EA046706B1 - SMC AND ATEZOLIZUMAB COMBINATION THERAPY FOR THE TREATMENT OF CANCER - Google Patents

Description

Field of technology to which the invention relates

Cell death by apoptosis (or programmed cell death) and other cell death pathways are regulated by various cellular mechanisms. Inhibitor of apoptosis proteins (IAPs), such as X-linked IAPs (XIAPs) or cellular IAP proteins 1 and 2 (cIAP1 and 2), are regulators of programmed cell death, including but not limited to the apoptotic pathway, such as in cancer cells. Other forms of cell death may include but are not limited to necroptosis, necrosis, pyroptosis, and immunogenic cell death. In addition, these IAPs regulate various cellular signaling pathways through E3 ubiquitin ligase activity, which may or may not be related to cell survival. Another regulator of apoptosis is the Smac polypeptide. Smac is a proapoptogenic protein released from mitochondria in association with cell death. Smac can bind to IAPs and antagonize their function. Smac mimetic compounds (SMCs) are non-endogenous pro-apoptotic compounds that can perform one or more functions or actions of endogenous Smac.

The prototypical protein XIAP directly inhibits the main initiator caspase and the executor caspase in apoptotic cascades.

Thus, XIAP may interfere with the termination of apoptotic programs. Cellular IAP proteins 1 and 2 are E3 ubiquitin ligases that regulate apoptotic signaling pathways recruited by immune cytokines. Dual loss of cIAP1 and 2 may render TNFa, TRAIL, and/or GE-1β toxic, for example, to most malignant cells. SMCs may inhibit XIAP, cIAP1, cIAP2, or other IAPs and/or promote other pro-apoptotic mechanisms.

Treatment of malignancy with SMC has been proposed. However, SMC alone may be insufficient to treat some types of malignancies. There is a need for malignancy treatment methods that improve the efficacy of SMC treatment in one or more types of malignancies.

Disclosure of the essence of the invention

The present invention relates to an antitumor composition comprising a monovalent Smac mimetic compound (SMC) and Atezolizumab.

In one aspect, the present invention is a combination wherein said monovalent SMC is AT-406/SM406/Debiol143/D1143.

Furthermore, the invention relates to the use of said combination 2 for treating a patient with a malignant neoplasm. The malignant neoplasm may be resistant to treatment with a Smac mimetic alone.

In one aspect, the patient is diagnosed with a cancer 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, digestive system cancer, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasm, renal cancer, laryngeal cancer, leukemia, liver cancer, liver metastasis, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cancer, ovarian cancer, childhood cancer, cancer pancreas, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, respiratory cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and urinary tract cancer.

Furthermore, the invention relates to a method for treating a malignant neoplasm, wherein said method comprises administering to a patient an effective amount of said combination, wherein SMC and Atezolizumab are administered simultaneously or at an interval of 28 days from each other in amounts that are together effective for treating said malignant neoplasm. SMC and Atezolizumab are administered within 14 days from each other, or within 10 days from each other, or within 5 days from each other, or within 24 hours from each other, or within 6 hours from each other, or substantially simultaneously. The malignant neoplasm may be resistant to treatment with a Smac mimetic alone.

In another embodiment, said method also comprises administering a therapeutic agent comprising an interferon, wherein said interferon is preferably interferon type 1. The malignant neoplasm may be 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, digestive system cancer, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasm, renal cancer, laryngeal cancer,

- 1,046,706 leukemia, liver cancer, liver metastases, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cancer, ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, respiratory cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and urinary tract cancer.

A neighboring cell is a cell that is located in sufficient proximity to a target cell to directly or indirectly receive an immune, inflammatory, or proapoptotic signal from the target cell.

Inducing apoptosis or cell death means increasing the likelihood that one or more cells will undergo apoptosis or die. A treatment may enhance cell death by increasing the likelihood that one or more treated cells will undergo apoptosis and/or by increasing the likelihood that one or more cells adjacent to a treated cell will undergo apoptosis or die.

Endogenous Smac activity refers to one or more biological functions of Smac that result in the promotion of apoptosis, including at least the inhibition of cIAP1 and cIAP2. It is not necessary that the biological function occur or be possible in all cells under all conditions, only that Smac be capable of performing the biological function in some cells under certain natural in vivo conditions.

A Smac mimetic compound or SMC means a composition of one or more components, such as a small molecule, a compound, a polypeptide, a protein, or any complex thereof, capable of inhibiting cIAP1 and/or inhibiting cIAP2.

Smac mimetic compounds include those 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 by increasing the amount, availability, or activity of one or more proteins capable of participating in an IAP-mediated apoptotic pathway, or by priming one or more proteins capable of participating in an IAP-mediated apoptotic pathway to participate in the activity of such a pathway.

Induction of the apoptotic program does not require stimulation of cell death per se: induction of the apoptotic program in a manner that does not result in cell death may have a synergistic effect along with SMC treatment, which stimulates apoptosis, leading to cell death.

The agent means a composition of one or more components capable of inducing an apoptotic or inflammatory program in one or more cells of a subject, and cell death following this program is inhibited by at least cIAP1 and cIAP2. The agent can be, for example, 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 of those listed in Table 4).

Treatment of a malignant neoplasm means inducing the death of one or more malignant cells in a subject or stimulating an immune response that may lead to tumor regression and blocking the spread of the tumor (metastasis).

Treatment of a malignant neoplasm may completely or partially relieve some or all of the signs and symptoms of a malignant neoplasm in a subject, reduce the severity of one or more symptoms of a malignant neoplasm in a subject, reduce the progression of one or more symptoms of a malignant neoplasm in a subject, or mediate the progression or severity of one or more symptoms that develop later.

A prodrug means a therapeutic agent that is produced in an inactive form that can be converted into an active form within the body of a subject, such as within the subject's cells, by the action of one or more enzymes, chemicals, or conditions existing in the subject.

By low dose or low concentration is meant a dose or concentration that is at least 5% lower (e.g. at least 10, 20, 50, 80, 90 or even 95% lower) than the lowest standard recommended dose or the lowest standard recommended concentration of a particular compound obtained for a given route of administration for the treatment of any disease or condition in humans.

A high dose is defined as a dose that is at least 5% higher (e.g., at least 10, 20, 50, 100, 200, or even 300% higher) than the highest standard recommended dose of a particular compound for the treatment of any human disease or condition.

An immune checkpoint inhibitor is a cancer drug that prevents cancer cells from turning off immune cells by antagonistically blocking their receptors or binding to their ligands, thereby restoring the immune system's ability to attack the tumor.

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Brief description of the drawings

Figures 1A-1F show a set of graphs and images demonstrating that SMC and oncolytic rhabdoviruses have synergistic activity in inducing cancer cell death. All panels in Figure 1 represent data from at least three independent experiments using biological replicates (n=3). Figure 1A is a pair of graphs showing the viability of LCL161-treated cells stained with Alamar blue and increasing MOI of VSYA51. Error bars, mean ± standard deviation. Figure 1B is a set of photomicrographs of cells treated with LCL161 and 0.1 MOI of VSVA51-GFP. Figure 1C is a pair of graphs showing the viability (Alamar Blue) of cells infected with VSVA51 (0.1 MOI) in the presence of increasing concentrations of LCL161. Error bars, mean ± standard deviation. Figure Figure 1D is a pair of graphs showing data obtained from cells infected with VSVA51 for 24 h. The cell culture supernatant was exposed to UV light to inactivate the virus, and the medium was then used for new cells in a viability assay (Alamar Blue) in the presence of LCL161. Error bars, mean ± SD. Figure 1E is a graph showing the viability of cells treated with both LCL161 and the non-spreading virus VSVA51AG (0.1 MOI). Error bars, mean ± SD. Figure Figure 1F shows a graph and a pair of images from cells coated with LCL161-containing agarose medium, inoculated with VSVA51-GFP in the middle of the well, and infectivity measured by fluorescence and cytotoxicity assessed by crystal violet staining (images overlaid; images without overlay are shown in Fig. 11). Error bars, mean ± standard deviation.

Figures 2A–2E are a set of graphs and images demonstrating that SMC treatment does not affect the response of cancer cells to oncolytic virus (OV) infection. All panels in Figure 2 represent data from at least three independent experiments using biological replicates. Figure 2A is a pair of graphs showing data obtained from cells that were pretreated with LCL161 and infected with the indicated MOI of VSVA51. Virus titers were assessed by standard plaque assay. Figure 2B is a pair of graphs and a set of time-lapse micrographs obtained from cells treated with LCL161 and VSVA51-GFP. Graphs represent the amount of GFP signal over time. Error bars, mean ± SD, n=12. Figure Figure 2C is a pair of graphs showing data from an experiment in which cell culture supernatants of cells treated with LCL161 and VSVA51 were probed for IFNe by ELISA. Error bars, mean ± SD, n=3. Figure 2D is a pair of graphs showing data from an experiment in which cells were treated with LCL161 and VSVA51 for 20 h and probed for interferon gene (ISG) expression by RT-qPCR. Error bars, mean ± SD, n=3. Figure 2E is a pair of images showing immunoblots of STAT1 pathway activation performed on cells that were pretreated with LCL161 and then stimulated with IFNe.

Fig. 3A-3H are a set of graphs showing that treatment of OV-infected cancer cells with SMC results in type 1 interferon (type 1 IFN) production and nuclear factor kappa B (NF-κB)-dependent proinflammatory cytokine production. All panels in Fig. 3 represent data from at least three independent experiments using biological replicates (n=3). Fig. 3A is a graph showing the viability of Alamar blur-stained cells transfected with combinations of non-targeting siRNA (NT), TNF-R1, and DR5 siRNA, and then treated with LCL161 and VSVA51 (0.1 MOI), or IFNe. Error bars, mean ± SD. Fig. 3B is a graph showing the viability of cells transfected with NTP or IFNAR1 siRNA and then treated with LCL161 and VSVA51AG. Fig. 3B is a graph showing the viability of cells transfected with NT or IFNAR1 siRNA and then treated with LCL161 and VSVA51AG. Error bars, mean ± standard deviation. Fig. 3D is a bar graph showing data collected from an experiment in which cytokine ELISA was performed on cells transfected with NTP or IFNAR1 siRNA and then treated with LCL161 and 0.1 MOI VSVA51. Error bars, mean ± standard deviation. Fig. 3E is a graph showing the viability of cells treated with both LCL161 and cytokines. Error bars, mean ± standard deviation. Fig. Figure 3F is a graph showing data from an experiment in which cells were pretreated with LCL161, stimulated with IFNe at 250 U/mL (~20 μg/mL), and cytokine mRNA levels were determined by RT-qPCR. Error bars, mean ± SD. Figure 3G is a pair of graphs showing the results of a cytokine ELISA performed on cells treated with LCL161 and 0.1 MOI VSVA51. Figure 3H is a graph showing the result of a cytokine ELISA performed on TKBβ-DN-expressing cells treated with LCL161 and VSVA51 or IFNe. Error bars, mean ± SD.

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Figures 4A–4G show a set of graphs and images demonstrating that treatment with the combination of SMC and OV is effective in vivo and dependent on cytokine signaling. Figure 4A shows a pair of graphs showing data from an experiment in which EMT6-F1uc tumors were treated with 50 mg/kg LCL161 (po) and 5x108 PFU VSVA51 (i.v.). The left panel shows tumor growth. The right panel shows the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM, n=5 for each group. Multiple comparison log-rank and Holm-Sydak tests: **, p<0.01; ***, p<0.001. Representative data from two independent experiments are shown. Figure 4B shows a series of representative IVIS images that were acquired in the experiment of Figure 4A. 4C and 4D show sets of immunofluorescence images of infection and apoptosis in tumors treated for 24 h with antibodies against α-VSV or α-c-caspase-3. Figure 4E is an image showing an immunoblot in which protein lysates of tumors from the respective treated mice were immunoblotted with the indicated antibodies. Figure 4F is a pair of graphs showing data from an experiment in which mice bearing EMT6-F1uc tumors were injected with neutralizing TNFa or isotype-matched antibodies and then treated with 50 mg/kg LCL161 (p.o.) and 5x108 PFU VSVA51 (i.v.). The left panel shows tumor growth. The right panel shows the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM. Carrier α-TNFa, n=5; SMC α-TNFa, n=5; Carrier+VSVΔ51, n=5; α-TNFa, n=5; SMC+VSVA51 α-TNFa, n=7; SMC+VSVA51 α-IgG, n=7. Multiple comparison log-rank test and Holm-Saydak test: ***, p<0.001. Figure 4G shows a set of representative IVIS images that were obtained in the experiment of Figure 4F.

Figures 5A–5E are a series of graphs and images demonstrating that small molecule immune stimulators enhance SMC therapy in mouse cancer models. Figure 5A is a graph showing the viability results by Alamar blue staining of EMT6 cells cocultured with splenocytes in a Traswell system and in which isolated splenocytes were treated with LCL161 and the indicated TLR agonists. Error bars, mean ± standard deviation. Representative data from at least three independent experiments using biological replicates (n=3) are shown. Figure 5B is a pair of graphs showing an experiment in which EMT6-Flue tumors were treated with SMC (50 mg/kg LCL161, p.o.) and poly(:C) (15 μg i.o. or 2.5 mg/kg i.p.). The left panel shows tumor growth. The right panel shows the Kaplan-Meier curve depicting mouse survival. Vehicle, vehicle+poly(:C) i.p., n=4; other groups, n=5. Error bars, mean ± SEM. Multiple comparisons of the log-rank and Holm-Saydak tests: **, p<0.01; ***, p<0.001. Figure 5C shows a series of representative IVIS images that were obtained in the experiment of Figure 5B. Figure 5B is a pair of graphs showing the results of an experiment in which EMT6-F1uc tumors were treated with LCL161 or a combination of 200 μg (i.o.) and/or 2.5 mg/kg (i.p.) CpG ODN 2216. The left panel shows tumor growth. The right panel shows the Kaplan-Meier curve depicting mouse survival. Carrier, n=5; SMC, n=5; Carrier+CpG i.p., n=5; SMC+CpG i.p., n=7; Carrier+CpG i.r., n=5; SMC+CpG i.r., n=8; Carrier+CpG i.p.+i.r., n=5; SMC+CpG i.p.+i.r., n=8. Error bars, mean ± SEM. Multiple comparisons of the log-rank test and the Holm-Saydak test: *, p<0.05; **, p<0.01; ***, p<0.001. Figure 5E shows a series of representative IVIS images that were obtained in the experiment of Fig. 5D.

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

Figure 7 shows a pair of graphs demonstrating that co-treatment with SMC and OV has highly synergistic activity against malignant cells. The graphs show data from an Alamar blue staining viability assay of cells treated with serial dilutions of a fixed ratio combination of VSVA51 and LCL161 (PFU:mkM LCL161). The additivity index (CI) was calculated using Calcusyn. Graphs represent the algebraic estimate of CI as a function of the fraction of cells involved (Fa). Error bars, mean ± SEM. Representative data from three independent experiments using biological replicates (n=3) are shown.

Figure 8 shows a pair of graphs demonstrating that monovalent and divalent SMCs together with OV have synergistic activity in inducing malignant cell death.

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Graphs show the result of Alamar blue staining viability assay of cells treated with 5 μM monovalent SMC (LCL161, SM-122) or 0.1 mM divalent SMC (AEG40730, OICR720, SM-164) and VSVA51 at different MOIs. Error bars, mean ± standard deviation. Representative data from three independent experiments using biological replicates (n=3) are shown.

Figures 9A and 9B are a set of images and graphs demonstrating that SMC-mediated killing of malignant cells is enhanced by oncolytic viruses. Figure 9A is a series of images showing the results of a virus spreading assay in cells coated with 0.7% agarose in the presence of vehicle or LCL161 and 500 PFU of the indicated viruses were distributed in the middle of the well. Cytotoxicity was assessed by crystal violet staining. The arrow indicates the expansion of the cell killing zone from the point of initiation of OV infection. Figure 9B is a set of graphs showing data from a viability assay with Alamar blue staining of cells treated with LCL161 and increasing MOIs of VSVA51 or Maraba-MG1. Error bars, mean ± standard deviation. Representative data from two independent experiments using biological replicates (n=3) are shown.

Figures 10A and 10B show a set of graphs and images demonstrating that cIAP1, cIAP2, and XIAP work together to protect cancer cells from OV-induced cell death. Figure 10A shows Alamar blue staining viability assay data from cells transfected with non-targeting (NT) siRNA or siRNA targeting cIAP1, cIAP2, or XIAP and then treated with LCL161 and 0.1 MOI VSVA51 for 48 h. Error bars, mean ± standard deviation. Representative data from three independent experiments using biological replicates (n=3) are shown. Figure 10B shows representative siRNA efficacy immunoblots for the experiment of Figure 10A.

Figure 11 shows the set of images used for the overlay images shown in Figure 1G. Cells were overlaid with agarose containing LCL161 inoculated with VSVA51-GFP in the middle of the well, and infectivity was measured by fluorescence and cytotoxicity was shown by crystal violet (CV) staining. Note: scale bars are the same size.

Figures 12A and 12B are a set of images and a graph showing that SMC treatment does not affect OV dissemination or replication in vivo. Figure 12A is a set of images showing images from an experiment in which EMT6 tumor-bearing mice were treated with 50 mg/kg LCL161 (p.o.) and 5x108 PFU of VSVA51-tagged firefly luciferase (VSVA51-Fluc) by i.v. injection. Virus dissemination and replication were visualized at 24 and 48 h using IVIS. Outline denotes tumor area. Representative data from two independent experiments are shown. Arrow points to VSVA51-Fluc-infected spleen. Figure 12B is a graph showing data from an experiment in which tumors and tissues at 48 h post infection were homogenized and virus titration was performed for each group. Error bars, mean ± standard error of the mean.

Figures 13A and 13B are images showing the study of siRNA-mediated knockdown of non-target (NT), TNFR1, DR5, and IFNAR1 by immunoblotting. Figure 13A is an immunoblot showing knockdown in samples from the experiment of Figure 3A. Figure 13B is an immunoblot showing knockdown in samples from the experiment of Figure 3B.

Figures 14A-14G are images and graphs demonstrating that SMC together with OV have synergistic activity in inducing caspase-8- and RIP-1-dependent apoptosis in cancer cells. All panels of Figure 14 are representative of data from three independent experiments using biological replicates. Figure 14A is a pair of images of immunoblots after immunoblotting for caspase and PARP activation was performed on cells pretreated with LCL161 and then treated with 1 MOI VSYA51. Figure 14B is a series of images showing micrographs of caspase activation that were obtained on cells cotreated with LCL161 and VSVA51 in the presence of the caspase-3/7 substrate DEVD-488. Figure 14C is a graph plotting the proportion of DEVD-488-positive cells in the experiment. 14B (n=12). Error bars, mean ± SD. Fig. 14D shows a series of images from an experiment in which apoptosis was assessed by photomicrographs of translocated phosphatidylserine (Annexin V-CF594) and loss of plasma membrane integrity (YOYO-1) in LCL161- and VSVA51-treated cells. Fig. 14E is a graph showing the proportion of Annexin V-CF594-positive and YOYO-1-negative apoptotic cells from the experiment in Fig. 14D (n=9). Error bars, mean ± SD. Fig. Figure 14F is a pair of graphs showing Alamar blue stained viability assay data from cells transfected with non-targeting (NT) siRNA or siRNA targeting caspase-8 or RIP1 and then treated with LCL161 and 0.1 MOI VSVA51 (n=3). Error bars, mean ± standard deviation. Figure 14G is an immunoblot image showing representative siRNA efficiency.

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Figures 15A and 15B are a set of graphs showing that OV-derived TNFa transgene expression enhances SMC-mediated cancer cell death. Figure 15A is a pair of graphs showing the viability of cells in the Alamar assay co-treated with 5 μM SMC and increasing MOIs of VSV-51-GFP or VSVA51-TNFa for 24 h. Error bars, mean ± SD. Figure 15B is a graph showing the representative EC50 shifts from the experiment in Figure 15A. The dose required to obtain 50% viable cells in the presence of SMC compared to vehicle was determined using nonlinear regression and shown as the log EC50 shift. Representative data from three independent experiments using biological replicates (n=3) are shown.

Figure 16 is a set of images demonstrating that oncolytic virus infection results in increased TNFa expression upon SMC treatment. EMT6 cells were treated with 5 μM SMC and 0.1 MOI VSVA51-GFP for 24 h, and the cells were visualized for intracellular TNFa by flow cytometry. Images show representative data from four independent experiments.

Figures 17A-17C are a pair of graphs and an image demonstrating that TNFa signaling is required for type I IFN-induced synergism with SMC treatment. All panels in Figure 17 show data from at least three independent experiments using biological replicates (n=3). Figure 17A is a graph showing the viability results in the Alamar blur assay of EMT6 cells transfected with non-targeting (NT) or TNF-R1 siRNA and then treated with LCL161 and VSVA51 (0.1 MOI) or IFNe. Error bars, mean ± standard deviation. Figure 17B shows the representative siRNA efficiency from the experiment in Figure 17A. Figure 17C shows a graph showing the viability of EMT6 cells that were pretreated with TNFa neutralizing antibodies and then treated with 5 μM SMC and VSVA51 or IFNe.

Figures 18A and 18B show schematically OV-induced type I IFN and SMC synergism in prostate cancer cell killing. Figure 18A shows schematically that viral infection of refractory cancer cells results in type 1 IFN production, which then induces the expression of IFN-stimulated genes such as TRAIL. Stimulation of type 1 IFN also results in NFκB-dependent TNFα production. IAP antagonism by SMC treatment results in upregulation of TNFα and TRAIL expression and apoptosis of adjacent tumor cells. Figure 18B shows schematically that infection of one tumor cell results in activation of the innate antiviral type 1 IFN pathway, resulting in type 1 IFN secretion by adjacent cells. Adjacent cells also produce the proinflammatory cytokines TNFα and TRAIL. Once infected, the cell undergoes oncolysis, while the rest of the tumor mass remains intact. On the other hand, neighboring cells undergo non-specific cell death upon SMC treatment as a result of SMC-mediated TNFa/TRAIL activation and increased apoptosis upon activation of proinflammatory cytokines.

Figures 19A and 19B are a graph and blot showing that SMC treatment causes minimal transient weight loss and results in cIAP1/2 inhibition. Figure 19A is a graph showing the weight of BALB/c mice treated with LCL161 (50 mg/kg LCL161, p.o.) recorded after a single treatment (day 0). n=5 per group. Error bars, mean ± SEM. Figure 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 times for Western blotting using the indicated antibodies.

Figures 20A-20C are a set of graphs showing that SMC treatment causes transient weight loss in a syngeneic mouse model of cancer. Figures 20A-20C are graphs showing mouse weight measurements upon co-treatment with SMC and oncolytic VSV (Figure 20A), poly(:C) (Figure 20B), or CpG (Figure 20C) in tumor-bearing animals from the experiments depicted in Figures 4A, 5B, and 5D, respectively. Error bars, mean ± SEM.

Figures 21A-21D are a series of graphs showing that VSVΔ51-induced cell death in HT-29 cells is enhanced by SMC treatment in vitro and in vivo. Figure 21A is a graph showing data from an experiment in which cells were infected with VSVA51, cell culture supernatant was exposed to UV light for 1 h, and coated with new cells at the indicated dose in the presence of LCL161. Viability was determined in the Alamar blue assay. Error bars, mean ± standard deviation. Figure 21B is a graph showing the viability of cells in the Alamar blue assay treated with both LCL161 and the non-spreading virus VSVA51AG (0.1 MOI). Error bars, mean ± standard deviation. 21A and 21B show representative data from three independent experiments using biological replicates (n=3). Fig. 21C is a pair of graphs showing the data from the experi

- 6,046,706 periment in which CD-1 nude mice bearing established HT-29 tumors were treated with 50 mg/kg LCL161 (p.o.) and 1x10 ⁸ PFU VSVA51 (i.o.). Vehicle, n = 5; VSVA51, n = 6; SMC, n = 6; VSVA51+SMC, n = 7. The left panel shows tumor growth relative to day 0 post-treatment. The right panel shows the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM. Multiple comparisons of the log-rank test and Holm-Sydak test: ***, p < 0.001. Fig. 21D is a graph showing mouse weight upon co-treatment with SMC and OV in tumor-bearing animals. Error bars, mean ± standard error of the mean.

Figure 22 is a blot showing that the synergistic activity of SMC and OV in vivo requires the type I IFN signaling pathway. EMT6 tumor-bearing mice were treated with vehicle or 50 mg/kg LCL161 for 4 h and then treated with IFNAR1 neutralizing or isotype antibodies for 20 h. The animals were then treated with PBS or VSVA51 for 18 h. Tumors were analyzed by Western blotting with the indicated antibodies.

Figures 23A and 23B are a pair of graphs showing that oncolytic infection of innate immune cells results in cancer cell death in the presence of SMC. Figure 23A is a graph showing data from an experiment in which immune subsets were sorted from splenocytes (CD11b+ F4/80+: macrophage; CD11b+ Gr1+: neutrophil; CD11b- CD49b+: NK cell, CD11b- CD49b-: T and B cells) and infected with 1 MOI of VSVA51 for 24 h. Cell culture supernatants were applied to SMC-treated EMT6 cells for 24 h, and EMT6 viability was assessed with Alamar Blue. Error bars, mean ± SD. 23B is a graph 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 μM SMC and viability was measured with Alamar Blue. Error bars, mean ± standard deviation

Fig. 24A-24H show a series of images of full-length immunoblots. The immunoblots in Fig. 24A-24H refer to (a) Fig. 2E, (b) Fig. 4E, (c) Fig. 10B, (d) Fig. 13, (e) Fig. 14A, (e) Fig. 14G, (g) Fig. 19, and (h) Fig. 17, respectively.

