeting the N F-KB pathway in MM contributes to the increase in efficacy of many standard therapeutics used in MM, such as the proteasome inhibitor bortezomib, immunomodulatory agents (IMiDs) thalidomide and lenalidomide and the synthetic glucocorticoid dexamethasone.

TNFa-mediated NF-κΒ signalling can be switched from a pro-survival signal to an apoptotic signal with the removal of the cellular inhibitors of apoptosis (clAPs); this process appears to be selective to cancer cells. clAP1 and clAP2 act interchangeably as E3 ligases in all members of the TNFa receptor superfamily, either ubiquitinating specific proteins to form a scaffold for signalling complexes, or targeting them for degradation. Examples of this can been seen in both arms of the NF-κΒ pathway: RI P1 is ubiquitinated via K63 linkages to form a scaffolding signalling complex that is required for the activation of the classical pathway whereas N IK receives a K48 linked ubiquitination targeting it for degradation, and keeping the alternative pathway inactive (FIG. 35). SMCs, are a novel class of anti-cancer therapeutics that mimic the endogenous Smac protein, which is involved in the activation of the intrinsic apoptotic pathway. Smac peptide and SMCs bind to the BI R domain of clAPs, which causes them to auto- ubiquitinate, targeting them for proteasomal degradation. When RIP1 is no longer ubiquitinated, it becomes free to form the ripoptosome, initiating the caspase cascade and cell death. SMCs have been shown to have strong synergy with TN Fa to induce NF-κΒ -mediated apoptosis in many cancer lines. SMCs also have synergistic cancer cell killing in combination other inflammatory cytokines such as I FNs, which can be induced by TLR agonists or oncolytic viruses. SMCs can even standardize therapeutics used for MM to enhance apoptosis of cancer cells. Several clinical trials that are currently being conducted for assessing the the efficacy of SMCs with chemotherapeutics in MM as well as other cancers have shown great therapeutic potential.

Activating the immune system increases cytokine production, which is advantageous for SMC- mediated MM cell killing. However, this cytokine production may have undesirable consequences on the MM cells. Many innate immune stimulants, such as I FNs and TLR agonists, have been shown to upregulate ligands of the immune checkpoint PD-1 . PD-1 is expressed on the surface of T cells and NK cells. When PD-1 binds its ligands, PD-L1 and PD-L2, it acts as a co-inhibitory signal for the T cell receptor to supress the cytotoxic ability of T cells. PD-L1 is expressed constitutively at low levels in many tissues and can be upregulated, presumably to prevent autoimmune reactions. However, PD-L1 is upregulated on cancer cells, leading to the cells evading detection by the adaptive immune system. In particular, PD-L1 can be upregulated in MM in response to I FNy and TLR agonists such as LPS. PD-L2 has a much more selective expression compared to PD-L1 . It is present in a subset of B cells and upregulated on select cells in response to strong NF-κΒ or STAT6 signalling.

SMCs can also affect the function of T cells of SMC-treated mice both in vitro and in vivo, e.g., increased proliferation, increased cytokine production of activated T cells extracted from mouse spleens after exposure to SMCs, and higher cytokine production from NKT and NK cells. Additionally, mice treated with SMC exhibit hyperresponsive T cells upon antigen stimulation. Therefore a SMC-based combination therapy could not only increase the apoptosis of MM cells but may also stimulate a selective adaptive response. Combining SMCs with innate immune stimulants or immune checkpoint inhibitors (ICIs) may be the best approach to overcome the strong pro-survival signals the MM cells receive.

Cancer cells are able to manipulate many of the pro-survival strategies healthy cells utilize in order to make them resistant to death-inducing signals. MM cells specifically are able to further amplify the constitutive N F-κΒ signalling used in plasma cells to make them resistant to apoptotic stimuli. This is accomplished by increased expression of pro-survival NF-κΒ target genes such as IL-6 and TNFa.

Additionally, MM cells are able to enhance expression of checkpoint inhibitors, which are presumably used to protect cells from inflammatory and cytotoxic environments; this helps them evade detection by T cells and NK cells. Targeting both apoptotic resistance and immune evasion in MM has the potential to overcome two of the major aspects of treatment resistance in this disease.

PD-1 blockade is effective at delaying MM disease progression and improving the survival time of mice significantly as shown using the syngeneic murine MM model. Using a monoclonal antibody against PD-1 has several advantages compared to alternative approaches for immune checkpoint blockade. Firstly, it is able to block binding of both PD-I ligands, PD-L1 and PD-L2. Many cancers are able to upregulate PD-L1 in response to interferon treatment, and PD-1 /PD-L1 are upregulated in MM patients after treatment. Additionally, a subset of immature B cells, called B1 cells, which secrete non-specific antibodies, have shown high expression of PD-L2. Furthermore, PD-L2 expression can increase in response to certain stimuli, such as NF-κΒ and STAT6 activation demonstrating the importance of examining expression levels of both ligands on MM cells. Human MM cells are able to upregulate both PD-1 ligands, making them unique in comparison to solid cancers. Although this suggests monoclonal antibody therapy targeting only PD-L1 (such as Bristol-Myers Squibb's BMS-936559/M DX-1 105, Genentech's M PDL3280A, Medlmmune's M EDI473, and EM D Serono's avelumab) would be less effective than treatments targeting PD-1 (such as Bristol-Myers Squibb's nivolumab. Merck's

pembrolizumab, and Curetech's pidilizumab), it shows the value of using anti-PD-1 antibodies in MM.

Secondly, PD-1 targeted approaches have the potential to have a more robust response against the cancer in comparison to other ICIs such as anti-CTLA-4. The differences in activity may be due to the particular roles of these molecules in T cell regulation. PD-1 is often found on CD8+ T cells and engagement with its ligand inhibits the cytotoxic response activated by TCR signalling. In contrast, CTLA-4 has a more prominent role in secondary lymphoid tissues on regulatory T cells. CTLA-4 engagement with its receptor, CD28, outcompetes and even down regulates the activating ligands for CD28, and causes dampening of T cell secondary clonal expansion. It is entirely possible that the lack of efficacy of anti-CTLA-4 treatment in Example 3 indicates MM invasion into secondary lymphoid organs. This could compromise anti-CTLA4 efficacy either by the CD4+ T cell population being proportionately lower within the germinal centres or T cell infiltration to the secondary lymphoid organs being hampered. In extramedullary MM, the cells can form plasmacytomas in the spleen and lymph nodes, which is often seen in late stages of the MM mouse model discussed in Example 3. Therefore, it is evident the germinal centres are compromised by the M PC-1 1 cells SMCs

An SMC of the present invention may be any small molecule, compound, polypeptide, protein, or any complex thereof, capable, or predicted of being capable, of inhibiting clAP1 , clAP2 and/or XIAP, and, optionally, one or more additional endogenous Smac activities. An SMC of the present invention is capable of potentiating apoptosis by mimicking one or more activities of endogenous Smac, including but not limited to, the inhibition of clAP1 and the inhibition of clAP2. An endogenous Smac activity may be, e.g., interaction with a particular protein, inhibition of a particular protein's function, or inhibition of a particular IAP. In particular embodiments, the SMC inhibits both clAP1 and clAP2. In some

embodiments, the SMC inhibits one or more other lAPs in addition to clAP1 and clAP2, such as XIAP or Livin/ML-IAP, the single BI R-containing IAP. In particular embodiments, the SMC inhibits clAP1 , clAP2, and XIAP. In any embodiment including an SMC and an immune stimulant, an SMC having particular activities may be selected for combination with one or more particular immune stimulants. In any embodiment of the present invention, the SMC may be capable of activities of which Smac is not capable. In some instances, these additional activities may contribute to the efficacy of the methods or compositions of the present invention.

Treatment with SMCs can deplete cells of clAP1 and clAP2, through, e.g., the induction of auto- or trans-ubiquitination and proteasomal-mediated degradation. SMCs can also de-repress XIAP's inhibition of caspases. SMCs may primarily function by targeting clAP1 and 2, and by converting TN Fa, and other cytokines or death ligands, from a survival signal to a death signal, e.g., for cancer cells.

Certain SMCs inhibit at least XIAP and the clAPs. Such "pan-IAP" SMCs can intervene at multiple distinct yet interrelated stages of programmed cell death inhibition. This characteristic minimizes opportunities for cancers to develop resistance to treatment with a pan-IAP SMC, as multiple death pathways are affected by such an SMC, and allows synergy with existing and emerging cancer therapeutics that activate various apoptotic pathways in which SMCs can intervene.

One or more inflammatory cytokines or death ligands, such as TNFa, TRAIL, and IL-1 β, potently synergize with SMC therapy in many tumor-derived cell lines. Strategies to increase death ligand concentrations in SMC-treated tumors, in particular using approaches that would limit the toxicities commonly associated with recombinant cytokine therapy, are thus very attractive. TNFa, TRAIL, and dozens of other cytokines and chemokines can be upregulated in response to pathogen recognition by the innate immune system of a subject. Importantly, this ancient response to microbial pathogens is usually self-limiting and safe for the subject, due to stringent negative regulation that limits the strength and duration of its activity.

SMCs may be rationally designed based on Smac. The ability of a compound to potentiate apoptosis by mimicking one or more functions or activities of endogenous Smac can be predicted based on similarity to endogenous Smac or known SMCs. An SMC may be a compound, polypeptide, protein, or a complex of two or more compounds, polypeptides, or proteins.

In some instances, SMCs are small molecule IAP antagonists based on an N-terminal tetrapeptide sequence (revealed after processing) of the polypeptide Smac. In some instances, an SMC is a monomer (monovalent) or dimer (bivalent). I n particular instances, an SMC includes 1 or 2 moieties that mimic the tetrapeptide sequence of AVPI from Smac/DIABLO, the second mitochondrial activator of caspases, or other similar IBMs (e.g., IAP-binding motifs from other proteins like casp9). A dimeric SMC of the present invention may be a homodimer or a heterodimer. In certain embodiments, the dimer subunits are tethered by various linkers. The linkers may be in the same defined spot of either subunit, but could also be located at different anchor points (which may be 'aa' position, P1 , P2, P3 or P4, with sometimes a P5 group available). I n various arrangements, the dimer subunits may be in different orientations, e.g., head to tail, head to head, or tail to tail. The heterodimers can include two different monomers with differing affinities for different BI R domains or different lAPs. Alternatively, a heterodimer can include a Smac monomer and a ligand for another receptor or target which is not an IAP. In some instances, an SMC can be cyclic. In some instances, an SMC can be trimeric or multimeric. A multimerized SMC can exhibit a fold increase in activity of 7,000-fold or more, such as 1 0-, 20-, 30-, 40-, 50-, 100-, 200-, 1 ,000-, 5,000-, 7,000-fold, or more (measured, e.g., by EC50 in vitro) over one or more corresponding monomers. This may occur, in some instances, e.g., because the tethering enhances the ubiquitination between lAPs or because the dual BI R binding enhances the stability of the interaction. Although multimers, such as dimers, may exhibit increased activity, monomers may be preferable in some embodiments. For example, in some instances, a low molecular weight SMC may be preferable, e.g., for reasons related to bioavailability.

In some instances of the present invention, an agent capable of inhibiting clAP1 /2 is a bestatin or

Me-bestatin analog. Bestatin or Me-bestatin analogs may induce clAP1 /2 autoubiquitination, mimicking the biological activity of Smac.

In certain embodiments of the present invention, an SMC combination treatment includes one or more SMCs and one or more interferon agents, such as an interferon type 1 agent, an interferon type 2 agent, and an interferon type 3 agent. Combination treatments including an interferon agent may be useful in the treatment of cancer, such as multiple myeloma. In some embodiments, a VSV expressing I FN, and optionally expressing a gene that enables imaging, such as N IS, the sodium-iodide symporter, is used in combination with an SMC. For instance, such a VSV may be used in combination with an SMC, such as the Ascentage Smac mimetic SM- 1387/APG-1387, the Novartis Smac mimetic LCL161 , or Birinapant. Such combinations may be useful in the treatment of cancer, such as hepatocellular carcinoma or liver metastases.

Various SMCs are known in the art. Non-limiting examples of SMCs are provided in Table 1 . While Table 1 includes suggested mechanisms by which various SMCs may function, methods and compositions of the present invention are not limited by or to these mechanisms.

Table 1 . Smac mimetic compounds

BI-75D2 Formula: C26H26N4O4S2 Preclinical Sanford-Burnham

Institute; J. Reed

I i

0 X : ^' ,

T5TR1 Crisostomo FR, Feng Y, Zhu X, Welsh K, An J, Reed JC, Huang Z. Design and synthesis Preclinical Sanford-Burnham of a simplified inhibitor for XIAP-BIR3 domain. Bioorg Med Chem Lett. 2009 Nov Institute (NIH?); J. 15;19(22):6413-8. doi: 0.1016/j.bmcl.2009.09.058. Epub 2009 Sep 17. PubMed PMID: Reed

19819692; PubMed Central PMCID: PMC3807767.

ML-101 Welsh K, Yuan H, Stonich D, Su Y, Garcia X, Cuddy M, Houghten R, Sergienko E, Reed Preclinical Sanford-Burnham

JC, Ardecky R, Ganji SR, Lopez M, Dad S, Chung TDY, Cosford N. Antagonists of IAP- Institute (NIH?); J. family anti-apoptotic proteins - Probe 1 . 2009 May 18 [updated 2010 Sep 2]. Probe Reed

Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National

Center for Biotechnology Information (US); 2010-. Available from

http://www.ncbi.nlm.nih.gov/books/NBK47341 /; Gonzalez-Lopez M, Welsh K, Finlay D,

Ardecky RJ, Ganji SR, Su Y, Yuan H, Teriete P, Mace PD, Riedl SJ, Vuori K, Reed JC,

Cosford ND. Design, synthesis and evaluation of monovalent Smac mimetics that bind to

the BIR2 domain of the anti-apoptotic protein XIAP. Bioorg Med Chem Lett. 201 1 Jul

15;21 (14):4332-6. doi: 10.1016/|.bmcl.201 1 .05.049. Epub 201 1 May 24.

MLS-0390866 Welsh K, Yuan H, Stonich D, Su Y, Garcia X, Cuddy M, Houghten R, Sergienko E, Reed Preclinical Sanford-Burnham

JC, Ardecky R, Ganji SR, Lopez M, Dad S, Chung TDY, Cosford N. Antagonists of IAP- Institute (NIH?); J. family anti-apoptotic proteins - Probe 1 . 2009 May 18 [updated 2010 Sep 2]. Probe Reed

Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National

Center for Biotechnology Information (US); 2010-. Available from

http://www.ncbi.nlm.nih.gov/books/NBK47341 /PubMed

Agents

An immunostimulatory or immunomodulatory agent of the present invention may be any agent capable of inducing a receptor-mediated apoptotic program that is inhibited by clAP1 and clAP2 in one or more cells of a subject. An immune stimulant of the present invention may induce an apoptotic program regulated by clAP1 (BIRC2), clAP2 (BI RC3 or API2), and optionally, one or more additional lAPs, e.g., one or more of the human IAP proteins NAI P (BI RC1 ), XIAP (BIRC4), survivin (BI RC5), Apollon/Bruce (BIRC6), ML-IAP (BI RC7 or livin), and ILP-2 (BI RC8). It is additionally known that various

immunomodulatory or agents, such as CpGs or IAP antagonists, can change immune cell contexts.

In some instances, an immune stimulant may be a TLR agonist, such as a TLR ligand. A TLR agonist of the present invention may be an agonist of one or more of TLR-1 , TLR-2, TLR-3, TLR-4, TLR- 5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-1 0 in humans or related proteins in other species (e.g., murine TLR-1 to TLR-9 and TLR-1 1 to TLR-13). TLRs can recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAM Ps), which are exclusively expressed by microbial pathogens, as well as danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells. PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipopeptides, as well as flagellin, bacterial DNA, and viral double-stranded RNA. DAM Ps include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix. Agonists of the present invention further include, for example, CpG oligodeoxynucleotides (CpG ODNs), such as Class A, B, and C CpG ODN's, base analogs, nucleic acids such as dsRNA or pathogen DNA, or pathogen or pathogen-like cells or virions. In certain embodiments, the agent is an agent that mimics a virus or bacteria or is a synthetic TLR agonist.

Various TLR agonists are known in the art. Non-limiting examples of TLR agonists are provided in Table 2. While Table 2 includes suggested mechanisms, uses, or TLR targets by which various TLR agonists may function, methods and compositions of the present invention are not limited by or to these mechanisms, uses, or targets.

Table 2. Agents: TLR Agonists

Compound Structure or Reference Compound Type or Application Agonist of :

oligonucleotides that combine B cell activation with high IFN-alpha

induction in plasmacytoid dendritic cells. Eur J Immunol 2003, 33:1633- 41

ODN 1018 Magone, M. T., Chan, C. C, Beck, L, Whitcup, S. M., Raz, E. (2000) Class B TLR-9 agonist

Systemic or mucosal administration of immunostimulatory DNA inhibits

early and late phases of murine allergic conjunctivitis Eur. J.

Immunol. 30,1841 -1850

CL401 Formula: C5 H92N8O4S Dual TLR agonist TLR-2 and TLR-7

J 1 ^'■ > ^0H

1

^H I II

0. .- . .-.

11

0

Adilipoline™ (CL413;) Formula: C81 H145N17O12S Dual TLR agonist TLR-2 and TLR-7

Ί _^

Compound Structure or Reference Compound Type or Application Agonist of:

CL531 Formula: C82H144N16O14S Dual TLR agonist TLR-2 and TLR-7

CL572 ( Formula: C41 H65N9O7S Dual TLR agonist Human TLR-2, mouse TLR-7, and human TLR-7

AdiFectin™ (CL347;) Formula: TLR agonist and nucleic acid carrier TLR-7

1

Imiquimod (InvivoGen) Imidazoquinoline compound; topical administration for treatment of basal cell carcinoma (see, e.g., Schulze HJ, Cribier B, Requena L, et al. Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from a randomized vehicle-controlled Phase III study in Europe. Br. J. Dermatol. 2005; 152 (5): 939-947; Quirk C, Gebauer K, Owens M, Stampone P. Two-year interim results from a 5-year study evaluating clinical recurrence of superficial basal cell carcinoma after treatment with imiquimod 5% cream daily for 6 weeks. Australas. J. Dermatol. 2006; 47(4):258-265.);

Topical administration for treatment of squamous cell carcinoma (see, e.g., Ondo AL, Mings SM, Pestak RM, Shanler SD. Topical combination therapy for cutaneous squamous cell carcinoma in situ with 5-fluorouracil cream and imiquimod cream in patients who have failed topical monotherapy. J. Am. Acad. Dermatol. 2006; 55(6): 1092-1094.)

Topical administration for treatment of melanoma (see, e.g., Turza K, Dengel LT, Harris RC, et al.

Effectiveness of imiquimod limited to dermal melanoma metastases, with simultaneous resistance of subcutaneous metastasis. J. Cutan. Pathol. 2009 DOI: 10.1 1 1 1 /j.1600- 0560.2009.01290.x. (Epub ahead of print); (see, e.g., Green DS, Dalgleish AG, Belonwu N, Fischer MP, Bodman-Smith MP. Topical

imiquimod and intralesional interleukin-2 increase activated lymphocytes and restore the Th1 /Th2 balance in patients with metastatic melanoma. Br. J.