Figures 25A and 25B are a set of graphs showing that non-replicating rhabdovirus-derived particles (NRRPs) have synergistic activity with SMC to induce cancer cell death. Figure 25A is a set of graphs showing data from an experiment in which EMT6, DBT, and CT-2A cancer cells were co-treated with LCL161 SMC (SMC, EMT6: 5 μM, DBT and CT-2A: 15 μM) and varying amounts of NRRP for 48 h (EMT6) or for 72 h (DBT, CT-2A) and cell viability was assessed using Alamar Blue. Figure 25B is a pair of graphs showing data from an experiment in which unfractionated mouse splenocytes were incubated with 1 particle/cell of NRRP or 250 μM CpG ODN 2216 for 24 h. The supernatant was then applied to EMT6 cells in a dose-dependent manner and 5 μM LCL161 was added. EMT6 viability was assessed 48 h after treatment with Alamar blue.

Figures 26A and 26B are a graph and a set of images showing that the vaccines have synergistic activity with SMC to induce cancer cell death. Figure 26A is a graph showing data from an experiment in which EMT6 cells were treated with vehicle or 5 μM LCL161 (SMC) and 1000 CFU/mL BCG or 1 ng/mL TNFa for 48 h and viability was assessed using Alamar blue. Figure 26B shows a set of representative IVIS images showing the survival of mammary fat pad tumor-bearing mice (EMT6-Flue) that were treated twice with vehicle or 50 mg/kg LCL161 (SMC) and PBS, intratumoral (i.t.), BCG (1x105 CFU), or BCG (1x105 CFU), intraperitoneally (i.p.) and subjected to real-time bioluminescence imaging with an IVIS CCD camera at different time points. Scale: p/sec/ ^cm2 /sr.

Figures 27A and 26B are a pair of graphs and a set of images showing that SMC has synergistic activity with type I IFN to induce mammary tumor regression. Figure 27A is a pair of graphs showing data from an experiment in which mice were injected with EMT6-F1uc tumors into mammary fat pads and treated for eight days post-implantation with combinations of vehicle or 50 mg/kg LCL161 (SMC), orally, and bovine serum albumin (BSA), 1 μg IFNa, intraperitoneally (i.p.), or 2 μg IFNa, intrauterine (i.m.). The left panel shows tumor growth. The right panel is a Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM. Figure 27B is a series of representative IVIS images from the experiment described in Figure 27B. 27A. Scale: r/sec/ ^cm2 /sr.

Figures 28A-28C are graphs showing that VSV-IFNe or VSV have synergistic activity with SMC to induce cancer cell death. Figure 28A shows data from an experiment in which EMT6 cells were co-treated with vehicle or 5 μM

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LCL161 (SMC) and various multiplicities of infection (MOI) of VSVA51-GFP, VSV-IFNe, or VSV-NIS-IFNe. Cell viability was assessed 48 h after treatment with Alamar blue. Figure 28B shows a pair of graphs in which EMT6 mammary tumor-bearing mice were treated twice with vehicle or 50 mg/kg LCL161 (SMC) p.o. and PBS or 1 x 10 ⁸ PFU VSV-IFNe-NIS i.m. Figure 28C shows a pair of graphs in which EMT6 tumor-bearing mice were treated twice with vehicle or 50 mg/kg LCL161 p.o. and 1 x 10 ⁸ PFU VSV i.m.

Figure 29 is a graph showing that nonviral and viral triggers induce robust TNFa expression in vivo. Mice were injected with 50 mg poly(:C), either intraperitoneally or by intravenous injection of ^5x108 PFU of VSVA51, VSV-mIFNe, or Maraba-MG1. At the indicated times, serum was isolated and analyzed by ELISA to quantify TNFa levels.

Figures 30A through 30C show a set of graphs and images demonstrating that virulently expressed proinflammatory cytokines have synergistic activity with SMC to induce mammary tumor regression. Figure 30A is a pair of graphs showing data from an experiment in which mice were injected with EMT6-F1uc tumors into the mammary fat pad and treated for seven days after implantation with combinations of vehicle or 50 mg/kg LCL161 (SMC) orally and PBS, 1 x 10 ⁸ PFU VSVA51-memTNFa (i.v.), or 1 x 10 ⁸ PFU VSVA51-solTNFa (i.v.). The left panel shows tumor growth. The right panel shows the Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM. Figure 30A 30B is a set of representative IVIS bioluminescence images that were obtained from the experiment described in Fig. 30A. Scale bar: p/sec/cm2/sr. Fig. 30C is a pair of graphs showing data from an experiment in which mice were injected with CT-26 tumor subcutaneously and, 10 days after implantation, were given combinations of vehicle or 50 mg/kg LCL161 orally and either PBS or 1 x 10 ⁸ PFU VSVA51-solTNFa intratumorally. The left panel shows tumor growth. The right panel is a Kaplan-Meier curve depicting mouse survival. Error bars, mean ± SEM.

Figures 31A and 31B are a set of images showing that SMC administration results in inhibition of cIAP1/2 protein in vivo in an orthotopic syngeneic mouse model of glioblastoma. Figure 31A is an image showing an immunoblot from an experiment in which CT-2A cells were implanted intracranially and treated orally with 50 mg/kg LCL161 (SMC) and the tumor was excised at the indicated time points and analyzed by Western blotting with antibodies against cIAP1/2, XIAP, and β-tubulin. Figure 31B is an image showing an immunoblot from an experiment in which CT-2A cells were implanted intracranially and injected with 10 μl of 100 μM LCL161, intrauterinely, and tumors were excised at the indicated time points and prepared for Western blotting using antibodies against cIAP1/2, XIAP, and β-tubulin.

Figures 32A-32E are a set of graphs and images showing that a transient proinflammatory response in the brain has synergistic activity with SMC to induce glioblastoma death. Figure 32A is a graph showing data from an experiment in which an ELISA was performed to determine soluble TNFa levels from 300 mg of crude brain protein extract prepared from mice that were injected intravenously (i.v.) with PBS or 50 mg of poly(:C) for 12 or 24 h. Brain extracts were prepared by mechanical homogenization in saline. Figure 32B is a graph showing Alamar blue viability assay data for murine glioblastoma cells (CT-2A, K1580) treated with 70 mg crude brain homogenate and 5 μM LCL161 (SMC) in culture for 48 h. Brain homogenates were prepared from mice that received 12 h i.p. injections of poly(I:C) or i.v. injections of 5 x 10 ⁸ PFU of VSVA51 or VSV-mIFNe. Fig. 32C is a Kaplan-Meier curve showing survival of mice that received three intracranial injections of 50 mg poly(I:C). The injections were performed on days 0, 3, and 7. Fig. Figure 32D is a Kaplan-Meier curve showing the survival of mice bearing CT-2A intracranial tumors that received combinations of SMC, VSVA51, or poly(:C). Mice received combinations of three administrations of vehicle, three administrations of 75 mg/kg LCL161 (po), three administrations of 5 x 10 ⁸ PFU VSVA51 (iv), or two administrations of 50 mg poly(I:C) (intracranial, i.c.). Mice were treated on days 7, 10, and 14 after tumor cell implantation under various conditions except for the poly(:C) group, which received i.c. injections on days 7 and 15. Numbers in parentheses indicate the number of mice per group. Figure 32E is a series of representative MRI images of mouse skulls from the experiments depicted in Figure 32A. 32D, which shows an animal at the endpoint and a representative mouse from the indicated groups at day 50 postimplantation. The dotted line represents the brain tumor.

Fig. 33 is a graph showing that SMC has synergistic activity with type I IFN to kill brain tumors. The graph shows the Kaplan-Meier curve showing the survival of CT-2A mice that received intracranial injections of

- 8 046706 carrier or 100 μM LCL161 (SMC) with PBS or 1 μg IFNa 7 days after implantation.

Figure 34 shows a general diagram of the NF-κB signaling pathway. Upon interaction of a ligand with a TNF family receptor, either the classical or alternative pathway is activated depending on the activity of cIAP1/2. In the classical NF-κB pathway, RIP1 receives K63-ubiquitin bonds from cIAP1/2 to form a signaling complex that mediates the phosphorylation of inhibitor of κB (IκB) after activation of IKB indase (IKK). Phosphorylated IκB is degraded, releasing the p50/p65 heterodimer. The alternative pathway remains inactive due to cIAP1/2 K48 associated with ubiquitination of NF-κB-inducing kinase (NIK). If NIK is stable, it mediates the phosphorylation of IKK and subsequently p100, which leads to the conversion of p100 to p52. The pathway culminates in NF-κB heterodimers translocating into the nucleus and acting as transcription factors to regulate the expression of target genes.

Figures 35A-35C depict the process of combining SMC with anti-PD-1 mAbs to delay disease progression and increase survival in a murine MM model. Figure 35A shows images of mice with Fluc MPC-11 cells that were treated with 250 μg ICI and 50 mg/kg three times a week for two weeks. Mice were treated with SMC and anti-PD-1 or anti-CTLA-4 mAbs. Mice treated with the combination of anti-PD-1 and SMC showed virtually no tumor burden as determined by IVIS bioluminescence imaging of tumor burden in the days following cell implantation. Figure 35B shows the treatment regimen with anti-PD-1, anti-CTLA-4, and SMC. Figure 35C is a graph showing the number of days mice survived after implantation with Fluc MPC-11 cells as determined by the Kaplan-Meier curve.

Figures 36A-36C are a series of graphs showing that innate immune stimulators have synergistic activity with SMC to induce MM cell death. Figure 36A is a series of histograms showing the viability of human U266, MM1R, and MM1S cell lines treated with 1 U/μl IFNa, IFNe, and IFNy in the presence of either vehicle or 5 μM SMC. Viability was determined by the absence of trypan blue staining after 24 h. Figures 36B and 36C are graphs showing the viability of the murine MM cell line MPC-11 treated with 5 μM SMC and varying multiplicities of infection (MOI) of VSVA51 and VSVmIFN, respectively. Viability was assessed after 24 h with Alamar Blue.

Figures 37A-37C show that IFN and SMC have synergistic activity in slowing MM disease progression in mice. Mice bearing Fluc MPC-11 cells were treated with 1 μg of recombinant IFNa and 50 mg/kg SMC three times. Figure 37A shows a series of IVIS bioluminescence images of a tumor taken at the indicated days after MM cell implantation. Figure 37B shows the Kaplan-Meier curve showing survival times. Figure 37C shows a diagram showing the treatment regimen.

Figures 38A-38C show that oncolytic virus can delay MM disease progression and increase survival. Figure 38A shows IVIS bioluminescence images obtained at the indicated days after implantation of mice bearing Fluc MPC-11 cells that were treated four times with 5x108 pfu VSVA51 and 50 mg/kg SMC. Figure 38B shows the Kaplan-Meier curve showing survival times. Figure 38C shows the treatment regimen.

Figures 39A-39C show that glucocorticoid receptor ligands have synergistic activity with SMC to sensitize resistant cell lines and mediate SMC cell death. Figure 39A is a schematic diagram showing that protein was extracted from MM1R and MM1S cells for Western blotting, using equal amounts of protein. Figures 39B and 39C are graphs showing that cells were treated with 5 μM SMC, 10 μM Dex, and 10 μM RU486 for the indicated time points, and dead cells were defined as YOYO-1 positive, a cell membrane impermeable DNA binding dye, and normalized to cell confluency within the well.

Figures 40A-40C show that SMC enhances NF-κB signaling and induces apoptosis. Human MM cell lines, MM1R and MM1S, were treated with 5 μM SMC and then harvested after 1, 16, or 48 h. Figure 40A shows Western blots for various components of the NF-κB pathway. Figures 40B and 40C show the quantification of the lanes in Figure 40A expressed as the ratios of p-p65 to p65 and p52:p100, respectively, which were normalized to the untreated control.

Figure 41 shows that combined treatment with SMC and IFNe increases NF-κB activity and induces apoptosis. Human U266, MM1R, and MM1S cell lines and mouse MPC-11 cell line and Fluc-tagged subline were treated with 5 μM SMC and 1 U/μl IFNe for 1 or 16 h. Cell pellets were collected and lysates were loaded in equal amounts for Western blotting.

Figures 42A-42C show that oncolytic virus in combination with SMC activates NF-κB signaling, leading to apoptosis of mouse MM cells. MPC-11 cells were treated with VSVA51 or VSVmIFN for 1, 12, or 24 h. Figure 42A shows a Western blot showing that

- 9 046706 cell pellets were collected and lysates were loaded in equal amounts for Western blotting. Figures 42B and 42C show protein levels quantified from the bands in Figure 42A and expressed as ratios of phospho-p65 to p65 or p52 to p100, respectively.

Fig. 43 shows that PD-L1 and PD-L2 expression is increased in human MM cell lines following IFNe treatment. PD-L1 and PD-L2 mRNA expression is increased at 6, 12, and 24 h following IFNe or IFNe plus SMC treatment compared to untreated controls.

Figures 44A-44D are graphs showing that the combination of SMC and immunomodulatory agents results in the death of malignant cells, which also include CD8+ T cells. Figures 44A and 44B are graphs showing data from an experiment in which mice cured after double treatment were re-injected intramammary fat pad with EMT6 cells (180 days after the initial postimplantation date) or intracranially injected with CT-2A cells (190 days after the initial postimplantation date). Figure 44C is a graph showing data from an experiment in which CT2A glioma cells or EMT6 breast cancer cells were trypsinized, surface stained with conjugated IgG isotype control or anti-PD-L1, and subjected to flow cytometry. Figure 44D is a graph showing data from an experiment in which CD8+ T cells were isolated from splenocytes (from naïve or EMT6 tumor-cured mice) using a CD8 T cell positive magnetic selection kit and subjected to an ELISpot assay for IFNy and Granzyme B. CD8+ T cells were cocultured with medium or cancer cells (12:1 cancer to CD8+ T cell ratio) and 10 mg control IgG or anti-PD-1 for 48 h. Three mice were used as independent biological replicates (previously EMT6 tumor-cured mice). 4T1 cells served as a negative control since 4T1 and EMT6 cells share the same MHC antigens.

Figures 45A-45D are graphs showing that SMC and immune checkpoint inhibitors are synergistic in orthotopic mouse cancer models. Figure 45A is a graph showing data from an assay in which EMT6 mammary tumor-bearing mice were injected intramuscularly with PBS or 1x108 PFU VSVD51 once and five days later the mice were injected with combinations of vehicle or 50 mg/kg LCL161 (SMC), orally, and 250 mg anti-PD, intraperitoneally (i.p.). Figure 45B and 45C are graphs showing data from an experiment in which mice bearing CT-2A or GL261 intracranial tumors were treated four times with vehicle or 75 mg/kg LCL161 (p.o.) and 250 mg (i.p.) control IgG, anti-PD-1, or anti-CTLA4. Fig. 45D is a graph showing data from an experiment in which athymic CD-1 nude mice bearing CT-2A intracranial tumors were treated with 75 mg/kg LCL161 (p.o.) and 250 mg (i.p.) anti-PD-1.

Figures 46A-46C are graphs demonstrating that SMC induces cell death of glioblastoma cells in the presence of cytokines or oncolytic viruses. Viability assessment in the Alamar blue assay of human (M059K, SNB75, U118) and mouse (CT-2A, GL261) glioblastoma cells treated with vehicle or 5 μM LCL161 (SMC) and 0.1 ηγ μλ ^-1 TNF-α or 0.01 MOI VSVA51 for 48 h (Figure 46A). Error bars, mean ± SD, n = 4. The indicated primary NF1-/+p53-/+ mouse cell lines were treated with vehicle or 5 μM LCL161 (SMC) and 0.01% BSA, 1 ηγ μλ ^-1 TNF-α, or the indicated MOI of the VSVA51 spreading variant (VSVA51AG) for 48 h, and viability was assessed in the Alamar blue assay (Fig. 46B). Error bars, mean ± SD, n = 4. Alamar blue assay viability assessment of tumor-initiating cells (BTIC) treated with vehicle or 5 μM LCL161 and 0.001 MOI of VSVA51 or Maraba-MG1 for 48 h (Fig. 46C). Error bars, mean, SD, n = 3. Fig. 46A and 46B show representative data from three independent experiments using biological replicates. Statistical significance was compared between vehicle and BSA treatments using ANOVA with Dunnett's multiple comparison test. Significance was indicated if p<0.0001 (*).

Figure 47 is a graph showing that SMC and TNF-α effectively synergize to induce glioblastoma cell death. Viability of CT-2A mouse glioblastoma cells treated with 0.01% BSA or 0.1 ηγ μλ ^-1 TNF-α and vehicle or 5 μM of the indicated monomeric or dimeric variant for 48 h. Viability was assessed by the Alamar blue assay. Error bars, mean ± standard deviation, n=4. Representative data from two independent experiments using biological replicates are shown. Statistical significance was compared with vehicle and BSA treatment using ANOVA with Dunnett's multiple comparison test. Significance was indicated if p<0.0001 (*).

Figures 48A and 48B show a series of graphs and an image demonstrating that resistance to SMK-based combinations in glioblastoma cells is caused by cFLIP inhibition. Primary NF1-/+p53-/+ mouse (K5001) or human (SF539) glioblastoma cells or non-transformed human (GM38) cells were transfected with non-targeting (NT) dsRNA or cFLIP dsRNA for 48

- 10,046,706 h and then treated for 48 h with vehicle or 5 μM LCL161 (SMC) and BSA, 0.1 ηγ μλ ^-1 TNF-α, or the indicated MOI of the VSVA51 spreading variant (VSVA51AG, Fig. 48A). Viability was determined by the Alamar blue assay. Error bars, mean ± standard deviation, n = 4. Representative data from three independent experiments using biological replicates are shown. Statistical significance was compared between vehicle and BSA treatment using ANOVA with Dunnett's multiple comparison test. Significance was indicated if p < 0.0001 (*). Efficiency of NT dsRNA or cFLIP-targeting dsRNA in the experiment on (Fig. 48B).

Figures 49A and 49B show images demonstrating the generation of a syngeneic orthotopic glioblastoma mouse model. Shown are MRI (Figure 49A) and granular (Figure 49B) images of a C57BL/6 mouse that was intracranially injected with PBS or 5 x 10 ⁴ CT-2A cells and sacrificed 35 days after implantation. Scale bar, 2 mm. Scale bar has centimeter divisions with mm markings.

Figures 50A and 50B are graphs showing that SMC and innate immune stimulants are synergistic in the treatment of glioblastoma. Scale bar, 2 mm. Alamar blue assay of viability of CT-2A cells treated with vehicle or 5 μM LCL161 and 0.01% BSA or 1 μg mL-1 IFN-aB/D. Error bars, means, standard deviations; n=4 (Figure 50A). Mice bearing 7-day-old intracranial CT-2A tumors were treated with combinations of 75 mg kg ^-1 LCL161 (po) and BSA or 1 μg IFN-aB/D (i.p.; Figure 50B). Figure 50B shows the data representing the Kaplan-Meier curve depicting the survival of mice. Multiple comparison of the log-rank test and the Holm-Sydak test: **, p<0.01; ***, p<0.001. The numbers in parentheses represent the number of mice per group.

Fig. 51 shows an image demonstrating that SMC treatment does not cause IAP inhibition in brain tissue from tumor-bearing mice. Mice were treated with 75 mg kg ^-1 LCL161 (SMC) for the indicated times, and tissues were analyzed by Western blotting using the indicated antibodies. n = 2 for each time point.

Figures 52A-52C are graphs demonstrating that SMC-based combination treatment results in long-term immunological antitumor memory. CT-2A cells were treated for 24 h with vehicle or 5 μM LCL161 (SMC) and 0.01% BSA, 1 ηγ μλ ^-1 TNF-alpha, 250 U ml-1 TNF-a, or 0.1 MOI VSVA51, and viable cells (Zombie Green negative) were analyzed by flow cytometry using the indicated antibodies (Figure 52A). Representative data from three independent experiments using biological replicates are shown. Naïve mice or mice previously cured by SMC-based treatment of mammary fat pad with EMT6 (mammary carcinoma, Fig. 52B) or intracranial CT-2A (glioblastoma, Fig. 55C) were re-challenged with EMT6 cells or mammary carcinoma cells in mammary fat pad or CT-2A cells, subcutaneously (s.c.) or intracranially (i.c.). Cells were implanted for 180 days after implantation. Data represent Kaplan-Meier curve depicting mouse survival. Multiple comparisons of log-rank test and Holm-Sydak test (compared with implantation method): **, 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 abolish the expression of checkpoint inhibitory molecules or MHC I/II proteins. SNB72 cells were treated for 24 h with vehicle or 5 μM LCL161 (SMC) and 1 ηγ μλ ^-1 TNF-alpha, 250 U ml-1 TNF-α, or 0.1 MOI VSVA51, and viable cells (Zombie Green negative) were analyzed by flow cytometry using the indicated antibodies. Representative data from three independent experiments are shown.

Figures 54A–54G are graphs showing that SMC and immune checkpoint-targeted antibodies are synergistic in a murine glioblastoma model. Splenic CD8+ T cells were isolated from naïve mice or mice previously cured of CT2A tumors and subjected to ELISpot assay for the detection of IFN-γ and GrzB. Malignant cells (CT-2A, LLC) were co-cultured with CD8+ cells (25:1 ratio) and 10 μg ml-1 control IgG or αPD-1 for 48 h, n=4 mice per group (Figure 55A). Significance was compared with naïve CD8+ T cells 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 CT-2A intracranial tumors were treated with 75 mg/kg LCL161 orally (SMC) on days 14, 16, 21, and 23 after implantation (Fig. 54B). Viable cells of the tumor mass 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 t-test. *, p<0.05; **, p<0.01 (Fig. 54C). Viable tumor cells from the experiment are shown (analyzed by flow cytometry using CD45 (PE) and PD-L1 (BV421, Fig. 54C) antibodies. n=6 mice per group. FMO, fluorescence minus one. Statistical significance was assessed by t-test. Mice with intracranial CT-2A (Figs. 54D, 54F, and 54G) or GL261 (Fig. 54E) were treated at the indicated times with combinations of vehicle, 75 mg kg ^-1 LCL161, p.o. (Figs. 54D, 54E, and 54G) or vehicle or 30 mg kg ^-1 birinapant, i.p.

- 11 046706 (i.p., Fig. 54F) and 250 μg IgG, a-PD-1, or a-CTLA4 (i.p.), or both (Fig. 54G). Data represent the Kaplan-Meier curve depicting mouse survival. Multiple comparison log-rank and Holm-Sydak tests: *, p<0.05; **, p<0.01; ***, p<0.001. Numbers in parentheses represent the number of mice per group. In Figs. 54A–54C, crosses indicate the mean, solid horizontal line depicts the median, box depicts the 25th to 75th percentile, and lines indicate the minimum range of values. Fig. 54D shows representative data from two independent experiments.

Figure 55 shows a series of graphs demonstrating that SMC treatment results in PD-1 activation in CD8 T cells. Mice bearing intracranial CT-2A tumors were orally treated with 75 mg/kg LCL161 (SMC) on days 14, 16, 21, and 23 postimplantation (Fig. 54B). Viable CT-2A tumor cells were analyzed by flow cytometry using CD45 (BV605), CD3 (APC-Cy7), CD8 (PE), and PD-1 (BV421) antibodies.

Figures 56A and 56B show graphs demonstrating that SMC and immune checkpoint inhibitors have synergistic treatment in a murine multiple myeloma model. MPC11 cells were treated with vehicle or 5 μM LCL161 (SMC) and 0.1 ηγ μλ ^-1 TNF-alpha, 250 U ml-1 IFN-α, or 250 U ml-1 TNF-α (Figure 55A). Viability was determined by the Alamar blue assay 48 h after treatment. Error bars, mean, standard deviation, n=4. Statistical significance was compared with vehicle and BSA treatment using ANOVA with Dunnett's multiple comparison test. Significance was indicated if p<0.0001 (***). Representative data from three independent experiments using biological replicates are shown. MPC-11 cells were dissociated and analyzed by flow cytometry with PE-Cy7-conjugated IgG or PD-L1 (Fig. 56B).

Figures 57A-57C are graphs showing that the combination of SMC and antibodies targeting immune checkpoint inhibitors in a mouse breast cancer model. Viability analysis of EMT6 cells treated with vehicle or 5 μM LCL161 (SMC) and 0.1 ηγ μλ ^-1 TNFα, 250 U ml-1 TNF-α, or 0.1 MOI VSVA51 for 48 h (Figure 57A). Error bars, mean ± standard deviation, n=4. Statistical significance was compared with vehicle and BSA treatment using ANOVA with Dunnett's multiple comparison test. Significance was indicated if p<0.0001 (***). Representative data from three independent experiments using biological replicates are shown. EMT6 cells were dissociated and prepared for flow cytometry with PE-Cy7-conjugated IgG or PD-L1 (Fig. 57B). Representative data from three independent experiments are shown. Mice bearing ~100 ^mm3 EMT6-F1uc tumors were treated at the indicated times postimplantation with PBS or 5x108 PFU VSVA51, intramuscularly, followed by vehicle or 50 mg/kg LCL161 (SMC), orally, and 250 μg IgG or a-PD-1, intraperitoneally (Fig. 55C). Left panel shows tumor growth. Error bars, mean, standard error, mean. Right panel shows Kaplan-Meier curve depicting mouse survival. Multiple comparisons of log-rank and Holm-Saydak tests: *, p<0.05; **, p<0.01. Numbers in parentheses represent the number of mice per group.