Dermatol. 2008; 159(3):606-614.);

Topical administration for treatment of vulvar intraepithelial neoplasia (see, e.g., Van Seters M, Van Beurden M, Ten Kate FJ, et al. Treatment of vulvar intraepithelial neoplasia with topical imiquimod. N. Engl. J. Med. 2008; 358(14):1465- 1473.);

Topical administration for treatment of cutaneous lymphoma (see, e.g., Stavrakoglou A, Brown VL, Coutts I. Successful treatment of primary cutaneous follicle centre lymphoma with topical 5% imiquimod. Br. J. Dermatol. 2007; 157(3): 620-622.);

Topical treatment as Human papillomavirus (HPV) vaccine (see, e.g., Daayana S, Elkord E, Winters U, et al. Phase II trial of imiquimod and HPV therapeutic vaccination in patients with vulval intraepithelial neoplasia. Br. J. Cancer. 2010; 102(7):1 129- 1 136.);

Subcutaneous/intramuscular administration: New York esophageal squamous cell carcinoma 1 cancer antigen (NY- ESO-1 ) protein vaccine for melanoma (see, e.g., Adams S, O'Neill DW, Nonaka D, et al. Immunization of malignant melanoma patients with full- length

NY-ESO-1 protein using TLR7

agonist imiquimod as vaccine

adjuvant. J. Immunol. 2008;

181 (1 )776-784.)

Monophosphoryl lipid A Subcutaneous/intramuscular TLR-4 (MPL) administration for vaccination

against HPV (see, e.g., Harper DM, Franco EL, Wheeler CM, et al.

Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle

vaccine against human

papillomavirus types 16 and 18:

follow-up from arandomised control trial. Lancet. 2006; 367(9518):1247- 1255.);

Subcutaneous/intramuscular

administration for vaccination

against non-small-cell lung cancer (see, e.g., Butts C, Murray N,

Maksymiuk A, et al. Randomized

Phase MB trial of BLP25 liposome vaccine in stage NIB and IV non- small-cell lung cancer. J. Clin.

Oncol. 2005; 23(27):6674-6681.)

CpG 7909 (i.e., PF- Subcutaneous/intramuscular TLR-9 3512676) administration for treatment of non- small-cell lung cancer (see, e.g.,

Manegold C, Gravenor D, Woytowitz D, et al. Randomized Phase II trial of a Toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage non-small-cell lung cancer. J. Clin. Oncol. 2008;

26(24):3979- 3986; Readett, D. ;

Denis, L. ; Krieg, A. ; Benner, R.;

Hanson, D. PF-3512676 (CPG

7909) a Toll-like receptor 9 agonist -

status of development for non-small cell lung cancer (NSCLC).

Presented at: 12th World Congress on Lung Cancer; Seoul, Korea. 2-6 September 2007);

Subcutaneous/intramuscular administration for treatment of metastatic melanoma (see, e.g., Pashenkov M, Goess G, Wagner C, et al. Phase II trial of a Toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma. J. Clin. Oncol. 2006; 24(36):5716-5724. ;

Subcutaneous/intramuscular administration; Melan-A peptide vaccine for melanoma (see, e.g., Speiser DE, Lienard D, Rufer N, et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J. Clin. Invest. 2005; 1 15(3):

739-746; Appay V, Jandus C, Voelter V, et al. New generation vaccine induces effective melanoma- specific CD8+ T cells in the circulation but not in the tumor site. J. Immunol. 2006; 177(3):1670- 1678.);

Subcutaneous/intramuscular administration; NY-ESO-1 protein vaccine (see, e.g., Valmori D, Souleimanian NE, Tosello V, et al. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-

In other instances, an immune stimulant may be a virus, e.g., an oncolytic virus. An oncolytic virus is a virus that selectively infects, replicates, and/or selectively kills cancer cells. Viruses of the present invention include, without limitation, adenoviruses, Herpes simplex viruses, measles viruses, Newcastle disease viruses, parvoviruses, polioviruses, reoviruses, Seneca Valley viruses, retroviruses, Vaccinia viruses, vesicular stomatitis viruses, lentiviruses, rhabdoviruses, sindvis viruses,

coxsackieviruses, poxviruses, and others. In particular embodiments of the present invention, the agent is a rhabodvirus, e.g., VSV. Rhabdoviruses can replicate quickly with high IFN production. In other particular embodiments, the agent is a feral member, such as Maraba virus, with the MG1 double mutation, Farmington virus, Carajas virus. Viral agents of the present invention include mutant viruses (e.g., VSV with a Δ51 mutation in the Matrix, or M, protein), transgene-modified viruses (e.g., VSV- hl FN ), viruses carrying -TNFa, -LTa/TNF , -TRAIL, FasL, -TL1 a, chimeric viruses (eg rabies), or pseudotyped viruses (e.g., viruses pseudotyped with G proteins from LCMV or other viruses). In some instances, the virus of the present invention will be selected to reduce neurotoxicity. Viruses in general, and in particular oncolytic viruses, are known in the art.

In certain embodiments, the agent is a killed VSV NRRP particle or a prime-and-boost tumor vaccine. NRRPs are wild type VSV that have been modified to produce an infectious vector that can no longer replicate or spread, but that retains oncolytic and immunostimulatory properties. NRRPs may be produced using gamma irradiation, UV, or busulfan. Particular combination therapies include prime-and- boost with adeno-MAGE3 (melanoma antigen) and/or Maraba-MG1 -MAGE3. Other particular combination therapies include UV-killed or gamma irradiation-killed wild-type VSV NRRPs. N RRPs may demonstrate low or absent neurotixicity. NRRPs may be useful, e.g., in the treatment of glioma, hematological (liquid) tumors, or multiple myeloma.

In some instances, the agent of the present invention is a vaccine strain, attenuated virus or microorganism, or killed virus or microorganism. In some instances, the agent may be, e.g., BCG, live or dead Rabies vaccines, or an influenza vaccine.

Non-limiting examples of viruses of the present invention, e.g., oncolytic viruses, are provided in Table 3. While Table 3 includes suggested mechanisms or uses for the provided viruses, methods and compositions of the present invention are not limited by or to these mechanisms or uses.

Table 3. Agents

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

(VRX-007) Patra D, Meyer JM, Shashkova EV, Kuppuswamy M, Dhar D, Thomas MA,

Tollefson AE, Zumstein LA, Wold WS, Toth K. An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector. Cancer Gene Ther. 2009 Aug;16(8):644-54. doi:

10.1038/cgt.2009.5. Epub 2009 Feb 6.

ColoAdl Ad3/1 1 p Adenovirus Phase 1 /2; CRC, HCC; ; Not open; Kuhn I, Harden P, Bauzon M, Chartier C,

Nye J, Thorne S, Reid T, Ni S, Lieber A, Fisher K, Seymour L, Rubanyi GM, Harkins RN, Hermiston TW. Directed evolution generates a novel oncolytic virus for the treatment of colon cancer. PLoS One. 2008 Jun 18;3(6):e2409. doi: 10.1371 /journal. pone.0002409.

CAVATAK Coxsackie virus Phase 1 ; Melanoma; IT; Completed

(CVA21 ) Phase 2; Melanoma; IT; Recruiting

Phase 1 ; SCCHN; IT; Terminated

Phase 1 ; Solid tumors; IV; Recruiting

Talimogene GM-CSF Herpes simplex Phase 1 ; Solid tumors; IT; Completed; Hu JC, Coffin RS, Davis CJ, Graham laherparepvec virus NJ, Groves N, Guest PJ, Harrington KJ, James ND, Love CA, McNeish I,

(OncoVEX) Medley LC, Michael A, Nutting CM, Pandha HS, Shorrock CA, Simpson J,

Steiner J, Steven NM, Wright D, Coombes RC. A phase I study of

OncoVEXGM-CSF, a second-generation oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor. Clin Cancer Res. 2006 Nov 15;12(22):6737-47.

Talimogene ICP34.5(-) Herpes simplex Phase 2; Melanoma; IT; Completed; Kaufman HL, Kim DW, DeRaffele G, laherparepvec virus Mitcham J, Coffin RS, Kim-Schulze S. Local and distant immunity induced by

(OncoVEX) intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage lllc and IV melanoma. Ann Surg Oncol. 2010

Mar;17(3):718-30. doi: 10.1245/s10434-009-0809-6; Senzer NN, Kaufman HL, Amatruda T, Nemunaitis M, Reid T, Daniels G, Gonzalez R, Glaspy J, Whitman E, Harrington K, Goldsweig H, Marshall T, Love C, Coffin R, Nemunaitis JJ. Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor- encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. J Clin Oncol. 2009 Dec 1 ;27(34):5763-71 . doi: 0.1200/JCO.2009.24.3675. Epub 2009 Nov 2.

Talimogene ICP47(-) Herpes simplex Phase 3; Melanoma; IT; Active

laherparepvec virus

(OncoVEX)

Talimogene Us1 1† Herpes simplex Phase 1 /2; SCCHN; IT; Completed; Harrington KJ, Hingorani M, Tanay MA, laherparepvec virus Hickey J, Bhide SA, Clarke PM, Renouf LC, Thway K, Sibtain A, McNeish IA,

(OncoVEX) Newbold KL, Goldsweig H, Coffin R, Nutting CM. Phase l/ll study of oncolytic

HSV GM-CSF in combination with radiotherapy and cisplatin in untreated stage lll/IV squamous cell cancer of the head and neck. Clin Cancer Res. 2010 Aug

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

JX-594 TK(-) Vaccinia (Wyeth Phase 1 ; Solid tumors; IV; Completed

strain) Phase 1 ; HCC; IT; Completed; Park BH, Hwang T, Liu TC, Sze DY, Kim JS,

Kwon HC, Oh SY, Han SY, Yoon JH, Hong SH, Moon A, Speth K, Park C, Ahn YJ, Daneshmand M, Rhee BG, Pinedo HM, Bell JC, Kirn DH. Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial. Lancet Oncol. 2008 Jun;9(6):533-42. doi:

10.1016/S1470-2045(08)70107-4. Epub 2008 May 19. Erratum in: Lancet Oncol. 2008 Jul;9(7):613.

Phase 1 ; Pediatric solid tumors; IT; Recruiting

Phase 1 ; Melanoma; IT; Completed; Hwang TH, Moon A, Burke J, Ribas A, Stephenson J, Breitbach CJ, Daneshmand M, De Silva N, Parato K, Diallo JS, Lee YS, Liu TC, Bell JC, Kirn DH. A mechanistic proof-of-concept clinical trial with JX-594, a targeted multi-mechanistic oncolytic poxvirus, in patients with metastatic melanoma. Mol Ther. 201 1 Oct;19(10):1913-22. doi:

10.1038/mt.201 1 .132. Epub 201 1 Jul 19.

Phase 1 /2; Melanoma; IT; Completed; Mastrangelo MJ, Maguire HC Jr, Eisenlohr LC, Laughlin CE, Monken CE, McCue PA, Kovatich AJ, Lattime EC. Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther. 1999 Sep-Oct;6(5):409-22.

Phase 2; HCC; IT; Not recruiting, analyzing data

Phase 2B; HCC; IV; Recruiting

Phase 1 /2; CRC; IV/IT; Recruiting

Phase 2; CRC; IT; Not yet recruiting

wDD-CDSR TK-, VGF-, LacZ, CD, Vaccinia Phase 1 ; Solid tumors; IT/IV; Recruiting; McCart JA, Mehta N, Scollard D,

Somatostatin R (Western Reilly RM, Carrasquillo JA, Tang N, Deng H, Miller M, Xu H, Libutti SK,

Reserve) Alexander HR, Bartlett DL. Oncolytic vaccinia virus expressing the human somatostatin receptor SSTR2: molecular imaging after systemic delivery using 1 1 1 ln-pentetreotide. Mol Ther. 2004 Sep;10(3):553-61 .

GL-ONC1 Renilla luciferase Vaccinia Phase 1 ; Solid tumors; IV; Recruiting, Gentschev I, Muller M, Adelfinger M,

Weibel S, Grummt F, Zimmermann M, Bitzer M, Heisig M, Zhang Q, Yu YA, Chen NG, Stritzker J, Lauer UM, Szalay AA. Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1 h68. PLoS One. 201 1 ;6(7):e22069. doi:

10.1371 /journal. pone.0022069. Epub 201 1 Jul 1 1 .

(GLV-h68) GFP, β-gal Vaccinia Phase 1 /2; Peritoneal carcinomatosis; IP; Recruiting

Lister β-glucoronidase Vaccinia Phase 1 /2; SCCHN; IV; Recruiting

VSV-hlFNp IFN-β Vesicular Phase 1 ; HCC; IT; Recruiting

stomatitis virus

(Indiana)

DNX-2401 DNAtrix Adenovirus See, e.g., Molecular Therapy 21 (10): 1814-1818, 2013 and Journal of

Vascular and Interventional Radiology 24(8) : 1 1 15-1 122, 2013

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

lab') M and L genes originate from Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic the San Juan strain; G gene virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: from the Orsay strain (both 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Whelan SP, Ball LA, Barr JN, Indiana serotype). Rarely used Wertz GT. Efficient recovery of infectious vesicular stomatitis virus entirely from in OV studies cDNA clones. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8388-92.)

VSV-WT-GFP, - WT VSV encoding reporter Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, RFP, -Luc, - genes (between G and L) to Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic LacZ track virus infection. Based on virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

pVSV-XN2. Toxicity similar to 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Fernadez et al., "Genetically VSV-WT Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for

Treatment of Malignant Disease", J Virol 76:895-904 (2002); Lan Wu, Tian-gui Huang, Marcia Meseck, Jennifer Altomonte, Oliver Ebert, Katsunori Shinozaki, Adolfo Garcia-Sastre, John Fallon, John Mandeli, and Savio L.C. Woo. Human Gene Therapy. June 2008, 19(6): 635-647)

VSV-G/GFP GFP sequence fused to VSV G Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, gene is inserted between the Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic WT G and L genes (in addition virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: to WT G). Toxicity similar to that 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Dalton, K. P. & Rose, J. K. (2001 ). of VSV-WT Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles. Virology 279, 414-421 .)

VSV-rp30 Derivative of VSV-G/GFP. Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

Generated by positive selection Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic on glioblastoma cells and virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: contains two silent mutations 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Wollmann, G., Tattersall, P. & van and two missense mutations, den Pol, A. N. (2005). Targeting human glioblastoma cells: comparison of nine one in P and one in L. 'rp30' viruses with oncolytic potential. J Virol 79, 6005-6022.)

indicates 30 repeated passages

VSV-pl -GFP, VSV expressing GFP or red Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, VSV-pl -RFP fluorescent protein (RFP or Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic dsRed) reporter gene at virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: position 1 . Attenuated because 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Wollmann, G., Rogulin, V., Simon, all VSV genes are moved I., Rose, J. K. & van den Pol, A. N. (2010). Some attenuated variants of downward, to positions 2-6. vesicular stomatitis virus show enhanced oncolytic activity against human Safe and still effective as an OV glioblastoma cells relative to normal brain cells. J Virol 84, 1563-1573.)

VSV-c!G-GFP Similar to VSV-p1 -GFP or VSV- Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, (or RFP) pl -RFP described above, but Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic (replication- with the G gene deleted. virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: defective) Cannot generate a second 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Wollmann, G., Rogulin, V., Simon, round of infection. Poor ability I., Rose, J. K. & van den Pol, A. N. (2010). Some attenuated variants of to kill tumor cells vesicular stomatitis virus show enhanced oncolytic activity against human

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

VSV-M6PY M mutant; the M51 R mutation Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, >A4-R34E and was introduced into the M gene, Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic other M mutants and, in addition, the mutations virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

in the PSAP motif (residues 37- 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Irie, T., Camera, E., Okumura, A., 40) of M Garci ^' a-Sastre, A. & Harty, R. N. (2007). Modifications of the PSAP region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo. J Gen Virol 88, 2559-2567.)

VSV-M(mut) M mutant; VSV M residues 52- Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

54 are mutated from DTY to Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic AAA. M(mut) cannot block virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: nuclear mRNA export 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Heiber, J. F. & Barber, G. N.

(201 1 ). Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent. J Virol 85, 10440-10450.)

VSV-G5, -G5R, G mutant; VSV-expressing Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, -G6, -G6R mutant G with amino acid Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic substitutions at various virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: positions (between residues 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Janelle, V., Brassard, F., Lapierre, 100 and 471 ). Triggers type I P., Lamarre, A. & Poliquin, L. (201 1 ). Mutations in the glycoprotein of vesicular IFN secretion as the M51 R, but stomatitis virus affect cytopathogenicity: potential for oncolytic virotherapy. J inhibits cellular transcription and Virol 85, 6513-6520.)

host protein translation like WT

VSV-CT1 G mutant; the cytoplasmic tail of Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, the G protein was truncated Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic from 29 to 1 aa. Decreased virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: neuropathology, but marginal 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Ozduman, K., Wollmann, G., oncolytic efficacy Ahmadi, S. A. & van den Pol, A. N. (2009). Peripheral immunization blocks lethal actions of vesicular stomatitis virus within the brain. J Virol 83, 1 1540— 1 1549.; Wollmann, G., Rogulin, V., Simon, I., Rose, J. K. & van den Pol, A. N. (2010). Some attenuated variants of vesicular stomatitis virus show enhanced oncolytic activity against human glioblastoma cells relative to normal brain cells. J Virol 84, 1563-1573.)

VSV-CT9-M51 G mutant; the cytoplasmic tail of Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

VSV-G was reduced from 29 to Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic 9 aa, also has ΔΜ51 mutation. virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: Attenuated neurotoxicity and 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Ozduman, K., Wollmann, G., good OV abilities Ahmadi, S. A. & van den Pol, A. N. (2009). Peripheral immunization blocks lethal actions of vesicular stomatitis virus within the brain. J Virol 83, 1 1540- 1 1549.; Wollmann, G., Rogulin, V., Simon, I., Rose, J. K. & van den Pol, A. N. (2010). Some attenuated variants of vesicular stomatitis virus show enhanced oncolytic activity against human glioblastoma cells relative to normal brain cells. J Virol 84, 1563-1573.)

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

vsv- Foreign glycoprotein; VSV Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

DV/F(L289A) expressing the NDV fusion Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic (same as rVSV- protein gene between G and L. virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: F) The L289A mutation in this 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Ebert, O., Shinozaki, K., Kournioti, protein allows it to induce C, Park, M. S., Garci ^' a-Sastre, A. & Woo, S. L. (2004). Syncytia induction syncytia alone (without NDV HN enhances the oncolytic potential of vesicular stomatitis virus in virotherapy for protein) cancer. Cancer Res 64, 3265-3270.)

VSV-S-GP Foreign glycoprotein; VSV with Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, the native G gene deleted and Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic replaced with a modified virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: glycoprotein protein (GP) from 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Bergman, I., Griffin, J. A., Gao, Y. Sindbis virus (SV). Also & Whitaker-Dowling, P. (2007). Treatment of implanted mammary tumors with expressing mouse GM-CSF recombinant vesicular stomatitis virus targeted to Her2/neu. Int J Cancer 121 , and GFP (between SV GP and 425-430.)

VSV L). The modified GP

protein recognizes the Her2

receptor, which is

overexpressed on many breast

cancer cells

VSV-AG-SV5-F Foreign glycoprotein; VSV G Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, gene is replaced with the Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic fusogenic simian parainfluenza virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: virus 5 fusion protein (SV5-F) 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Chang, G., Xu, S., Watanabe, M., gene Jayakar, H. R., Whitt, M. A. & Gingrich, J. R. (2010). Enhanced oncolytic activity of vesicular stomatitis virus encoding SV5-F protein against prostate cancer. J Urol 183, 161 1 -1618.)

VSV-FAST, Foreign glycoprotein; VSV or Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

VSV-(AM51 )- VSV-MA51 expressing the p14 Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic

FAST FAST protein of reptilian virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

reovirus (between VSV G and 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Brown, C. W., Stephenson, K. B., L) Hanson, S., Kucharczyk, M., Duncan, R., Bell, J. C. & Lichty, B. D. (2009). The p14 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis. J Virol 83, 552-561.)

VSV-LCMV-GP Foreign glycoprotein; VSV Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, (replication- lacking the G gene was Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic defective) pseudotyped with the non- virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

neurotropic glycoprotein of 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Muik, A., Kneiske, I., Werbizki, M., LMCV Wilflingseder, D., Giroglou, T., Ebert, O., Kraft, A., Dietrich, U., Zimmer, G. & other authors (201 1 ). Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism. J Virol 85, 5679-5684.)