Figures 58A and 58B show graphs demonstrating that SMC enhances the immune response in the presence of glioblastoma cells. Expression of these factors was detected by ELISA on cell culture supernatants of CT-2A cells that were co-incubated for 48 h with splenocytes isolated from naïve mice or from mice previously cured of CT-2A intracranial tumors by co-administration of SMC and anti-PD-1 (CT-2A cell to splenocyte ratio 1:20, Figure 58A). Crosses indicate the mean, solid horizontal line represents the mean, rectangle represents the 25th to 75th percentile, and lines represent the minimum range of values. Statistical significance was compared with naïve CD8+ T cells, which was 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 obtained from naïve or cured mice and treated with vehicle or 5 μM LCL161 (SMC) for 48 h (Fig. 58B). Crosses indicate the mean, solid horizontal line depicts the median, rectangle depicts the 25th to 75th percentile, and lines indicate the minimum range of values. Statistical significance was compared for vehicle and IgG-treated T cells as assessed by ANOVA with Dunnett's multiple comparison test. ** p<0.01; ***, p<0.001.

Figures 59A–59E are images and graphs showing that CD8+ T cells are required for the synergism of SMC and immune checkpoint inhibitors for the treatment of glioblastoma. Expression of these immune factors was detected by ELISA on cell culture supernatants of CT-2A cells that were co-incubated for 48 h with splenocytes isolated from naïve mice or from mice previously cured of CT-2A intracranial tumors by co-administration of SMC and anti-PD-1 (CT-2A cell to splenocyte ratio 1:20, Figure 59A). Data were plotted as heat maps using normalized scaling. Box and line maps are shown in Figure 58A. Quantification of these factors

- 12 046706 was determined by ELISA from CT-2A cells that were co-cultured with splenocytes from naïve or cured mice and treated with vehicle or 5 μM LCL161 (SMC) for 48 h (Fig. 59B). Splenocytes from naïve or cured mice were co-cultured with CT-2A cells labeled with mKAte2 (CT-2A-mKate2) in the presence of 20 μg ml-1 control IgG or anti-PD1 and 5 μM of the indicated SMC (Fig. 59C). CT-2A-mKate2 cells were numbered using Incucyte Zoom. Crosses indicate the mean, solid horizontal line depicts the median, box depicts the 25th to 75th percentile, and lines indicate the minimum range of values. Significance was compared with naïve splenocytes as assessed by ANOVA with Dunnett's multiple comparison test. Significance was indicated as * if <0.0001. n=6 for naïve mice and n=6 for cured mice. Scale bar, 100 μm. C57BL/6 mice bearing CT-2A intracranial tumors were treated on the indicated days with combinations of IgG (i.p.) and vehicle (p.o.) or a-PD-1 (i.p.) and 75 mg kg ^-1 LCL161 (p.o.) and i.p. either IgG, a-CD4, or a-CD8 (all antibodies were 250 μg, Fig. 59D). CD-1 nude mice bearing CT-2A intracranial tumors were treated at the indicated times with combinations of vehicle or 75 mg kg ^-1 LCL161, p.o., and PBS or 250 μg IgG or a-PD-1, intraperitoneally (i.p., Fig. 59E). Data represent Kaplan-Meier curves depicting mouse survival. Multiple comparison log-rank and Holm-Saydak tests: *, p <0.05; **, p < 0.01. Numbers in parentheses represent the number of mice per group.

Figure 60 shows a series of graphs demonstrating that combination treatment with SMC and an immune checkpoint inhibitor results in increased systemic levels of proinflammatory cytokines. Mouse sera were analyzed by multiplex ELISA to quantify the indicated proteins. Crosses indicate the mean, solid horizontal lines represent the median, boxes represent the 25th to 75th percentile, and lines represent the minimum range of values. Significance was compared between vehicle- and IgG-treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p<0.05. n=6 for each treatment group.

Figures 61A-61G are graphs showing that administration of SMC and an immune checkpoint inhibitor to mouse glioblastoma models results in changes in immune effector cell infiltration. Mice bearing intracranial CT-2A tumors received vehicle or 75 mg kg ^-1 LCL161, orally (SMC), and 250 μg IgG or a-PD-1, intraperitoneally at the indicated times (Figure 61A). Mice were sacrificed on day 27 postimplantation. Viable T cells isolated from tumors were analyzed by flow cytometry using the following antibodies: CD45 (PECy5), CD3 (APC), CD4 (PE-Cy7), CD8 (BV786), CD25 (BV605), and PD-1 (BV421; Figs. 61B–61E). Viable cells from the experiment described in (a) were analyzed by flow cytometry using the following antibodies: CD45 (BV605), CD11b (APC-Cy7), Gr1 (BV786), F4/80 (PE), and CD3 (APC; and Figs. 61F and 61G). In all panels, crosses indicate mean, solid horizontal line represents median, box represents 25th to 75th percentile, and lines represent minimum range. Significance was compared between 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.

Figures 62A-62G are graphs and images showing that the combination of SMC and an immune checkpoint inhibitor elicits a proinflammatory cytokine response and the efficacy is dependent on the type I IFN signaling pathway. Viable brain tumor cells were isolated and prepared for cytometry using the following antibodies: CD45 (BV605), CD3 (APCCy7), Cd4 (PE-Cy7), CD8 (BV786/0), IFN-γ (BV421), TNF-a (PE) and GrzB (AF647; Figures 62A-62D). Crosses indicate the mean, solid horizontal line represents the median, box represents the 25th to 75th percentile, and lines represent the minimum range of values. Significance was compared between vehicle- and IgG-treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05. n = 6 for each treatment group. Mouse sera were analyzed by multiplex ELISA to quantify the indicated proteins (Fig. 62E). Data were plotted as heat maps using normalized scaling. n = 6 for each treatment group. Mice were treated and CT-2A intracranial tumors were subjected to quantification of 176 cytokine and chemokine genes by RT-qPCR (Fig. 62F). Normalized heat maps of the two major groups identified by hierarchical clustering are shown. n = 4 for each treatment group. Mice bearing CT-2A intracranial tumors were treated with vehicle or 75 mg kg ^-1 LCL161 (po) or intraperitoneally at the indicated days post-implantation, along with the corresponding IgG isotype control or 2.5 mg a-IFNAR1, 350 μg a-IFN-γ, or 250 μg a-PD-1 (Fig. 62G). Significance was compared between vehicle- and IgG-treated mice as assessed by ANOVA with Dunnett's multiple comparison test. *, p < 0.05. Numbers in parentheses represent the number of mice per group.

Fig. 63 is an image showing that proinflammatory cytokine and chemoattractant chemokine genes are upregulated by combined treatment with SMC and an immune inhibitor.

- 13 046706 control point. Intracranial CT-2A tumors were subjected to quantitative analysis of 176 cytokine and chemokine genes by RT-qPCR. Normalized heat maps of major groups identified by hierarchical clustering are shown. n=4 for each treatment group.

Figure 64 shows a series of graphs demonstrating that SMC enhance the clonal expansion of CD8+ T cells in the presence of glioblastoma target cells. Splenic CD8+ T cells from mice previously cured of CT-2A tumors were plated on CFSE and co-incubated with CT-2A cells (10:1 ratio) for 96 h in the presence of vehicle or 5 μM LCL161 (SMC) or 20 μg ml-1 control IgG or anti-PD1. Viable cells were analyzed by flow cytometry. Significance was compared between 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.

Figures 65A–65C are graphs and images showing that the proinflammatory cytokine TNF-α is required for T cell-mediated killing of glioblastoma cells upon treatment with a Smac mimetic and an immune checkpoint inhibitor. Isolated CD8 T cells obtained from the spleen and lymph nodes of mice previously cured of CT2A intracranial tumors were cocultured with CT-2A cells in the presence of vehicle or 5 μM LCL161 and 20 μg mL-1 of IgG or a-PD-1 for 24 h. Viable T cells were analyzed by flow cytometry using the following antibodies: CD3 (APC-Cy7), CD8 (BV711), GrzB (AF647), and TNF-α (PE; Figure 65A). Crosses indicate the mean, solid horizontal line depicts the median, box depicts the 25th to 75th percentile, and lines indicate the minimum range of values. Significance was compared between 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. CD8+ T cells were cocultured with mKate-tagged CT-2A cells (CT-2A-mKate2) for 72 h in the presence of vehicle or 5 μM LCL161 and 20 μg/ml control IgG, a-PD-1, or aTNF-a (Fig. 65B). mKate2-positive cells were enumerated using Incucyte Zoom software. Crosses indicate the mean, solid horizontal line depicts the median, box depicts the 25th to 75th percentile, and lines indicate the minimum range of values. Significance was compared between vehicle- and IgG-treated mice as assessed by ANOVA with Dunnett's multiple comparison test. p<0.01; ***, p<0.001. n=6 for each treatment group. Scale bar, 100 μm. Mice bearing CT-2A intracranial tumors were treated with vehicle or 75 mg kg ^-1 LCL161 (po) or intraperitoneally on the indicated day after implantation, together with the corresponding IgG isotype control or 500 μg a-PD-1 (Fig. 65C). Significance was compared between vehicle- and IgG-treated mice as assessed by ANOVA with Dunnett's multiple comparison test. **, p<0.01. Numbers in parentheses represent the number of mice per group.

Fig. 66 schematically shows that SMCs are immunoregulatory drugs that act on tumor and immune cells to kill malignancy via the innate and adaptive immune system. A model is shown depicting the single agent and combinatorial immunomodulatory effects of Smac mimetics based on the inventors' findings. The effect of IAP antagonism on these immune or tumor cells is described as follows: (1) SMCs stimulate the production of cytokines and chemokines by various immune cells such as macrophages or T cells, resulting in the infiltration of immune cells into the tumor microenvironment. (2) SMC treatment reduces the population of immunosuppressive M2 macrophages while increasing the proinflammatory M1 population. (3) SMCs reduce the amount of cIAP1 and cIAP2 and sensitize tumors to killing by immune ligands such as TNF-a or TRAIL1. Tumor cell death is sensed by the immune system, resulting in priming of the cytotoxic T cell (CTL) response. (4) SMCs stimulate the CD40L/CD40 signaling pathway, a member of the TNF/TNFR family, on antigen presenting cells (APCs) to accelerate the differentiation and maturation of dendritic cells (DCs) and macrophages. APCs present tumor antigens to the immune system and then release cytotoxic inflammatory cytokines. (5) As a result of the degradation of cIAP1 and cIAP2 by SMC treatment, SMCs activate the alternative NF-κB pathway, eliminating the requirement for a TNF superfamily ligand (e.g., 4-1BB) and thus providing a co-simulatory T cell signal. (6) SMCs have been shown to enhance 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 reversed by release of Smac from mitochondria or its mimic drug SMC13-15.

Fig. 67 schematically illustrates the cooperative and complementary mechanisms for the synergistic effect of SMC and immune checkpoint inhibitors (ICIs). (1) The presence of therapeutic recombinant antibodies that block the PD-1/PD-L1 targeting mediates the CD8+ T cell T cell receptor (TCR) signaling pathway upon binding to antigen presented by malignant cells via the major histocompatibility complex I (MHC-I) molecule. Pa

- 14 046706 Concurrent depletion of IAP by SMP treatment may enhance T cell activation, likely by inducing a concomitant tumor necrosis factor receptor superfamily (TNFRSF) stimulatory response (similar to 4-1BB 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, may result in enhanced tumor cell killing by GrzB. (3) Depletion of cIAP1 and cIAP2 by SMC results in increased local TNF-a production by T cells in the tumor microenvironment, likely mediated by activation of the NF-κB alternative pathway. (4) As a result of cIAP1/2 loss, SMC-treated malignant cells become susceptible to cell death induced by the presence of proinflammatory cytokines such as TNF-α.

Fig. 68A-68D show images showing full-size Western blots.

Detailed description of the invention

The present invention relates to methods and compositions for enhancing the efficacy of Smac mimetic compounds (SMC) in the treatment of malignant neoplasm. In particular, the present invention relates to methods and compositions for combination therapy that includes SMC and a second agent that stimulates one or more cell death pathways that are inhibited by cIAP1 and/or cIAP2. The second agent can be, for example, a TLR agonist of a virus, such as an oncolytic virus, or an interferon or an agent related thereto.

The data presented herein demonstrate that therapy with the agent and SMC results in tumor regression and long-term cure in vivo (see, for example, Example 1). This combination therapy was well tolerated by the mice, with body weight returning to pre-treatment levels shortly after cessation of therapy. The tested combination therapy is capable of treating several mouse models of refractory aggressive cancers. One of skill in the art, based on the disclosure and the data presented herein, will appreciate that any one or more of a plurality of SMCs and any one or more of a plurality of agents, such as a TLR agonist, a pathogen, or a pathogen mimetic, can be combined in one or more embodiments of the present invention such that apoptosis is stimulated and a cancer is treated.

Although other approaches have been taken to improve SMC therapy, complete responses have been observed very rarely, particularly in aggressive immunocompetent model systems. Certain embodiments of the present invention, including treatment of a cancer with a pathogen mimetic, such as a pathogen mimetic with a mechanism of action partially dependent on TRAIL, may have certain advantages. First, this approach may induce TNF-alpha-mediated apoptosis and necroptosis: given the plasticity and heterogeneity of some cancers in the progression stage, therapies that simultaneously induce several different cell death mechanisms may be more effective than those that do not. Second, pathogen mimetics may induce an integrated innate immune response that includes layers of negative feedback. These feedback mechanisms may impact cytokine responses in a manner that is difficult to replicate using recombinant proteins and thus may act to warrant a combination therapy approach.

Multiple myeloma (MM) is an incurable malignancy characterized by the rapid proliferation of plasma cells in the bone marrow. MM is the second most common hematological malignancy, with a median survival of only three to five years after diagnosis. MM cells cause bone resorption, leading to fractures and immunosuppression when they invade the bone marrow compartment. MM cells can disseminate to other tissues to form plasmacytomas, and the disease can progress to an aggressive leukemic stage. Current therapies can prolong life and alleviate symptoms, but are not a cure. New treatments are urgently needed to combat treatment resistance and inevitable relapse.

Early in the disease, malignant cells are dependent on the bone marrow microenvironment, particularly on TNFa and interleukin-6 (IL-6) from bone marrow microenvironmental cells. As the disease progresses, cells become independent of their environment and survive with high autocrine production of TNFa. At all stages, cells have high levels of NFκB signaling, which enhances their survival, in part due to intrinsic mutations in key components of the pathway. Targeting the NF-κB pathway in MM has been shown to improve the efficacy of many standard therapeutics used in MM, such as the proteasome inhibitor bortezomab, the immunomodulatory agents (IMiDs) thalidomide and lenalidomide, and the synthetic glucocorticoid dexamethasone.

TNFa-mediated NF-κB signaling pathway can be switched from a pre-survival signaling pathway to an apoptotic signaling pathway along with the removal of cellular inhibitors.

- 15 046706 apoptosis pathways (cIAP); this process appears to be selective for malignant cells. cIAP1 and cIAP2 act interchangeably as E3 ligases in all members of the TNFa receptor superfamily, either ubiquitinating specific proteins to scaffold signaling complexes or targeting them for degradation. Examples of this can be seen in both branches of the NF-κB pathway: RIP1 is ubiquitinated via K63 linkages to scaffold a signaling complex that is required for activation of the classical pathway, and NIK is signaled for K48-linked ubiquitination and degradation and keeps the alternative pathway inactive (Fig. 35). SMCs are a new class of anticancer therapeutics that mimic the endogenous protein Smac, which is involved in activation of the intrinsic apoptotic pathway. Smac and SMC peptides bind to the BIR domain of cIAP, which results in their auto-ubiquitination, targeting them for proteasomal degradation. When RIP1 is no longer ubiquitinated, it becomes free and forms ripoptosomes, initiating the caspadic cascade and cell death.

SMC and TNFa have been shown to have a strong synergistic effect and induce NF-kB-mediated apoptosis in many cancer cell lines. SMC also has a synergistic effect on cancer cell killing when combined with other inflammatory cytokines such as IFN, which can be induced by TLR agonists or oncolytic viruses. SMC may even standardize therapeutic agents used to treat MM to enhance cancer cell apoptosis. Several clinical trials currently underway to evaluate the efficacy of SMC along with chemotherapy in MM as well as other types of malignancies have shown great therapeutic potential.

Immune activation increases cytokine production, which is beneficial for SMC-mediated killing of MM cells. However, such cytokine production may have undesirable consequences for MM cells. Many stimulators of the innate immune system, such as IFNs and TLR agonists, have been shown to activate the PD-1 immune checkpoint ligand. PD-1 is expressed on the surface of T cells and NK cells. When PD-1 binds to its ligands, PD-L1 and PD-L2, it acts as a coinhibitory signal for the T cell receptor and suppresses the cytotoxic capacity of T cells. PD-L1 is expressed constitutively at low levels in many tissues and can be activated, presumably to prevent autoimmune reactions. However, PD-L1 is upregulated on malignant cells, resulting in cell evasion from recognition by the adaptive immune system. In particular, PD-L1 can be activated in MM in response to IFNy and TLR agonists such as LPS. PD-L2 has a much more selective expression compared to PD-L1. It is present on a subset of B cells and is activated on selected cells in response to strong NF-κB or STAT6 signaling.

SMCs can also influence T cell function in SMC-treated mice both in vitro and in vivo, such as increasing proliferation, enhancing cytokine production of activated T cells extracted from mouse spleen after SMC treatment, and increasing NKT and NK cell cytokine production. In addition, SMC-treated mice exhibit hyperresponsive T cells upon antigen stimulation. Therefore, SMC-based combination therapy may not only enhance cell apoptosis in MM but also stimulate selective adaptive response. Combining SMCs and innate immune response stimulators or immune checkpoint inhibitors (ICIs) may be a better approach to overcome the strong survival signaling pathway that MM cells receive.

Malignant cells are able to manipulate many of the survival approaches used by healthy cells, rendering them resistant to death signals. MM cells, in particular, are able to further enhance the constitutive NF-κB signaling used in plasma cells, rendering them resistant to apoptotic signals. This is achieved by increasing the expression of NF-κB pro-survival signaling pathway target genes, such as IL-6 and TNFa. In addition, MM cells are able to upregulate the expression of checkpoint inhibitors, which appear to be used to protect cells from inflammatory and cytotoxic environmental insults; helping them to evade detection by T cells and NK cells. Targeting both apoptotic resistance and immune evasion in MM offers the potential to overcome two major aspects of treatment resistance in this disease.

PD-1 blockade is effective in slowing MM progression and significantly prolonging survival in mice, as demonstrated in a syngeneic MM mouse model. The use of an anti-PD-1 monoclonal antibody has several advantages over alternative immune checkpoint blockade approaches. First, it is able to block the binding of both PD-L ligands, PD-L1 and PD-L2. A large number of malignancies are able to upregulate PD-L1 in response to interferon treatment, and PD-1/PD-L1 are upregulated in MM patients following treatment. In addition, a subset of immature B cells called B1 cells, which secrete non-specific antibodies, have been shown to highly express PD-L2. Moreover, PD-L2 expression can be upregulated in response to certain stimuli, such as NF-κB and STAT6 activation, suggesting

- 16 046706 highlights the importance of examining the expression levels of both ligands on MM cells. Human MM cells are capable of activating both PD-1 ligands, making them unique compared to solid cancers. While this suggests that monoclonal antibody therapy targeting only PD-L1 (eg, Bristol-Myers Squibb’s BMS-936559/MDX-1105, Genentech’s MPDL3280A, MedImmune’s MEDI4000, and EMD Serono’s alvelumab) will be less effective than PD-1-targeted therapies (eg, Bristol-Myers Squibb’s nivolumab, Merck’s pembrolizumab, and Curetech’s pidilizumab), the use of anti-PD-1 antibodies in MM has been shown to have value.

Second, PD-1-targeting approaches may have the effect of eliciting a more potent anti-malignancy response compared to other ICIs such as anti-CTLA-4. The difference in potency may be due to the distinct roles of these molecules in T cell regulation. PD-1 is abundant on CD8+ T cells, and engagement with its ligand inhibits the cytotoxic response activated by TCR signaling. In contrast, CTLA-4 plays a more prominent role in secondary lymphoid tissues on regulatory T cells. Binding of CTLA-4 to its receptor, CD28, outcompetes and even inhibits CD28 activating ligands and causes attenuation of secondary T cell clone expansion. It is possible that the lack of efficacy of anti-CTLA-4 treatment in Example 3 indicates MM infiltration into secondary lymphoid organs. This may compromise the efficacy of anti-CTLA4 either by the CD4+ T cell population being proportionally lower in the germinal centers or by the difficulty of T cell infiltration into secondary lymphoid organs. In extramedullary MM, cells may form plasmacytomas in the spleen and lymph nodes, which is often seen in late MM in the mouse model described in Example 3. Thus, it is clear that the germinal centers are compromised by MPC-11 cells.

SMC

The SMC of the present invention can be any small molecule, compound, polypeptide, protein, or any complex thereof that is capable of, or is believed to be capable of, inhibiting cIAP1, cIAP2, and/or XIAP and, optionally, exhibiting one or more additional endogenous Smac activities. The SMC of the present invention is capable of enhancing apoptosis by mimicking one or more endogenous Smac activities, including, but not limited to, inhibition of cIAP1 and inhibition of cIAP2. An endogenous Smac activity can be, for example, interaction with a particular protein, inhibition of the function of a particular protein, or inhibition of a particular IAP. In particular embodiments, the SMC inhibits both cIAP1 and cIAP2. In some embodiments, the SMC inhibits one or more other IAPs besides cIAP1 and cIAP2, such as XIAP or Livin/ML-IAP, which contains a single BIR IAP. In particular embodiments, SMC inhibits cIAP1, cIAP2, and XIAP. In any embodiment comprising SMC and an immune stimulator, SMC having a particular activity can be selected to form a combination with one or more particular immune stimulators. In any embodiment of the present invention, SMC can be capable of actions that Smac is not capable of. In some cases, these additional activities can enhance the effectiveness of the methods or compositions of the present invention.

SMC treatment can deplete cells of cIAP1 and cIAP2, for example, by inducing auto- or trans-ubiquitination and proteasome-mediated degradation. SMC can also suppress XASAP caspase downregulation. SMC may primarily function by targeting cIAP1 and 2, and by converting TNFa and other cytokines or death ligands of the pro-survival signaling pathway into a death signal, for example in malignant cells.

Some SMCs inhibit at least XIAP and cIAP. Such pan-IAP SMCs may interfere with several distinct but interrelated steps in programmed cell death. This characteristic minimizes the potential for resistance to panIAP SMC cancer treatments because such SMCs affect a wide range of death pathways, providing synergistic interactions with existing and emerging cancer therapies that activate multiple apoptotic pathways in which SMCs may be involved.

One or more inflammatory cytokines or death ligands, such as TNFa, TRAIL, and IL1β, have strong synergistic effects with SMC therapy on many tumor-derived cell lines. Thus, strategies to increase death ligand concentrations in SMC-treated tumors, particularly using approaches that limit the toxicity typically associated with recombinant cytokine therapy, are very attractive. TNFa, TRAIL, and dozens of other cytokines and chemokines can be activated in response to pathogen recognition by an individual’s innate immune system. Importantly, this basal response to microbial pathogens is typically self-limiting and safe for the individual due to tight down-regulation that limits the strength and duration of its activity.

SMCs can be rationally engineered based on Smac. The ability of a compound to enhance apoptosis by mimicking one or more functions or actions of endogenous Smac may be

- 17 046706 is 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 cases, the SMC are small molecule IAP antagonist molecules based on the N-terminal tetrapeptide sequence (revealed after processing) of the Smac polypeptide. In some cases, the SMC is a monomer (monovalent) or a dimer (bivalent). In particular, the SMC includes 1 or 2 moieties that mimic the AVPI tetrapeptide sequence of Smac/DIABLO, the second mitochondrial activator of caspases, or other IBM-like (e.g., IAP-binding motifs of other proteins such as casp9). The dimeric SMC of the present invention can be a homodimer or a heterodimer. In some embodiments, the subunits of the dimer are connected by different linkers. The linkers can be at the same specific location of any subunit, and can also be located at different attachment points (which can be positions a.a., P1, P2, P3 or P4, sometimes there is a P5 group). In different arrangements, the subunits of the dimer may be in different orientations, such as head-to-tail, head-to-head, or tail-to-tail. Heterodimers may comprise two different monomers with different affinities for different BIR domains or different IAPs. Alternatively, a heterodimer may comprise a Smac monomer and a ligand for another receptor or target that is not an IAP. In some cases, SMC may be cyclic. In some cases, SMC may be trimeric or multimeric. Multimerized SMC may have an increased potency of 7,000-fold or more, such as 10, 20, 30, 40, 50, 100, 200, 1000, 5000, 7000-fold or more (as measured by, for example, EC ₅₀ in vitro) compared to one or more corresponding monomers. This may occur in several cases, for example, because the binding enhances ubiquitination among IAPs or because dual binding of BIR increases the stability of the interaction. Although multimers, such as dimers, may exhibit increased activity, monomers may be preferred in some embodiments. For example, in some cases, a low molecular weight SMC may be preferred, for example, for reasons related to bioavailability.

In some instances of the present invention, the agent capable of inhibiting cIAP1/2 is a bestatin analog or Me-bestatin. Bestatin or Me-bestatin analogs can induce autoubiquitination of cIAP1/2 by mimicking the biological activity of Smac.