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

L. & Hiscott, J. (201 1 ). Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy. Cancer Gene Ther 18, 435-443.)

VSV-(MA51 )- Suicide gene; VSV-MA51 Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, NIS expressing the human NIS Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic gene (for 'radiovirotherapy' virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: with 131 1) 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Goel, A., Carlson, S. K., Classic, K.

L, Greiner, S., Naik, S., Power, A. T., Bell, J. C. & Russell, S. J. (2007). Radioiodide imaging and radiovirotherapy of multiple myeloma using

VSV(D51 )-NIS, an attenuated vesicular stomatitis virus encoding the sodium iodide symporter gene. Blood 1 10, 2342-2350.)

VSV-TK Suicide gene; VSV expressing Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E,

TK; can improve oncolysis if Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic used with non-toxic prodrug virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: ganciclovir 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Fernandez, M., Porosnicu, M.,

Markovic, D. & Barber, G. N. (2002). Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. J Virol 76, 895-904.)

VSV-mlFNp, - Immunomodulation; VSV Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, hlFNp, VSV- expressing the murine (m), Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic rlFNp human (h) or rat (r) IFN-β gene virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

10.1099/vir.0.046672-0. Epub 2012 Oct 10; Jenks, N., Myers, R., Greiner, S. M., Thompson, J., Mader, E. K., Greenslade, A., Griesmann, G. E., Federspiel, M. J., Rakela, J. & other authors (2010). Safety studies on intrahepatic or intratumoral injection of oncolytic vesicular stomatitis virus expressing interferonb in rodents and nonhuman primates. Hum Gene Ther 21 , 451- 462. ; Obuchi, M., Fernandez, M. & Barber, G. N. (2003). Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity. J Virol 77, 8843-8856.)

VSV-IL4 Immunomodulation; VSV Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, expressing IL-4 Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Fernandez, M., Porosnicu, M., Markovic, D. & Barber, G. N. (2002). Genetically engineered vesicular stomatitis virus in gene therapy: application for treatment of malignant disease. J Virol 76, 895-904.)

VSV-IFN-NIS VSV expressing IFNb and Naik S, Nace R, Federspiel MJ, Barber GN, Peng KW, Russell SJ. Curative thyroidal sodium iodide one-shot systemic virotherapy in murine myeloma. Leukemia. 2012 sym porter Aug;26(8):1870-8. doi: 10.1038/leu.2012.70. Epub 2012 Mar 19.

VSV-IL12 Immunomodulation; VSV Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, expressing IL-12 Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi:

Strain Modification(s)/Description Virus Clinical Trial; Indication; Route; Status; Reference

VSV-(A51 )-M3 Immunomodulation; VSV-MA51 Recombinant VSV used as oncolytic agent against cancer (see, e.g., Hastie E, expressing the murine Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic gammaherpesvirus-68 virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: chemokine-binding protein M3 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Wu, L, Huang, T. G.,Meseck, M.,

Altomonte, J., Ebert, O., Shinozaki, K., Garci ^' a-Sastre, A., Fallon, J., Mandeli, J. & Woo, S. L. (2008). rVSV(MD51 )-M3 is an effective and safe oncolytic virus for cancer therapy. Hum Gene Ther 19, 635-647.)

HSV-1 Genome and Structure: ds Herpesviridae Clinical phase l/ll; Glioma; Wollmann et al. Oncolytic virus therapy for

DNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Human Feb;18(1 ):69-81

NDV Genome and Structure: ss (-) Paramyxoviridae Clinical phase l/ll; Glioma; Wollmann et al. Oncolytic virus therapy for

RNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Avian Feb;18(1 ):69-81

Adeno Genome and Structure: ds Adenoviridae Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for

DNA; Naked glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-

Representative Host: Human Feb;18(1 ):69-81

Reo Genome and Structure: ds Reoviridae Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for

RNA; Naked glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Feb;18(1 ):69-81

Mammalian

Vaccinia Genome and Structure: ds Poxviridae Preclinical in vivo; Glioma; Wollmann et al. Oncolytic virus therapy for

DNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Feb;18(1 ):69-81

Cow/horse, others

Polio Genome and Structure: ss (+) Picornaviridae Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for

RNA; Naked glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-

Representative Host: Human Feb;18(1 ):69-81

VSV Genome and Structure: ss (-) Rhabdoviridae Preclinical in vivo; Glioma; Wollmann et al. Oncolytic virus therapy for

RNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Feb;18(1 ):69-81

Livestock/mosquito

MVM Genome and Structure: ss Parvoviridae Preclinical in vitro; Glioma; Wollmann et al. Oncolytic virus therapy for

DNA; Naked glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-

Representative Host: Mouse Feb;18(1 ):69-81

Sindbis Genome and Structure: ss (+) Togaviridae Preclinical in vitro; Glioma; Wollmann et al. Oncolytic virus therapy for

RNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Feb;18(1 ):69-81

Mammalian/mosquito

PRV Genome and Structure: ds Herpesviridae Preclinical in vitro; Glioma; Wollmann et al. Oncolytic virus therapy for

DNA; Enveloped glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Representative Host: Pig Feb;18(1 ):69-81

Table 4. List of immune checkpoint inhibitor biologies approved by the US Food and Drug Administration or in clinical development,

Cancers

The methods and compositions of the present invention may be used to treat a wide variety of cancer types. One of skill in the art will appreciate that, since cells of many if not all cancers are capable of receptor-mediated apoptosis, the methods and compositions of the present invention are broadly applicable to many if not all cancers. The combinatorial approach of the present invention is efficacious in various aggressive, treatment refractory tumor models. In particular embodiments, for example, the cancer treated by a method of the present invention may be adrenal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and other central nervous system (CNS) cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, cancer of the head and neck, hepatocellular carcinoma, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphomas including Hodgkin's and non-Hodgkin's lymphomas, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplasia syndrome, multiple myeloma, oral cavity cancer (e.g. lip, tongue, mouth, and pharynx), ovarian cancer, paediatric cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenomathymoma, prostate cancer, renal cell carcinoma, cancer of the respiratory system, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, cancer of the urinary system, and other carcinomas and sarcomas. Other cancers are known in the art.

The cancer may be a cancer that is refractory to treatment by SMCs alone. The methods and compositions of the present invention may be particularly useful in cancers that are refractory to treatment by SMCs alone. Typically, a cancer refractory to treatment with SMCs alone may be a cancer in which lAP-mediated apoptotic pathways are not significantly induced. In particular embodiments, a cancer of the present invention is a cancer in which one or more apoptotic pathways are not significantly induced, i.e., is not activated in a manner such that treatment with SMCs alone is sufficient to effectively treat the cancer. For instance, a cancer of the present invention can be a cancer in which a clAP1 /2-mediated apoptotic pathway is not significantly induced.

A cancer of the present invention may be a cancer refractory to treatment by one or more agents.

In particular embodiments, a cancer of the present invention may be a cancer refractory to treatment by one or more agents (absent an SMC) and also refractory to treatment by one or more SMCs (absent an agent). Formulations and Administration

In some instances, delivery of a naked, i.e. native form, of an SMC and/or agent may be sufficient to potentiate apoptosis and/or treat cancer. SMCs and/or agents may be administered in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitably pharmacologically effective, e.g., capable of potentiating apoptosis and/or treating cancer.

Salts, esters, amides, prodrugs and other derivatives of an SMC or agent can be prepared using standard procedures known in the art of synthetic organic chemistry. For example, an acid salt of SMCs and/or agents may be prepared from a free base form of the SMC or agent using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the SMC or agent is dissolved in a polar organic solvent, such as methanol or ethanol, and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to, both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain typical acid addition salts of SMCs and/or agents, for example, halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of SMCs and/or agents of the present invention may be prepared in a similar manner using a pharmaceutically acceptable base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Certain typical basic salts include, but are not limited to, alkali metal salts, e.g., sodium salt, and copper salts.

Preparation of esters may involve functionalization of, e.g., hydroxyl and/or carboxyl groups that are present within the molecular structure of SMCs and/or agents. In certain embodiments, the esters are acyl-substituted derivatives of free alcohol groups, i.e., moieties derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters may be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

Amides may also be prepared using techniques known in the art. For example, an amide may be prepared from an ester using suitable amine reactants or prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.

An SMC or agent of the present invention may be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, e.g., to stabilize the composition, increase or decrease the absorption of the SMC or agent, or improve penetration of the blood brain barrier (where appropriate). Physiologically acceptable compounds may include, e.g., carbohydrates (e.g., glucose, sucrose, or dextrans), antioxidants (e.g. ascorbic acid or glutathione), chelating agents, low molecular weight proteins, protection and uptake enhancers (e.g., lipids), compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds, particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, suspending agents, and the like. In certain embodiments, a pharmaceutical formulation may enhance delivery or efficacy of an SMC or agent.

In various embodiments, an SMC or agent of the present invention may be prepared for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration. Administration may occur, for example, transdermal^, prophylactically, or by aerosol.

A pharmaceutical composition of the present invention may be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, and lipid complexes.

In certain embodiments, an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrator (e.g. calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone, etc.), a binder (e.g. alpha-starch, gum arabic, microcrystalline cellulose,

carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), or an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.) may be added to an SMC or agent and the resulting composition may be compressed to manufacture an oral dosage form (e.g., a tablet). In particular embodiments, a compressed product may be coated, e.g., to mask the taste of the compressed product, to promote enteric dissolution of the compressed product, or to promote sustained release of the SMC or agent. Suitable coating materials include, but are not limited to, ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate,

hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).

Other physiologically acceptable compounds that may be included in a pharmaceutical composition including an SMC or agent may include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound, depends, e.g., on the route of administration of the SMC or agent and on the particular physio-chemical characteristics of the SMC or agent.

In certain embodiments, one or more excipients for use in a pharmaceutical composition including an SMC or agent may be sterile and/or substantially free of undesirable matter. Such compositions may be sterilized by conventional techniques known in the art. For various oral dosage form excipients, such as tablets and capsules, sterility is not required. Standards are known in the art, e.g., the USP/NF standard.

An SMC or agent pharmaceutical composition of the present invention may be administered in a single or in multiple administrations depending on the dosage, the required frequency of administration, and the known or anticipated tolerance of the subject for the pharmaceutical composition with respect to dosages and frequency of administration. I n various embodiments, the composition may provide a sufficient quantity of an SMC or agent of the present invention to effectively treat cancer.

The amount and/or concentration of an SMC or agent to be administered to a subject may vary widely, and will typically be selected primarily based on activity of the SMC or agent and the

characteristics of the subject, e.g., species and body weight, as well as the particular mode of administration and the needs of the subject, e.g., with respect to a type of cancer. Dosages may be varied to optimize a therapeutic and/or prophylactic regimen in a particular subject or group of subjects.

In certain embodiments, an SMC or agent of the present invention is administered to the oral cavity, e.g. , by the use of a lozenge, aersol spray, mouthwash, coated swab, or other mechanism known in the art.

In certain embodiments, an SMC or agent of the present invention is administered using a slow- release solid wafer inserted in the brain cavity left upon tumor resection at the time of surgery. The wafer may be a biodegradable polyanhydride wafer containing an SMC or poly(l :C). The number of wafers placed may depend on the size of the resection cavity following surgical excision of the primary brain tumor. Delivery of drug from a slow-release wafer directly to brain tissue bypasses the problem of delivering systemic treatment across the blood-brain barrier. The polymer matrix may be comprised of a copolymer of 1 ,3-bis-(p-carboxyphenoxy) propane and sebacic acid (PCPP-SA; 80:20 molar ratio) that is dissolved in an organic solvent with drug, spraydried into microparticles ranging from 1 -20 μηι, and compression molded into wafers. In certain embodiments, the rigid wafers degrade in a two-step process wherein water penetration hydrolyzes the anyhydride bonds during the first 1 0 hours followed by erosion of the copolymer into the surrounding aqueous environment.

In certain embodiments, an SMC or agent of the present invention may be administered systemically (e.g., orally or as an injectable) in accordance with standard methods known in the art. In certain embodiments, the SMC or agent may be delivered through the skin using a transdermal drug delivery systems, i.e., transdermal "patches," wherein the SMCs or agents are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer or reservoir underlying an upper backing layer. The reservoir of a transdermal patch includes a quantity of an SMC or agent that is ultimately available for delivery to the surface of the skin. Thus, the reservoir may include, e.g., an SMC or agent of the present invention in an adhesive on a backing layer of the patch or in any of a variety of different matrix formulations known in the art. The patch may contain a single reservoir or multiple reservoirs.

In particular transdermal patch embodiments, a reservoir may comprise a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, and polyurethanes. Alternatively, the SMC and/or agent-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, a liquid or hydrogel reservoir, or another form of reservoir known in the art. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the patch and provides the device with a substantial portion of flexibility. The material selected for the backing layer is preferably substantially impermeable to the SMC and/or agent and to any other materials that are present.

Additional formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams including an SMC or agent are typically viscous liquids or semisolid emulsions, e.g. oil-in-water or water-in-oil emulsions. Cream bases are typically water-washable and include an oil phase, an emulsifier, and an aqueous phase. The oil phase, also sometimes called the "internal" phase, of a cream base is generally comprised of petrolatum and a fatty alcohol, e.g., cetyl alcohol or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant. The specific ointment or cream base to be used may be selected to provide for optimum drug delivery according to the art. As with other carriers or vehicles, an ointment base may be inert, stable, non-irritating, and non-sensitizing.

Various buccal and sublingual formulations are also contemplated.

In certain embodiments, administration of an SMC or agent of the present invention may be parenteral. Parenteral administration may include intraspinal, epidural, intrathecal, subcutaneous, or intravenous administration. Means of parenteral administration are known in the art. In particular embodiments, parenteral administration may include a subcutaneously implanted device.

In certain embodiments, it may be desirable to deliver an SMC or agent to the brain. In embodiments including system administration, this could require that the SMC or agent cross the blood brain barrier. In various embodiments this may be facilitated by co-administering an SMC or agent with carrier molecules, such as cationic dendrimers or arginine-rich peptides, which may carry an SMC or agent over the blood brain barrier.

In certain embodiments, an SMC or agent may be delivered directly to the brain by administration through the implantation of a biocompatible release system (e.g., a reservoir), by direct administration through an implanted cannula, by administration through an implanted or partially implanted drug pump, or mechanisms of similar function known the art. I n certain embodiments, an SMC or agent may be systemically administered (e.g., injected into a vein). In certain embodiments, it is expected that the SMC or agent will be transported across the blood brain barrier without the use of additional compounds included in a pharmaceutical composition to enhance transport across the blood brain barrier.

In certain embodiments, one or more an SMCs or agents of the present invention may be provided as a concentrate, e.g., in a storage container or soluble capsule ready for dilution or addition to a volume of water, alcohol, hydrogen peroxide, or other diluent. A concentrate of the present invention may be provided in a particular amount of an SMC or agent and/or a particular total volume. The concentrate may be formulated for dilution in a particular volume of diluents prior to administration.

An SMC or agent may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. The compound may also be administered topically in the form of foams, lotions, drops, creams, ointments, emollients, or gels. Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer, such as ethanol, can be applied. Other suitable formulations and modes of administration are known or may be derived from the art.

An SMC or agent of the present invention may be administered to a mammal in need thereof, such as a mammal diagnosed as having cancer. An SMC or agent of the present invention may be administered to potentiate apoptosis and/or treat cancer.

A therapeutically effective dose of a pharmaceutical composition of the present invention may depend upon the age of the subject, the gender of the subject, the species of the subject, the particular pathology, the severity of the symptoms, and the general state of the subject's health.

The present invention includes compositions and methods for the treatment of a human subject, such as a human subject having been diagnosed with cancer. In addition, a pharmaceutical composition of the present invention may be suitable for administration to an animal, e.g., for veterinary use. Certain embodiments of the present invention may include administration of a pharmaceutical composition of the present invention to non-human organisms, e.g., a non-human primates, canine, equine, feline, porcine, ungulate, or lagomorphs organism or other vertebrate species.

Therapy according to the invention may be performed alone or in conjunction with another therapy, e.g., another cancer therapy, and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment optionally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed or it may begin on an outpatient basis. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the subject, the stage and type of the subject's disease, and how the patient responds to the treatment.

In certain embodiments, the combination of therapy of the present invention further includes treatment with a recombinant interferon, such as I FN-a, I FN-β, I FN-γ, pegylated I FN, or liposomal interferon. In some embodiments, the combination of therapy of the present invention further includes treatment with recombinant TN F-a, e.g., for isolated-limb perfusion. In particular embodiments, the combination therapy of the present invention further includes treatment with one or more of a TN F-a or IFN-inducing compound, such as DMXAA, Ribavirin, or the like. Additional cancer immunotherapies that may be used in combination with present invention include antibodies, e.g., monoclonal antibodies, targeting CTLA-4, PD-1 , PD-L1 , PD-L2, or other checkpoint inhibitors. Cyclic dinucleotides (CDNs) [cyclic di-GM P (guanosine 5'-monophosphate) (CDG), cyclic di-AM P (adenosine 5'-monophosphate) (CDA), and cyclic GM P-AM P (cGAM P)] are a class of pathogen-associated molecular pattern molecules (PAM Ps) that activate the TBK1 /interferon regulatory factor 3 (I RF3)/type 1 interferon (I FN) signaling axis via the cytoplasmic pattern recognition receptor stimulator of interferon genes (STING). In certain embodiments, STING agonists can be combined with an SMC to treat cancer.

Routes of administration for the various embodiments include, but are not limited to, topical, transdermal, nasal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, or oral administration). As used herein, "systemic administration" refers to all nondermal routes of administration, and specifically excludes topical and transdermal routes of administration.

In any of the above embodiments, the route of administration may be optimized based on the characteristics of the SMC or agent. In some instances, the SMC or agent is a small molecule or compound. In other instances, the SMC or agent is a nucleic acid. In still other instances, the agent may be a cell or virus. In any of these or other embodiments, appropriate formulations and routes of administration will be selected in accordance with the art.

In the embodiments of the present invention, an SMC and an agent are administered to a subject in need thereof, e.g., a subject having cancer. In some instances, the SMC and agent will be administered simultaneously. I n some embodiments, the SMC and agent may be present in a single therapeutic dosage form. In other embodiments, the SMC and agent may be administered separately to the subject in need thereof. When administered separately, the SMC and agent may be administered simultaneously or at different times. In some instances, a subject will receive a single dosage of an SMC and a single dosage of an agent. In certain embodiments, one or more of the SMC and agent will be administered to a subject in two or more doses. I n certain embodiments, the frequency of administration of an SMC and the frequency of administration of an agent are non-identical, i.e., the SMC is administered at a first frequence and the agent is administered at a second frequency.

In some embodiments, an SMC is administered within one week of the administration of an agent. In particular embodiments, an SMC is administered within 3 days (72 hours) of the administration of an agent. In still more particular embodiments, an SMC is administered within 1 day (24 hours) of the administration of an agent.

In particular embodiments of any of the methods of the present invention, the SMC and agent are administered within 28 days of each other or less, e.g., within 14 days of each other. In certain embodiments of any of the methods of the present invention, the SMC and agent are administered, e.g., simultaneously or within 1 minute, 5 minutes, 1 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 1 2 hours, 1 8 hours, 24 hours, 36 hours, 2 days, 4 days, 8 days, 1 0 days, 12 days, 16 days, 20 days, 24 days, or 28 days of each other. In any of these embodiments, the first administration of an SMC of the present invention may precede the first administration of an agent of the present invention. Alternatively, in any of these embodiments, the first administration of an SMC of the present invention may follow the first administration of an agent of the present invention. Because an SMC and/or agent of the present invention may be administered to a subject in two more doses, and because, in such instances, doses of the SMC and agent of the present invention may be administered at different frequencies, it is not required that the period of time between the administration of an SMC and the administration of an agent remain constant within a given course of treatment or for a given subject.

One or both of the SMC and the agent may be administered in a low dosage or in a high dosage. In embodiments in which the SMC and agent are formulated separately, the pharmacokinetic profiles for each agent can be suitably matched to the formulation, dosage, and route of administration, etc. I n some instances, the SMC is administered at a standard or high dosage and the agent is administered at a low dosage. In some instances, the SMC is administered at a low dosage and the agent is administered at a standard or high dosage. I n some instances, both of the SMC and the agent are administered at a standard or high dosage. I n some instances, both of the SMC and the agent are administered at a low dosage.

The dosage and frequency of administration of each component of the combination can be controlled independently. For example, one component may be administered three times per day, while the second component may be administered once per day or one component may be administered once per week, while the second component may be administered once per two weeks. Combination therapy may be given in on-and-off cycles that include rest periods so that the subject's body has a chance to recover from effects of treatment.

Kits

In general, kits of the invention contain one or more SMCs and one or more agents. These can be provided in the kit as separate compositions, or combined into a single composition as described above. The kits of the invention can also contain instructions for the administration of one or more SMCs and one or more agents.

Kits of the invention can also contain instructions for administering an additional

pharmacologically acceptable substance, such as an agent known to treat cancer that is not an SMC or agent of the present invention.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include kits that contain, e.g. , two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, ointments, foams etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dosage regimen or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the disease (e.g. , a type of cancer) to be treated, the severity of the disease, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect the dosage regimen or other aspects of administration.

EXAMPLES

Example 1: Smac mimetics prime tumors for destruction by the innate immune system

Smac mimetic compounds are a class of apoptosis sensitizing drugs that have proven safe in cancer patient Phase I trials. Stimulating an innate anti-pathogen response may generate a potent yet safe inflammatory "cytokine storm" that would trigger death of tumors treated with Smac mimetics. The present example demonstrates that activation of innate immune responses via oncolytic viruses and adjuvants, such as poly(l:C) and CpG, induces bystander death of cancer cells treated with Smac mimetics in a manner mediated by I FN , TNFa or TRAIL. This therapeutic strategy may lead to durable cures, e.g., in several aggressive mouse models of cancer. With these and other innate immune stimulants having demonstrated safety in human clinical trials, the data provided herein points strongly towards their combined use with Smac mimetics for treating cancer.

The present example examines whether stimulating the innate immune system using pathogen mimetics would be a safe and effective strategy to generate a cytokine milieu necessary to initiate apoptosis in tumors treated with an SMC. We report here that non-pathogenic oncolytic viruses, as well as mimetics of microbial RNA or DNA, such as poly (l :C) and CpG, induce bystander killing of cancer cells treated with an SMC that is dependent either upon I FN , TNFa, or TRAIL production. Importantly, this therapeutic strategy was tolerable in vivo and led to durable cures in several aggressive mouse models of cancer.

SMC therapy sensitizes cancer cells to bystander cell death during oncolytic virus infection

Oncolytic viruses (OVs) are emerging biotherapies for cancer currently in phase l-l l l clinical evaluation. One barrier to OV therapy may be the induction of type I I FN- and NFKB-responsive cytokines by the host, which orchestrate an antiviral state in tumors. It was examined whether we could harness those innate immune cytokines to induce apoptosis in cancer cells pretreated with an SMC. To begin, a small panel of tumor-derived and normal cell lines (n=30) was screened for responsiveness to the SMC LCL161 and the oncolytic rhabdovirus VSVA51 . We chose LCL1 61 because this compound is the most clinically advanced drug in the SMC class, and VSVA51 because it is known to induce a robust antiviral cytokine response. In 1 5 of the 28 cancer cell lines tested (54%), SMC treatment enhanced sensitivity the EC50 of VSVA51 by 1 0 - 1 0,000 fold (FIG. 6, and representative examples in FIGS. 1 A and 1 B). Similarly, low dose of VSVA51 reduced the EC50 of SMC therapy from undetermined levels (>2500 nM) to 4.5 and 21 .9 nM in two representative cell lines: the mouse mammary carcinoma EMT6 and the human glioblastoma SNB75 cells, respectively (FIG. 1 C). Combination index analyses determined that the interaction between SMC therapy and VSVA51 was synergistic (FIG. 7).

Experiments using four other SMCs and five other oncolytic viruses showed that a spectrum of monovalent and bivalent SMCs synergize with VSVA51 (FIG. 8). We find that the oncolytic

rhabdoviruses, VSVA51 and Maraba-MG1 , are superior in eliciting bystander killing in synergizing with SMCs, compared to HSV, reovirus, vaccinia and wild-type VSV platforms, all of which have elaborate mechanisms to disarm aspects of innate immune signalling (FIGS. 9A and 9B). Genetic experiments using RNAi-mediated silencing demonstrated that both XIAP and the clAPs must be inhibited to obtain synergy with VSVA51 (FIGS. 1 0A, 10B, and 24C). In stark contrast to the results in tumor-derived cell lines, non-cancer GM38 primary human skin fibroblasts and HSkM human skeletal myoblasts were unaffected by VSVA51 and SMC combination therapy (FIG. 6). Taken together, these data indicate that oncolytic VSV synergizes with SMC therapy in a tumor-selective fashion.

To determine if VSVA51 elicits bystander cell death in IAP-depleted neighbouring cells not infected by the virus, cells were treated with SMCs prior to infection with a low dose of VSVA51 (MOI = 0.01 infectious particles per cell). We assessed whether conditioned media derived from cells infected with VSVA51 (which was subsequently inactivated by UV light) could induce death when transferred to a plate of virus naive cancer cells treated with an SMC. The conditioned media induced cell death only when the cells were co-treated with an SMC (FIG. 1 D). We also found that a low-dose of a pseudo-typed G-less strain of VSVA51 (MOI = 0.1 ), containing a deletion of the gene encoding for its glycoprotein (VSVA51 AG) that limits the virus to a single round of infection, was toxic to an entire plate of cancer cells treated with an SMC (FIG. 1 E). Finally, we performed a cytotoxicity assay in cells overlaid with agarose, used to retard the spread of VSVA51 expressing a fluorescent tag, and observed dramatic cell death in SMC treated cells outside of the zone of virus infection (FIGS. 1 F and 1 1 ). Overall, these results indicate that VSVA51 infection leads to the release of at least one soluble factor that can potently induce bystander cell death in neighboring, uninfected, cancer cells treated with SMCs.

SMC therapy does not impair the cellular innate immune response to oncolytic VSV

The cellular innate immune response to an RNA virus infection in mammalian tumor cells can be initiated by members of a family of cytosolic (RIG-l-like receptors, RLRs) and endosomal (toll-like receptors, TLRs) viral RNA sensors. Once triggered, these receptors can seed parallel I FN-response factor (I RF) 3/7 and nuclear-factor kappa B (NF-κΒ) cell signalling cascades. These signals can culminate in the production of IFNs and their responsive genes as well as an array of inflammatory chemokines and cytokines. This prompts neighboring cells to preemptively express an armament of antiviral genes and also aids in the recruitment and activation of cells within the innate and adaptive immune systems to ultimately clear the virus infection. The clAP proteins have recently been implicated in numerous signalling pathways downstream of pathogen recognition, including those emanating from RLRs and TLRs. Accordingly, it was examined whether SMC therapy alters the antiviral response to oncolytic VSV infection in tumor cells and in mice. To begin, the effect of SMC therapy on VSVA51 productivity and spread was evaluated. Single-step and multi-step growth curves of VSVA51 productivity revealed that SMC treatment does not affect the growth kinetics of VSVA51 in EMT6 or SNB75 cells in vitro (FIG. 2A). Moreover, analysis through time-lapse microscopy demonstrates that SMC treatment does not alter VSVA51 infectivity in or spread through tumor cells (FIG. 2B). Furthermore, viral replication and spread in vivo were analyzed by determining tumor load using I VIS imaging and tissue virus titration. No differences in viral kinetics were found upon SMC treatment in EMT6 tumor-bearing mice (FIGS. 1 2A and 12B). As EMT6 and SNB75 cells both have functional type I I FN responses that regulate the VSV life cycle, these data provide strong, albeit indirect, evidence that SMC therapy does not affect the antiviral signalling cascades in cancer cells.

To probe deeper, I FN production was measured in EMT6 and SNB75 cells treated with VSVA51 and SMCs. This experiment revealed that the SMC treated cancer cells respond to VSVA51 by secreting IFN (FIG. 2C), although at slightly lower levels as compared to VSVA51 alone. It was asked whether the dampened IFN secretion from SMC treated cells had any bearing on the induction of downstream IFN stimulated genes (ISGs). Quantitative RT-PCR analyses of a small panel of ISGs in cells treated with VSVA51 and SMC revealed that IAP inhibition had no bearing on ISG gene expression in response to an oncolytic VSV infection (FIG. 2D). Consistent with this finding, western blot analyses indicated that SMCs do not alter the activation of Jak/Stat signalling downstream of I FN (FIGS. 2E and 24A). Collectively, these data suggest that SMCs do not impede the ability of tumor cells to sense and respond to an infection from VSVA51 .

/ΡΛ/β orchestrates bystander cell death during SMC and oncolytic VSV co-therapy

SMCs sensitize a number of cancer cell lines towards caspase 8-dependant apoptosis induced by TNFa, TRAIL, and IL-1 β. As RNA viruses can trigger the production of these cytokines as part of the cellular antiviral response, the involvement of cytokine signaling in SMC and OV induced cell death was investigated. To start, the TNF receptor (TNF-R1 ) and/or the TRAIL receptor (DR5) were silenced and synergy between SMC and VSVA51 was assayed. This experiment revealed that TN Fa and TRAIL are not only involved, but collectively are indispensable for bystander cell death (FIGS. 3A-3H, 13A, and 24D). Consistent with this finding, western blot and immunofluorescence experiments revealed strong activation of the extrinsic apoptosis pathway, and RNAi knockdown experiments demonstrated a requirement for both caspase-8 and Rip1 in the synergy response (FIGS. 14A-14G, 24E, and 24F). Moreover, engineering TNFa into VSVA51 improved synergy with SMC therapy by an order of magnitude (FIGS. 15A and 15B).

Next, the type I I FN receptor (I FNAR1 ) was silenced and it was found, unexpectedly, that I FNAR1 knockdown prevented the synergy between SMC therapy and oncolytic VSV (FIGS. 3B, 13B, and 24D). It was predicted that I FNAR1 knockdown would dampen but not completely suppress bystander killing, as TRAIL is a well-established ISG that is responsive to type I I FN28. TN Fa and IL-1 β are considered to be independent of IFN signaling, but they are nevertheless responsive to N F-κΒ signaling downstream of virus detection. This result suggests the possibility of a non-canonical type I I FN-dependant pathway for the production of TNFa and/or IL-1 β. Indeed, when the m RNA expression of IFN , TRAIL, TN Fa, and I L- 1 β were probed during an oncolytic VSV infection, a significant temporal lag was found between the induction of I FN and that of both TRAIL and TNFa (FIG. 3C). This data also suggests that TNFa - like TRAIL - may be induced secondary to I FN . To prove this concept, I FNAR1 was silenced before treating cells with VSVA51 . I FNAR1 knockdown completely abrogated the induction of both TRAIL and TN Fa by oncolytic VSV (FIG. 3D). Moreover, synergy with SMC was recapitulated using recombinant type I I FNs (I FNo/β) and type II I FN (I FNy), but not type I I I I FNs (IL28/29) (FIG. 3E). Taken together, these data indicate that type I I FN is required for the induction of TNFa and TRAIL during a VSVA51 infection of tumor cells. Moreover, the production of these cytokines is responsible for bystander killing of neighboring, uninfected SMC-treated cells.

To explore the non-canonical induction of TNFa further, the m RNA expression levels of TRAIL and TNFa in SNB75 cells treated with recombinant I FN were measured. Both cytokines were induced by I FN treatment (FIG. 3F), and ELISA experiments confirmed the production of their respective protein products in the cell culture media (FIG. 3G). Interestingly, there was a significant time lag between the induction of TRAIL and that of TNFa. As TRAIL is a bona fide ISG and TNFa is not, this result raised the possibility that TNFa is not induced by I FN directly, but responds to a downstream ISG up-regulated by IFN . Thus, quantitative RT-PCR was performed on 1 76 cytokines in SNB75 cells and 70 that were significantly up-regulated by I FN were identified (Table 5). The role of these ISGs in the induction of TNFa by I FN is currently being investigated. It is also intriguing that SMC treatment potentiated the induction of both TRAIL and TNFa by I FN in SNB75 cells (FIGS. 3F and 3G). Furthermore, using a dominant-negative construct of IKK, it was found that the production of these inflammatory cytokines downstream of I FN was dependent, at least in part, on classical NF-κΒ signalling (FIG. 3H). In EMT6 cells, SMC treatment was found to enhance cellular production of TN Fa (5- to 7-fold percentage increase) upon VSV infection (FIG. 1 6). Finally, it was also demonstrated that blocking TNF-R1 signalling (with antibodies or siRNA) prevents EMT6 cell death in the presence of SMC and VSVA51 or IFN (FIGS. 17A- 17C and 24H). The relationship between type I IFN and TNFa is complex, having either complimentary or inhibitory effects depending on the biological context. However, without limiting the present invention to any particular mechanism of action, a simple working model can be proposed as follows: Tumor cells infected by an oncolytic RNA virus up-regulate type I I FN, and this process is not affected by SMC antagonism of the IAP proteins. Those IFNs in turn signal to neighboring, uninfected cancer cells to express and secrete TNFa and TRAIL, a process that is enhanced by SMC treatment, which

consequently induces autocrine and paracrine programmed cell death in uninfected tumor cells exposed to SMC (FIGS. 1 8A and 1 8B). Table 5

IFNp Gene Name Gene Identification

5.8 IL22 Interleukin 22

9.7 IL1 F5 Interleukin 1 family, member 5 (delta)

2.4 IFNW1 Interferon, omega 1

12.6 IL1 1 Interleukin 1 1

25.1 IL1 F8 Interleukin 1 family, member 8 (eta)

-1 .3 EDA Ectodysplasin A

8 FGF5 Fibroblast growth factor 5

5 VEGFC Vascular endothelial growth factor C

4.9 LIF Leukemia inhibitory factor

1 .3 CCL25 Chemokine (C-C motif) ligand 25

8.3 BMP3 Bone morphogenetic protein 3

1 .6 IL17C Interleukin 17C

-2.3 TNFSF7 CD70 molecule

2.5 TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8

2.5 FASLG Fas ligand (TNF superfamily, member 6)

2.7 BMP8B Bone morphogenetic protein 8b

6 IL7 Interleukin 7

5.2 CCL24 Chemokine (C-C motif) ligand 24

-2.2 INHBE Inhibin, beta E

5.8 IL23A Interleukin 23, alpha subunit p19

-1 .1 IL17F Interleukin 17F

2.9 CCL21 Chemokine (C-C motif) ligand 21

8.5 CSF1 Colony stimulating factor 1 (macrophage)

3 IL15 Interleukin 15

5.7 NRG2 Neuregulin 2

N/A INHBB Inhibin, beta B

N/A LTB Lymphotoxin beta (TNF superfamily, member 3)

N/A BMP7 Bone morphogenetic protein 7

-3.8 IL1 F9 Interleukin 1 family, member 9

6.1 IL12B Interleukin 12B

6.2 FLT3LG Fms-related tyrosine kinase 3 ligand

3 FGF1 Fibroblast growth factor 1 (acidic)

-2 CXCL13 Chemokine (C-X-C motif) ligand 13

2.2 IL17B Interleukin 17B

7.8 GDNF Glial cell derived neurotrophic factor

-1 .7 GDF7 Growth differentiation factor 7

-2.4 LTA Lymphotoxin alpha (TNF superfamily, member 1 )

1 .7 LEFTY2 Left-right determination factor 2

5 FGF19 Fibroblast growth factor 19

9.8 FGF23 Fibroblast growth factor 23

4.8 CLC Cardiotrophin-like cytokine factor 1

3 ANGPT1 Angiopoietin 1 vsv IFNp Gene Name Gene Identification

2 10.6 TPO Thyroid peroxidase

2 2.1 EFNA5 Ephrin-A5

1.9 6.4 IL1 F10 Interleukin 1 family, member 10 (theta)

1.9 7.6 LEP Leptin (obesity homolog, mouse)

1.8 3 IL5 Interleukin 5 (colony-stimulating factor, eosinophil)

1.8 5.7 IFNE1 Interferon epsilon 1

1.8 2.7 EGF Epidermal growth factor (beta-urogastrone)

1.7 3.4 CTF1 Cardiotrophin 1

1.7 -1 .9 BMP2 Bone morphogenetic protein 2

1.7 3 EFNB2 Ephrin-B2

1.6 1 FGF8 Fibroblast growth factor 8 (androgen-induced)

1.6 -2 TGFB2 Transforming growth factor, beta 2

1.5 -1 .6 BMP8A Bone morphogenetic protein 8a

1.5 3.3 NTF5 Neurotrophin 5 (neurotrophin 4/5)

1.5 1 GDF10 Growth differentiation factor 10

1.5 1 .5 TNFSF13B Tumor necrosis factor (ligand) superfamily, member 13b

1.5 2.5 IFNA1 Interferon, alpha 1

1.4 -1 .3 INHBC Inhibin, beta C

1.4 2.8 FGF7 Galactokinase 2

1.4 3.3 IL24 Interleukin 24

1.4 -1 .1 CCL27 Chemokine (C-C motif) ligand 27

1.3 1 .9 FGF13 Fibroblast growth factor 13

1.3 1 .4 IFNK Interferon, kappa

1.3 2 ANGPT2 Angiopoietin 2

1.3 7.6 IL18 Interleukin 18 (interferon-gamma-inducing factor)

1.3 7 NRG1 Neuregulin 1

1.3 4.9 NTF3 Neurotrophin 3

1.2 15 FGF10 Fibroblast growth factor 10

1.2 1 .9 KITLG KIT ligand

1.2 -1 .3 IL17D Interleukin 17D

1.2 1 .1 TNFSF4 Tumor necrosis factor (ligand) superfamily, member 4

1.2 1 .3 VEGFA Vascular endothelial growth factor

1.1 2.4 FGF1 1 Fibroblast growth factor 1 1

1.1 -1 .4 IL17E Interleukin 17E

1.1 -2.1 TGFB1 Transforming growth factor, beta 1

1 3.1 GH1 Growth hormone 1

- ¹ 6.1 IL9 Interleukin 9

- ¹ -2.5 EFNB3 Ephrin-B3

- ¹ 1 .8 VEGFB Vascular endothelial growth factor B

- ¹ -1 .2 IL1 F7 Interleukin 1 family, member 7 (zeta)

- ¹ -2.1 GDF1 1 Growth differentiation factor 1 1

-1 .1 1 .3 ZFP91 Zinc finger protein 91 homolog (mouse) vsv IFNp Gene Name Gene Identification

-1 .2 -1 .1 BMP6 Bone morphogenetic protein 6

-1 .2 -1 .2 AMH Anti-Mullerian hormone

-1 .3 -1 LEFTY1 Left-right determination factor 1

-1 .3 2.4 EFNA3 Ephrin-A3

-1 .3 -1 .3 LASS1 LAG1 longevity assurance homolog 1

-1 .5 1 EFNA4 Ephrin-A4

-1 .8 1 .3 PDGFD DNA-damage inducible protein 1

-1 .8 1 .8 IL10 Interleukin 10

-1 .9 1 .6 GDF5 Growth differentiation factor 5

-1 .9 1 .3 EFNA2 Ephrin-A2

-1 .9 -1 .5 EFNB1 Ephrin-B1

-1 .9 -1 .4 GDF8 Growth differentiation factor 8

-1 .9 1 .6 PDGFC Platelet derived growth factor C

-2.2 2.4 TSLP Thymic stromal lymphopoietin

-2.3 -1 .5 BMP10 Bone morphogenetic protein 10

-2.4 -4.6 CXCL12 Chemokine (C-X-C motif) ligand 12

-2.5 4 IFNG Interferon, gamma

-2.6 1 .2 EPO Erythropoietin

-2.7 -2.1 GAS6 Growth arrest-specific 6

-2.9 2.9 PRL Prolactin

-2.9 -2.1 BMP4 Bone morphogenetic protein 4

-2.9 -5.7 INHA Inhibin, alpha

-3 -1 .3 GDF9 Growth differentiation factor 9

-3.1 -1 .5 FGF18 Fibroblast growth factor 18

-3.2 N/A IL17 Interleukin 17

-3.2 -1 .1 IL26 Interleukin 26

-3.4 1 .2 EFNA1 Ephrin-A1

-3.8 -1 .1 FGF12 Fibroblast growth factor 12

-4 -2.3 FGF9 Fibroblast growth factor 9 (glia-activating factor)