In some embodiments of the present invention, the SMC-based combination therapy comprises one or more SMC agents and one or more interferon-based agents, such as interferon type 1, interferon type 2 and/or interferon type 3. Combination therapies, including an interferon-based agent, can be used to treat a malignancy, such as multiple myeloma.

In some embodiments, a VSV expressing IFN and, optionally, expressing a gene that allows for imaging, such as NIS, a sodium iodide symporter, is used in combination with SMC. For example, such a VSV can be used in combination with SMC, such as Smac mimetic SM-1387/APG-1387 from Ascentage, Smac mimetic LCL161 from Novartis, or Birinapant. Such combinations can be effective for the treatment of malignancy, such as hepatocellular carcinoma or liver metastases.

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

- 18 046706

Table 1

Smac Mimetic Compounds

Connection Structure or link Clinical status Organization; author/inventor GDC0152/RG7419 Baker JE, Boerboom LE, Olinger GN. Cardioplegiainduced damage to ischemic immature myocardium is independent of oxygen availability. Ann Thorac Surg. 1990 Dec;50(6):934-9. Clinical trials Genentech/Roche; W. Fairbrother GDC-0145 Clinical trials Genentech/Roche; W. Fairbrother AEG40826/ HGS1029 Clinical trials Aegera/Pharmascien ce (Canada); J. Jaquith LCL-161 Chen KF, Lin JP, Shiau CW, Tai WT, Liu CY, Yu HC, Chen PJ, Cheng AL. Inhibition of Bcl-2 improves the effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells. Biochem Pharmacol. 2012 Aug 1,-84(3):268-77. doi: 10.1016/j.bcp.2012.04.023. Epub 2012 May 9. Clinical trials Novartis; L. Zawel AT-406/ SM406/ Debioll43/ D1143 Cai Q, Sun H, Peng Y, Lu J, Nikolovska-Coleska Z, McEachern D, Liu L, Qiu S, Yang CY, Miller R, Yi H, Zhang T, Sun D, Kang S, Guo M, Leopold L, Yang D, Wang S. A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment. J Med Chern. 2011 Apr 28,-54(8):2714-26. doi: 10.1021/jml01505d. Epub 2011 Mar 28. Clinical trials Ascenta (USA)/DebioPharma (Switzerland); Shaomeng Wang (University of Michigan) TL32711/ Birinapant (formerly Dubrez L, Berthelet J, Glorian V. IAP proteins as targets for drug development in oncology. Onco Targets Ther. 2013 Sep 16,-9:1285-1304. Clinical trials Tetralogic (USA, formerly Gentara with GTI cpd TL32711) eCollection 2013. Review. designations); S. Condon GDC-0917/CUDC-427 Wong H, Gould SE, Budha N, Darbonne WC, Kadel EE 3rd, La H, Alicke B, Halladay JS, Erickson R, Portera C, Tolcher AW, Infante JR, Mamounas M, Flygare JA, Hop CE, Fairbrother WJ. Learning and confirming with preclinical studies: modeling and simulation in the discovery of GDC-0917, an inhibitor of apoptosis proteins antagonist. Drug Metab Dispos. 2013 Dec;41(12):2104-13. doi: 10.1124/dmd.113.053926. Epub 2013 Sep 16. Clinical trials Curis (Genentech); W. Fairbrother APG-1387/SM-1387 Clinical trials Ascenta (USA)/Ascentage (China); Shaomeng Wang AZD5582 Hennessy EJ, Adam A, Aquila BM, Castriotta LM, Cook D, Hattersley M, Hird AW, Huntington C, Kamhi VM, Laing NM, Li D, MacIntyre T, Omer CA, Oza V, Patterson T, Repik G, Rooney MT , Saeh JC, Sha L, Vasbinder MM, Wang H, Whitston D. Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP Antagonists Resulting in a Clinical Candidate for the Treatment of Cancer (AZD5582) . J Med Chern. 2013 Dec 27,-56(24):9897919. doi: 10.1021/jm401075x. Epub 2013 Dec 13. Clinical trial candidate AstraZeneca; E. Hennessy T-3256336 Sumi H, Yabuki M, Iwai K, Morimoto M, Hibino R, Inazuka M, Hashimoto K, Kosugi Y, Aoyama K, Yamamoto S, Yoshimatsu M, Yamasaki H, Tozawa R, Ishikawa T, Yoshida S. Antitumor activity and pharmacodynamic biomarkers of a novel and orally Clinical trial candidate Takeda (Japan); D. Dougan, T. Ishikawa

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available small-molecule antagonist of inhibitor of apoptosis proteins. Mol Cancer Ther. 2013 Feb;12 (2):230-40. doi: 10.1158/1535-7163.MCT-120699. Epub 2012 Dec 12. JP1584 Clinical trial candidate Joyant (GeminX, USA); Xiaodong Wang, Patrick Harran JP1201 Clinical trial candidate Joyant (GeminX, USA); Xiaodong Wang, Patrick Harran GT-A Clinical trial candidate Joyant (GeminX, USA); Xiaodong Wang, Patrick Harran AT-IAP Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, and Alison Woolford. Abstract 2944: AT-IAP, a dual cIAPl and XIAP antagonist with oral antitumor activity in melanoma models. Cancer Research: April 15, Clinical trial candidate Astex (UK)/Otsuka (Japan); G. Chessari 2013; Volume 73, Issue 8, Supplement 1 doi: 10.1158/1538-7445.AM2013-2944 Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC inhibl Park CM, Sun C, Olejniczak ET, Wilson AE, Meadows RP, Betz SF, Elmore SW, Fesik SW. Nonpeptidic small molecule inhibitors of XIAP. Bioorg Med Chern Lett. 2005 Feb 1;15(3):771-5. Pfizer (IDUN acquired cpds from Abbott collaboration); SW Fesik, KJ Tomaselli inhib2 Park CM, Sun C, Olejniczak ET, Wilson AE, Meadows RP, Betz SF, Elmore SW, Fesik SW. Nonpeptidic small molecule inhibitors of XIAP. Bioorg Med Chern Lett. 2005 Feb 1;15(3):771-5. Pfizer (IDUN acquired cpds from Abbott collaboration); SW Fesik, KJ Tomaselli BI-75D2 Formula: C ₂₆ H ₂₆ N ₄ O ₄ S ₂ Preclinical trials Sanford-Burnham Institute; J. Reed / o 'ch o /—( O 'Guch ho ⁷ ty· T5TR1 Crisdstomo FR, Feng Y, Zhu X, Welsh K, An J, Reed JC, Huang Z. Design and synthesis of a simplified inhibitor for the XIAP-BIR3 domain. Bioorg Med Chern Lett. 2009 Nov 15,-19(22):6413-8. doi: 0.1016/j.bmcl.2009.09.058. Epub 2009 Sep 17. PubMed PMID: 19819692; PubMed Central PMCID: PMC3807767. Preclinical trials Sanford-Burnham Institute (NIH?); J. Reed ML-101 Welsh K, Yuan H, Stonich D, Su Y, Garcia X, Cuddy M, Houghten R, Sergienko E, Reed JC, Ardecky R, Ganji SR, Lopez M, Dad S, Chung TDY, Cosford N. Antagonists of ΙΑΡ-family antiapoptotic proteins - Probe 1. 2009 May 18 [updated 2010 Sep 2]. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. Available from Preclinical trials Sanford-Burnham Institute (NIH?); J. Reed

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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 Chern Lett. 2011 Jul 15;21(14):4332-6. doi: 10.1016/j.bmcl.2011.05.049. Epub 2011 May 24. MLS-0390866 Welsh K, Yuan H, Stonich D, Su Y, Garcia X, Cuddy M, Houghten R, Sergienko E, Reed JC, Ardecky R, Ganji SR, Lopez M, Dad S, Chung TDY, Cosford N. Antagonists of ΙΑΡ-family antiapoptotic proteins - Probe 1. 2009 May 18 [updated 2010 Sep 2]. Probe 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/NBK4 7 341/PubMe d Preclinical trials Sanford-Burnham Institute (NIH?); J. Reed MLS- Finlay D, Vamos M, Gonzalez-Lopez M, Ardecky RJ, Ganji SR, Yuan H, Su Y, Cooley TR, Hauser CT, Welsh K, Reed JC, Cosford ND, Vuori K. SmallMolecule IAP Antagonists Sensitize Cancer Cells to TRAIL- Induced Apoptosis: Roles of XIAPs and cIAPs. Mol Cancer Ther. 2014 Jan;13(1):5-15. doi: 10.1158/1535-7163.MCT-13-0153. Epub 2013 Nov 5. Preclinical trials Sanford-Burnham Institute (NIH?); J. Reed ML183 Ardecky RJ, Welsh K, Finlay D, Lee PS, GonzalezLopez M, Ganji SR, Ravanan P, Mace PD, Riedl SJ, Preclinical trials Sanford-Burnham Institute (NIH?); Vuori K, Reed JC, Cosford ND. Design, synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP. Bioorg Med Chern Lett. 2013 Jul 15,-23(14):4253-7. doi: 10.1016/j.bmcl.2013.04.096. Epub 2013 May 14;Lopez M, Welsh K, Yuan H, Stonich D, Su Y, Garcia X, Cuddy M, Houghten R, Sergienko E, Reed JC, Ardecky R, Reddy S, Finlay D, Vuori K, Dad S, Chung TDY, Cosford NDP. Antagonists of IAPfamily anti-apoptotic proteins - Probe 2. 2009 Sep 1 [updated 2011 Feb 10]. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. available http://www.ncbi.nlm.nih.gov/books/NBK550 68/ J. Reed SM-8 3 Gatti L, De Cesare M, Ciusani E, Corna E, Arrighetti N, Cominetti D, Belvisi L, Potenza D, Moroni E, Vasile F, Lecis D, Delia D, Castiglioni V, Scanziani E, Seneci P, Zaffaroni N, Perego P. Antitumor Activity of a Novel Homodimeric SMAC Mimetic in Ovarian Carcinoma. Mol Pharm. 2014 Jan 6,-11(1):283-93. doi: 10.1021/mp4004578. Epub 2013 Nov 27. Preclinical trials University of Milan; M. Bolognesi SMAC037/SM37 Mastrangelo E, Cossu F, Milani M, Sorrentino G, Lecis D, Delia D, Manzoni L, Drago C, Seneci P, Scolastico C, Rizzo V, Bolognesi M. Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo [5.3.0]alkane smac mimetics. Structure, activity, and Preclinical trials University of Milan; M. Bolognesi

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recognition principles. J Mol Biol. 2008 Dec 19,-384(3): 673-89. doi: 10.1016/j.jmb.2008.09.064. pub 2008 Oct 7. SMAC066 Cossu F, Malvezzi F, Canevari G, Mastrangelo E, Lecis D, Delia D, Seneci P, Scolastico C, Bolognesi M, Milani M. Recognition of Smacmimetic compounds by the BIR domain of cIAPl. Protein Sci. 2010 ec;19(12):2418-29. doi:10.1002/pro.523. Preclinical trials University of Milan; M. Bolognesi SMC 9 a Monomer: Seneci P, Bianchi A, Battaglia C, Belvisi L, Bolognesi M, Caprini A, Cossu F, Franco Ed, Matteo Md, Delia D, Drago C, Khaled A, Lecis D, Manzoni L, Marizzoni M, Mastrangelo E, Milani M, Motto I, Moroni E, Potenza D, Rizzo V, Servida F, Turlizzi E, Varrone M, Vasile F, Scolastico C. Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy. Bioorg Med Chern. 2009 Aug 15,-17 (16):5834-56. doi: 10.1016/j.bmc.2009.07.009. Epub 2009 Jul 10. Dimer in different configurations: Ferrari V, Cutler DJ. Uptake of chloroquine by human erythrocytes. Biochem Pharmacol. 1990 Feb 15,-39(4):753-62. PubMed PMID: 2306282.; Cossu F, Milani M, Vachette P, Malvezzi F, Grassi S, Lecis D, Delia D, Drago C, Seneci P, Bolognesi M, Mastrangelo E. Structural insight into inhibitor of apoptosis protein recognition by a potent divalent smac-mimetic. PLoS One. 2012,-7(11):e49527. doi: 10.1371/journal.pone.0049527. Epub 2012 Nov 15. Preclinical trials University of Milan; M. Bolognesi OICR-720 Enwere EK, Holbrook J, Lejmi-Mrad R, Vineham J, Timusk K, Sivaraj B, Isaac M, Uehling D, Al-awar R, LaCasse E, Korneluk RG. TWEAK and cIAPl regulate myoblast fusion through the noncanonical NF-κB signaling pathway. Sci Signal. 2012 Oct 16;5 (246) :ra75. doi: 10.1126/scisignal.2003086. Preclinical trials Ontario Institute for Cancer Research; R. Korneluk SM-164 Sun H, Nikolovska-Coleska Z, Lu J, Meagher JL, Yang CY, Qiu S, Tomita Y, Ueda Y, Jiang S, Krajewski K, Roller PP, Stuckey JA, Wang S. Design, synthesis, and characterization of a potent , nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP. J Am Chern Soc. 2007 Dec 12,-129(49):15279-94. Epub 2007 Nov 14. Preclinical trials Ascenta SM1200 Sheng R, Sun H, Liu L, Lu J, McEachern D, Wang G, Wen J, Min P, Du Z, Lu H, Kang S, Guo M, Yang D, Wang S. A potent bivalent Smac mimetic (SM1200) achieving rapid, complete, and durable tumor regression in mice. J Med Chern. 2013 May 23,-56(10):3969-79. doi: 10.1021/jm400216d. Epub 2013 May 7. Preclinical trials Ascenta SM-173 Lu J, Bai L, Sun H, Nikolovska-Coleska Z, McEachern D, Qiu S, Miller RS, Yi H, Shangary S, Sun Y, Meagher JL, Stuckey JA, Wang S. SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP. Preclinical trials Ascenta

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Cancer Res. 2008 Nov 5; 68(22):9384-93. doi: 10.1158/0008-5472.CAN-08-2655. Connection 21 Sun H, Stuckey JA, Nikolovska-Coleska Z, Qin D, Meagher JL, Qiu S, Lu J, Yang CY, Saito NG, Wang S. Structure-based design, synthesis, evaluation, and crystallographic studies of conformationally constrained Smac mimetics as inhibitors of the X-linked inhibitor of apoptosis protein (XIAP). J Med Chern. 2008 Nov 27,-51(22):7169-80. doi:10.1021/jm8006849. Preclinical trials Ascenta WS-5 Zhang B, Nikolovska-Coleska Z, Zhang Y, Bai L, Qiu S, Yang CY, Sun H, Wang S, Wu Y. Design, synthesis, and evaluation of tricyclic, conformationally constrained small-molecule mimetics of second mitochondria-derived activator of caspases. J Med Chern. 2008 Dec 11,-51(23):7352-5. doi: 0.1021/jm801146d. Preclinical trials Ascenta SH-130 Dai Y, Liu M, Tang W, DeSano J, Burstein E, Davis M, Pienta K, Lawrence T, Xu L. Molecularly targeted radiosensitization of human prostate cancer by modulating inhibitor of apoptosis. Clin Cancer Res. 2008 Dec 1,-14(23):7701-10. doi: 10.1158/1078-0432.CCR-08-0188. Preclinical trials Ascenta SMI 62 Sun H, Liu L, Lu J, Qiu S, Yang CY, Yi H, Wang S. Cyclopeptide Smac mimetics as antagonists of TAP proteins. Bioorg Med Chern Lett. 2010 May 5;20 (10):3043-6. Preclinical trials Ascenta SM163 (connection 3) Sun H, Liu L, Lu J, Qiu S, Yang CY, Yi H, Wang S. Cyclopeptide Smac mimetics as antagonists of TAP proteins. Bioorg Med Chern Lett. May 2010 Preclinical trials Ascenta 15,-20 (10): 3043-6. SM337 Wang S. Design of small-molecule Smac mimetics as IAP antagonists. Curr Top Microbiol Immunol. 2011; 348:89-113. doi: 10.1007/82_20У_1И · Preclinical trials Ascenta SM122 (or SH122 ) Lu J, Bai L, Sun H, Nikolovska-Coleska Z, McEachern D, Qiu S, Miller RS, Yi H, Shangary S, Sun Y, Meagher JL, Stuckey JA, Wang S. SM-164: a novel, bivalent Smac mimetic that induces apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP. Cancer Res. 2008 Nov 15,-68(22):9384-93. doi: 10.1158/0008-5472.CAN-08-2655. Preclinical trials Ascenta AEG40730 Bertrand MJ, Milutinovic S, Dickson KM, Ho WC, Boudreault A, Durkin J, Gillard JW, Jaquith JB, Morris SJ, Barker PA. cIAPl and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell. 2008 Jun 20,-30(6):689-700. doi: 10.1016/j.molcel.2008.05.014. Preclinical trials Aegera LBW242 Keating J, Tsoli M, Hallahan AR, Ingram WJ, Haber M, Ziegler DS. Targeting the inhibitor of apoptosis proteins as a novel therapeutic strategy in medulloblastoma. Mol Cancer Ther. 2012 Dec,-11(12):2654-63. doi: 10.1158/15357163.MCT-12-0352. Epub 2012 Sep 25. Preclinical trials Novartis BV6 Muller-Sienerth N, Dietz L, Holtz P, Kapp M, Grigoleit GU, Schmuck C, Wajant H, Siegmund D. SMAC mimetic BV6 induces cell death in monocytes and maturation of monocyte-derived dendritic cells. PLoS One. 2011;6 ( 6):e21556. doi: Preclinical trials Genentech

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10.1371/journal.pone.0021556. Epub 2011 Jun 30. MVl Monomeric version of BV6: Fulda S, Vucic D. Targeting IAP proteins for therapeutic intervention in cancer. Nat Rev Drug Discov. 2012 Feb 1,-11(2):109-24. doi: 10.1038/nrd3627. Review. Erratum in: Nat Rev Drug Discov. 2012 Apr;11(4):331. Preclinical trials Genentech ATRA hybrid Itoh Y, Ishikawa M, Kitaguchi R, Okuhira K, Naito M, Hashimoto Y. Double protein knockdown of cIAPl and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist. Bioorg Med Chern Lett. 2012 Jul 1;22(13):4453-7. doi: 10.1016/j.bmcl.2012.04.134. Epub 2012 May 23. Preclinical trials Genentech SNIPER (fusion construct of bestatin and estrogen receptor ligand) Okuhira K, Demizu Y, Hattori T, Ohoka N, Shibata N, Nishimaki-Mogami T, Okuda H, Kurihara M, Naito M. Development of hybrid small molecules that induce degradation of estrogen receptoralpha and necrotic cell death in breast cancer cells. Cancer Sci. 2013 Aug 30. doi: 10.1111/cas.12272. [Online publication ahead of print] Preclinical trials RMT5265 Ramachandiran S, Cain J, Liao A, He Y, Guo X, Boise LH, Fu H, Ratner L, Khoury HJ, BernalMizrachi L. The Smac mimetic RMT5265.2HCL induces apoptosis in EBV and HTLV-I associated lymphoma cells by inhibiting XIAP and promoting the mitochondrial release of cytochrome C and Smac. Leuk Res. 2012 Jun;36(6):784-90. doi: 10.1016/j.leukres.2011.12.024. Epub 2012 Feb 10; Li L, Thomas RM, Suzuki H, De Brabander JK, Wang Preclinical trials Joyant (USA) X, Harran PG. A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death. Science. 2004 Sep 3; 305(5689):1471-4. JP1010 Probst BL, Liu L, Ramesh V, Li L, Sun H, Minna JD, Wang L. Smac mimetics increase cancer cell response to chemotherapeutics in a TNF-dependent manner. Cell Death Differ. Oct 2010; 17(10):1645-54. doi: 10.1038/cdd.2010.44. Epub 2010 Apr 30. Preclinical trials Joyant (USA) JP1400 Probst BL, Liu L, Ramesh V, Li L, Sun H, Minna JD, Wang L. Smac mimetics increase cancer cell response to chemotherapeutics in a TNF-dependent manner. Cell Death Differ. 2010 Oct;17(10):1645-54. doi: 10.1038/cdd.2010.44. Epub 2010 Apr 30. Preclinical trials Joyant (USA) AVT-10 Preclinical trials Abbott A-410099.1 Oost TK, Sun C, Armstrong RC, Al-Assaad AS, Betz SF, Deckwerth TL, Ding H, Elmore SW, Meadows RP, Olejniczak ET, Oleksijew A, Oltersdorf T, Rosenberg SH, Shoemaker AR, Tomaselli KJ, Zou H, Fesik SW. Discovery of potent antagonists of the antiapoptotic protein XIAP for the treatment of cancer. J Med Chern. 2004 Aug 26; 47(18):4417-26. Preclinical trials Abbott 822B Jae Sik Shin, Seung-Woo Hong, Dong-Hoon Jin, InHwan Bae, Maeng-Sup Kim, Young-Soon Na, Jae-Lyun Lee, Yong Sang Hong, and Tae-Won Kim. Abstract 592: Novel IAP antagonist (822B) induces apoptosis through degradation of IAP proteins which have a BIR3 domain in human pancreatic Preclinical trials Hanmi (Korea) cancer cells. Cancer Research: April 15, 2011; Volume 71, Issue 8, Supplement 1 doi: 10.1158/1538-7445.AM2011-592 Proceedings: AACR 102nd Annual Meeting 2011-- Apr 2-6, 2011; Orlando, FL GT13402 Preclinical trials Tetralogic SW iii -123 (sigma2I ligand hybrid) Zeng C, Vangveravong S, McDunn JE, Hawkins WG, Mach RH. Sigma-2 receptor ligand as a novel method for delivering a SMAC mimetic drug for treating ovarian cancer. Br J Cancer. 2013 Oct 29,-109(9):2368-77. doi: 10.1038/bj c . 2013.593. Epub 2013 Oct 8. Preclinical trials (RH Mach) Preclinical trials Apoptos (USA) Preclinical trials Sanofi- Aventis/Synthelabo (EU)

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Agents

The immunostimulatory or immunomodulatory agent of the present invention may be any agent capable of inducing a receptor-mediated apoptotic program that is inhibited by cIAP 1 and cIAP2 in one or more cells of an individual. The immune stimulator of the present invention may induce an apoptotic program regulated by cIAP1 (BIRC2), cIAP2 (BIRC3 or API2) and, optionally, one or more additional IAPs, such as one or more human IAP proteins NAIP (BIRC1) XIAP (BIRC4), survivin (BIRC5), Apollon/Bruce (BIRC6), ML-IAP (BIRC7 or livin) and ILP-2 (BIRC8). In addition, it is known that various immunomodulators or agents such as CpG or IAP antagonists can alter the context of immune cells.

In some cases, the immune stimulator may be a TLR agonist, such as a TLR ligand. The TLR agonist of the present invention may be an agonist of one or more of human TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, and TLR-10 or related proteins of other species (e.g., mouse TLR-1-TLR-9 and TLR-11-TLR-13). TLRs can recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are expressed only by microbial pathogens, as well as distress-associated molecular patterns (DAMPs), which are endogenous molecules released by necrotic or destructive cells. PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptide glycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. DAMPs 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 ODN), such as ODN, class A, B and C CpG, base analogs, nucleic acids such as dsRNA or DNA of a pathogen, or pathogenic or pathogen-like cells, or virions. In some 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. Although Table 2 includes suggested mechanisms, uses, or TLR targets by which various TLR agonists may act, the methods and compositions of the present invention are not limited to these mechanisms, uses, or targets.

Table 2

Agents: TLR agonists

Connection Structure or link Connection type or application Agonist: Poly-ICLC (polyinosinic: polycytidic acid, poly(1:C)) Levy NV. Historical overview of the use of polynucleotides in cancer. J Biol Response Mod. 1985;4:475-480. 7. Levy HB. Induction of interferon in vivo by polynucleotides. Tex Rep Biol Med. 1977;35:91-98. Intracavitary administration for the treatment of mesothelioma (see, for example, Currie AJ, Van Der Most RG, Broomfield SA, Prosser AC, Tovey MG, Robinson BW. Targeting the effector site with ΙΕΝ-αβinducing TLR ligands reactivates tumor-resident CD8 T cell responses to eradicate established solid tumors. J. Immunol. 2008; 180 (3) :1535–1544.) Toll-like receptor (TLR)-3 Poly(A:U) polyadenylic polyuridylic acid Ducret JP, Caille P, Sancho Garnier H, et al. A phase I clinical tolerance study of polyadenylic-polyuridylic acid in cancer patients. J Biol Response Mod 1985;4:129-133. Polyadenylic. polyuridylic acid in the cotreatment of cancer. Michelson AM, Lacour F, Lacour J. Proc Soc Exp Biol Med. 1985 May;179(1):1-8. Synthetic double-stranded RNA molecule TLR-3 CL075 Gorden KB. et al., 2005. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol. 174(3):125 9-68, InvivoGen, InvivoGen Insight (Company Newsletter) Spring 2013: 8 pp. Formula: C ₁₃ H ₁₃ N ₃ S Triazoquinoline compound TRL-7 or TLR7/8

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nh ₂ AA s ^z M CL097 Salio M. et al., 2007. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. PNAS 104:20490–20495. Butchi nJ. et al., 2008. Analysis of the Neuroinflammatory Response to TLR7 Stimulation in the Brain: Comparison of Multiple TLR7 and/or TLR8 Agonists. J Immunol 180:7604–7612 Imidazoquinoline compound TRL-7 or TLR7/8 CL264 US Patent Publication No. 20110077263 Formula: C ₁₉ H ₂ 3K ₇ O ₄ ' nh ₂ Ί AVoh ^λ ν ^ν η 1 - Αγ ΗΝ Ο^ΟΗ Adenine analogue TRL-7 or TLR7/8 CL307 Basic analogue TRL-7 or TLR7/8 Gardiquimod™ US Patent Publication No. 20110077263 Formula: C ₁₇ H ₂₃ N ₅ O Imidazoquinoline compound TRL-7 or TLR7/8 G j AC | he Loxoribin Gorden KV. et al., 2005. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol. 174(3):1259-68. 2. Schindler U. & Baichwal V.R., 1994. Three NF-kB binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol, 14(9):5820–5831. Formula: C ₁₃ H ₁₇ N ₅ O ₆ ho N nh ₂ OH OH Analogue of guanosine TRL-7 or TLR7/8 Poly(dT) Jurk M. et al., 2006. Modulating the responsiveness of human TLR7 and 8 to small molecule ligands with T-rich phosphorothiate oligodeoxynucleotides. Eur J Immunol. 36(7):1815-26. 2. Gorden KKB. et al., 2006. Oligodeoxynucleotides Differentially Modulate Activation of TLR7 and TLR8 by Imidazoquinolines. J. Immunol. 177:8164-8170. 3. Gorden KKB. et al., 2006. Cutting Edge: Activation of Murine TLR8 by a Combination of Thymidine homopolymer ODN (17 mer) TRL-7 or TLR7/8

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Imidazoquinoline Immune Response Modifiers and PolyT OligodeoxynucleotidesJ. Immunol., 177: 6584–6587. R848 Hemmi H. et al. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3(2):196–200. 2. Jurk m. et al. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R848. Nat Immunol 3(6):499. 3. Gorden KKB. et al., 2006. Cutting Edge: Activation of Murine TLR8 by a Combination of Imidazoquinoline Immune Response Modifiers and PolyT Oligodeoxynucleotides J. Immunol., 177: 65846587 Formula: C ₁₇ H ₂ 2N ₄ O2, HC1 NH ₃ CI mMRX Xx ron Imidazoquinoline compound TRL-7 or TLR7/8 ODN 1585 Ballas ZK. et al., 2001. Divergent therapeutic and immunological effects of oligodeoxynucleotides with distinct CpG motifs. J Immunol. 167(9):4878-86 Class A CpG ODN TLR-9 ODN 2216 Class A CpG ODN TLR-9 ODN 2336 Ballas ZK. et al., 2001. Divergent therapeutic and immunological effects of oligodeoxynucleotides with distinct CpG motifs. J Immunol. 167(9):4878-86 Class A CpG ODN TLR-9 ODN 1668 Heit A. et al., 2004. CpG-DNA aided cross- Class B CpG ODN TLR-9 priming by cross-presenting in cells. J Immunol. 172(3):1501-7 ODN 1826 Z Moldoveanu, L Love-Homan, W.Q Huang, A.M Krieg CpG DNA, a novel immune enhancer for systemic and mucosal immunization with influenza virus Vaccine, 16 (1998), pp. 12161224 Class B CpG ODN TLR-9 ODN 2006 (ODN 7909 or PF3512676) Z Moldoveanu, L Love-Homan, W.Q Huang, A.M Krieg CpG DNA, a novel immune enhancer for systemic and mucosal immunization with influenza virus Vaccine, 16 (1998), pp. 12161224 Class B CpG ODN TLR-9 ODN 2007 Krieg, A; CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002, 20: 709 Class B CpG ODN TLR-9 ODN 2395 Roda J.M. et al., 2005. CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol. 175(3):1619-27. Class C CpG ODN TLR-9 ODN M3 62 Hartmann G, Battiany J, Poeck H, et al.: Rational design of new CpG oligonucleotides that combine B cell activation with high IFNalpha induction in plasmacytoid dendritic cells. Eur J Immunol 2003, 33:1633-41 Class C CpG ODN TLR-9 ODN 1018 Magone, M. T., Chan, S. C., Beck, L., Whitcup, S. M., Raz, E. (2000) Systemic or mucosal administration of immunostimulatory DNA inhibits early and late phases of murine allergic conjunctivitis Eur. J. Immunol. 30.1841-1850 Class B TLR-9 agonist CL401 _{Formula : C54H92N8O4S} Dual TLR agonist TLR-2 and TLR-7

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NH; p| ^{Ν Ν} _ο ο η J Adilipoline™ (CL413;) Formula: C ₈₁ H ₁₄₅ N ₁₇ O ₁₂ S «On NHs ^Η Ou 0 _μ ''О „ NHi V l! _g ν Dual TLR agonist TLR-2 and TLR-7 CL531 Formula: C ₈ 2H ₁₄ 4N ₁₆ O ₁₄ S O _o Az\z^/S/'S/\Kv4 NH; °^NH, ^ΰ n^n HO' _r ^NH „ 1 > ^OH Λ NNN HN Ό ^HL Y% η “γΝχΟκΗ, О HN^O NH HO'% Dual TLR agonist TLR-2 and TLR-7 CL572 Formula: C ₄₁ H ₆₅ N ₉ O ₇ S nh ₂ μΆν Ί X 9оn ^H Vl a. ⁿ Г 1 u E Ίΐ''''^'O С^Нгу ^H A O O 'O Dual TLR agonist Human TLR-2, mouse TLR-7 and human TLR-7 AdiFectin™ (CL347;) Formula: C7 ₂ H ₁₃₄ N ₁₁ O ₆ P TLR agonist and nucleic acid carrier TLR-7 U X J CL419 Formula: C ₄₈ H ₉₇ N ₅ O ₅ S TLR agonist and nucleic acid carrier TLR-2 PamadiFectin™ (CL553;) Formula: C ₆₇ H ₁₁₈ N ₁₂ O ₈ S NH, ΡΌ OH -'V'NV-N 0 H 1 JI ^H ^Д_ η H ^NHb Xi pKzwvw TLR agonist and nucleic acid carrier TLR-2 and TLR-7 Peptidoglycan TLR ligand; cell surface location (Expert Rev Clin Pharmacol 4(2): 275289, 2011) TLR-1/2; TLR-2/6 Diacylated lipopeptide Buwitt-Beckmann u. et al., 2005. Toll-like receptor 6-independent signaling by diacylated lipopeptides. Eur J Immunol. 35(1):282-9 TLR ligand; cell surface location TLR-2/6 Triacylated lipopeptide Alirantis ao et al., 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science .285(5428):736-9. Ozinsky a. et al., 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS. 97(25):13766-71. 3 TLR ligand; cell surface location TLR-1/2 Lipopolysaccharide (LPS) No data TLR ligand; cell surface location; intratumoral administration for the treatment of glioma, (see, for example, Mariani CL, Raj on D, Bova FJ, Streit WJ. TLR-4

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Nonspecific immunotherapy with intratumoral lipopolysaccharide and zymosan A but not GM-CSF leads to an effective antitumor response in subcutaneous RG-2 gliomas. J. Neurooncol. 2007; 85(3):231240. ) CpG 7909 Intravenous administration for the treatment of non-Hodgkin's lymphoma (see, for example, Link BK, Ballas ZK, Weisdorf D, et al. Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion demonstrates immunologic modulation in patients with previously treated non-Hodgkin lymphoma. J. Immunother. 2006; 29(5):558-568. ) TLR-9 852A Intravenous administration for the treatment of malonoma and other malignancies [12,55]; (see, for example, Dudek AZ, Yunis C, Harrison LI, et al. First in human Phase I trial of 852A, a novel systemic Toll-like receptor 7 agonist, to activate innate immune responses in patients with advanced cancer. Clin. Cancer TLR-7 Res. 2007; 13 (2 3 ) : 7119-7125'; Dummer R, Hauschild A, Becker JC, et al. An exploratory study of systemic administration of the Tolllike receptor-7 agonist 852A in patients with refractory metastatic melanoma. Clin. Cancer Res. 2008; 14(3):856864 . Intravenous administration for the treatment of chronic lymphocytic leukemia (see, for example, Spaner DE, Shi Y, White D, et al. A Phase I/II trial of TLR7 agonist immunotherapy in chronic lymphocytic leukemia. Leukemia. 2010; 24(1):222- 226. ) Ampligen Intravenous administration for the treatment of chronic fatigue syndrome [60]; intravenous administration for the treatment of HIV (see, for example, Thompson КА, Strayer DR, Salvato PD, et al. Results of a double-blind placebo-controlled study of the double-stranded RNA drug polyl:polyC12U in the treatment of HIV infection. Eur. J. Clin. Microbiol. Infect. Dis. 1996; 15(7):580- TLR-3

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587. [PubMed: 8874076]) Resiquimod Oral administration for the treatment of hepatitis C ((See, for example, Pockros PJ, Guyader D, Patton H, et al. Oral resiquimod in chronic HCV infection: safety and efficacy in 2 placebocontrolled, double-blind Phase I studies. J. Hepatol. 2007; 47(2):174182 . ) ; Topical application for the treatment of herpes simplex virus type 2 (See, for example, Mark KE, Corey L, Meng TC, et al. Topical resiquimod 0.01% gel decreases herpes simplex virus type 2 genital shedding: a randomized, controlled trial. J. Infect. Dis. 2007; 195(9):1342-1331.) TLR-7/8 ANA975 Oral administration for the treatment of hepatitis (see, for example, Fletcher S, Steffy K, Averett D. Masked oral prodrugs of Toll-like receptor 7 agonists: a new approach for the treatment of infectious disease. Curr. Opin. Investigure Drugs. 2006; 7(8):702-708.) TLR-7 Imiquimod Imidazoquinoline TLR-7 (InvivoGen) compound; topical application for the treatment of basal cell carcinoma (see, for example, 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. 2 0 05; 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 use for the treatment of squamous cell carcinoma (see, for example, 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.

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Dermatol. 2006; 55(6):10921094.) Topical 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: 10.1111/j.16000560.2009.01290.x. (Epub link for print); (see, e.g., Green DS, Dalgleish AG, Belonwu N, Fischer MD, Bodman-Smith MD. Topical imiquimod and intralesional interleukin-2 increase activated lymphocytes and restore the Thl/Th2 balance in patients with metastatic melanoma. Br. J. Dermatol. 2008; 159 (3) :606-614.); Topical use for the treatment of vaginal intraepithelial neoplasia (see, for example, 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 use for the treatment of cutaneous lymphomas (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):620622 . ) ; Topical use as a vaccine against human papillomavirus (HPV) (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):1129- 1136.); 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 fulllength NY-ESO-1 protein using TLR7 agonist imiquimod as

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vaccine adjuvant. J. Immunol. 2008; 181(1):776-784.) Monophosphoryl lipid A (MPL) Subcutaneous/intramuscular 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 LI viruslike particle vaccine against human papillomavirus types 16 and 18: follow-up from arandomised control trial. Lancet. 2006; 367(9518):12471255.); Subcutaneous/intramuscular administration for vaccination against non-small cell lung cancer (see, e.g., Butts C, Murray N, Maksymiuk A, et al. Randomized Phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J. Clin. Oncol. 2005; 23(27):6674-6681.) TLR-4 CpG 7909 (i.e. PF3512676) Subcutaneous/intramuscular administration for the treatment of non-small cell lung cancer (see, for example, Manegold C, Gravenor D, Woytowitz D, et al. Randomized Phase II trial TLR-9 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. September 2-6, 2007); Subcutaneous/intramuscular administration for the treatment of metastatic melanoma (see, for example, 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 against melanoma (see,

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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; 115(3): 739-746; Arrau 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/Thl responses and CD8 T cells through cross-priming. Proc. Natl Acad. Sci. USA. 2007; 104 (21) :8947-8952.) CpG 1018 ISS Subcutaneous/intramuscular administration for the treatment of lymphoma (see, e.g., Friedberg JW, Kim H, McCauley M, et al. Combination immunotherapy with a CpG oligonucleotide TLR-9 (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-a/p-inducible gene expression, without significant toxicity. Blood. 2005; 105(2):489-495; Friedberg JW, Kelly JL, Neuberg D, et al. Phase II study of a TLR-9 agonist (1018 ISS) with rituximab in patients with relapsed or refractory follicular lymphoma. Br. J. Haematol. 2009; 146(3):282-291.) Bacillus No data Intratumoral administration for TLR-2 Calmette-Guerin bladder cancer treatment (BCG) (See, for example, Simons MR, O'Donnell MA. Griffith TS. Role of neutrophils in BCG immunotherapy for bladder cancer. Urol. Oncol. 2008; 26(4): 341-345.) Zimosan A — Intratumoral administration for the treatment of glioma (see, for example, Mariani CL, Raj on D, Bova FJ, Streit WJ. Nonspecific immunotherapy with intratumoral lipopolysaccharide and zymosan A but not GM-CSF leads to an effective antitumor response in subcutaneous RG-2 gliomas. J. Neurooncol. 2007; 85(3):231240 . ) TLR-2

In other cases, the immune stimulator can be a virus, such as 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, but are not limited to, 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, coxsackie viruses, poxviruses, and others. In specific embodiments of the present invention, the agent is a rhabdovirus, such as VSV. Rhabdoviruses can replicate rapidly with high IFN content. In other specific embodiments, the agent is a wild type, such as Maraba virus, MG1 double mutation, Farmington virus, Carajas virus. Viral agents of the present invention include mutant viruses (e.g., VSV with the A51 mutation in the matrix or M protein), transgene-modified viruses (e.g., VSV-hIFNp), viruses carrying -TNFa, -LTo/TNFp, -TRAIL, FasL, -TL1a, chimeric viruses (e.g., rabies), or pseudotyped viruses (e.g., viruses pseudotyped with G proteins from LCMV or other viruses). In some cases, the virus is actually

- 33 046706 the present invention will be selected to reduce neurotoxicity. Viruses in general and oncolytic viruses in particular are known in the art.

In some embodiments, the agent is a killed VSV NRRP particle or a prime-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 retains oncolytic and immunostimulatory properties. NRRPs can be produced using gamma irradiation, UV irradiation, or busulfan. Specific combination therapies include prime-boost with adeno-MAGE3 (melanoma antigen) and/or Maraba-MG1MAGE3. Other specific combination therapies include UV-killed or gamma-irradiated killed wild-type VSR NRRPs. NRRPs can exhibit low or no neurotoxicity. NRRPs can be used, for example, in the treatment of glioma, hematologic (liquid) tumors, or multiple myeloma.

In some cases, the agent of the present invention is a vaccine strain, an attenuated virus or microorganism, or a killed virus or microorganism. In some cases, the agent may be, for example, BCG, live or dead rabies vaccines, or an influenza vaccine.

Non-limiting examples of viruses of the present invention, such as oncolytic viruses, are provided in Table 3. Although Table 3 includes suggested mechanisms or uses for the suggested viruses, the methods and compositions of the present invention are not limited to these mechanisms or their uses.

Table 3

Agents

Strain Modification/Description Virus Clinical Trial; Indication; Route; Status; Link Oncorin (H101) E1B-55k- Adenovirus Phase II; SCCHN; intratumoral (IT); completed; Xu RH, Yuan ZY, Guan ZZ, Cao Y, Wang HQ, Hu XH, Feng JF, Zhang Y, Li F, Chen ZT, Wang JJ, Huang JJ, Zhou QH, Song ST. [Phase II clinical study of intratumoral H101, an E1B deleted adenovirus, in combination with chemotherapy in patients with cancer]. Ai Zheng. 2003 Dec;22(12):1307-10. Chinese. Oncorin (H101) EZ- Adenovirus Phase 3; SCCHN; B.O.; completed; Xia ZJ, Chang JH, Zhang L, Jiang WQ, Guan ZZ, Liu JW, Zhang Y, Hu XH, Wu GH, Wang HQ, Chen ZC, Chen JC, Zhou QH, Lu JW, Fan QX, Huang JJ, Zheng X [Phase III randomized clinical trial of intratumoral injection of E1B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus]. Ai Zheng. 2004 Dec;23(12):1666-70. Chinese Opukh-015 E1B-55k- Adenovirus Phase 1; Lung metastases; intravenous (IV); Completed; Nemunaitis J, Cunningham C, Buchanan A, Blackburn A, Edelman G, Maples P, Netto G, Tong A, Randlev B, Olson S, Kirn D. Intravenous infusion of a replication-selective adenovirus

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(ONYX-015) in cancer patients: safety, feasibility and biological activity. Gene Ther. 2001 May;8(10):746-59. Onyx-015 EZV- Adenovirus Phase 1; Glioma; Intracavitary; Completed; Chiocca EA, Abbed KM, Tatter S, Louis DN, Hochberg EH, Barker F, Kracher J, Grossman SA, Fisher JD, Carson K, Rosenblum M, Mikkelsen T, Olson J, Markert J, Rosenfeld S, Nabors LB, Brem S, Phuphanich S, Freeman S, Kaplan R, Zwiebel J. A phase I open-label, dose-escalation, multi-institutional trial of injection with an E1BAttenuated adenovirus, ONYX-015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol Ther. 2004 Nov;10(5):958-66. Phase 1; Ovarian Cancer; Intraperitoneal (IP); Completed; Vasey RA, Shulman LN, Campos S, Davis J, Gore M, Johnston S, Kirn DH, O'Neill V, Siddiqui N, Seiden MV, Kaye SB. Phase I trial of intraperitoneal injection of the ElB-55-kd-gene-deleted adenovirus ONYX-015 (dll520) given on days 1 through 5 every 3 weeks in patients with recurrent/refractory epithelial ovarian cancer. J Clin Oncol. 2002 Mar 15; 20(6):1562-9. Phase 1; SCCHN; B.O.; Completed; Ganly I, Kirn D, Eckhardt G, Rodriguez GT, Soutar DS, Otto R, Robertson AG, Park 0, Gulley ML, Heise C, Von Hoff DD, Kaye SB. A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res. 2000 Mar;6(3):798-806. Erratum in: Clin Cancer Res 2000 May;6(5):2120. Clin Cancer Res 2001 Mar;7(3):754. Eckhardt SG [corrected to Eckhardt G]. Phase 1; Solid tumors; B.B.; Completed; Nemunaitis J, Senzer N, Sarmiento S, Zhang YA, Arzaga R, Sands B, Maples P, Tong AW. A phase I trial of intravenous infusion of ONYX-015 and enbrel in solid tumor patients. Cancer Gene Ther. 2007 Nov;14(11):885-93. Epub 2007 Aug 17. Phase 1; Sarcoma; B.O.; Completed; Galanis E, Okuno SH, Nascimento AG, Lewis BD, Lee RA, Oliveira AM, Sloan JA, Atherton P, Edmonson JH, Erlichman C, Randlev B, Wang Q, Freeman S, Rubin J. Phase I-II trial of ONYX-015 in combination with MAP chemotherapy in patients with advanced sarcomas. Gene Ther. 2005 Mar;12(5):437-45. Phase 1/2; PanCa; B.O.; Completed;

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Hecht JR, Bedford R, Abbruzzese JL, Lahoti S, Reid TR, Soetikno RM, Kirn DH, Freeman SM. A phase I/II trial of intratumoral endoscopic ultrasound injection of ONYX-015 with intravenous gemcitabine in unresectable pancreatic carcinoma. Clin Cancer Res. 2003 Feb;9(2):55561.

Phase 2; CRC; B.B.; Completed; Hamid O, Varterasian ML, Wadler S, Hecht JR, Benson A 3rd, Galanis E, Uprichard M, Omer C, Bycott P, Hackman RC, Shields AF. Phase II trial of intravenous CI-1042 in patients with metastatic colorectal cancer. J Clin Oncol. 2003 Apr 15; 21(8):1498-504.

Phase 2; Hepatobiliary; VV; Completed; Makower D, Rozenblit A, Kaufman H, Edelman M, Lane ME, Zwiebel J, Haynes H, Wadler S. Phase II clinical trial of intralesional administration of the oncolytic adenovirus ONYX-015 in patients with hepatobiliary tumors with correlative p53 studies. Clin Cancer Res. 2003 Feb;9(2):693-702.

Phase 2; CRC, PanCa; intra-arterial (IA); Completed; Reid T, Galanis E, Abbruzzese J, Sze D, Wein LM, Andrews J, Randlev B, Heise C, Uprichard M, Hatfield M, Rome L, Rubin J, Kirn D. Hepatic arterial infusion of a replicationselective oncolytic adenovirus (dll520) : phase II viral, immunologic, and clinical endpoints. Cancer Res. 2002 Nov 1, -62(21):60709.

Phase 2; SCCHN; B.O.; Completed; Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E, Kuhn J, McCarty T, Landers S, Blackburn A, Romel L, Randlev B, Kaye S, Kirn D. Phase II trial of intratumoral administration of ONYX -015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol. 2001 Jan 15,-19(2):289-98.

Phase 2; SCCHN; B.O.; Completed; Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH. a controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med. 2000

Aug;6(8):879-85.

Phase 2; CRC; B.B.; Completed; Reid TR, Freeman S, Post L, McCormick F,

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Sze DY. Effects of Onyx-015 among metastatic colorectal cancer patients that have failed prior treatment with 5-FU/leucovorin. Cancer Gene Ther. 2005 Aug;12(8):673-81. CG7060 PSA control Adenovirus Phase 1; Prostate cancer; B.O.; Completed; DeWeese TL, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R, Detorie N, Rodriguez R, Haulk T, DeMarzo AM, Piantadosi S, Yu DC, Chen Y, Henderson DR, Carducci MA, Nelson WG, Simons JW. A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res. 2001 Oct 15; 61(20):7464-72. CG7870/CV787 Probasin-E1A rats Adenovirus Phase 1/2; Prostate cancer; V.V.; Completed; Small EJ, Carducci MA, Burke JM, Rodriguez R, Fong L, van Ummersen L, Yu DC, Aimi J, Ando D, Working P, Kirn D, Wilding G. A phase I trial of intravenous CG7870, a replication-selective, prostatespecific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer. Mol Ther. 2006 Jul;14(1):107-17. Epub 2006 May 9. CG7870/CV787 hPSA-ΕΙΒ, EZ+ Adenovirus Phase 1/2; Prostate Cancer; V.V.; Discontinued in 2005 CG0070 E2F-1, GM-CSF Adenovirus Phase 2/3; Bladder cancer; Intracavitary; Not yet initiated; Ramesh N, Ge Y, Ennist DL, Zhu M, Mina M, Ganesh S, Reddy PS, Yu DC. CG0070, a conditionally replicating granulocyte macrophage colonystimulating factor-armed oncolytic adenovirus for the treatment of bladder cancer. Clin Cancer Res. 2006 Jan 1; 12 (1) :305-13. Telomelysin hTERT Adenovirus Phase 1; Solid tumors; B.O.; Completed; Nemunaitis J, Tong AW, Nemunaitis M, Senzer N, Phadke AP, Bedell C, Adams N, Zhang YA, Maples PB, Chen S, Pappen B, Burke J, Ichimaru D, Urata Y, Fujiwara T. A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors. Mol Ther. 2010 Feb;18(2):429-34. doi: 10.1038/mt.2009.262. Epub 2009 Nov 24. Ad5-CD/TKrep CD/TK Adenovirus Phase 1; Prostate cancer; B.O.; completed; Freytag SO, Khil M, Stricker H, Peabody J, Menon M, DePeralta-Venturina M, Nafziger D, Pegg J, Paielli D, Brown S, Barton K, Lu M, Aguilar-Cordova E, Kim JH. Phase I study of replicationcompetent adenovirus-mediated double suicide gene therapy for the

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treatment of locally recurrent prostate cancer. Cancer Res. 2002 Sep 1;62(17):4968-76. Phase 1; Prostate cancer; B.O.; Completed; Freytag SO, Stricker H, Pegg J, Paielli D, Pradhan DG, Peabody J, DePeralta-Venturina M, Xia X, Brown S, Lu M, Kim JH. Phase I study of replication-competent adenovirus-mediated double-suicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate- to high-risk prostate cancer. Cancer Res. 2003 Nov 1,-63(21):7497-506. Ad5-D24-RGD RGD, Delta-24 Adenovirus Phase 1; Ovarian Cancer; B. B.; Completed; Kimball KJ, Preuss MA, Barnes MN, Wang M, Siegal GP, Wan W, Kuo H, Saddekni S, Stockard CR, Grizzle WE, Harris RD, Aurigemma R, Curiel DT, Alvarez RD. A phase I study of a tropism-modified conditionally replicative adenovirus for recurrent malignant gynecologic diseases. Clin Cancer Res. 2010 Nov 1,-16(21):5277-87. doi: 10.1158/10780432.CCR-10-0791. Epub 2010 Oct 26. Phase 1; Glioma; B. O.; Recruiting Phase 1/2; Glioma; B. O.; Recruiting. Ad5-SSTR/TK-RGD SSTR, TK, RGD Adenovirus Phase 1; Ovarian Cancer; VB; Active; Ramesh N, Ge Y, Ennist DL, Zhu M, Mina M, Ganesh S, Reddy PS, Yu DC. CG0070, a conditionally replicating granulocyte macrophage colonystimulating factor-armed oncolytic adenovirus for the treatment of bladder cancer. Clin Cancer Res. 2006 Jan 1,-12(1):305-13. CGTG-102 Ad5/3, GM-CSF Adenovirus Phase 1/2; Solid tumors; B.O.; Not open; Koski A, Kangasniemi L, Escutenaire S, Pesonen S, Cerullo V, Diaconu I, Nokisalmi P, Raki M, Rajecki M, Guse K, Ranki T, Oksanen M, Holm SL, Haavisto E, KariojaKallio A, Laasonen L, Partanen K , Ugolini M, Helminen A, Karli E, Hannuksela P, Pesonen S, Joensuu T, Kanerva A, Hemminki A. Treatment of cancer patients with a serotype 5/3 chimeric oncolytic adenovirus expressing GMCSF. Mol Ther. 2010 Oct;18(10):1874-84. doi: 10.1038/mt.2010.161. Epub 2010 Jul 27. CGTG-102 Delta-24 Adenovirus Phase 1; Solid Tumors; IT/B.B.; Recruiting INGN-007 (VRX-007) wtEla, ADP Adenovirus Phase 1; Solid tumors; BO; not open; Lichtenstein DL, Spencer JF, Doronin K, 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