-4.5 1 .4 CCL26 Chemokine (C-C motif) ligand 26

-8 9.7 CCL19 Chemokine (C-C motif) ligand 19

N/A N/A BMP15 Bone morphogenetic protein 15

N/A N/A CCL15 Chemokine (C-C motif) ligand 14

N/A N/A CCL16 Chemokine (C-C motif) ligand 16

N/A N/A CCL18 Chemokine (C-C motif) ligand 18

N/A N/A CCL23 Chemokine (C-C motif) ligand 23

N/A N/A CD40LG CD40 ligand (TNF superfamily)

N/A N/A CSF3 Colony stimulating factor 3 (granulocyte)

N/A N/A CXCL5 Chemokine (C-X-C motif) ligand 5

N/A N/A FGF4 Fibroblast growth factor 4

N/A N/A FGF6 Fibroblast growth factor 6

N/A N/A GH2 Growth hormone 2 vsv IFNp Gene Name Gene Identification

N/A N/A IL2 Interleukin 2

N/A N/A IL21 Interleukin 21

N/A N/A IL28A Interleukin 28A (interferon, lambda 2)

N/A N/A INHBA Inhibin, beta A

N/A N/A NRG3 Neuregulin 3

N/A N/A TNFSF1 1 Tumor necrosis factor (ligand) superfamily, member 1 1

N/A N/A TNFSF13 Tumor necrosis factor (ligand) superfamily, member 13

N/A 6.5 NRG4 Neuregulin 4

N/A 6.1 IL3 Interleukin 3 (colony-stimulating factor, multiple)

N/A 1 .8 TNFSF9 Tumor necrosis factor (ligand) superfamily, member 9

Oncolytic VSV potentiates SMC therapy in preclinical animal models of cancer

To evaluate SMC and oncolytic VSV co-therapy in vivo, the EMT6 mammary carcinoma was used as a syngeneic, orthotopic model. Preliminary safety and pharmacodynamic experiments revealed that a dose of 50 mg/kg LCL1 61 delivered by oral gavage was well tolerated and induced clAP112 knockdown in tumors for at least 24 hrs, and up to 48-72 hours in some cases (FIGS. 1 9A, 1 9B, and 24G). When tumors reached ~100 mm ³ , we began treating mice twice weekly with SMC and VSVA51 , delivered systemically. As single agents, SMC therapy led to a decrease in the rate of tumor growth and a modest extension in survival, while VSVA51 treatments had no bearing on tumor size or survival (FIGS. 4A and 4B). In stark contrast, combined SMC and VSVA51 treatment induced dramatic tumor regressions and led to durable cures in 40% of the treated mice. Consistent with the bystander killing mechanism elucidated in vitro, immunofluorescence analyses revealed that the infectivity of VSVA51 was transient and limited to small foci within the tumor (FIG. 4C), whereas caspase-3 activation was widespread in the SMC and VSVA51 co-treated tumors (FIG. 4D). Furthermore, immunoblots with tumor lysates demonstrated activation of caspase-8 and -3 in doubly-treated tumors (FIGS. 4E, 24B, and 24G). While the animals in the combination treatment cohort experienced weight loss, the mice fully recovered following the last treatment (FIG. 20A).

To confirm these in vivo data in another model system, the human HT-29 colorectal adenocarcinoma xenograft model was tested in nude (athymic) mice. HT-29 is a cell line that is highly responsive to bystander killing by SMC and VSVA51 co-treatment in vitro (FIGS. 21 A and 21 B). Similar to our findings in the EMT6 model system, combination therapy with SMC and VSVA51 induced tumor regression and a significant extension of mouse survival (FIG. 21 C). In contrast, neither monotherapy had any effect on HT-29 tumors. Furthermore, there was no additional weight loss in the double treated mice compared to SMC treated mice (FIG. 21 D). These results indicate that the synergy is highly efficacious in a refractory xenograft model and that the adaptive immune response does not have a major role initially in the efficacy of SMC and OV co-therapy.

Role of the innate antiviral responses and immune effectors in co-treatment synergy

It was next determined whether oncolytic VSV infection coupled with SMC treatment leads to

TNFa- or I FNp-mediated cell death in vivo. It was investigated whether blocking TNFa signalling via neutralizing antibodies would affect SMC and VSVA51 synergy in the EMT6 tumor model. Compared to isotype matched antibody controls, the application of TN Fa neutralizing antibodies reverted the tumor regression and decreased the survival rate to values close to the control and single treatment groups (FIGS. 4F and 4G). This demonstrates that TNFa is required in vivo for the anti-tumor combination efficacy of SMC and oncolytic VSV.

To investigate the role of I FN signaling in the SMC and OV combination paradigm, Balb/c mice bearing EMT6 tumors were treated with I FNAR1 blocking antibodies. Mice treated with the I FNAR1 blocking antibody succumbed to viremia within 24-48 hours post infection. Prior to death, tumors were collected at 1 8-20 hours after virus infection, and the tumors were analyzed for caspase activity. Even though these animals with defective type I I FN signaling were ill due to a large viral burden, the excised tumors did not demonstrate signs of caspase-8 activity and only showed minimal signs of caspase-3 activity (FIG. 22) in contrast to the control group, which showed the expected activation of caspases within the tumor (FIG. 22). These results support the hypothesis that intact type I I FN signaling is required to mediate the anti-tumor effects of the combination approach.

To assess the contribution of innate immune cells or other immune mediators to the efficacy of

OV/SMC combination therapy, treating EMT6 tumors was first attempted in immunodeficient NOD-scid or NSG (NOD-scid-IL2Rgamma ^nu ") mice. However, similar to the I FNAR1 depletion signaling studies, these mice also died rapidly due to viremia. Therefore, the contribution of innate immune cells was addressed by employing an ex vivo splenocyte culture system as a surrogate model. Innate immune populations that have the capacity to produce TNFa were positively selected and further sorted from naive splenocytes. Macrophages (CD1 1 b+ F4/80+), neutrophils (CD1 1 b+ Gr1 +), NK cells (CD1 1 b- CD49b+) and myeloid-negative (lymphoid) population (CD1 1 b- CD49-) were stimulated with VSVA51 , and the conditioned medium was transferred to EMT6 cells to measure cytotoxicity in the presence of SMC. These results show that VSVA51 -stimulated macrophages and neutrophils, but not NK cells, are capable of producing factors that lead to cancer cell death in the presence of SMCs (FIG. 23A). Primary macrophages from bone marrow were also isolated and these macrophages also responded to oncolytic VSV infection in a dose-dependent manner to produce factors which kill EMT6 cells (FIG. 23B).

Altogether, these findings demonstrate that multiple innate immune cell populations can respond to mediate the observed anti-tumor effects, and that macrophages are the most likely effectors of this response.

Immune adjuvants poly(l:C) and CpG potentiate SMC therapy in vivo

It was next investigated whether synthetic TLR agonists, which are known to induce an innate proinflammatory response, would synergize with SMC therapy. EMT6 cells were co-cultured with mouse splenocytes in a transwell insert system, and the splenocytes were treated with SMC and agonists of TLR 3, 4, 7 or 9. All of the tested TLR agonists were found to induce the bystander death of SMC treated EMT6 cells (FIG. 5A). The TLR4, 7, and 9 agonists LPS, imiquimod, and CpG, respectively, required splenocytes to induce bystander killing of EMT6 cells, presumably because their target TLR receptors are not expressed in EMT6 cells. However, the TLR3 agonist poly(l :C) led to EMT6 cell death directly in the presence of SMCs. Poly(l:C) and CpG were next tested in combination with SMC therapy in vivo. These agonists were chosen as they have proven to be safe in humans and are currently in numerous mid to late stage clinical trials for cancer. EMT6 tumors were established and treated as described above. While poly(l :C) treatment had no bearing on tumor growth as a single agent, combination with SMCs induced substantial tumor regression and, when delivered intraperitoneally, led to durable cures in 60% of the treated mice (FIGS. 5B and 5C). Similarly, CpG monotherapy had no bearing on tumor size or survival, but when combined with SMC therapy led to tumor regressions and durable cures in 88% of the treated mice (FIGS. 5D and 5E). Importantly, these combination therapies were well tolerated by the mice, and their body weight returned to pre-treatment levels shortly after the cessation of therapy (FIGS. 20B and 20C). Taken together with the oncolytic VSV results, the data demonstrate that a series of clinically advanced innate immune adjuvants strongly and safely synergize with SMC therapy in vivo, inducing tumor regression and durable cures in several treatment refractory, aggressive mouse models of cancer.

Example 2: Inactivated viral particles, cancer vaccines, and stimulatory cytokines synergize with SMCs to kill tumors

The use of current cancer immunotherapies, such as BCG (Bacillus Calmette-Guerin), recombinant interferon (e.g. I FNa), and recombinant Tumor Necrosis Factor (e.g. TNFa used in isolated limb perfusion for example), and the recent clinical use of biologies (e.g. blocking antibodies) to immune checkpoint inhibitors that overcome tumor-mediated suppression of the immune system (such as anti- CTLA-4 and anti-PD-1 or PDL-1 monoclonal antibodies) highlight the potential of 'cancer immunotherapy' as an effective treatment modality. As shown in Example 1 , we have demonstrated the robust potential of non-viral immune stimulants to synergize with SMCs (FIG. 5). To expand on these studies, we also examined for the potential of SMCs to synergize with non-replicating rhabdovirus-derived particles (called NRRPs), which are UV-irradiated VSV particles that retain their infectious and immunostimulatory properties without the ability to replicate and spread. To assess if NRRPs directly synergize with SMCs, we co-treated various cancer cell lines, EMT6, DBT, and CT-2A, with SMCs and differing levels of NRRPs, and assessed cell viability by Alamar blue. We observed that NRRPs synergize with SMCs in these cancer cell lines (FIG. 25A). To assess if NRRPs can induce a potent proinflammatory response, we treated fractionated mouse splenocytes with NRRPs (or synthetic CpG ODN 221 6 as a positive control), transferred the cell culture supernatants to EMT6 cells in culture in a dose-response fashion, and treated the cells with vehicle or SMC. We observed that the immunogenicity of N RRPs is at a similar level of CpG, as there was a considerable proinflammatory response, which led to a high degree of EMT6 cell death in the presence of SMCs (FIG. 25B). As the treatment of CpG and SMC in the EMT6 tumor model resulted in a 88% cure rate (FIG. 5D), these findings suggest that the combination of SMCs and NRRPs can be highly synergistic in vivo.

Our success in finding synergy between SMCs and live or inactivated single-stranded RNA oncolytic rhabdoviruses (e.g., VSVA51 , Maraba-MG1 , and N RRPs) suggested that a clinic approved attenuated vaccine may be able to synergize with SMCs. To test this possibility, we assessed the ability to synergize with SMCs of the cancer biologic, the vaccine for tuberculosis mycobacterium, BCG, which is typically used to treat bladder cancer in situ due to the high local production of TNFa. Indeed, the combination of SMC and BCG potently synergises to kill EMT6 cells in vitro (FIG. 26A). These findings were similarly extended in vivo; we observed significant tumor regression with combined treatment of an oral SMC and BCG administered locally or systemically (i.e., either given intratumorally or

intraperitoneally, respectively) (FIG. 26B). These findings attest to the applicability of approved vaccines for combination cancer immunotherapies with SMCs.

Type I IFN synergizes with SMCs in vivo

The effects of viruses, and likely other TLR agonists and vaccines, appear to be mediated, in part, by type I I FN production, which is controlled by various signaling mechanism, including m RNA translation. Our findings raised the distinct possibility of combining SMC treatment with existing immunotherapies, such as recombinant I FN, as an effective approach to treat cancer. To explore the potential of this combination, we conducted two treatment regimens of SMC and either intraperitoneal or intratumoral injections of recombinant I FNa in the syngeneic orthotopic EMT6 mammary carcinoma model. While treatment of IFNa had no effect on EMT6 tumor growth or overall survival, SMC treatment slightly extended mouse survival and had a cure rate of 17% (FIG. 27). However, the combined treatment of SMC and intraperitoneal or intratumoral injections of I FNa significantly delayed tumor growth and extended survival of tumor-bearing mice, resulting in cure rates of 57% and 86%, respectively (FIG. 27) These results support the hypothesis that direct stimulation with type I I FN can synergize with SMCs to eradicate tumors in vivo.

Assessment of additional oncolytic rhabdoviruses for the potential of synergy with SMCs

While VSVA51 is a preclinical candidate, the oncolytic rhabdoviruses VSV-I FN and Maraba- MG1 are currently undergoing clinical testing in cancer patients. As shown in Example 1 , we have demonstrated that Maraba-MG1 synergizes with SMCs in vitro (FIG. 9). We also confirmed that SMCs synergized with the clinical candidates, VSV-I FN and VSV-NIS-I FN (i.e. carrying the imaging gene, NIS, sodium iodide symporter), in EMT6 cells (FIG. 28). To assess whether these viruses can induce a profinflammatory state in vivo, we treated infected mice i.v. with 5x1 0 ⁸ PFU of VSVA51 , VSV-I FN , and Maraba-MG1 and measured the level of TNFa from the serum of infected mice. In all cases, there was a transient, but robust increase of TNFa from oncolytic virus infection at 12 hrs post-infection, which was barely detectable by 24 hr (FIG. 29). This makes sense as these infections are self-limiting in immunocompetent hosts. These results suggest that the clinical candidate oncolytic rhabdoviruses have the potential to synergize with SMCs in a fashion similar to VSVA51 .

As shown in Example 1 , we documented that a form of VSVA51 that was engineered to express full-length TNFa can enhance oncolytic virus induced death in the presence of SMC (FIG. 1 5). To expand on these findings, we also engineered VSVA51 to express a form of TNFa that had its intracellular and transmembrane components replaced with the secretory signal from human serum albumin (VSVA51 -solTNFa). Compared to full-length TN Fa (memTNFa), solTNFa is constitutively secreted from host cells, while the memTNFa form may be anchored on plasma membrane (and still capable of inducing cell death in a juxtacrine manner) or is released due to endogenous processing by metalloproteases (such as ADAM 17) to kill cells in a paracrine fashion. We assessed whether either forms of TN Fa from oncolytic VSV infected cells will synergize with SMC in the orthotopic syngeneic mammary cancer model, EMT6. As expected, treatment with SMC slightly delayed EMT6 tumor growth rates and slightly extended the survival of tumor bearing mice, and the combination of vehicle with either VSVA51 -memTN Fa or VSVA51 -solTNFa had no impact on overall survival or tumor growth rates (FIGS. 30A and 30B). On the other hand, virally expressed TN Fa significantly slowed tumor growth rates and led to increases in the survival rates of 30% and 70%, respectively. Notably, the 40% tumor cure rate from combined SMC and VSVA51 (FIG. 4A) required four treatments and a dose of 5x1 0 ⁸ PFU of VSVA51 . However, the combination of TNFa-expressing oncolytic VSV and SMC resulted in a higher cure rate and was accomplished with two treatment regimens at a virus dose of 1 x1 0 ⁸ PFU. To assess whether this treatment strategy can be applied to other refractory syngeneic models, we assessed whether VSVA51 -solTNFa synergizes with SMCs in a subcutaneous model of the mouse colon carcinoma cell line, CT-26. As expected, we did not observe an impact of tumor growth rates or survival with VSVA51 -solTNFa and observed a modest decrease of the tumor growth rate and a slight extension of survival (FIG. 30C). However, we were able to further delay tumor growth and extend survival of these tumor bearing mice with the combined treatment of SMC and VSVA51 -solTNFa. Hence, the inclusion of a TN Fa transgene within oncolytic viruses is a significant advantage for the combination of SMC. One could easily envisage the inclusion of other death ligand transgenes, such as TRAIL, FasL, or lymphotoxin, into viruses to synergize with SMCs. Exploring the potential of SMCs to eradicate brain tumors

The combination of SMCs with immune stimulatory agents is applicable to many different types of cancer, including brain malignancies for which effective therapies are lacking and for which

immunotherapies hold promise. As a first step, we determined whether SMCs can cross the blood-brain- barrier (BBB) in a mouse model of brain tumors, as the BBB is a significant barrier to drug entry into the brain. We observed the SMC-induced degradation of clAP112 proteins in intracranial CT-2A tumors several hours after drug administration, indicative that SMCs are capable of crossing the BBB to antagonize clAP1 /2 and potentially XIAP within brain tumors (FIG. 31 A). We also demonstrated that the direct injection of SMC (1 0 μΙ_ of a 1 00 μΜ solution) intracranially can result in the potent down-regulation of both clAPI /2 and XIAP proteins (FIG. 31 B), which is a direct consequence of SMC-induced autoubiquitination of the lAPs or the result of tumor cell death induction in the case of XIAP loss. As a second step, we wished to determine whether systemic stimulation of immune stimulants can led to a proinflammatory response in the brain of naive mice. Indeed, we observed marked up-regulation of TNFa levels from the brain from mice that were intraperitoneally injected with the viral mimic, poly(l :C), a TLR3 agonist (FIG. 32A). We followed up this finding by extracting crude protein lysates from the brains of mice that were treated with poly(l :C) or with the clinical candidate oncolytic rhabdoviruses VSVA51 , VSV-I FN , or Maraba-MG1 , and then applied these lysates onto CT-2A or K1 580 glioblastoma cells in the presence of SMCs. We observed that the stimulation of an innate immune response with these non-viral synthetic or biologic viral agents resulted in enhanced cell death in the presence of SMCs with these two glioblastoma cell lines (FIG. 32B). As a third step, we also confirmed that poly(l :C) could be directly administered intracranially without overt toxicities, which may provide an even increased cytokine induction at the site of tumors (FIG. 32C). Finally, we assessed whether the direct immune stimulation within the brain or systemic stimulation would lead to durable cures in SMC-treated mouse models of brain cancer. The combination of SMCs orally and poly(l :C) intracranially or VSVA51 i.v. results in the near complete survival of CT-2A bearing mouse gliomas (FIGS. 32D and 32E), with an expected survival rate of 86 and 1 00%, respectively. As a follow-up to the observed synergy between SMC and intracranial treatment of poly(l :C), we also assessed the potential for treatment of CT-2A gliomas with direct, simultaneous intracranial injections of SMC and recombinant human I FNa (B/D). Indeed, we observed a marked positive impact of mouse survival with the combined treatment, with a cure rate of 50% (FIG. 33). Importantly, the single or combined SMC or I FNa treatment did not result in any overt neurotoxicity in these tumor bearing mice. Overall, these results reveal that multiple modes of SMC treatment can synergize with a multitude of locally or systemically administered innate immunostimulants to kill cancer cell in vitro and to eradicate tumors in animal models of cancer.

METHODS

Reagents

Novartis provided LCL1 61 (Houghton, P. J. et al. Initial testing (stage 1 ) of LCL1 61 , a SMAC mimetic, by the Pediatric Preclinical Testing Program. Pediatr Blood Cancer 58: 636-639 (201 2); Chen, K. F. et al. Inhibition of Bcl-2 improves effect of LCL161 , a SMAC mimetic, in

hepatocellular carcinoma cells. Biochemical Pharmacology 84: 268-277 (201 2)). SM-1 22 and SM-1 64 were provided by Dr. Shaomeng Wang (University of Michigan, USA) (Sun, H. et al. Design, synthesis, and characterization of a potent, nonpeptide, cellpermeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BI R3 domains in XIAP. J Am Chem Sod 29: 15279-1 5294 (2007)).