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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/llp 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/j ournal.pone .0002 40 9 . CAVATAK Coxsackievirus (CVA21) Phase 1; Melanoma; VO; Completed Phase 2; Melanoma; VO; Recruiting Phase 1; SCCHN; VO; Completed Phase 1; Solid Tumors; VO; Recruiting Talimogen lagerparepvec (OncoVEX) GM-CSF Herpes simplex virus Phase 1; Solid tumors; IN.; Completed; Hu JC, Coffin RS, Davis CJ, Graham NJ, Groves N, Guest PJ, Harrington KJ, James ND, Love CA, McNeish I, 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 secondgeneration oncolytic herpes simplex virus expressing granulocyte macrophage colony-stimulating factor. Clin Cancer Res. 2006 Nov 15; 12(22):6737-47. Talimogen lagerparepvec (OncoVEX) ICP34.5(-) Herpes simplex virus Phase 2; Melanoma; B.O.; Completed; Kaufman HL, Kim DW, DeRaffele G, Mitcham J, Coffin RS, Kim-Schulze S. Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GMCSF in patients with stage Tile and TV melanoma. Ann Surg Oncol. 2010 Mar;17(3):718-30. doi: 10.12 45/S10 43 4-009-080 9-6; 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 IT clinical trial of a granulocyte-macrophage colonystimulating factor-encoding, secondgeneration 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. Talimogen lagerparepvec (OncoVEX) ICP47(-) Herpes simplex virus Phase 3; Melanoma; B.O.; Actively Talimogen lagerparepvec (OncoVEX) Usll ΐ Herpes simplex virus Phase 1/2; SCCHN; VO; Completed; Harrington KJ, Hingorani M, Tanay MA, Hickey J, Bhide SA, Clarke PM, Renouf LC, Thway K, Sibtain A,

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McNeish ΙΑ, Newbold KL, Goldsweig H, Coffin R, Nutting CM. Phase I/II study of oncolytic HSV GM-CSF in combination with radiotherapy and cisplatin in untreated stage III/IV squamous cell cancer of the head and neck. Clin Cancer Res. 2010 Aug 1,-16(15):4005-15. doi: 10.1158/10780432.CCR-10-0196. G207 ICP34.5(-), ICP6(-) Herpes simplex virus Phase 1/2; Glioma; In.; Completed; Markert JM, Liechty PG, Wang W, Gaston S, Braz E, Karrasch M, Nabors LB, Markiewicz M, Lakeman AD, Palmer CA, Parker JN, Whitley RJ, Gillespie GY. Phase lb trial of mutant herpes simplex virus G207 inoculated preand post-tumor resection for recurrent GBM. Mol Ther. 2009 Jan;17(1):199-207. doi: 10.1038/mt.2008.228. Epub 2008 Oct 28; Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD, Palmer CA, Feigenbaum F, Tornatore C, Tufaro F, Martuza RL. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther. 2000 May;7(10):867-74. G207 LacZ(+) Herpes simplex virus Phase 1; Glioma; B.O.; Completed G47Delta From G207, ICP47- Herpes simplex virus Phase 1; Glioma; V.O.; Recruitment ongoing; Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing. Proc Natl Acad Sci USA. 2001 May 22,-98(11):6396-401. Epub 2001 May 15. PubMed PMID: 11353831; PubMed Central PMCID: PMC33479. HSV 1716 (Seprevir) ICP34.5(-) Herpes simplex virus Phase 1; Solid tumors, excluding CNS; B.O.; Recruiting Phase 1; SCCHN; B.O.; Completed; Mace AT, Ganly I, Soutar DS, Brown SM. Potential for efficacy of the oncolytic Herpes simplex virus 1716 in patients with oral squamous cell carcinoma. Head Neck. 2008 Aug;30(8):1045-51. doi: 10.1002/hed.20840. Phase 1; Glioma; B.O.; Completed; Harrow S, Papanastassiou V, Harland J, Mabbs R, Petty R, Fraser M, Hadley D, Patterson J, Brown SM, Rampling R. HSV1716 injection into the brain adjacent to tumor following surgical resection of high-grade glioma: safety data and long-term survival. Gene Ther. 2004 Nov;ll(22) :1648-58,- Papanastassiou V, Rampling R, Fraser M, Petty R, Hadley D, Nicoll J, Harland J, Mabbs R, Brown M. The potential for efficacy of the modified (ICP 34.5()) herpes simplex virus HSV1716 following intratumoral injection

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into human malignant glioma: a proof of principle study. Gene Ther. 2002 Mar;9(6):398-406. Phase 1; Melanoma; B.O.; MacKie RM, Stewart B, Brown SM. Intralesional injection of herpes simplex virus 1716 in metastatic melanoma. Lancet. 2001 Feb 17,-357(9255):525-6. Phase 1; Mesothelioma; V.B.; not active HF10 HSV-1 HF strain Herpes simplex virus Phase 1; Solid Tumors; ED; recruiting Phase 1; Pancreatic Cancer; ED; Completed; Nakao A, Kasuya H, Sahin TT, Nomura N, Kanzaki A, Misawa M, Shirota T, Yamada S, Fujii T, Sugimoto H, Shikano T, Nomoto S, Takeda S, Kodera Y, Nishiyama Y. A phase I dose-escalation clinical trial of intraoperative direct intratumoral injection of HF10 oncolytic virus in non-resectable patients with advanced pancreatic cancer. Cancer Gene Ther. 2011 Mar;18(3):167-75. doi: 10.1038/cgt.2010.65. Epub 2010 Nov 19. Phase 1; Breast Cancer; ED; Completed; Kimata N, Imai T, Kikumori T, Teshigahara 0, Nagasaka T, Goshima F, Nishiyama Y, Nakao A. Pilot study of oncolytic viral therapy using mutant herpes simplex virus (HF10) against recurrent metastatic breast cancer. Ann Surg Oncol. 2006 Aug;13(8):1078-84. Epub 2006 Jul 24. Phase 1; SCCHN; B.O.; Completed; Fujimoto Y, Mizuno T, Sugiura S, Goshima F, Kohno S, Nakashima T, Nishiyama Y. Intratumoral injection of herpes simplex virus HF10 in recurrent head and neck squamous cell carcinoma. Acta Otolaryngol. 2006 Oct;126(10):1115-7. NV1020 Herpes simplex virus Phase 1; Liver Metastasis CRC; ΙΑ; Completed; Fong Y, Kim T, Bhargava A, Schwartz L, Brown K, Brody L, Covey A, Karrasch M, Getrajdman G, Mescheder A, Jarnagin W, Kemeny N. A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer. Mol Ther. 2009 Feb;17(2) :389-94. doi: 10.1038/mt.2008.240. Epub 2008 Nov 18 . MV-CEA CEA Measles virus (Edmonston) Phase 1; Ovarian cancer; V.B.; Completed; Galanis E, Hartmann LC, Cliby WA, Long HJ, Peethambaram PP, Barrette BA, Kaur JS, Haluska PJ Jr, Aderca I, Zollman PJ, Sloan JA, Keeney G, Atherton PJ, Podratz KS, Dowdy SC, Stanhope CR, Wilson TO, Federspiel MJ, Peng KW, Russell SJ. Phase I trial of intraperitoneal

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administration of an oncolytic measles virus strain engineered to express carcinoembryonic antigen for recurrent ovarian cancer. Cancer Res. 2010 Feb 1,-70(3):875-82. doi: 10.115 8/0 00 8-5 4 72.CAN-0 9-27 62 . Epub 2010 Jan 26. Phase 1; Glioma; B.O.; Recruitment in progress MV-NIS NIS Measles virus (Edmonston) Phase 1; Myeloma; V.V.; Recruiting Phase 1; Ovarian Cancer; V.B.; Recruiting Phase 1; Mesothelioma; V.B.; Recruiting Phase 1; SCCHN; V.O.; Not Started NDV-HUJ Newcastle disease virus Phase 1/2; Glioma; V.V.; Completed; Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L, Greenbaum E, Rozenman-Yair S, Panet A, Libson E, Irving CS, Galun E, Siegal T. Phase l/ll trial of intravenous NDVHUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther. 2006 Jan;13(1):221-8. Epub 2005 Oct 28; Pecora AL, Rizvi N, Cohen GI, Meropol NJ, Sterman D, Marshall JL, Goldberg S, Gross P, O'Neil JD, Groene WS, Roberts MS, Rabin H, Bamat MK, Lorence RM. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J Clin Oncol. 2002 May l;20(9):2251-66. PV7 01 Newcastle disease virus Phase 1; Solid Tumors; VV; Completed; Laurie SA, Bell JC, Atkins HL, Roach J, Bamat MK, O’Neil JD, Roberts MS, Groene WS, Lorence RM. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization. Clin Cancer Res. 2006 Apr 15,-12(8):2555-62. MTH-68/H Newcastle disease virus Phase 2; Solid tumors; Inhalational; Completed; Csatary LK, Eckhardt S, Bukosza I, Czegledi F, Fenyvesi C, Gergely P, Bodey B, Csatary CM. Attenuated veterinary virus vaccine for the treatment of cancer. Cancer Detect Prev. 1993;17(6):619-27. H-1PV Parvovirus Phase 1/2; Glioma; IT/B.B.; Recruitment in progress; Geletneky K, Kiprianova I, Ayache A, Koch R, Herrero Y Calle M, Deleu L, Sommer C, Thomas N, Rommelaere J, Schlehofer JR. Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models. Neuro Oncol. 2010 Aug;12(8):804-14. doi: 10.1093/neuonc/noq023. Epub 2010 Mar 18. PVS-RIPO IRES Poliovirus (Sabin) Phase 1; Glioma; B.O.; Recruiting; Goetz C, Gromeier M. Preparing an oncolytic poliovirus recombinant for clinical application against

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glioblastoma multiforme. Cytokine Growth Factor Rev. 2010 AprJun; 21 (2-3): 197-203. doi: 10.1016/j.cytogfr.2010.02.005. Epub 2010 Mar 17. Review. Reolysin Reovirus (Dearing) Phase 1/2; Glioma; B.O.; Completed; Forsyth P, Roldan G, George D, Wallace C, Palmer CA, Morris D, Cairncross G, Matthews MV, Markert J, Gillespie Y, Coffey M, Thompson B, Hamilton M. A phase I trial of intratumoral administration of reovirus in patients with histologically confirmed recurrent malignant gliomas. Mol Ther. 2008 Mar;16(3):627-32. doi: 10.1038/sj.mt.6300403. Epub 2008 Feb 5 . Phase 1; Peritoneal cancer; V.B.; Recruiting Phase 1; Solid tumors; V.V.; Completed; Vidal L, Pandha HS, Yap TA, White CL, Twigger K, Vile RG, Melcher A, Coffey M, Harrington KJ, DeBono JS. A phase I study of intravenous oncolytic reovirus type 3 Dearing in patients with advanced cancer. Clin Cancer Res. 2008 Nov 1,-14(21):7127-37. doi: 10.1158/10780432.CCR-08-0524 . Phase 1; Solid tumors; VV; Recruiting Phase 1; CRC; VV; Recruiting Phase 2; Sarcoma; VV; Completed Phase 2; Melanoma; VV; Postponed Phase 2; Peritoneal cancer, ovarian; VV; Recruiting Phase 2; Pancreatic Cancer; V.V.; Recruiting Phase 2; SCCHN; V.V.; Not Recruiting Phase 2; Melanoma; V.V.; Recruiting Phase 2; Pancreatic Cancer; V.V.; Recruiting Phase 2; Lung Cancer; V.V.; Recruiting Phase 3; SCCHN; V.V.; Recruiting NTX-010 Seneca Valley Virus Phase 2; small cell lung cancer; V.V.; recruiting; PMID: 17971529 Tosa 511 CD Retrovirus Phase 1/2; Glioma; VO; Recruiting; Tai SK, Wang WJ, Chen TC, Kasahara N. Single-shot, multicycle suicide gene therapy by replicationcompetent retrovirus vectors achieves long-term survival benefit in experimental glioma. Mol Ther. 2005 Nov;12(5):842-51. JX-594 GM-CSF Vaccinia virus (Wyeth strain) Phase 1; CRC; B.B.; Recruitment in progress JX-594 TK(-) Vaccinia virus (Wyeth strain) Phase 1; Solid Tumors; V.V.; Completed Phase 1; NCC; V.O.; 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,

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Pinedo HM, Bell JC, Kirn DH. Use of a targeted oncolytic poxvirus, JX594, in patients with refractory primary or metastatic liver cancer: a phase I trial. Lancet Oncol. 2008 Jun;9(6):533-42. doi: 10.1016/S14702045(08)70107-4. Epub 2008 May 19. Erratum in: Lancet Oncol. 2008 Jul;9(7):613. Phase 1; Pediatric Solid Tumors ; VA; Recruiting Phase 1; Melanoma; VA; 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. 2011 Oct;19(10):1913-22. doi: 10.1038/mt.2011.132. Epub 2011 Jul 19. Phase 1/2; Melanoma; B.O.; Completed; Mastrangelo MJ, Maguire HC Jr, Eisenlohr LC, Laughlin CE, Monken CE, McCue PA, Kovatich AJ, Lattime EC. Intratumoral recombinant GM-CSFencoding virus as gene therapy in patients with cutaneous melanoma. Cancer Gene Ther. 1999 SepOct; 6(5):409-22. Phase 2; HCC; B.O.; Not recruiting, data analysis Phase 2B; HCC; B.V.; Recruiting Phase 1/2; CRC; IV/B.O.; Recruiting Phase 2; CRC; B.O.; Not yet recruiting wDD-CDSR TK-, VGF-, LacZ, CD, Somatostatin R Vaccinia virus (Western Reserve) Phase 1; Solid tumors; IT/B.B.; Recruiting; McCart JA, Mehta N, Scollard D, Reilly RM, Carrasquillo JA, Tang N, Deng H, Miller M, Xu H, Libutti SK, Alexander HR, Bartlett DL. Oncolytic vaccinia virus expressing the human somatostatin receptor SSTR2: molecular imaging after systemic delivery using llllnpentetreotide. Mol Ther. 2004 Sep;10(3):553-61. GL-ONC1 Renilla luciferase Vaccinia virus Phase 1; Solid tumors; VV; Recruiting, Gentschev I, Muller Μ, 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-lh68. PLoS One. 2011;6(7) :e22069. doi: 10.1371/j ournal. pone .0022 069. Epub 2011 Jul 11. (GLV-h68) GFP, β-gal Vaccinia virus Phase 1/2; Peritoneal carcinomatosis; V.B.; Recruitment ongoing Lister β-glucuronidase Vaccinia virus Phase 1/2; SCCHN; V.V.; Recruitment in progress

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VSV-hlFNP IFN-β Vesicular stomatitis virus (Indiana) Phase 1; NSS; V.O.; Recruitment in progress DNX-2401 DNAtrix Adenovirus See, for example, Molecular Therapy 21(10): 1814–1818, 2013 and Journal of Vascular and Interventional Radiology 24(8): 1115–1122, 2013 Toca511 Tocagen Lentivirus See, for example, Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular and Interventional Radiology 24(8): 1115-1122, 2013 HSV T-VEC HSV See, for example, Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular and Interventional Radiology 24(8): 1115-1122, 2013 H-1 ParvOryx Parvovirus See, for example, Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular and Interventional Radiology 24(8): 1115-1122, 2013 VACV-TRAIL (see the work of Karolina Autio and Suvi Parvainen, Helsinki) Vaccinia virus See, for example, Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular and Interventional Radiology 24(8): 1115-1122, 2013 VACV-CD40L (see the work of Karolina Autio and Suvi Parvainen, Helsinki) Vaccinia virus See, for example, Molecular Therapy 21(10): 1814-1818, 2013 and Journal of Vascular and Interventional Radiology 24 (8): 1115-1122, 2013 Maraba (See the work of Dave Stojdl and John Bell) Rhabdovirus Candidates for preclinical/clinical studies Maraba-MGl (See the work of Dave Stojdl and John Bell) Rhabdovirus Maraba MGl-hMAGE-A3 (See the work of Dave Stojdl, Brian Litchy and John Bell) Rhabdovirus Candidates for preclinical/clinical studies Sindbis virus Candidates for preclinical/clinical studies Coxsackievirus A21 Candidates for preclinical/clinical studies MYXV Poxvirus Preclinical/clinical candidate Chan WM, Rahman MM, McFadden G. Oncolytic myxoma virus: the path to clinic. Vaccine. 2013 Sep 6,-31(39) : 4252-8 . doi: 10.1016/j.vaccine.2013.05.056. Epub 2013 May 29. WT VSV ('Rose lab') Parental rWT VSV for most VSV-based OVs. The L gene and 49 residues at the N-terminus of the N gene are from the Mudd-Summers strain, the remainder from the San Juan strain (both are Indiana serotypes) Recombinant VSV has been used as an oncolytic anticancer agent (see, e.g., J Gen Virol 93(12): 2529–2545, 2012; Lawson ND, Stillman EA, Whitt MA, Rose JK. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci USA. 1995 May 9,-92(10):4477–81. Erratum in: Proc Natl Acad Sci USA 1995 Sep 12; 92 (19) : 9009.) VSV-WT-XN2 (or XN1) A derivative of rWT VSV ('Rose lab'). Generated using pVSV-XN2 (or pVSV-XNl), a full-length VSV plasmid containing sites Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, Grdzelishvili VZ. Vesicular stomatitis virus as a

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uniqueXhoI and Nhel flanked by VSV transcriptional start and stop signals between the G and L genes. pVSV-XN2 (or pVSV-XNl) was commonly used to produce recombinant VSVs encoding the extra gene 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; Schnell MJ, Buonocore L, Kretzschmar E, Johnson E, Rose JK. Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles. Proc Natl Acad Sci USA. 1996 Oct 15;93(21):11359-65.) WT VSV ('Wertz lab') Alternative rWT VSV. Genes N, P, M, and L are from the San Juan strain; gene G is from the Orsay strain (both are Indiana serotypes). Rarely used in OV research Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Whelan SP, Ball LA, Barr JN, Wertz GT. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci USA. 1995 Aug 29; 92 (18) :8388-92.) VSV-WT-GFP, -RFP, Luc, -LacZ WT VSV encoding reporter genes (between G and L) to monitor viral infection. Based on pVSV-XN2. Similar toxicity to VSV-WT Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Fernadez et al., Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease, J Virol 76:895904 (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 gene inserted between WT G and L genes (in addition to WT G). Toxicity similar to VSV-WT Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Dalton, K. P. & Rose, J. K. (2001). 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 VSV-G/GFP derivative. Obtained with positive Recombinant VSV was used as an oncolytic

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selected on glioblastoma cells and contains two silent mutations and two missense mutations, one in P and one in L. 'gr30' indicates 30 repeat passages anticancer agent (see, for example, Hastie E, 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; G., Tattersall, P. & van den Pol, A. N. (2005). Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential. J Virol 79, 6005-6022. VSV-pl-GFP, VSV-plRFP VSV expressing GFP, or red fluorescent protein (RFP or dsRed) reporter gene at position 1. Attenuated because all VSV genes are moved downward, to positions 2-6. Safe and still effective as OV Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; 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-dG-GFP (or RFP) (replication defective) Similar to VSV-pl-GFP or VSVpl-RFP as described above, but with deletion of the G gene. Fails Recombinant VSV has been used as an oncolytic anticancer agent (see, conduct a second round of infection. Low ability to kill malignant cells for example, Hastie E, 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; 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-ΔΡ, -AL, -AG (semi-replication competent) The virus itself cannot replicate due to the fact that one VSV gene is deleted, but when the viruses are simultaneously infected, efficient replication, safety and oncolysis occur (especially the VSVAG/VSVAL combination). VSVAP and VSVAL contain dsRed instead of the corresponding viral gene. VSVAG contains the GFP gene instead of G Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Muik, A., Dold, C., GeiL, Y., Volk, A., Werbizki, M., Dietrich, U. & von Laer, D. (2012). Semireplication-competent vesicular stomatitis virus as a novel platform for oncolytic virotherapy. J Mol Med (Berl) 90, 959-970.) VSV-M51R Mutant; mutation M51R introduced into M Recombinant VSV has been used as an oncolytic anticancer agent (see,

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for example, Hastie E, 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; Kopecky, S. A., Willingham, M. C., & Lyles, D. S. (2001). Matrix protein and another viral component contribute to induction of apoptosis in cells infected with vesicular stomatitis virus. J Virol 75, 12169-12181.) VSV-AM51, VSV-AM51GFP, - RFP, -FLuc, -Luc, - LacZ M-mutant; mutation ΔΜ51 is introduced into M. In addition, some recombinants encode a reporter gene between G and L Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Stojdl, D. F., Lichty, B. D., tenOever, B. R., Paterson, J. M., Power, A. T., Knowles, S., Marius, R., Reynard, J., Poliquin, L. & other authors (2003). VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4, 263-275.; Power, A. T. & Bell, J. C. (2007) . Cell-based delivery of oncolytic viruses: a new strategic alliance for a biological strike against cancer. Mol Ther 15, 660-665.; Wu, L., Huang, T. G., Meseck, M., Altomonte, J., Ebert, 0., 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.) VSV-*Mmut M mutant; VSV in which one mutation or combination of mutations has been introduced into the following positions of M: M33A, M51R, V221F and S226R Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Hoffmann, M., Wu, Y. J., Gerber, M., Berger-Rentsch, M., Heimrich, B., Schwemmle, M. & Zimmer, G. (2010). Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shutoff activity. J Gen Virol 91, 27822793.) VSV-M6PY >A4-R34E and other M mutants M-mutant; mutation M51R is introduced into the M gene, and, in addition, mutations in the PSAP motif (residues 37-40) of M Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, Grdzelishvili VZ. Vesicular stomatitis virus as a

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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; Irie, 1., Carnero, E., Okumura, A., 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, 25592567.) VSV-M(mut) M mutant; VSV M residues 52-54 are mutated from DTY to AAA. M(mut) cannot block nuclear mRNA export Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Heiber, J. F. & Barber, G. N. (2011). Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent. J Virol 85, 1044010450.) VSV-G5, -G5R, -G6, -G6R G mutant; VSV-expressing mutant G with amino acid substitutions at different positions (between residues 100 and 471). Triggers type I IFN secretion as M51R, but Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic inhibits cellular transcription and translation of host proteins such as WT virotherapy against cancer. J Gen Virol. 2012 Dec;93(Pt 12):2529-45. doi: 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Janelle, V., Brassard, F., Lapierre, P., Lamarre, A., & Poliquin, L. (2011). Mutations in the glycoprotein of vesicular stomatitis virus affect cytopathogenicity: potential for oncolytic virotherapy. J Virol 85, 6513-6520.) VSV-CT1 G mutant; cytoplasmic tail of G protein is truncated from 29 to 1 aa. Reduction of neuropathology, but high colitic effect Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Ozduman, K., Wollmann, G., Ahmadi, S. A. & van den Pol, A. N. (2009). Peripheral immunization blocks lethal actions of vesicular stomatitis virus within the brain. J Virol 83, 11540-11549.; 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,

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1563-1573.) VSV-CT9-M51 G-mutant, the cytoplasmic tail of VSV-G was truncated from 29 to 9 aa, also contains the ΔΜ51 mutation. Reduced neurotoxicity and good OV abilities Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Ozduman, K., Wollmann, G., Ahmadi, S. A. & van den Pol, A. N. (2009). Peripheral immunization blocks lethal actions of vesicular stomatitis virus within the brain. J Virol 83, 11540-11549.; 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-DV/F(L289A) (same as rVSV-F) Foreign glycoprotein; VSV expression of the NDV fusion protein gene between G and L. The L289A mutation in this protein allows induction of syncytium only (without the NDV HN protein) Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Ebert, 0., Shinozaki, K., Kournioti, C., Park, M. S., Garci'a-Sastre, A. & Woo, S. L. (2004). Syncytia induction enhances the oncolytic potential of vesicular stomatitis virus in virotherapy for cancer. Cancer Res 64, 3265-3270.) VSV-S-GP Foreign glycoprotein; VSV with native G gene removed and replaced with modified Sindbis virus (SV) glycoprotein protein (GP). Also expression of mouse GM-CSF and GFP (between SV GP and VSV L). Modified GP protein recognizes the Neg2 receptor, which is overexpressed on a large number of breast cancer cells Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Bergman, I., Griffin, J. A., Gao, Y. & Whitaker-Dowling, P. (2007) . Treatment of implanted mammary tumors with recombinant vesicular stomatitis virus targeted to Her2/neu. Int J Cancer 121, 425430. ) VSV-AG-SV5-F Foreign glycoprotein; VSV G gene replaced by simian parainfluenza virus 5 fusion protein (SVS-F) gene Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Chang, G., Xu, S.,

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Watanabe, M., Jayakar, N. 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, 1611-1618.) VSV-FAST, VSV(ΔΜ51)-FAST Foreign glycoprotein; VSV or VSV-MA51 expressing pl4 FAST protein of reptile reovirus (between G and L of VSV) Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Brown, C. W., Stephenson, K. B., Hanson, S., Kucharczyk, M., Duncan, R., Bell, J. C. & Lichty, B. D. (2009). The pl4 FAST protein of reptilian reovirus increases vesicular stomatitis virus neuropathogenesis. J Virol 83, 552561. ) VSV-LCMV-GP (replicationdefective) Foreign glycoprotein; VSV lacking the G gene was pseudotyped with the neurotrophic glycoprotein LMCV Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Muik, A., Kneiske, I., Werbizki, M., Wilflingseder, D., Giroglou, T., Ebert, 0., Kraft, A., Dietrich, U., Zimmer, G. & other authors (2011). Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism. J Virol 85, 56795684.) VSV-H/F, -aEGFR, aFR,-aPSMA (replicationdefective) Foreign glycoproteins; G gene-less VSV pseudotyped F and H MVs displaying single-chain antibodies (scFv) specific for epidermal growth factor receptor, folate receptor, or prostate cell membrane antigen Retargeted VSVs to cells expressing target receptor Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Ayala-Breton, C., Barber, G. N., Russell, S. J. & Peng, K. W. (2012). Retargeting vesicular stomatitis virus using measles virus envelope glycoproteins. Hum Gene Ther 23, 484-491.) VSV-let-7wt microRNA target; let-7 microRNA is inserted into the 3'UTR of M VSV Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, Grdzelishvili VZ. Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer. J Gen

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Virol. 2012 Dec;93(Pt 12):2529-45. doi: 10.1099/vir.0.046672-0. Epub 2012 Oct 10; Edge, R. E., Falls, T. J., Brown, C. W., Lichty, B. D., Atkins, H. & Bell, J. C. (2008). A let-7 microRNA-sensitive vesicular stomatitis virus demonstrates specific tumor replication. Mol Ther 16, 1437-1443.) VSV-124, -125, 128, -134 (M or L mRNA) microRNA targets; VSV recombinants with neuron-specific microRNAs (miRNA-124, 125, 128 or 134) targets were inserted into the 3’-UTR of M or L VSV mRNA Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Kelly, E. J., Nace, R., Barber, G. N. & Russell, S. J. (2010). Attenuation of vesicular stomatitis virus encephalitis through microRNA targeting. J Virol 84, 1550-1562.) VSV-mp53, VSV- M(mut)-mp53 Malignancy suppressor; BCB [WT or M(mut)] expressing the mouse p53 gene. M(mut) with residues 52-54 of the M protein altered from DTY to AAA Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Heiber, J. F., & Barber, G. N. (2011). Vesicular stomatitis virus expressing tumor suppressor p53 is a highly attenuated, potent oncolytic agent. J Virol 85, 1044010450. ) VSV-C : U Suicide gene; VSV expressing CD/UPRT E.coli catalyzing the modification of 5-fluorocytosine to chemotherapeutic 5FU Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Porosnicu, M., Mian, A. & Barber, G. N. (2003). The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene. Cancer Res 63, 8366-8376.) vsv-c Suicide gene; VSV-MA51 expressing CD/UPRT Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Leveille, S., Samuel,

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S., Goulet, M. L., & Hiscott, J. (2011). Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy. Cancer Gene Ther 18, 435-443.) VSV-(ΜΔ51)-NIS Suicide gene; VSV-MA51, expressing human NIS gene (for radioviral therapy with 1311) Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; 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 110, 23422350.) VSV-TK Suicide gene; VSV expressing TK; may improve oncolysis with nontoxic prodrug ganciclovir Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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-mIFNp, -ΗΙΕΝβ, VSV-rlFNp Immunomodulation; VSV expressing the mouse (m), human (h), or rat (g) ΙΓΝ--β gene Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; 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, 451462.; 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.)