AEG40730 (Bertrand, M. J. et al. clAP1 and clAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RI P1 ubiquitination. Mol Cell 30: 689-700 (2008)) was synthesized by Vibrant Pharma Inc (Brantford, Canada). OICR720 was synthesized by the Ontario Institute for Cancer Research (Toronto, Canada) (Enwere, E. K. et al. TWEAK and clAP1 regulate myoblast fusion through the noncanonical NF-kappaB signalling pathway. Sci Signal 5: ra75 (2013)). I FNa, IFN , IL28 and IL29 were obtained from PBL Interferonsource (Piscataway, USA). All siRNAs were obtained from Dharmacon (Ottawa, Canada; ON TARGETplus SMARTpool). CpG-ODN 2216 was synthesized by I DT (5'- gggGGACGATCGTCgggggg-3' (SEQ I D NO: 1 ), lowercase indicates phosphorothioate linkages between these nucleotides, while italics identify three CpG motifs with phosphodiester linkages). Imiquimod was purchased from BioVision Inc. (Milpitas, USA). poly(l:C) was obtained from InvivoGen (San Diego, USA). LPS was from Sigma (Oakville, Canada). Cell culture

Cells were maintained at 37 °C and 5% C02 in DM EM media supplemented with 10% heat inactivated fetal calf serum, penicillin, streptomycin, and 1 % non-essential amino acids (Invitrogen, Burlington, USA). All of the cell lines were obtained from ATCC, with the following exceptions: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Cell lines were regularly tested for mycoplasma contamination. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) or DharmaFECT I (Dharmacon) for 48 hours as per the manufacturer's protocol.

Viruses

The Indiana serotype of VSVA51 (Stojdl, D. F. et al. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4(4), 263-275 (2003)) was used in this study and was propagated in Vero cells. VSVA51 -GFP is a recombinant derivative of VSVA51 expressing jellyfish green fluorescent protein. VSVA51 -Fluc expresses firefly luciferase.

VSVA51 with the deletion of the gene encoding for glycoprotein (VSVA51 AG) was propagated in HEK293T cells that were transfected with pMD2-G using Lipofectamine2000 (Invitrogen). To generate the VSVA51 -TNFa construct, full-length human TNFa gene was inserted between the G and L viral genes. All VSVA51 viruses were purified on a sucrose cushion. Maraba-MG1 , VVDD-B1 8R-, Reovirus and HSV1 ICP34.5 were generated as previously described (Brun, J. et al. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol Ther 1 8, 1440-1449 (201 0); Le Boeuf, F. et al. Synergistic interaction between oncolytic viruses augments tumor killing. Mol Ther 1 8, 888-895 (201 1 ); Lun, X. et al. Efficacy and safety/toxicity study of recombinant vaccinia virus JX-594 in two

immunocompetent animal models of glioma. Mol Ther 18, 1 927-1 936 (2010)). Generation of adenoviral vectors expressing GFP or co-expressing GFP and dominant negative ΙΚΚβ was as previously describedl 6.

In vitro viability assay

Cell lines were seeded in 96-well plates and incubated overnight. Cells were treated with vehicle (0.05% DMSO) or 5 μΜ LCL1 61 and infected with the indicated MOI of OV or treated with 250 U/mL IFN , 500 U/mL I FNa, 500 U/mL IFNy, 1 0 ng/mL IL28, or 1 0 ng/mL IL29 for 48 hours. Cell viability was determined by Alamar blue (Resazurin sodium salt (Sigma)) and data was normalized to vehicle treatment. The chosen sample size is consistent with previous reports that used similar analyses for viability assays. For combination indices, cells were seeded overnight, treated with serial dilutions of a fixed combination mixture of VSVA51 and LCL1 61 (5000:1 , 1 000:1 and 400:1 ratios of PFU VSVA51 : μΜ LCL1 61 ) for 48 hours and cell viability was assessed by Alamar blue. Combination indices (CI) were calculated according to the method of Chou and Talalay using Calcusyn (Chou, T. C. & Talaly, P. A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 252, 6438-6442 (1 977)). An n=3 of biological replicates was used to determine statistical measures (mean with standard deviation or standard error). Spreading assay

A confluent monolayer of 786-0 cells was overlaid with 0.7% agarose in complete media. A small hole was made with a pipette in the agarose overlay in the middle of the well where 5x10 ³ PFU of VSVA51 -GFP was administered. Media containing vehicle or 5 μΜ LCL1 61 was added on top of the overlay, cells were incubated for 4 days, fluorescent images were acquired, and cells were stained with crystal violet. Splenocyte co-culture

EMT6 cells were cultured in multiwell plates and overlaid with cell culture inserts containing unfractionated splenocytes. Briefly, single-cell suspensions were obtained by passing mouse spleens through 70 μηι nylon mesh and red blood cells were lysed with ACK lysis buffer. Splenocytes were treated for 24 hr with either 0.1 MOI of VSVA51 AG, 1 Mg/mL poly(l:C), 1 Mg/mL LPS, 2 μΜ imiquimod, or 0.25 MM CpG prior in the presence of 1 μΜ LCL161 . EMT6 cell viability was determined by crystal violet staining. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation).

Cytokine responsiveness bioassay

Cells were infected with the indicated MOI of VSVA51 for 24 hours and the cell culture supernatant was exposed to UV light for 1 hour to inactive VSVA51 particles. Subsequently, the UV- inactivated supernatant was applied to naive cells in the presence of 5 MM LCL1 61 for 48 hours. Cell viability was assessed by Alamar blue. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation).

Microscopy

To measure caspase-3/7 activation, 5 MM LCL161 , the indicated MOI of VSVA51 , and 5 MM CellPlayer Apoptosis Caspase-3/7 reagent (Essen Bioscience, Ann Arbor, USA) were added to the cells. Cells were placed in an incubator outfitted with an IncuCyte Zoom microscope with a 10X objective and phase-contrast and fluorescence images were acquired over a span of 48 hours. Alternatively, cells were treated with 5 μΜ LCL161 and 0.1 MOI of VSVA51 -GFP and SMC for 36 hours and labeled with the Magic Red Caspase-3/7 Assay Kit (ImmunoChemsitry Technologies, Bloomington, USA). To measure the proportion of apoptotic cells, 1 Mg/mL Annexin V-CF594 (Biotium, Hayward, USA) and 0.2 MM YOYO- 1 (Invitrogen) was added to SMC and VSVA51 treated cells. Images were acquired 24 hours post- treatment using the IncuCyte Zoom. Enumeration of fluorescence signals was processed using the integrated object counting algorithm within the IncuCyte Zoom software. An n=12 (caspase-3/7) or n=9 (Annexin V, YOYO-1 ) of biological replicates was used to determine statistical measures (mean, standard deviation).

Multiple step growth curves

Cells were treated with vehicle or 5 MM LCL161 for 2 hours and subsequently infected at the indicated MOI of VSVA51 for 1 hour. Cells were washed with PBS, and cells were replenished with vehicle or 5 MM LCL161 and incubated at 37°C. Aliquots were obtained at the indicated times and viral titers assessed by a standard plaque assay using African green monkey VERO cells.

Western immunoblotting

Cells were scraped, collected by centrifugation and lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche, Laval, Canada). Equal amounts of soluble protein were separated on polyacrylamide gels followed by transfer to nitrocellulose membranes. Individual proteins were detected by western immunoblotting using the following antibodies: pSTATI (9171 ), caspase-3 (9661 ), caspase-8 (9746), caspase-9 (9508), DR5 (3696), TNF-R1 (3736), cFLIP (321 0), and PARP (9541 ) from Cell Signalling Technology (Danvers, USA); caspase-8 (161 2) from Enzo Life Sciences (Farmingdale, USA); IFNAR1 (EP899) and TNF-R1 (19139) from Abeam (Cambridge, USA) ; caspase-8 (AHZ0502) from

Invitrogen; cFLI P (clone NF6) from Alexis Biochemicals (Lausen, Switzerland); RI P1 (clone 38) from BD Biosciences (Franklin Lakes, USA); and E7 from Developmental Studies Hybridoma Bank (Iowa City, USA). Our rabbit anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect human and mouse CIAP1 /2 and XIAP, respectively. AlexaFluor680 (Invitrogen) or I RDye800 (Li-Cor, Lincoln, USA) were used to detect the primary antibodies, and infrared fluorescent signals were detected using the Odyssey Infrared Imaging System (Li-Cor).

RT-qPCR

Total RNA was isolated from cells using the RNAEasy Mini Plus kit (Qiagen, Toronto, Canada). Two-step RT-qPCR was performed using Superscript I I I (Invitrogen) and SsoAdvanced SYBR Green supermix (BioRad, Mississauga, Canada) on a Mastercycler ep realplex (Eppendorf, Mississauga, Canada). All primers were obtained from realtimeprimers.com. An n=3 of biological replicates was used to determine statistical measures (mean, standard deviation). ELISA

Cells were infected with virus at the indicated MOI or treated with I FN for 24 hours and clarified cell culture supernatants were concentrated using Amicon Ultra filtration units. Cytokines were measured with the TN Fa Quantikine high sensitivity, TN Fa DuoSet, TRAIL DuoSet (R&D Systems, Minneapolis, USA) and VeriKine I FN (PBL I nterferonsource) assay kits. An n=3 of biological replicates was used to determine statistical analysis.

EMT6 mammary tumor model

Mammary tumors were established by injecting 1 x1 0 ⁵ wild-type EMT6 or firefly luciferase-tagged EMT6 (EMT6-Fluc) cells in the mammary fat pad of 6-week old female BALB/c mice. Mice with palpable tumors (-100 mm ³ ) were co-treated with either vehicle (30% 0.1 M HCI, 70% 0.1 M NaOAc pH 4.63) or 50 mg/kg LCL1 61 per os and either i.v. injections of either PBS or 5x1 0 ⁸ PFU of VSVA51 twice weekly for two weeks. For poly(l :C) 25 and SMC treatments, animals were treated with LCL161 twice a week and either BSA (i.t.), 20 ug poly(l :C) i.t. or 2.5 mg/kg poly(l :C) i.p. four times a week. The SMC and CpG group was injected with 2 mg/kg CpG (i.p.) and the next day was followed with CpG and SMC treatments. The CpG and SMC treatments were repeated 4 days later. Treatment groups were assigned by cages and each group had min n=4-8 for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment. Blinding was not possible. Animals were euthanized when tumors metastasized intraperitoneally or when the tumor burden exceeded 2000 mm ³ . Tumor volume was calculated using (TT)(W) ² (L)/4 where W = tumor width and L = tumor length. Tumor bioluminescence imaging was captured with a Xenogen 2000 I VIS CCD-camera system (Caliper Life Sciences

Massachusetts, USA) following i.p. injection of 4 mg luciferin (Gold Biotechnology, St. Louis, USA).

HT-29 subcutaneous tumor model

Subcutaneous tumors were established by injecting 3x1 0 ⁶ HT-29 cells in the right flank of 6-week old female CD-1 nude mice. Palpable tumors (~200 mm3) were treated with five intratumoral injections (i.t.) of PBS or 1 x10 ⁸ PFU of VSVA51 . Four hours later, mice were administered vehicle or 50 mg/kg LCL1 61 per os. Treatment groups were assigned by cages and each group had min n=5-7 for statistical measures (mean, standard error; Kaplan-Meier with log rank analysis). The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment.

Blinding was not possible. Animals were euthanized when tumor burden exceeded 2000 mm ³ . Tumor volume was calculated using (TT)(W) ² (L)/4 where W = tumor width and L = tumor length.

All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service in concordance with guidelines established by the Canadian Council on Animal Care.

Antibody-mediated cytokine neutralization

For neutralizing TNFa signaling in vitro, 25 Mg/mL of a-TNFa (XT3.1 1 ) or isotype control (HRPN) was added to EMT6 cells for 1 hour prior to LCL1 61 and VSVA51 or I FN co-treatment for 48 hours. Viability was assessed by Alamar blue. For neutralizing TNFa in the EMT6-Fluc tumor model, 0.5 mg of a-TNFa or a-HRPN was administered 8, 10 and 12 days post-implantation. Mice were treated with 50 mg/kg LCL1 61 (p.o.) on 8, 10 and 1 2 days post-implantation and were infected with 5x10 ⁸ PFU VSVA51 i.v. on days 9, 1 1 and 13. For neutralization of type I I FN signalling, 2.5 mg of a-I FNAR1 (MAR1 -5A3) or isotype control (MOPC-21 ) were injected into EMT6-tumor bearing mice and treated with 50 mg/kg LCL1 61 (p.o.) for 20 hours. Mice were infected with 5x1 0 ⁸ PFU VSVA51 (i.v.) for 1 8-20 hours and tumors were processed for Western blotting. All antibodies were from BioXCell (West Lebanon, USA).

Flow cytometry and sorting

EMT6 cells were co-treated with 0.1 MOI of VSVA51 -GFP and 5 μΜ LCL1 61 for 20 hours. Cells were trypsinized, permeabilized with the CytoFix/CytoPerm kit (BD Biosciences) and stained with APC- TNFa (MP6-XT22) (BD Biosciences). Cells were analyzed on a Cyan ADP 9 flow cytometer (Beckman Coulter, Mississauga, Canada) and data was analyzed with FlowJo (Tree Star, Ashland, USA).

Splenocytes were enriched for CD1 1 b using the EasySep CD1 1 b positive selection kit (StemCell Technologies, Vancouver, Canada). CD49+ cells were enriched using the EasySep CD49b positive selection kit (StemCell Technologies) from the CD1 1 b- fraction. CD1 1 b+ cells were stained with F4/80- PE-Cy5 (BM8, eBioscience) and GM -FITC (RB6-8C5, BD Biosciences) and further sorted with MoFlo Astrios (Beckman Coulter). Flow cytometry data was analyzed using Kaluza (Beckman Coulter). Isolated cells were infected with VSVA51 for 24 hours and clarified cell culture supernatants were applied to EMT6 cells for 24 hours in the presence of 5 μΜ LCL1 61 . Bone marrow derived macrophages

Mouse femurs and radius were removed and flushed to remove bone marrow. Cells were cultured in RPMI with 8% FBS and 5 ng/ml of M-CSF for 7 days. Flow cytometry was used to confirm the purity of macrophages (F4/80+ CD1 1 b+).

Immunohistochemistry

Excised tumors were fixed in 4% PFA, embedded in a 1 :1 mixture of OCT compound and 30% sucrose, and sectioned on a cryostat at 1 2 μηι. Sections were permeablized with 0.1 % Triton X-100 in blocking solution (50 mM Tris-HCI pH 7.4, 100 mM L-lysine, 145 mM NaCI and 1 % BSA, 1 0% goat serum), a-cleaved caspase 3 (C92-605, BD Pharmingen, Mississauga, Canada) and polyclonal antiserum VSV (Dr. Earl Brown, University of Ottawa, Canada) were incubated overnight followed by secondary incubation with AlexaFluor-coupled secondary antibodies (Invitrogen).

Statistical analysis

Comparison of Kaplan-Meier survival plots was conducted by log-rank analysis and subsequent pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot, San Jose, USA). Calculation of EC ₅₀ values was performed in Graph Pad Prism using normalized nonlinear regression analysis. The EC ₅₀ shift was calculated by subtracting the log ₁₀ EC ₅₀ of SMC-treated and VSVA51 -infected cells from log ₁₀ EC ₅₀ of vehicle treated cells infected by VSVA51 . To normalize the degree of SMC synergy, the EC ₅₀ value was normalized to 1 00% to compensate for cell death induced by SMC treatment alone.

Example 3: SMC-containing immunotherapies demonstrate anti-myeloma activity Immune checkpoint blockade synergizes with SMC treatment to delay disease progression in MM

MPC-1 1 cells stably expressing a luciferase transgene were implanted via intravenous injection in to BALB/c mice. This in vivo MM model mimics the human disease well and follows predictable disease progression. MPC-1 1 cells are obtained from a murine plasmacytoma. Following two rounds of treatment with SMC and monoclonal antibodies against either PD-1 or CTLA-4, only anti-PD-1 based treatments showed response in terms of delayed disease progression. Mice treated with the combination of anti-PD-1 and SMC showed the best response, with almost no tumour burden as determined by luminescence signal (FIG. 35). This combination also significantly prolonged survival of the mice compared to the control group (p=0.01 ) and compared to PD-1 treatment alone (p=0.0163). Type 1 interferons synergize with SMCs to cause MM cell death

In vitro work examining the effects of various cytokines in combination with SMC highlighted the potential of type 1 I FNs. Specifically, I FNa and I FN showed very strong synergistic killing of MM cells with SMC in most cell lines tested (FIG. 36A). Using the same MPC-1 1 mouse model, mice were treated with recombinant I FNa and SMC at three different time points (FIG. 37). Oncolytic viruses synergize with SMCs to cause MM cell death

An oncolytic virus derived from vesicular somatic virus, VSVA51 , synergizes well with SMC in vitro to cause cell death in MPC-1 1 cells (FIGS. 36B and 36C). SMC-containing combinations were also tested in the MPC-1 1 syngeneic mouse model. The combination treatment did not reduce tumour burden as effectively as VSVA51 alone and was not well tolerated by the mice. Treatment of VSVA51 alone did delay disease progression however, and the increase in survival was significant compared to an untreated control group (p=0.0379, log rank analysis) (FIG. 38).

SMC synergizes with standard MM therapeutics

In vitro viability assays showed synergistic cell killing of MM cells in a SMC-based combination with the synthetic glucocorticoid dexamethasone (Dex) (FIG. 39B). When SMC was combined with the glucocorticoid receptor antagonist RU486, there were comparable levels of cell death, suggesting synergy may not be due to activation of GCR, but rather to its inhibitory effects on NF- κΒ. SMC based combination treatments activate NF-κΒ signalling and cause apoptosis in MM cells

SMC treatment effectively caused rapid degradation of clAP1 and clAP2 (FIG. 40A). As a single agent, SMC treatment increased NF-κΒ signalling; beginning with a slight short-term boost in the classical pathway, as evidenced by a higher ratio of phosphorylated-p65 to p65, followed by prolonged reduction (FIG. 40B). As the activation of the classical pathway waned, the alternative N F-κΒ pathway was very strongly activated, shown by an increased ratio of p52 to p1 00 (FIG. 40C). Apoptosis in the cells was confirmed by the presence of cleaved poly(ADP-ribose) polymerase (PARP). Cleavage of PARP is often used as an apoptotic marker because it is a substrate of caspases in early stages of apoptosis.

Combining a SMC with either I FN (FIG. 41 ) or with VSVA5 or with VSVm lFN (containing an inserted gene for murine I FN ) (FIG. 42) had many of the same features as SMC treatment alone. For instance, the classical pathway was eventually down- regulated and the alternative pathway was upregulated. There was also apoptosis, as evidenced by both PARP cleavage and caspase 8 cleavage. The I FN receptor, I FNAR1 , was also down-regulated with I FN treatment, which is intriguing since it would be necessary for continued response to IFN . With the VSV treatments, RI P1 was almost completely degraded in late time points; this is yet another signal of apoptosis as it is degraded by caspase 8 after the ripoptosome is formed.

Sensitivity to SMC in MM1 R and MM1S is related to glucocorticoid receptor expression

Responsiveness to SMC-mediated cell death varies drastically between the related human MM cell lines MM 1 R and MM1 S, which are derived from the same parent line and differ only in expression of GCR. MM1 R, which has no detectable expression of GCR (FIG. 39A), is very sensitive to SMC (FIG. 39C), while MM1 S, which has high GCR expression, is resistant. MM 1 S can become sensitive to SMC treatments when treated with either Dex, or with a GCR antagonist RU486 (FIG. 39B). Innate immune stimulants upregulate inhibitors of the adaptive immune response

Human MM cell lines U266, MM 1 R and MM1 S strongly upregulated PD-L1 in response to IFN treatment. Comparable upregulation was also seen with a combination of SMC and I FN . The other Iigand for PD-1 , PD-L2, was similarly upregulated with I FN -based treatments. This effect was noticeable at both early and late time points for both proteins (FIG. 43). This suggests any immune stimulants that activate type 1 I FNs would result in the upregulation of T cell co-inhibitory molecules.