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VSV-IL4 Immunomodulation; VSV expressing IL-4 Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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 thyroid sodium iodide symporter Naik S, Nace R, Federspiel MJ, Barber GN, Peng KW, Russell SJ. Curative one-shot systemic virotherapy in murine myeloma. Leukemia. 2012 Aug;26(8):1870-8. doi:10.1038/leu.2012.70. Epub 2012 Mar 19. VSV-IL12 Immunomodulation; VSV expressing IL-12 Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Shin, E. J., Wanna, G. B., Choi, B., Aguila, D., Ill, Ebert, 0., Genden, E. M., & Woo, S. L. (2007a). Interleukin-12 expression enhances vesicular stomatitis virus oncolytic therapy in murine squamous cell carcinoma. Laryngoscope 117, 210-214.) VSV-IL23 Immunomodulation; VSV expressing IL-23 Significantly attenuated in CNS but effective OV Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Miller, J. M., Bidula, S. M., Jensen, T. M. & Reiss, C. S. (2010). Vesicular stomatitis virus modified with single chain IL-23 exhibits oncolytic activity against tumor cells in vitro and in vivo. Int J Inferon Cytokine Mediator Res 2010, 63-72.) VSV-IL28 Immunomodulation; VSV expressing IL-28, a member of the IFN type III (IFN-λ) family Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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

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2012 Oct 10; Wongthida, R., Diaz, R. M., Galivo, F., Kottke, T., Thompson, J., Pulido, J., Pavelko, K., Pease, L., Melcher, A. & Vile, R. (2010 ). Type III IFN interleukin28 mediates the antitumor efficacy of oncolytic virus VSV in immune competent mouse models of cancer. Cancer Res 70, 4539-4549.) VSV-opt.hIL-15 Immunomodulation; VSV-MA51 expressing a highly secreted variant of human IL-15 Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Stephenson, K. B., Barra, N. G., Davies, E., Ashkar, A. A. & Lichty, B. D. (2012) . Expressing human interleukin-15 from oncolytic vesicular stomatitis virus improves survival in a murine metastatic colon adenocarcinoma model through the enhancement of antitumor immunity. Cancer Gene Ther 19, 238-246.) VSV-CD40L Immunomodulation; VSV expressing CD40L, a member of the tumor necrosis factor (TNF) family Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Galivo, F., Diaz, R. M., Thanarajasingam, U., Jevremovic, D., Wongthida, P., Thompson, J., Kottke, T., Barber, G. N., Melcher, A. & Vile, R. G. (2010). Interference of CD4OL-mediated tumor immunotherapy by oncolytic vesicular stomatitis virus. Hum Gene Ther 21, 439-450.) VSV-Flt3L Immunomodulation; VSV-MA51 expressing soluble form of Flt3L, a DC-activating growth factor Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Leveille, S., Goulet, M. L., Lichty, B. D. & Hiscott, J. (2011). Vesicular stomatitis virus oncolytic treatment interferes with tumor-associated dendritic cell functions and abrogates tumor antigen presentation. J Virol 85, 12160-12169.) VSV/hDCT Immunomodulation; VSV-MA51 expressing hDCT Recombinant VSV has been used as an oncolytic anticancer agent (see,

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for example, Hastie E, 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; Boudreau, J. E., Bridle, B. W., Stephenson, K. B., Jenkins, K. M., Brunellie're, J., Bramson, J. L., Lichty, B. D., & Wan, Y. (2009). Recombinant vesicular stomatitis virus transduction of dendritic cells enhances their ability to prime innate and adaptive antitumor immunity. Mol Ther 17, 1465-1472.) VSV-hgplOO Immunomodulation; VSV expressing hgplOO, a modified self-TAA agonist, tolerated in C57BL/6 mice Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Wongthida, P., Diaz, R. M., Galivo, F., Kottke, T., Thompson, J., Melcher, A. & Vile, R. (2011). VSV oncolytic virotherapy in the B16 model depends upon intact MyD88 signaling. Mol Ther 19, ISO158 . ) VSV-ova Immunomodulation; VSV, Recombinant VSV was used in expressing chicken ovalbumin (in the Bl6ova cancer model) as an oncolytic anticancer agent (see, for example, Hastie E, 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; Diaz, R. M., Galivo, F., Kottke, T., Wongthida, P., Qiao, J., Thompson, J., Valdes, M., Barber, G. & Vile, R. G. (2007) . Oncolytic immunovirotherapy for melanoma using vesicular stomatitis virus. Cancer Res 67, 2840-2848.) VSV-gG Immunomodulation; VSV expressing EHV-1 glycoprotein G, a broad spectrum of viral chemokine-binding proteins Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Altomonte, J., Wu, L., Chen, L., Meseck, M., Ebert, 0., Garcra-Sastre, A., Fallon, J. & Woo, S. L. (2008). Exponential enhancement of oncolytic vesicular stomatitis virus potency by vectormediated suppression of inflammatory responses in vivo. Mol Ther 16, 146-153. )

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VSV-UL141 Immunomodulation; VSV expressing a secreted form of the human cytomegalovirus UL141 protein is known to inhibit NK cell function by blocking the ligand of the NK cell-activating receptor Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Altomonte, J., Wu, L., Meseck, M., Chen, L., Ebert, 0., Garcia-Sastre, A., Fallon, J., Mandeli, J. & Woo, S. L. (2009). Enhanced oncolytic potency of vesicular stomatitis virus through vector-mediated inhibition of NK and NKT cells. Cancer Gene Ther 16, 266278 . ) VSV-(Δ51)-M3 Immunomodulation; VSV-ΜΔδ1 expressing gammahepressvirus-68 chemokine-binding protein M3 Recombinant VSV has been used as an oncolytic anticancer agent (see, for example, Hastie E, 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; Wu, L., Huang, T. G.,Meseck, M., Altomonte, J., Ebert, 0., 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: dsDNA; enveloped Characteristic host: Human Herpesviruses Clinical phase I/II; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 NDV Genome and structure: ds(-)RNA; enveloped Characteristic host: Birds Paramyxoviruses Clinical phase I/II; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 Adeno Genome and Structure: dsDNA; naked Characteristic host: Human Adenoviruses Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 JanFeb; 18 (1):69-81 Reo Genome and Structure: dsDNA; naked Characteristic host: Mammals Reoviruses Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 JanFeb; 18 (1):69-81 Vaccinia virus Genome and Structure: dsDNA; enveloped Characteristic Host: Cows/horses, others Poxviruses Preclinical in vivo; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81 Polio Genome and structure: ds(+)RNA; naked Characteristic host: Human Picornaviruses Clinical phase I; Glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 JanFeb; 18 (1):69-81 VSV Genome and structure: ds (-) Rhabdoviruses Preclinical in vivo; Glioma;

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RNA; enveloped Wollmann et al. Oncolytic virus Characteristic owner: therapy for glioblastoma multiforme: Cattle/mosquitoes concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 MVM Genome and Structure: dsDNA; Parvoviruses Preclinical in vitro; Glioma; naked Wollmann et al. Oncolytic virus Typical owner: Mouse therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 Sindbis Genome and structure: ds (+) Togaviruses Preclinical in vitro; Glioma; RNA; enveloped Wollmann et al. Oncolytic virus Characteristic owner: therapy for glioblastoma multiforme: Mammals/Mosquitoes concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 PRV Genome and Structure: dsDNA; Herpesviruses Preclinical in vitro; Glioma; shell Wollmann et al. Oncolytic virus Characteristic owner: Pig therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 Measles Genome and structure: ds (-) Paramyxovirus Clinical phase I; Glioma; Wollmann RNA; enveloped y et al. Oncolytic virus therapy for Characteristic host: Human glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Feb;18 (1) :69-81 Myxoma Genome and Structure: dsDNA; Poxviruses Preclinical in vivo; Glioma; shell Wollmann et al. Oncolytic virus Typical owner: Rabbit therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 H1PV Genome and Structure: dsDNA; Parvoviruses Clinical phase I; Glioma; Wollmann naked et al. Oncolytic virus therapy for Typical owner: Rat glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan- Feb;18 (1) :69-81 SVV Genome and structure: ds (+) Picornaviruses Preclinical in vitro; Glioma; RNA; naked Wollmann et al. Oncolytic virus Characteristic owner: Pig therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 HSV (G207)I Phase I; Malignant glioma; B.O. Introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Markert JM, Medlock MD, Rabkin SD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther. 2000;7:867Y874. Phase I; Malignant glioma; B.O. Introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Markert JM, Liechty PG, WangW, et al. Phase lb trial of mutant herpes simplex virus G207 inoculated pre- and post-tumor resection for recurrent GBM. Mol Ther. 2009;17:199Y207. Phase I; Malignant glioma; B.O. Introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81

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HSV (1716) Phase II; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 JanFeb; 18(1):69-81; Rampling R, Cruickshank G, Papanastassiou V, et al. Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther. 2000;7:859Y866. Phase I; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81; Papanastassiou V, Rampling R, Fraser M, et al. The potential for efficacy of the modified (ICP 34.5 (j)) herpes simplex virus HSV1716 following intratumoral injection into human malignant glioma: a proof of principle study. Gene Ther. 2002;9:398Y406. Phase I; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Harrow S, Papanastassiou V, Harland J, et al. HSV1716 injection into the brain adjacent to tumor following surgical resection of high-grade glioma: safety data and long-term survival. Gene Ther. 2004;11:1648Y1658. Phase II; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81 HSV (G47Z1) Phase I; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 HSV (M032) Phase I; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 AdV (ONYX-015) Phase I; Malignant glioma; injection to tumor resection cavity; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Chiocca EA, Abbed KM, Tatter S, et al. A phase I open-label, dose escalation, multi-institutional trial of injection with an ElBAttenuated adenovirus, ONYX-015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol Ther. 2004;

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10:958Y966. AdV (Delta24-RGD) Phase I; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 ReoV Phase I; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Forsyth P, Roldan G, George D, et al. A phase I trial of intratumoral administration of reovirus in patients with histologically confirmed recurrent malignant gliomas. Mol Ther. 2008;16:627Y632. Phase I; Malignant glioma; Convection enhanced; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 JanFeb; 18 (1):69-81 NDV(HUJ) Phase I/II; Malignant glioma; V.V.; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J 2012 Jan-Feb;18(1):69-81; Freeman AI, Zakay-Rones Z, Gomori JM, et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther. 2006;13:221Y228. Phase I/II; Malignant glioma; V.V.; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 NDV (MTH-68) Case study/Series; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81; Csatary LK, Bakacs T. Use of Newcastle disease virus vaccine (MTH- 68/H) in a patient with high-grade glioblastoma. JAMA. 1999;281: 1588Y1589. Case study/Series; Malignant glioma; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81; Csatary LK, Gosztonyi G, Szeberenyi J, et al. MTH-68/H oncolytic viral treatment in human high-grade gliomas . J Neurooncol. 2004;67:83Y93. Case study/Series; Malignant glioma; V.V.; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81; Wagner S, Csatary CM, Gosztonyi G, et al. Combined treatment of pediatric high-grade glioma with the oncolytic viral strain MTH-68/H and oral valproic acid. APMIS. 2006;114:731Y743. Measles (MV-CEA) Phase I; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 Hl H1PV Phase I; Malignant glioma; B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81 Polio (PVS-RIPO) Phase I; Malignant glioma; convection-enahnced B.O. introduction; Wollmann et al. Oncolytic virus therapy for glioblastoma multiforme: concepts and candidates. Cancer J. 2012 Jan-Feb;18(1):69-81

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Table 4

List of biologic checkpoint inhibitors approved by the US Food and Drug Administration or in clinical trials

Target receptor or ligand class Common name or name of the medicinal biological product (alias or description) Company 1 CTLA4 Ipilimumab (MDX-010, 10D1) BMS ² CTLA4 Tremelimumab (SR-675,206, ticilimumab) Pfizer 1 PD1 Pembrolizumab (lambrolizumab, MK-3475) Merck 2 PD1 Nivolumab (MDX-1106, BMS-936558, ONO4538) BMS 3 PD1 Pidilizumab (ST-011, MDV9300) Meditation (Curetech) 4 PD1 AMP-224 (fusion protein) GSK/ Amp1immune 5 PD1 AMP-514 (MEDI0680) GSK/ Arnplimmune 6 PD1 AUNP 12 (a peptide) Aurigene/ Pierre Fabre 7 PD1 PDR001 Novartis 8 PD1 BGB-A317 BeiGene 9 PD1 REGN2810 Regeneron 10 PD-L1 Av elumab (MSB0010718C) Pfizer/Merck Serono 11 PD-L1 BMS-935559 (MDX-1105) BMS/Medarex 12 PD-L1 Atezolizumab (MPDL3280A, RG7446) Roche- Genentech 13 PD-L1 Durvalumab (MEDI4736) AZ/Medimmune 14 PD-L1 Novartis (CoStim) 1 LAG3 BMS-986016 BMS 2 LAG3 LAG525 Novartis 3 LAG3 IMP321 ImmuTep 1 TIM3 MBG453 Novartis 1 KIRs Lirilumab (IPH2102/BMS-986015) BMS 1 B7NZ/ CD276 MGA271 Macrogenics

Malignant tumor

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

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The malignancy may be a malignancy that is not suitable for treatment with SMC alone. The methods and compositions of the present invention may be particularly effective for malignancies that are not amenable to treatment with SMC alone. Typically, a malignancy that is resistant to treatment with SMC alone may be a malignancy in which the IAP-mediated apoptotic pathways are not significantly induced. In particular embodiments, a malignancy of the present invention is a malignancy in which one or more apoptotic pathways are not significantly induced, i.e., not activated, such that treatment with SMC alone is sufficient to effectively treat the malignancy. For example, a malignancy of the present invention may be a malignancy in which the cIAP1/2-mediated apoptotic pathway is not significantly induced.

A cancer of the present invention may be a cancer refractory to treatment with one or more agents. In particular embodiments, a cancer of the present invention may be a cancer refractory to treatment with one or more agents (not SMC), and also refractory to treatment with one or more SMC (not another agent).

Composition and introduction

In some cases, delivery of the naked, i.e., native form, SMC and/or agent may be sufficient to enhance apoptosis and/or treat cancer. SMC and/or agents may be administered in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided that the salt, ester, amide, prodrug, or derivative is suitably pharmacologically effective, such as capable of enhancing apoptosis and/or treating a malignancy.

Salts, esters, amides, prodrugs and other derivatives of SMC or the agent can be prepared using standard procedures known in the art of synthetic organic chemistry. For example, an acid salt of SMC and/or agents can be prepared from the free base form of SMC or the agent using a conventional procedure that typically involves reaction with a suitable acid. Typically, the base form of SMC or the 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 adding a less polar solvent. Suitable acids for forming acid addition salts include, but are not limited to, organic acids such as 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, ptoluenesulfonic acid, salicylic acid, and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

An acid addition salt can be converted into a free base by treatment with a suitable base. Some typical acid addition salts of SMC and/or agents, for example halogen salts, as such can be prepared using hydrochloric acid or hydrobromic acid. Conversely, the preparation of basic salts of SMC and/or agents of the present invention can be carried out in a similar manner using a pharmaceutically acceptable base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like. Some typical basic salts include, but are not limited to, alkali metal salts, for example, sodium salt and copper salt.

The preparation of esters may include functionalization of, for example, hydroxyl and/or carboxyl groups that are present in the molecular structure of the SMC and/or agents. In some 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 alkyl and preferably is lower alkyl. If desired, the esters can be converted to free acids using conventional hydrogenolysis or hydrolysis procedures.

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

The SMC or agent of the present invention may be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition, increase or decrease the absorption of the SMC or agent, or improve penetration through the blood-brain barrier (if necessary). Physiologists

- 62 046706 physiologically acceptable compounds may include, for example, carbohydrates (e.g., glucose, sucrose, or dextrans), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, protective agents and uptake enhancers (e.g., lipids), compositions that reduce the clearance or hydrolysis of active agents or excipients or other stabilizers and/or buffers. Other physiologically acceptable compounds, particularly when using tablets, capsules, gelcaps, and the like, include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, suspending agents, and the like. In some embodiments, the pharmaceutical composition may enhance the delivery or effectiveness of the SMC or agent.

In various embodiments, the SMC or agent of the present invention may be formulated for parenteral, topical, oral, nasal (or other), rectal, or local administration. Administration may be, for example, transdermal, prophylactic, or aerosol.

The pharmaceutical composition of the present invention can be administered in various unit dosage forms depending on the route of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injection formulations, implantable sustained release formulations, and lipid complexes.

In some embodiments, an excipient (e.g., lactose, sucrose, starch, mannitol, and the like), an optional disintegrant (e.g., calcium carbonate, calcium carboxymethylcellulose, sodium starch glycollate, crospovidone, and the like), a binder (e.g., alpha starch, gum arabic, microcrystalline cellulose, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, cyclodextrin, and the like), or optional lubricants (e.g., talc, magnesium stearate, polyethyleneglycol 6000, and the like) can be added to the SMC or agent, and the resulting composition can be compressed to form an oral dosage form (e.g., a tablet). In particular embodiments, the compressed product may be coated, for example, to mask the taste of the compressed product, to improve enteric dissolution of the compressed product, or to promote sustained release of the SMC or agent. Suitable coating agents include, but are not limited to, ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, and Eudragit (Rohm & Haas, Germany; methacrylic acrylic copolymer).

Other physiologically acceptable compounds that may be included in a pharmaceutical composition containing the SMC or agent may include wetting agents, emulsifiers, dispersing agents, or preservatives that are particularly effective in 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, for example, on the route of administration of the SMC or agent and on the particular physiochemical characteristics of the SMC or agent.

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

The pharmaceutical composition of SMC or the agent of the present invention can be administered as a single or multiple administration depending on the dosage, the desired frequency of administration and the known or expected tolerance of the individual to the pharmaceutical composition in terms of dose and frequency of administration. In various embodiments, the composition can provide a sufficient amount of SMC or the agent of the present invention to effectively treat cancer.

The amount and/or concentration of SMC or agent to be administered to an individual may vary widely and is typically selected based on the activity of the SMC or agent and the characteristics of the individual, such as body type and weight, as well as the particular route of administration and the individual's need, such as the type of malignancy. The dosage may be varied to optimize the therapeutic and/or prophylactic regimen for a particular individual or group of subjects.

In some embodiments, the SMC or agent of the present invention is administered to the oral cavity, such as via a lozenge, aerosol spray, mouth rinse, coated swab, or other mechanism known in the art.

In some embodiments, the SMC or agent of the present invention is administered via a solid, sustained-release implant wafer inserted into the brain cavity following tumor resection during surgery. The implant wafer may be a biodegradable polyanhydride implant containing SMC or poly(I:C). The number of implant capsules placed may depend

- 63 046706 network from the size of the resection cavity after surgical removal of the primary brain tumor. Delivery of the drug from the implant with slow release directly into the brain tissue eliminates the problem of systemic delivery through the blood-brain barrier. The polymer matrix can consist of a copolymer of 1,3-bis-(p-carboxyphenoxy)propane and sebacic acid (PCPP-SA, molar ratio 80:20), which is dissolved in an organic solvent together with the drug, spray dried into microparticles in the range of 1 to 20 μm and pressed into implants. In some embodiments, the rigid implants are degraded in a two-stage process in which water penetration hydrolyzes the anhydride bonds during the first 10 hours, followed by destruction of the copolymer into the surrounding aqueous medium.

In some embodiments, the SMC or agent of the present invention can be administered systemically (e.g., orally or by injection) according to standard methods known in the art. In some embodiments, the SMC or agent can be delivered through the skin using transdermal drug delivery systems, i.e., a transdermal patch, in which the SMC or agents are typically in a laminar structure that serves as a drug delivery device to be attached to the skin. In such a structure, the drug composition is typically contained in a layer or reservoir underlying the upper support layer. The reservoir of the transdermal patch includes an amount of the SMC or agent that is ultimately available for delivery to the skin surface. Thus, the reservoir can include, for example, the SMC or agent of the present invention as an adhesive on a backing layer of the patch or in any of a variety of different matrix compositions known in the art. The patch can contain one reservoir or several reservoirs.

In particular embodiments of the transdermal patch, the reservoir may comprise a polymer matrix of a pharmaceutically acceptable contact adhesive material that serves to attach 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 in separate and distinct layers, with the adhesive underlying the reservoir, which in this case may be either a polymer matrix as described above, a liquid or hydrogel reservoir, or another form known in the art. The backing layer in these laminates, which serves as the top surface of the device, preferably acts as the main structural element of the patch and imparts significant flexibility to the device. The material selected for the backing layer is preferably substantially impermeable to the SMC and/or agent and any other materials that are present.

Additional formulations for topical delivery include, but are not limited to, ointments, gels, sprays, liquids, and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum products. Creams, which include the SMC or agent, are typically viscous liquids or semisolid emulsions, such as 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 typically composed of petrolatum and a fatty alcohol such as cetyl alcohol or stearyl alcohol; the aqueous phase is usually, but not necessarily, larger in volume than the oil phase and typically contains a humectant. The emulsifier in a cream base is typically a nonionic, anionic, cationic, or amphoteric surfactant. The specific ointment or cream base to be used can be selected to ensure optimal drug delivery in accordance with the state of the art. As with other carriers, the ointment base may be inert, stable, non-irritating, and non-sensitizing.

Various buccal and sublingual preparations are also considered.

In some embodiments, administration of the 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 implantable device.

In some embodiments, it may be desirable to deliver SMC or an agent to the brain. In embodiments including systemic administration, this may require passage of the SMC or agent through the blood-brain barrier. In various embodiments, this may be facilitated by co-administering the SMC or agent with carrier molecules such as cationic dendrimers or arginine-rich peptides that can transport the SMC or agent through the blood-brain barrier.

In some embodiments, the SMC or agent may be delivered directly to the brain by administration via implantation of a biocompatible release system (e.g., a reservoir), by direct administration via an implanted cannula, by administration via an implanted or partially implanted drug pump, or by mechanisms

- 64 046 706 similar function known in the art. In some embodiments, the SMC or agent may be administered systemically (e.g., administered intravenously). In some embodiments, the SMC or agent is expected to be transported across the blood-brain barrier without the use of additional compounds included in the pharmaceutical composition to enhance transport across the blood-brain barrier.

In some embodiments, one or more SMCs or agents of the present invention may be provided as a concentrate, such as in a storage container or dissolvable capsule, ready for dilution or addition to a volume of water, alcohol, hydrogen peroxide or other diluent. The concentrate of the present invention may be provided in a specific amount of SMC or agent and/or a specific total volume. The concentrate may be prepared for dilution in a specific volume of diluents prior to administration.

The SMC or agent can be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. The compound can also be administered topically in the form of foam, lotion, drops, creams, ointments, emollients or gels. Parenteral administration of the compound is advantageously carried out, for example, in the form of saline solutions or together with the compound introduced into liposomes. In cases where the compound itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be used. Other suitable compositions and methods of administration are known or can be taken from the state of the art.

SMC or the agent of the present invention can be administered to a mammal in need thereof, such as a mammal diagnosed with a malignant neoplasm. SMC or the agent of the present invention can be administered to enhance apoptosis and/or treat a malignant neoplasm.

A therapeutically effective dose of the pharmaceutical composition of the present invention may depend on the age of the individual, the sex of the individual, the type of individual, the specific pathology, the severity of the symptoms and the general health of the individual.

The present invention includes compositions and methods for treating a human, such as a human diagnosed with a malignant neoplasm. In addition, the pharmaceutical composition of the present invention may be suitable for administration to an animal, such as for veterinary use. Some embodiments of the present invention may include administering the pharmaceutical composition of the present invention to non-humans, such as primates, dogs, horses, cats, pigs, ungulates or lagomorphs, or other vertebrate species.

The therapy according to the invention may be carried out independently or in combination with another therapy, for example, another therapy for a malignant neoplasm, and may be carried out at home, in a doctor's office, in a clinic, in a hospital outpatient department, or in a hospital. The treatment does not necessarily begin in a hospital so that the doctor can closely monitor the effects of the therapy and make any adjustments that are necessary, or it may begin in an outpatient setting. The duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the individual, the stage and type of the individual's disease, and how the patient responds to the treatment.

In some embodiments, the combination therapy of the present invention further comprises treatment with a recombinant interferon, such as IFN-α, IFN-β, IFN-γ, pegylated IFN, or liposomal interferon. In some embodiments, the combination therapy of the present invention further comprises treatment with recombinant TNF-α, such as for isolated limb perfusion. In specific embodiments, the combination therapy of the present invention further comprises treatment with one or more TNF-α or IFN inducing compounds, such as DMXAA, Ribavirin, or the like. Additional cancer immunotherapy that can be used in combination with the present invention includes antibodies, such as monoclonal antibodies targeting CTLA-4, PD-1, PD-L1, PD-L2, or other checkpoint inhibitors. Cyclic dinucleotides (CDN) [cyclic di-GMP (guanosine 5'-monophosphate) (CDG), cyclic di-AMP (adenosine 5'-monophosphate) (CDA), and cyclic GMP-AMP (cGAMP)] are pathogen-associated molecules (PAMPs) that activate part of the TBK1/interferon regulatory factor 3 (IRF'3)/interferon type 1 signaling pathway by stimulating the cytoplasmic recognition domain of the interferon receptor (STING). In some embodiments, STING agonists can be combined with SMC to treat malignancy.

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

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

In embodiments of the present invention, the SMC and the agent are administered to a subject in need thereof, such as a patient with a cancer. In some cases, the SMC and the agent are administered simultaneously. In some embodiments, the SMC and the agent may be present in a single therapeutic dosage form. In other embodiments, the SMC and the agent may be administered separately to a subject in need thereof. When administered separately, the SMC and the agent may be administered simultaneously or at different times. In some cases, the subject receives a single dose of the SMC and a single dose of the agent. In some embodiments, one or more SMC and agents are administered to the subject in two or more doses. In some embodiments, the frequency of administration of the SMC and the frequency of administration of the agent are not identical, i.e., the SMC is administered at a first frequency and the agent is administered at a second frequency.

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

In particular embodiments of any of the methods of the present invention, the SMC and the agent are administered within 28 days of each other or less, such as within 14 days of each other. In some embodiments of any of the methods of the present invention, the SMC and the agent are administered, for example, simultaneously or within 1, 5, 10, 15, 30 minutes, 1, 2, 4, 6, 12, 18, 24, 36 hours, 2, 4, 8, 10, 12, 16, 20, 24 or 28 days of each other. In any of these embodiments, the first administration of the SMC of the present invention may precede the first administration of the agent of the present invention. Alternatively, in any of these embodiments, the first administration of the SMC of the present invention may follow the first administration of the agent of the present invention. Because SMC and/or the agent of the present invention may be administered to an individual in two additional doses, and because in such cases the doses of SMC and the agent of the present invention may be administered at different frequencies, it is not necessary that the time period between administration of SMC and administration of the agent be maintained constant during a given course of treatment or for a given individual.

One or both of the SMC and the agent may be administered at a low dose or at a high dose. In embodiments in which the SMC and the agent are separately formulated, the pharmacokinetic profiles for each agent may be appropriately matched to the formulation, dosage, and route of administration, and the like. In some cases, the SMC is administered at a standard or high dose, and the agent is administered at a low dose. In some cases, the SMC is administered at a low dose, and the agent is administered at a standard or high dose. In some cases, both the SMC and the agent are administered at a standard or high dose. In some cases, both the SMC and the agent are administered at a low dose.

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

Sets

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

Kits of the invention may also contain instructions for administering an additional pharmacologically acceptable substance, such as an agent known to treat a cancer, that is not SMC or an agent of the present invention.

Individually or separately formulated agents may be packaged together as a kit. Non-limiting examples include kits that contain, for example, two tablets, a tablet and a powder, a suppository and a liquid in a vial, two topical creams, ointments, foams, and the like. A kit may include optional components that facilitate administration of a single dose to patients, such as vials for reconstituting powder forms, syringes for injection, individual delivery systems, inhalers, and the like. In addition, a unit dose kit may contain instructions for preparing and administering the compositions. The kit may be prepared as a single dose for a single individual, multiple uses for a specific individual (in a constant dosing regimen or where individual compounds may vary in effectiveness as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple individuals (bulk packaging). The components of the kit may be assembled

- 66 046706 in cardboard boxes, blister packs, bottles, test tubes and the like.

The dose of each compound of the claimed combinations will depend on several factors, including: the route of administration, the disease (e.g., type of malignancy) being treated, the severity of the disease, and the age, weight, and general condition of the patient being treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic agent) information about a particular individual may influence the dosage regimen or other aspects of administration.

Examples

Example 1. Smac mimetics prime tumors for destruction via the innate immune system.

Smac mimetics are a class of apoptosis-sensitizing agents that have proven safe for the treatment of cancer in phase I studies. Stimulation of the innate antipathogen response can trigger a potent but harmless inflammatory cytokine storm that can lead to the death of Smac mimetic-treated tumors. In this example, we show that activation of innate immune responses by oncolytic viruses and adjuvants such as poly(:C) and CpG induces non-specific cell death of Smac mimetic-treated cancer cells in a manner mediated by IFNe, TNFa or TRAIL. This therapeutic strategy may lead to durable cures, for example, in several mouse models of aggressive cancer. With these and other innate immune stimulators having demonstrated safety in human clinical trials, the data presented here strongly suggest their combination use with Smac mimetics for the treatment of malignancy.

The present case study examines whether stimulation of the innate immune system using pathogen mimetics is a safe and effective strategy to generate the cytokine environment required to initiate apoptosis in SMC-treated tumors. The authors argue here that nonpathogenic oncolytic viruses, as well as microbial RNA or DNA mimetics such as poly(I:C) and CpG, induce nonspecific killing of SMC-treated malignant cells that is dependent on either IFNe, TNFa, or TRAIL. Importantly, this therapeutic strategy was feasible in vivo and resulted in durable cure in several mouse models of aggressive malignancy.

SMC therapy sensitizes malignant cells to non-specific cell death during oncolytic viral infection.

Oncolytic viruses (OVs) are novel biotherapeutic agents for the treatment of cancer that are currently in phase I-III clinical trials. One barrier to OV therapy may be the induction of type I IFN-γ and NFkB cytokines by the host, which orchestrate an antiviral state in tumors. We examined whether these innate immune cytokines could be used to induce apoptosis in SMC-pretreated cancer cells. We initially recruited a small panel of tumor and normal cell lines (n=30) to screen for response to the SMC LCL161 and the oncolytic rhabdovirus VSVA51. We selected LCL161 because it is a compound in late-stage clinical trials in the SMC class and VSVA51 because it is known to induce a robust antiviral cytokine response. In 15 of 28 cell lines tested (54%), SMC treatment increased the sensitivity of the _EC50 to VSVA51 by 10- to 10,000-fold (Fig. 6 and examples in Figs. 1A and 1B). Similarly, low dose VSVA51 reduced the EC50 of SMC therapy from undefined levels (>2500 nM) to 4.5 and 21.9 nM in two representative cell lines: EMT6 mouse mammary carcinoma and SNB75 human glioblastoma cells, respectively (Fig. 1C). Combination index analysis showed that the interaction between SMC therapy and VSVA51 is synergistic (Fig. 7). Experiments using four other SMCs and five other oncolytic viruses showed that a range of monovalent and divalent SMCs synergized with VSVA51 (Fig. 8). We found that the oncolytic rhabdoviruses VSVA51 and Maraba-MG1 were highly effective in inducing non-specific killing in synergy with SMC, compared with HSV, reovirus, vaccine, and wild-type VSV platforms, all of which have sophisticated mechanisms to disarm innate immune signaling (Figs. 9A and 9B). Genetic experiments using RNAi-mediated silencing revealed that both XIAP and CIAP must be inhibited to synergize with VSVA51 (Figs. 10A, 10B, and 24C). In sharp contrast to the results obtained in tumor-derived cell lines, combination therapy with VSVA51 and SMC had no effect on non-malignant primary human dermal fibroblasts GM38 and human skeletal myoblasts HSKM (Fig. 6). Taken together, these data indicate that oncolytic VSV synergizes with SMC therapy in a tumor-selective manner.

To determine whether VSVA51 induces non-specific cell death of IAP-depleted neighboring cells not infected with virus, cells were treated with SMC prior to infection with a low dose of

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VSVA51 (MOI=0.01 infectious particles/cell). We assessed whether conditioned medium obtained from VSVA51-infected cells (which were subsequently inactivated with UV light) could induce cell death when transferred to a layer of naïve cancer cells treated with SMC. The conditioned medium induced cell death only when the cells were co-treated with SMC (Fig. 1D). We also found that a low dose of a pseudotyped G gene-reduced strain of VSVA51 (MOI=0.1) containing a deletion of the gene encoding its glycoprotein (VSVA51AG), which limits the virus to a single round of infection, was toxic to the entire SMC-treated cancer cell layer (Fig. 1E). Finally, we performed a cytotoxicity assay in cells with agarose overlay used to inhibit the spread of VSYA51 expressing a fluorescent label and observed significant cell death in SMC-treated cells outside the viral infection zone (Figs. 1F and 11). Overall, these results indicate that VSVA51 infection results in the release of at least one soluble factor that can actively induce cell death in neighboring, uninfected, SMC-treated cancer cells at the border.

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

The innate cellular immune response to RNA virus infection in mammalian tumor cells can be activated by members of the cytosolic (RIG-I-like receptors, RLRs) and endosomal (toll-like receptors, TLRs) families of viral RNA sensors. Once triggered, these receptors can induce the IFN response factor (IRF) 3/7 and nuclear factor kappa B (NF-κΒ) cell signaling cascades. These signals can culminate in the production of IFNs and their response genes, as well as a range of inflammatory chemokines and cytokines. This instructs neighboring cells to preemptively express an armamentarium of antiviral genes, and helps recruit and activate cells of the innate and adaptive immune systems to ultimately clear the viral infection. It has recently been shown that cIAP proteins are involved in multiple signaling pathways following pathogen detection, including those derived from RLRs and TLRs. Accordingly, we examined whether SMC treatment affects the antiviral response to oncolytic VSV infection in tumor cells and mice. First, the effect of SMC treatment on VSVA51 productivity and dissemination was assessed. Single-step and multistep growth curves of VSVA51 productivity showed that SMC treatment did not affect the growth kinetics of VSVA51 in EMT6 or SNB75 cells in vitro (Fig. 2A). Moreover, time-lapse microscopy analysis showed that SMC treatment did not alter VSVA51 infectivity or dissemination in tumor cells (Fig. 2B). Furthermore, viral replication and dissemination in vivo were analyzed by determining tumor burden using IVIS imaging and titrating viral tissues. No differences in viral kinetics were detected with SMC treatment in EMT6 tumor-bearing mice (Figs. 12A and 12B). Because EMT6 and SNB75 cells possess functional type I IF responses that regulate the VSV life cycle, these data provide strong, albeit indirect, evidence that SMC therapy does not affect antiviral signaling cascades in malignant cells.

To investigate this further, IFNe production was measured in VSVA51- and SMC-treated EMT6 and SNB75 cells. This experiment showed that SMC-treated cancer cells respond to VSVA51 by secreting IFNe (Fig. 2C), albeit at slightly lower levels compared to YSYΔ51. The question was raised whether the attenuated IFNe secretion of corresponding SMC-treated cells affects the induction of downstream IFN stimulatory gene (ISG) genes. Quantitative RT-PCR analyses of a small subset of ISGs in VSVA51- and SMC-treated cells showed that IAP inhibition does not affect ISG gene expression in response to oncolytic VSV infection (Fig. 2D). Consistent with this, Western blot analysis showed that SMCs did not alter activation of Jak/Stat signaling downstream of IFNe (Figs. 2E and 24A). Together, these data suggest that SMCs do not interfere with the ability of tumor cells to be sensitive and responsive to YSYΔ51 infection.

IFNe orchestrates nonspecific cell death during SMC therapy and treatment with the oncolytic virus VSV.

SMCs sensitize a variety of cancer cell lines to caspase 8-dependent apoptosis induced by TNFa, TRAIL, and GE-1β. Since RNA viruses can initiate 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. First, the TNF receptor (TNF-R1) and/or TRAIL receptor (DR5) were silenced and the synergy between SMCs and YSYΔ51 was analyzed. This experiment revealed that TNFa and TRAIL are not only involved but also required together for nonspecific cell death (Figs. 3A–3H, 13A, and 24D). Consistent with these data are the Western blot and immunofluorescence experiments that showed strong activation of the extrinsic apoptotic pathway and the RNAi knockout experiments that showed a requirement for both

- 68 046706 caspase-8 and Rip1 in a synergistic response (Figs. 14A-14G, 24E, and 24F). Furthermore, engineered TNFa in VSVΔ51 improved synergy with SMC therapy by an order of magnitude (Figs. 15A and 15B).

The type I IFN receptor (IFNAR1) was then silenced and, surprisingly, IFNAR1 knockdown was found to not synergize SMC therapy with oncolytic VSV (Figs. 3B, 13B, and 24D). IFNAR1 knockdown was predicted to attenuate, but not completely suppress, nonspecific killing since TRAIL is a well-known ISG that responds to type I IFN28. TNFa and IL-1e are thought to be independent of the IFN signaling pathway but nevertheless respond to the final steps of the NF-κB signaling pathway following viral detection. This result suggests the possibility of a noncanonical type I IFN-γ pathway for TNFa and/or IL-1e production. Indeed, when IFNe, TRAIL, TNFa, and IL-III mRNA expression was examined during oncolytic VSV infection, a significant time lag was found between the induction of IFNe and that of TRAIL and TNFa (Fig. 3C). These data further suggest that TNFa, similar to TRAIL, may be induced secondary to IFNe. To prove this concept, IFNAR1 was silenced prior to treatment of cells with VSVΔ51. Knockdown of IFNAR1 completely abolished the induction of both TRAIL and TNFa by oncolytic VSV (Fig. 3D). Moreover, the synergism with SMC was recapitulated using IFN (IFNa/β) and type II IFN (IFNy), but not type III IFN (IL28/29) (Fig. 3E). Taken together, these data indicate that type I IFN is required for the induction of TNFa and TRAIL during VSVA51 infection of tumor cells. Moreover, the production of these cytokines is responsible for the non-specific death of surrounding, uninfected SMC-treated cells.

To investigate non-canonical TNFa induction, TRAIL and TNFa mRNA expression levels were further measured in SNB75 cells treated with recombinant IFNe. Both cytokines were induced by IFNe treatment (Fig. 3F), and ELISA experiments confirmed the production of their respective protein products in the cell culture medium (Fig. 3G). Interestingly, there was a significant time lag between TRAIL and TNFa induction. Since TRAIL is a full-fledged ISG and TNFa is not, this result raises the possibility that TNFa is not induced by IFNe directly but in response to subsequent steps in the ISG signaling pathway activated by IFNp. Thus, qRT-PCR was performed on 176 cytokines in SNB75 cells and 70 were identified that were significantly activated by IFNe (Table 5). The role of these ISGs in TNFa induction by IFNp is currently under investigation. Also of interest is the finding that SMC treatment enhanced both TRAIL and TNFa induction by IFNe in SNB75 cells (Figs. 3F and 3G). Furthermore, using a dominant negative IKK construct, it was found that the production of these inflammatory cytokines downstream of IFNe signaling was dependent, at least in part, on classical NF-κB signaling (Fig. 3H). In EMT6 cells, SMC treatment was found to enhance cellular TNFa production (5-7-fold percent increase) upon VSV infection (Fig. 1B). Finally, it has also been shown that blocking TNF-R1 signaling (using antibodies or dsRNA) prevents EMT6 cell death in the presence of SMC and VSVΔ51 or IFN3 (Figs. 17A-17C and 24H). The relationship between type I IFN and TNFa is complex, with either complementary 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 with oncolytic virus RNA activate type I IFN, and this process is not affected by SMC antagonism of IAP proteins. These IFNs, in turn, signal neighboring uninfected malignant 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 (Fig. 18A and 18B).

Table 5

VSV ΙΕΝβ The genes are named Gene identification 25465,4 1017, 8 CCL8 Chemokine (C-C motif) ligand 8 13388,9 44.9 IL29 Interleukin 29 (interferon, lambda 1) 5629,3 24.3 IFNB1 Interferon, beta 1, fibroblast 1526, 8 16, 2 TNFSF15 Superfamily of factors (ligands)

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tumor necrosis, member 15 847 24, 6 CCL5 Chemokine (C-C motif), ligand 5 747.7 17.2 CCL3 Chemokine (C-C motif), ligand 3 650, 9 60, 6 TNFSF10 Tumor necrosis factor (ligand) superfamily, member 10 421.3 296, 1 IL12A Interleukin 12A 289, 3 10.7 TNFSF18 Tumor necrosis factor (ligand) superfamily, member 18 255, 3 18, 8 CCL7 Chemokine (C-C motif), ligand 7 154.2 19, 2 IL6 Interleukin 6 (interferon, beta 2) 150, 8 12.9 IL1RN Interleukin 1 receptor antagonist 108, 1 25, 5 CCL20 Chemokine (C-C motif), ligand 20 78, 6 6, 2 CXCL1 Chemokine (C-X-C motif), ligand 1 64.7 14, 8 CCL2 Chemokine (C-C motif), ligand 2 62.5 14.5 CCL4 Chemokine (C-C motif), ligand 4 55, 6 1,2 CXCL3 Chemokine (C-X-C motif), ligand 3 55, 2 4.3 TNF Tumor necrosis factor (TNF superfamily, member 2) 48, 8 4.3 IGF1 Insulin-like growth factor 1 (somatomedin C) 48.4 2.8 CXCL2 Chemokine (C-X-C motif), ligand 2 38,5 3, 8 CCL11 Chemokine (C-C motif), ligand 11 37,5 3, 8 HGF Hepatocyte growth factor 36, 5 75, 1 NGFB Nerve growth factor, beta-polypeptide 32.9 4 FGF14 Fibroblast growth factor 14 24.7 25, 6 FGF2 0 Fibroblast growth factor 20 21,5 16.4 IL1B Interleukin 1, beta 20 36, 3 CSF2 Colony stimulating factor 2 (granulocyte-macrophage) 18.3 2, 6 GDF3 Growth Differentiation Factor 3 17.2 2 CCL28 Chemokine (C-C motif), ligand 28 12 2,1 CCL22 Chemokine (C-C motif), ligand 22 11.3 2,5 CCL17 Chemokine (C-C motif), ligand 17 10,5 2 CCL13 Chemokine (C-C motif), ligand 13

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10,5 15, 3 IL20 Interleukin 20 9, 7 22.8 FGF16 Fibroblast growth factor 16 8, 8 3, 6 TNFSF14 Tumor necrosis factor (ligand) superfamily, member 14 8.2 2.7 FGF2 Fibroblast growth factor 2 (main) 7, 1 8, 1 BDNF Brain-derived neurotrophic factor 7, 1 9, 7 ILIA Interleukin 1, alpha 7, 1 10, 9 ANGPT4 Angiopoietin 4 7 1,5 TGFB3 Growth Transformation Factor Beta 3 7 5, 8 IL22 Interleukin 22 6, 9 9, 7 IL1F5 Interleukin 1 superfamily, member 5 (delta) 6, 7 2,4 IFNW1 Interferon, omega 1 6, 6 12, 6 IL11 Interleukin 11 6, 6 25, 1 IL1F8 Interleukin 1 superfamily, member 8 (eta) 6, 3 -1.3 EDA Ectodysplasin A 5, 9 8 FGF5 Fibroblast growth factor 5 5, 8 5 VEG FC Vascular endothelial growth factor C 5, 2 4.9 LIF Leukemia inhibitory factor 5 1,3 CCL25 Chemokine (C-C motif), ligand 25 4, 9 8.3 BMP3 Bone morphogenetic protein 3 4, 9 1, 6 IL17C Interleukin 17C 4, 8 -2.3 TNFSF7 molecule CD70 4.3 2,5 TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8 4.3 2,5 FAS LG Fas ligand (TNF superfamily member 6) 4.2 2.7 BMP 8V Bone morphogenetic protein 8b 4.2 6 IL7 Interleukin 7 4, 1 5.2 CCL24 Chemokine (C-C motif), ligand 24 4 -2.2 INHBE Inhibin, beta E 4 5, 8 IL23A Interleukin 23, alpha subunit

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pl 9 3, 8 -1,1 IL17F Interleukin 17F 3, 7 2.9 CCL21 Chemokine (C-C motif), ligand 21 3, 5 8.5 CSF1 Colony stimulating factor 1 (macrophage) 3, 5 3 IL15 Interleukin 15 3, 4 5, 7 NRG2 Neuregulin 2 3, 3 No data INHBB Inhibin, beta B 3, 3 No data LTB Lymphotoxin beta (superfamily TNF member 3) 3, 3 No data BMP 7 Bone morphogenetic protein 7 3 -3, 8 IL1F9 Interleukin family 1, member 9 2, 9 6, 1 IL12B Interleukin 12B 2, 8 6, 2 FLT3LG Fms-related tyrosine kinase ligand 2.7 3 FGF1 Fibroblast growth factor 1 (acidic) 2,5 -2 CXCL13 Chemokine (C-X-C motif), ligand 13 2,4 2,2 IL17B Interleukin 17B 2,3 7, 8 GDNF Glial cell derived neurotrophic factor 2,3 -1.7 GDF7 Growth Differentiation Factor 7 2,3 -2.4 LTA Lymphotoxin alpha (TNF superfamily, member 1) 2,2 1,7 LEFTY2 Left-Right Determination Factor 2 2, 1 5 FGF19 Fibroblast growth factor 19 2, 1 9, 8 FGF2 3 Fibroblast growth factor 23 2, 1 4, 8 CLC Cardiotrophin-like cytokine factor 1 2, 1 3 ANGPT1 Angiopoietin 1 2 10, 6 TRO Thyroid peroxidase

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2 2,1 EFNA5 Efrin-A5 1, 9 6, 4 IL1F10 Interleukin family 1 member 10 (theta) 1, 9 7, 6 LEP Leptin (obesity homologue, 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 (betaurogastron) 1,7 3,4 CTF1 Cardiotrophin 1 1,7 -1.9 BMP 2 Bone morphogenetic protein 2 1,7 3 EFNB2 Ephrin-B2 1, 6 1 FGF8 Fibroblast growth factor 8 (androgen-induced) 1, 6 -2 TGFB2 Growth Transformation Factor Beta 2 1,5 -1, 6 BMP 8 A Bone morphogenetic protein 8a 1,5 3,3 NTF5 Neurotrophin 5 (neurotrophin 4/5) 1,5 1 GDF10 Growth Differentiation Factor 7 1,5 1,5 TNFSF13 B 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 I ENK 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

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1,2 -1.3 IL17D Interleukin 17D 1,2 1, 1 TNFSF4 Tumor necrosis factor (ligand) superfamily, member 4 1,2 1,3 VEG FA Vascular endothelial growth factor 1, 1 2,4 FGF11 Fibroblast growth factor 11 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 Efrin-VZ -1 1, 8 VEGFB Vascular endothelial growth factor B -1 -1,2 IL1F7 Interleukin family 1, member 7 (zeta) -1 -2.1 GDF11 Growth Differentiation Factor 11 -1,1 1,3 ZFP91 Zinc finger protein homolog 91 (mouse) -1,2 -ι,ι BMP 6 Bone morphogenetic protein 6 -1,2 -1,2 AMN Antimulleric hormone -1.3 -1 LEFTY1 Left-Right Determination Factor 