Combination of SMCs and immunomodulatory agents leads to cancer cell death that also involves CD8+ T cells

FIGS. 44A and 44B are graphs showing data from an experiment in which double treated cured mice were re-injected with EMT6 cells in the mammary fatpad (1 80 days from the initial post-implantation date) or reinjected with CT-2A cells intracranially (1 90 days from the initial post-implantation date). FIG. 44C is a graph showing data from an experiment in which CT-2A glioma or EMT6 breast cancer cells were trypsinized, surface stained with conjugated isotype control IgG or anti-PD-L1 and processed for flow cytometry. FIG. 44D is a graph showing data from an experiment in which CD8+ T-cells were enriched from splenocytes (from naive mice or mice previously cured of EMT6 tumours) using a CD8 T- cell positive magnetic selection kit, and subjected to ELISpot assays for the detection of I FNy and Granzyme B. CD8+ T-cells were co-cultured with media or cancer cells (12:1 ratio of cancer cells to CD8+ T-cells) and 1 0 mg of control IgG or anti-PD-1 for 48 hr. Three mice were used as independent biological replicates (were previously cured of EMT6 tumors). 4T1 cells serve as a negative control as 4T1 and EMT6 cells carry the same major histocompatibility antigens.

SMCs synergize with immune checkpoint inhibitors in orthotopic mouse models of cancer

FIG. 45A is graph showing data in which EMT6 mammary tumor bearing mice were treated once with PBS or 1 x1 0 ⁸ PFU VSVD51 intratumorally, and five days later, the mice were treated with combinations of vehicle or 50 mg/kg LCL1 61 (SMC) orally and 250 mg of anti-PD- intraperitoneally (i.p.). FIGS. 45B and 45C are graphs showing data in which mice bearing intracranial CT-2A or GL261 tumors were treated four times with vehicle or 75 mg/kg LCL1 61 (oral) and 250 mg (i.p.) of control IgG, anti-PD-1 or anti-CTLA-4. FIG. 45D is a graph showing data in which athymic CD-1 nude mice bearing CT-2A intracranial tumors were treated with 75 mg/kg LCL1 61 (oral) and 250 mg (i.p.) anti-PD-1 .

Example 4: Smac mimetics synergize with immune checkpoint inhibitors to promote tumor immunity Cell culture

Cell lines RPMI-8226, U266, MM1 R, MM1 S, M PC-1 1 were acquired from ATCC. MPC-1 1 was cultured in DM EM (Hyclone) with 10% FBS (Hyclone), U266 was cultured in RPMI-1 640 (Hyclone) with 15% FBS, all other lines were cultured in RPM I-1 640 with 1 0% FBS.

Cells were maintained at 37 °C and 5% C02 in DM EM media supplemented with 10% heat- inactivated fetal calf serum and 1 % non-essential amino acids (Invitrogen). All of the cell lines were obtained from ATCC, with the following exceptions: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Primary NF1 -/+p53-/+ cells were derived from C57BI/6J p53+/-/NF1+/- mice. Cell lines were regularly tested for mycoplasma contamination. BTICs were cultured in serum-free culture medium supplemented with EGF and FGF- 250. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 h as per the manufacturer's protocol. Cell lines were regularly tested for mycoplasma

contamination. BTICs were cultured in serum-free culture medium supplemented with EGF and FGF-2. For siRNA transfections, cells were reverse transfected with Lipofectamine RNAiMAX (Invitrogen) for 48 h as per the manufacturer's protocol. Antibodies and reagents

In vivo: LCL1 61 was a generous gift from Novartis. Anti-PD-1 (clone J43) was purchased from BioXcell. Poly(l :C) (HMW vaccigrade, Invivogen). I FNa (for in vivo use) was a generous gift from Dr Peter Staeheli in Germany. Tetralogic Pharmaceuticals provided Birinapant.

In vitro: I FNs were obtained from PBL assay science; Dexamethasone and RU486 were purchased from Sigma Aldrich.

Antibodies used include RIAP1 (in house), PD-L1 (Abeam), PD-L2 (R&D Systems), GCR (Santa Cruz), P1 00 (Cell Signalling), P65 (cell signalling), p-P65 (cell signalling), IFNAR1 (Abeam), PARP (Cell Signalling), tubulin (Developmental Studies Hybridoma Bank), RI P1 (R&D Systems), capsase 8 (R&D Systems).

AT-406, GDC-0917, and AZD-5582 were purchased from Active Biochem. TNF-a was purchased from Enzo. I FN-β was obtained from PBL Assay Science. Broad host range I FN-aB/D was produced in yeast and purified by affinity immunochromatography. Nontargeting siRNA or siRNA targeting cFLI P were obtained from Dharmacon (ON-TARGETplus SMARTpool). High molecular weight poly(l :C) was obtained from Invivogen.

Animal work

4-5 week old BALB/c mice were purchased from Charles River and injected IV with 1 x1 0 ⁶ M PC- 1 1 Flue cells stably expressing a firefly luciferase (Flue) transgene. Treatments include 50mg/kg LCL161 , 250 Mg anti-PD-1 , 250 Mg anti-CTLA4, 25Mg poly(l :C), 5x1 0 ⁸ pfu VSVA51 , 1 ug I FNa. Imaging of mice was done with the in vivo imaging system I VIS, after I P injection of 200 ί of luciferin to measure luminescence.

Viruses

The Indiana serotype of VSV was used in this study. VSV-EGFP, VSVA51 (lacking amino acid 51 in the M gene) and Maraba-MG1 were propagated in Vero cells and purified on an OptiPrep gradient. VSVA51 with the deletion of the gene encoding for glycoprotein (VSVA51 AG) was propagated in HEK293T-cells that were transfected with pM D2-G using Lipofectamine2000 (I nvitrogen), and purified on a sucrose cushion. NRRPs were generated by exposing VSV-EGFP to UV (250mJ cm-2) using a XL- 1000 UV crosslinker (Spectrolinker). In vitro viability assay

Cell lines were seeded in 96-well plates and incubated overnight. Cells were treated with vehicle (0.05% DMSO) or LCL161 and infected with the indicated MOI of virus or treated with 1 μg ml_ ^"1 I FN- aB/D, 0.1 ng ml_ ^"1 TN F-a, or the indicated of NRRPs for 48 h. Cell viability was determined by Alamar blue (Resazurin sodium salt (Sigma)), and data were normalized to vehicle treatment. The chosen sample size is consistent with previous reports that used similar analyses for viability assays, but no statistical methods were used to determine sample size.

Western blotting

Cells were scraped, collected by centrifugation, and lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche). Tumors were excised, minced, and lysed as above. Equal amounts of soluble protein were separated on polyacrylamide gels followed by transfer to nitrocellulose membranes. Individual proteins were detected by Western blotting using for cFLI P (7F1 0, 1 :500, from Alexis Biochemicals) and β-tubulin (1 :1 000, E7 from Developmental Studies Hybridoma Bank). Rabbit anti-rat IAP1 and IAP3 polyclonal antibodies were used to detect human and mouse clAP1 /2 and XIAP, respectively (1 :5000; Cyclex Co.). AlexaFluor680 (Invitrogen) or I RDye800 (Li-Cor) (1 :2500) were used to detect the primary antibodies, and infrared fluorescent signals were detected using the Odyssey Infrared Imaging System (Li-Cor). Full-length blots are shown in FIGS. 68A-68D. ELISA

For detection of TNF-a in vivo, mice were treated with 50 μg poly(l :C) intraperitoneally (i.p.) or 5x1 0 ⁸ PFU of VSVA51 intravenously (i.v.). Brains were homogenized in 20 mM HEPES-KOH (pH 7.4), 150 mM NaCI, 10% glycerol and 1 mM MgCI2, supplemented with EDTA-free protease inhibitor cocktail (Roche). N P-40 was added to final concentration of 0.1 % and clarified through centrifugation. Equal amounts were processed for the detection of TNF-a with the TNF-a Quantikine assay kits (R&D Systems).

To assess the specificity of the adaptive immune response, mice cured of CT-2A tumors by SMC and anti-PD-1 treatment and age-matched control (nal ^' ve) C57BL/6 female mice were injected subcutaneously with 1 x1 0 ⁶ CT-2A cells. After seven days, splenocytes were isolated and cocultured with CT-2A cells for 48 hours (20:1 ratio of splenocytes to cancer cells) in the presence of vehicle or 5 μΜ SMC or 20 Mg mL ^"1 of the indicated antibodies. The secretion of I FN-γ, GrzB, TNF-a, IL-1 7, IL-6, and IL- 10 was determined by ELISA (kits are from R&D Systems).

CT-2A and GL261 brain tumor models

Female 5-week old C57BL/6 or CD-1 nude mice were anesthetized with isofluorane and the surgical site was shaved and prepared with 70% ethanol. 5x10 ⁴ cells were stereotactically injected in a 10-μί volume into the left striatum over 1 minute into the following coordinates: 0.5 mm anterior, 2 mm lateral from bregma, and 3.5 mm deep. The skin was closed using surgical glue. Mice were treated with either vehicle (30% 0.1 M HCI, 70% 0.1 M NaOAc pH 4.63) or 75 mg kg-1 LCL161 orally and intratumorally (i.t.) in 1 0 μί with 50 μg poly(l :C), intravenously (i.v.) with 5x10 ⁸ VSVA51 or intraperitoneally (i.p.) with 250 Mg of anti-CD4 (GK1 .5), anti-CD8 (YTS1 69.4), anti-PD1 (J43), or CTLA-4 (9H 10).

For treatment with birinapant, mice were treated with vehicle (12.5% Captisol) or 30 mg kg ^"1 birinapant (i.p.). In some cases, animals were treated with anti-IFNAR1 (MAR1 -5A3), anti-I FN-γ (R4-6A2) or anti-TNF-a (XT3.1 1 ). Isotype control IgG antibodies were used as appropriately: BE0091 , BE0087, BP0090, MOPC-21 , or H PRN. All neutralizing and control antibodies were from BioXCell. For intracranial cotreatment of SMC and type I IFN, mice were injected 1 0 μΙ_ i.t. with combinations of vehicle (0.5% DMSO), 1 00 μΜ LCL161 , 0.01 % BSA, or 1 Mg I FN-aB/D. Alternatively, mice were treated orally with vehicle or 75 mg kg-1 LCL1 61 and 1 [ig I FN-a B/D (i.p.). Animals were euthanized when they showed predetermined signs of neurologic deficits (failure to ambulate, weight loss >20% body mass, lethargy, hunched posture). Treatment groups were assigned by cages and each group had 5 to 9 mice for statistical measures (Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.

The sample size is consistent with previous reports that examined tumor growth and mouse survival following cancer treatment but no statistical methods were used to determine sample size.

MRI

Live mouse brain MRI was performed at the University of Ottawa pre-clinical imaging core using a 7 Tesla GE/Agilent M R 901 . Mice were anaesthetized for the M RI procedure using isoflurane. A 2D fast spin echo sequence (FSE) pulse sequence was used for the imaging, with the following parameters: 1 5 prescribed slices, slice thickness = 0.7 mm, spacing = 0 mm, field of view = 2 cm, matrix = 256x256, echo time = 25 ms, repetition time = 3,000 ms, echo train length = 8, bandwidth = 16 kHz, 1 average, and fat saturation. The FSE sequence was performed in both transverse and coronal planes, for a total imaging time of about 5 minutes.

EMT6 mammary tumor model

Mammary tumors were established by injecting 1 x1 05 EMT6 cells in the mammary fat pad of 5- week old female BALB/c mice. Mice with palpable tumors (~1 00 mm3) were cotreated with either vehicle

(30% 0.1 M HCI, 70% 0.1 M NaOAc pH 4.63) or 50 mg kg-1 LCL1 61 orally and either i.t. injections of 5x1 0 ⁸ PFU of VSVA51 or i.p. injections of control IgG (BE0091 ) or anti-PD-1 (J43). Animals were euthanized when tumors metastasized intraperitoneally or when the tumor burden exceeded 2,000 mm ³ .

Tumor volume was calculated using (TT)(W)2(L)/4 where W = tumor width and L = tumor length.

Treatment groups were assigned by cages and each group had 4 to 5 mice for statistical measures

(mean, standard error; Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.

MPC-11 multiple myeloma model

A mouse model of multiple myeloma and plasmacytoma was established by injecting 1 x1 0 ⁶ luciferase-tagged MPC-1 1 cells (i.v.) into female 4-5 week old BALB/c mice. Mice were treated with vehicle (30% 0.1 M HCI, 70% 0.1 M NaOAc pH 4.63) or 75 mg kg-1 LCL1 61 orally and with 250 Mg of control IgG or a-PD-1 antibodies (i.p). Bioluminescence imaging was captured with a Xenogen2000 I VIS CCD-camera system (Caliper Life Sciences) following i.p. injection of 4 mg luciferin (Gold Biotechnology). Treatment groups were assigned by cages and each group had 3 to 4 mice for statistical measures (Kaplan-Meier with log rank analysis). There was no randomization and the lead investigator was blinded to group allocation.

Tumor rechallenge

Nal ^' ve age-matched female C57BL/6 mice or mice previously cured of intracranial CT-2A tumors by SMC-based combination treatment with immunostimulants (minimum of 1 80 days post-implantation) were reinjected with CT-2A cells i.e. as described above or with 5x1 0 ⁵ cells subcutaneously. Naive BALB/c or mice previously cured of luciferase-tagged EMT6 mammary tumors with SMC and VSVA51 combination treatment (90 to 1 20 d post-implantation) were reinjected with 5x1 0 ⁵ untagged EMT6 cells in the fat pad. Animals were euthanized as described above. Blinding or randomization was not possible. All animal experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service in accordance with guidelines established by the Canadian Council on Animal Care.

Flow cytometry

For in vitro analysis, cells were treated with vehicle (0.01 % DMSO) or 5 μΜ LCL1 61 and 0.01 % BSA, 1 ng mL-1 TN F-a, 250 U mL-1 I FN-β or 0.1 MOI of VSVA51 for 24 hr. Cells were released from plates with enzyme-free dissociation buffer (Gibco) and stained with Zombie Green and the indicated antibodies. For analysis of tumor immune infiltrates, intracranial CT-2A tumors were mechanically dissociated, RBCs lysed in ACK lysis buffer and stained with Zombie Green and the indicated antibodies. In some cases, cells were stimulated with 5 ng/ml PMA and 500 ng/ml lonomycin in the presence of Brefeldin A for 5 h, and intracellular antigens were processed using BD Cytofix/Cytoperm kit. Antibodies include Fc Block (1 0131 9, 1 :500), PD-L1 (1 0F.9G2, 1 :250), PD-L2 (TY25, 1 :1 00), l-A/l-E (M5/1 14.15.2, 1 :200) and H-2Kd/H-2Dd- (34-1 -2S, 1 :200), CD45 (30-F1 1 , 1 :300), CD3 (17A2, 1 :500), CD4 (GK1 .5, 1 :500), CD8 (53-6.7, 1 :500), PD-1 (29.1 A1 2, 1 :200), CD25 (PC61 , 1 :1 50), Gr1 (RB6-AC5, 1 :200), F4/80 (BM8, 1 :200), GrzB (GB1 1 , 1 :1 50) and I FN-γ (XMG1 .2, 1 :200). All antibodies were from BioLegend except for TNF-a (MP6-XT22, 1 :200) and CD1 1 b (M 1 /70, 1 :1 00) where from BD Biosciences. Cells were analyzed on a Cyan ADP 9 (Beckman Coulter) or BD Fortessa (BD Biosciences) and data was analyzed with FlowJo (Tree Star).

Microscopy

Detection of mKate2-CT-2A cells was performed in an incubator outfitted with an Incucyte Zoom microscope equipped with a 1 0X objective. Enumeration of fluorescent signals from the Incucyte Zoom was processed using the integrated object counting algorithm within the Incucyte Zoom software. Multiplex ELISA

The detection of serum proteins following combinatorial SMC and anti-PD-1 treatment was analyzed by a flow cytometry-based multiplex kit (LEGEN Dplex inflammation panel from Biolegend). Hierarchical analysis was determined using Morpheus (https://software.broadinstitute.org/morpheus).

RT-qPCR

Total RNA was extracted from cells using the RNeasy mini prep kit (Qiagen). Two step RT-qPCR was performed using iScript and SsoAdvanced SYBR Green supermix (BioRad) on a Mastercycler ep realplex (Eppendorf). qPCR was done with PD-L1 and PD-L2 primers (Qiagen) and SI BR green reagent (Bio-Rad). Relative expression was calculated as ΔΔΟί using RPL13A as a control.

The library panel of cytokine and chemokine genes was from realtimeprimers.com. A n = 4 was performed for each treatment conditioned and data was normalized to eight different reference genes and compared to each vehicle and IgG sample. The data was analyzed by hierarchical analysis using Morpheus.

ELISpot

CD8+ T-cells were enriched from splenocytes of female age-matched nal ^' ve mice or mice previously cured of intracranial CT-2A (1 80 days post-implantation) or mammary EMT6 tumors (120 days post-implantation) using a CD8 magnetic selection kit (Stemcell Technologies). CD8+ cells were co- cultured with cancer cells (1 :20 for CT-2A, LLC, and 1 :1 2.5 for EMT6 or 4T1 cells) and with 1 0 ig mL-1 IgG (BE0091 ) or anti-PD-1 (J43) for 48 h using the IFN-γ or Granzyme B ELISpot kits (R&D Systems).

Statistics

For all animal studies, survival was calculated from the number of days post implantation of MM cells, and plotted as Kaplan Meier curves. From those, log rank test was used to determine significance. For in vitro viability assays, error is presented at standard deviation. Subsequent pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot). Comparison between multiple treatment groups was analyzed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test with adjustments for multiple comparison (GraphPad). Estimate of variation was analyzed with GraphPad. Comparison of treatment pairs was analyzed by two-sided t-tests (GraphPad).

Example 5: Combining immunostimulatory agents for glioblastoma therapy

We show here that cultured and primary glioblastoma cell lines are killed with SMC when combined with exogenous TNF-a, the oncolytic virus VSVA51 , or with an infectious but non-replicating virus, VSVA51 AG (FIGS. 46A and 46B). We confirmed that the synergistic effects between the SMC,

LCL1 61 , and TNF-a is a general phenomena within this drug class, as we observed death of glioblastoma cells with the combination of TNF-a and different SMCs (FIG. 47). Furthermore, we also observed potentiation of SMC efficacy with the oncolytic rhabdoviruses, VSVA51 or Maraba-MG1 , for human brain tumor initiating cells (BTICs) (FIG. 46C). Non-replicating rhabdovirus particles (N RRPs), which retain their infectious and immunostimulatory properties without the ability to replicate21 , similarly were found to synergize with SMCs to induce glioblastoma cell death. Notably, only approximately 50% of profiled cancer cell lines are sensitized to death in combination of SMC and TNF-a or TNF-related apoptosis- inducing ligand (TRAIL); the majority of resistant cell lines are further sensitized to death with the downregulation of the caspase-8 inhibitor, cFLI P (cellular FLICE-like inhibitory protein). Consistent with these previous findings, two glioblastoma lines that are refractory to combined treatment with SMC and TNF-a or VSVA51 AG were killed upon silencing of cFLIP (FIGS. 48A and 48B). Normal diploid human fibroblasts, in contrast, were not sensitized to cell death with the downregulation of cFLI P and combined treatment. These findings suggest that an I FN and/or cytokine response, and not direct virus-induced cytolysis, are responsible for the SMC-induced death of glioblastoma cells.

Since VSVA51 is neurotoxic, and since issues remain about the 'immune privileged' brain microenvironment and penetration of drugs across the blood-brain barrier (BBB), we set out to test the effects of systemic and intracranial immunotherapy agent delivery. Following the establishment of intracranial CT-2A tumors (FIGS. 49A and 49B), we tested whether the systemic administration by oral gavage of the SMC, LCL1 61 , could cause the transient degradation of its primary targets proteins, clAP1 and clAP2, within the intracranial murine tumors. In contrast, we did not observe downregulation of the clAPs in neighboring non-tumorous brain tissue nor in the cortex or cerebellum in non-tumor bearing mice (FIG. 51 ). Therefore, SMCs have the capacity to reach tumors within the brain that have a compromised BBB. The systemic administration of immunostimulatory agents, such as the synthetic TLR3 agonist poly(l :C) injected intraperitoneally (i.p.) or the oncolytic virus VSVA51 administered intravenously (i.v.), induced the production of cytokine TN F-a in the serum and brain of non-tumor bearing mice.

When mice bearing intracranial CT-2A glioblastoma were treated singly with SMC (oral gavage), VSVA51 (i.v.)m or poly(l :C) (intracranially, i.e.), the extension of mouse survival was minimal for this aggressive cancer (1 7% survival rate) (FIG. 51 C). However, the combination of systemic SMC with an immunostimulatory trigger, VSVA51 or poly(l :C), significantly extended survival and resulted in durable cures for 71 % or 86% of the mice, respectively. Tumors (which were not tagged with a foreign protein to avoid enhanced immunity) were imaged at day 40 post-implantation by MRI to confirm the observed treatment outcomes.

The virus-induced immune effects are mediated in part by type I IFNs. We show here that CT-2A cells are partially sensitive to combined SMC and recombinant IFN-a in vitro (FIG. 50A). We observed that the intracranial administration of SMC resulted in even more profound degradation of the IAP proteins in CT-2A brain tumors (FIG. 53). For in vivo studies, we used a form of recombinant I FN-a that consists of a hybrid of human isoforms IFN-a B and IFN-a D, which displays potent antiviral activity among a broad range of species. A single coadministration of SMC and IFN-a significantly extended mouse survival and resulted in a 50% durable cure rate. Long-term survivors displayed no overt physical or behavioral defects from the single or combined intracranial treatments of SMC, poly(l :C) or I FN-a (FIG. 54). Furthermore, as we observed a transient increase of intracranial TNF-a within the brain upon systemic VSVA51 infection or treatment with poly(l :C), we sought to determine whether systemic administration of recombinant I FN-a alongside with SMC treatment would be efficacious in the CT-2A glioblastoma model. Similar to the combination of SMC and VSVA51 , the combination of IFN-a administered i.p. with oral gavage of SMC resulted in durable cures in 55% of the mice (FIG. 50B). These results suggest that the presence of a transient inflammatory environment in the brain is tolerable and indicate that indirect and other direct (intracranial) routes of combination treatment administration may be feasible. Example 6: Generation of long-term tumor immunity in cured mice

The innate immune system is a key player in the SMC-mediated death of tumor cells.

Nevertheless, fundamental questions remain as to the contributory role of the adaptive immune system in this SMC combination approach. Furthermore, a potential pitfall of the proposed use of oncolytic viruses or other immunostimulatory agents in combination with SMC treatment could be the increase in expression of checkpoint inhibitor ligands on cancer cells, thereby negating CTL-mediated attack of tumors. Flow cytometry analysis revealed that treatment of glioma cells with recombinant type I I FN or infection with VSV7_51 , but not treatment with TNF-a, resulted in the increased surface expression of PD- L1 and major histocompatibility complex (M HC) I markers. Moreover, there was no significant impact on the expression of these tumor surface molecules by SMC treatment (FIGS. 52A and 56).

Interestingly, mice previously cured of orthotopic EMT6 mammary carcinomas by combined SMC treatments were completely resistant to tumor engraftment when rechallenged with EMT6 cells (FIG. 52B). However, another syngeneic cell line, 4T1 , that shares the major histocompatibility proteins, was not rejected from these cured mice. We found that mice cured with intracranial CT-2A tumors were also resistant to tumor engraftment of CT-2A cells injected either subcutaneously or intracranially (FIG. 52C). We next evaluated the cytotoxic potential of CD8 T-cells from cured mice via an ELISpot assay.

Stimulation of CD8+ T-cells from cured mice, but not cells isolated from naive mice, with CT-2A cells revealed the presence of specific reactive T-cells, as demonstrated by enhanced I FN-γ and Granzyme B (GrzB) production (FIG. 54A). The inclusion of anti-PD-1 blocking antibodies further increased the expression of IFN-γ and GrzB. Similar results were observed with mice cured of EMT6 tumors (FIG. 44D). Collectively, these results suggest the generation of a robust and specific long term tumor immunity using SMC combination therapy.

Example7: Immune checkpoint inhibitors synergize with IAP antagonists

We next investigated whether a current class of cancer immunotherapy, known as immune checkpoint inhibitors or ICIs, could enhance SMC efficacy. It has been recently reported that ICI treatment of glioblastoma in mice results in at least a partial extension of survival. We first sought to determine whether SMC treatment influences PD-1 expression in a subset of infiltrating immune cells within CT-2A brain tumors. While there was no statistical difference between the levels of infiltrating CD3+ or CD3+ CD8+ cells within intracranial CT-2A tumors, we observed a robust increase of CD3+ and CD3+ CD8+ cells expressing the immune checkpoint, PD-1 (FIG. 54B and FIG. 55). Although there was a general increase in the expression of PD-L1 in CD25- cells, which are predominantly CT-2A cells, the trend did not reach statistical significance (FIG. 54C).

To determine whether the increased levels of PD-1 + CD8 T-cells may be a negative modulator for SMC efficacy, we assessed blocking the checkpoint target, PD-1 , as well as CTLA-4, in combination with SMC using two mouse models of glioblastoma. The systemic administration of anti-PD-1 or anti- CTLA4 antibodies demonstrated no activity on their own (FIGS. 54D and 54E). In contrast, the combination of anti-PD-1 and SMC significantly extended survival and resulted in 71 % and 33% durable cure rates in the CT-2A and GL261 models, respectively. Furthermore, when combined with a SMC, the anti-PD-1 biologic was superior to the anti-CTLA-4 biologic in the CT-2A model (71 % versus 43%; FIG. 54D).

There are two structural classes of SMCs: monomers and dimers. Monomeric SMCs consist of a single chemical molecule that binds to the BI R domains of the lAPs while dimeric SMCs consist of two SMC molecules connected by a linker allowing for cooperative binding and/or tethering of lAPs. A clinically advanced SMC, LCL161 , is the focus of most of our studies, and is a potent monomer. We next sought to assess whether another clinically advanced dimeric SMC similarly synergizes with an ICI for the treatment of glioblastoma. We observed a significant increase in survival of mice bearing intracranial CT- 2A tumors when treated with anti-PD-1 and the dimer SMC, Birinapant (FIG. 54F). As the combined blockade of PD-1 or CTLA-4 are beneficial for patients with melanoma, we sought to determine whether the combination of PD-1 and CTLA-4 would similarly significantly enhance SMC therapy. The combination of antibodies targeting PD-1 and CTLA-4 was effective at inducing durable cures in a mouse model of cancer, we observed an overall survival rate of 67% (FIG. 54G). Strikingly, the inclusion of SMC treatment with anti-PD-1 and anti-CTLA-4 together resulted in a 1 00% durable cure rate.

The synergistic effect between SMC and ICIs is not restricted to brain tumors. We also observed a significant extension of the survival of mice bearing a highly aggressive and treatment refractory model of multiple myeloma using MPC-1 1 cells (FIGS. 56A and 56B). A durable cure rate of 75% was also obtained in mice harboring mammary EMT6 tumors, which was further increased to 1 00% with the inclusion of an immune stimulant (FIGS. 57A-57C).

Example 8: CD8+ T-cells are required for efficacy of SMCs and ICIs

To provide an initial insight into the cellular mechanism of action, we profiled the production of immune factors from CT-2A cells that were co-cultured with splenocytes derived from mice cured of intracranial CT-2A tumors using combined SMC and anti-PD-1 treatment. We observed a significant increase in the production of I FN-γ and GrzB from CT-2A cells co-incubated with splenocytes derived from surviving mice (FIGS. 58A and 59A). Notably, there was an increase in the production of Interleukin 17 (IL-1 7). We also observed a reduction in the expression of the proinflammatory cytokines IL-6 and TNF-a, which was unexpected, given that IL-1 7 has been previously found to stimulate the NF-KB pathway.

However, the expression of I FN-γ and IL-1 7 from splenocytes isolated from cured mice significantly increased with anti-PD-1 or PD-L1 treatment, suggesting that a T-cell-based immune response can be augmented upon checkpoint inhibition through the PD-1 axis. We next sought to determine whether this gene response is affected by SMC treatment. Among the previously analyzed cytokines, the inclusion of SMC in these cocultures along with ant-PD-1 blockade increased the secretion of I FN-Y, GrzB, IL-1 7, and TN F-a (FIGS. 59B and 59B). Notably, the level of IL-6 in the supernatant was not affected by SMC treatment. Furthermore, the immunosuppressive cytokine IL-1 0 had a general trend of decreased secretion with combined SMC and anti-PD-1 treatment. As there is an increase in the levels of GrzB, a cytotoxic factor that is partially blocked by XIAP31 - 33 and TNF-a, we next assessed whether co-cultures of glioblastoma cells with splenocytes from naive mice or mice previously cured of CT-2A intracranial tumors would lead to death of CT-2A cells. Using various differently structured SMCs, we saw a statistically significant increase in the death of CT-2A cells in the presence of SMCs, and this response was increased with the inclusion of anti-PD-1 antibodies (FIG. 59C).

Collectively, these results indicate that a robust effector T-cell response is elicited with the combination treatment of ICI and SMC. To further elucidate the cellular mechanism of action, we undertook the depletion of immune cells using specific CD4 or CD8 targeting antibodies. We found that the 71 % cure rate induced by the combination therapy is completely abrogated upon depletion of CD8+ T-cells (FIG. 59D). Interestingly, the depletion of CD4+ T-cells resulted in a 100% cure rate with the combination of SMC and anti-PD-1 , and a 17% cure rate in the control group. These results suggest that removal of CD4+ immunosuppressive cells (such as regulatory T-cells) aids with the induction of tumor regression and that CD4+ cells are not required for efficacy of the combined treatment approach. In a second approach, intracranial CT-2A tumors were established in CD1 nude mice, which lack functional T- cells, and then treated with the combination of anti-PD-1 antibodies with vehicle or SMC. The survival advantage provided by the SMC and anti-PD-1 combination was lost in these T-cell deficient mice (FIG. 59E). Overall, the synergistic effect between SMC and anti-PD-1 is dependent on a functional adaptive immune response and thus implicates CD8+ T-cells as the primary immune cell mediators for in vivo efficacy.

Example 9: SMC treatment affects intratumoral immune cell infiltration

To understand the immune cellular aspect of the synergy between SMC and ICI treatment, we evaluated the profiles of infiltrating CD45+ immune cells of mice bearing glioblastoma. In these studies, we evaluated the infiltrating immune cells in later stage glioblastoma tumors following anti-PD-1 and SMC cotreatment (FIG. 61 A). A flow cytometry analysis of tumor infiltrating Tcells revealed a statistically insignificant trend in the proportion of CD4+ and CD8+ T-cells between the vehicle and IgG control treatment group and all single and double treated mice (FIG. 61 B). However, an analysis of CD4+ and CD25+ T-cells, indicative of a regulatory T-cell (Treg) population, revealed a significant decrease of this cell population with SMC treatment alone or combination of SMC and ICI (FIG. 61 C).

Next, we characterized the surface presentation of PD-1 in T-cells following single and combinatorial treatment. We noted a significant increase in CD8+ T-cells expressing PD-1 in mice treated with SMC alone, and the treatment of anti-PD-1 or combined treatment of SMC and anti-PD-1 resulted in less detectable surface presentation of PD-1 (FIG. 61 D). In addition, we observed a trend in the decreased presentation of PD-1 in CD4+ T-cells in SMC or anti-PD-1 treatment groups. However, the detectable level of surface PD-1 was abrogated with combinatorial treatment of SMC and anti-PD-1 (FIG. 61 E).

In addition to the observed T-cell infiltration of intracranial glioblastoma tumors, we next characterized the presence of myeloid-derived suppressor cells (M DSC) and astrocytes/microglia. In contrast to a previous report, we did not detect differences in the M DSC population (CD1 1 b+ Gr1 +) in any treatment cohorts (FIG. 61 F). However we noted that the astrocyte/microglia population was significantly decreased in the treatment cohorts that included anti-PD-1 (FIG. 61 G). Overall, these results indicate that the consequence of combinatorial treatment is the decrease of an immunosuppressive CD4 T-cell population with a concomitant decrease of PD-1 presentation in T-cells and a reduction of astrocytes and/or microglia.

Example 10: SMC synergy with ICIs is dependent on TNF-a

We next characterized the tumoral cellular cytokine and chemokine profiles of mice bearing intracranial glioblastoma tumors treated with combinations of SMC and anti-PD-1 . Flow cytometry analysis revealed that there was an increase of CD8+ cells expressing GrzB with the inclusion of anti-PD- 1 antibodies. The ratio of cytotoxic CD8+ (FIG. 62A) and CD4+ Treg ratio was also increased in the anti- PD-1 and SMC and anti-PD-1 treatment cohorts (FIG. 62B). In addition to assessing GrzB expression, we analyzed the levels of I FN-γ and TNF-a in T-cells. Unexpectedly, we observed a decrease in the proportion of CD4+ cells expressing I FN-γ upon SMC treatment (even in inclusion of antibodies targeting PD-1 ), but saw no change in the expression level of I FN-γ in any treatment cohort within CD8+ cells (FIG. 62C). We then analyzed the expression level of TNF-a in T-cells. In this context, we observed a significant increase of TNF-a expressing CD4+ and CD8+ T-cells (FIG. 62D), indicating that these T-cells can directly induce SMC-mediated tumor cell death.

We also evaluated the effect of combined SMC treatment and anti-PD-1 blockade on serum concentration and gene expression levels of cytokines and chemokines in the intracranial CT-2A glioblastoma model. We detected statistically significant increases in the proinflammatory cytokines I FN- , IL-1 -a, IL-1 β, and IL-1 7 and the multifaceted cytokines I FN-γ, IL-27, and GM-CSF (FIGS. 60 and 62E). Notably, there was no difference in the presence of anti-inflammatory cytokines, such as IL-1 0. Similarly, an analysis of the cytokine and chemokine expression profiles within intracranial CT-2A tumors following combined SMC and ICI treatment revealed clustering of proinflammatory cytokines and chemokines

(FIGS. 62F and 63). Among these candidates from SMC or combined SMC and ICI treatment were the proinflammatory cytokines I FN-β, IL-1 β, IL-17, Osm, and TNF-a, the chemokines Ccl2 (also known as MCP-1 ), Ccl5, Ccl7, Ccl22, Cxcl9, CscM 0, and CxcM 1 , and multifaceted factors, such as FasL, IL-2, IL-1 2 and I FN-γ.

As we observed a consistent increase in the levels of I FN-β and I FN-γ, we next sought to characterize the functional role of these signaling molecules with the use of blocking/neutralizing antibodies in mice bearing intracranial CT-2A tumors and treated with SMC and anti-PD-1 . Abrogation of type I I FN signaling by using an antibody that blocks the I FNAR1 receptor negated the synergistic effects towards increasing survival of mice bearing intracranial CT-2A tumors following combined SMC and anti- PD1 treatment (FIG. 62G). In contrast, antagonism of I FN-γ function by employing an anti-I FN-γ antibody partially inhibited the synergistic effects of combined SMC and ICI treatment. Overall, these results indicate that each treatment agent, including when combined, results in the generation of different gene and protein signatures, but overall, is dependent on intact type I I FN signaling.

Overall, our results indicate that the synergistic effects between SMC and ICI can be primarily attributed towards enhancing a CTL-mediated attack against glioblastoma cells, and this involves a proinflammatory response that includes type I I FN. The coculture of CT-2A cells and CD8+ Tcells isolated from mice previously cured of intracranial tumors resulted in an increase of GrzB positive CD8+ T-cells, which was not increased with SMC treatment alone (FIG. 65A). However, there was only a slight decrease of viable CT-2A cells when co-incubated with the same CTLs, even when the PD-1 /PD-L1 axis was abrogated (FIG. 65B). As we previously noted that the type I I FN response also leads to the production of TN F-a, we assessed the ability of T-cells to produce TN F-a following SMC treatment in the presence of glioblastoma cells. Accordingly, we next evaluated the production of TN F-a.

The inclusion of SMC significantly increased the proportion of CD8+ T-cells expressing TNF-a, regardless of inclusion of antibodies targeting PD-1 (FIG. 65A). In accordance with the increased expression level of TN F-a from CD8 T-cells, we observed significant decrease of CT-2A cells in a coculture system using CT-2A cells and CD8 T-cells from cured mice (FIG. 65B). Notably, the SMC- mediated effects on eliciting death of CT-2A cells were mainly dependent on TNF-a (the primary mediator of SMC induced tumor killing). Next, we evaluated whether SMC treatment enhances T-cell proliferation. Indeed, we observed a significant decrease of CFSE-loaded CD8+ T-cells, along with the appearance of a new population of faintly labeled CFSE-cells, following co-incubation of CT-2A cells, and this effect was pronounced with the inclusion of SMC and anti-PD-1 (FIG. 64).

These results indicate that cytotoxic T-cells, in response to SMC and anti-PD-1 treatment, may lead to enhanced tumor cell death due to the increased production of GrzB and TNF-a, pro-death factors that induce tumor cell death due to the antagonism of the lAPs. We functionally characterized the role of TNF-a by employing blocking antibodies targeting TN F-a. When systemic blockade of TNF-a was applied, we observed almost a complete reversal of the efficacy of combined SMC and ICI treatment (FIG. 65C), highlighting the importance of TNF-a for the synergistic effect of these disparate agents.

The immunomodulatory anti-cancer effects of SMCs are multimodal (FIGS. 66 and 67). SMCs can polarize macrophages away from the immunosuppressive M2 type towards the inflammatory TNF-a- producing M1 phenotype. Moreover, SMC anticancer effects are highly potentiated by proinflammatory cytokines, and the presence of these cytokines, such as TNF-a or TRAIL, within the tumor

microenvironment leads to tumor cell death. Specifically, SMC mediated depletion of the clAPs converts the TNF-a-mediated survival response into a death pathway in cancer cells.

Our current studies demonstrate that SMCs can cooperate and dramatically intensify the action of ICIs, including anti-PD-1 or anti-CTLA4 antibodies, allowing for durable cures of mice bearing aggressive intracranial tumors. The multiplicity and complexity of mechanisms involved with SMC therapy make it difficult to isolate the individual roles for the varied immunomodulatory actions in the combination synergy. However, it is clear that TNF-a cytotoxicity is involved. Moreover, the current study further demonstrates that CD8+ T-cells are also required for anti-cancer activity when an ICI is combined with an SMC.

In summary, we have shown for the first time that SMCs can potentiate the activity of ICIs in mouse tumor models. Furthermore, this combination effect depends on the presence of CD8+ T-cells with a concomitant decrease of immunosuppressive CD4+ T-cells, and type I and I I I FN and TNF-a signaling pathways, clearly implicating the role of adaptive immunity for SMC-mediated cures in mice. Thus, SMC-mediated T-cell co-stimulatory signals provide the drive for adaptive immune responses that develop against the tumor and this is fully realized when the brakes imposed by co-inhibitory signals, such as PD-1 or PD-L1 , are removed with IC Is.

Other Embodiments

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

While the invention has been described in connection with the specific embodiments, it will be understood that it is capable of further modifications. Therefore, this application is intended to cover any variations, uses, or adaptations of the invention that follow, in general, the principles of the invention, including departures from the present disclosure that come within known or customary practice within the art.

What is claimed is: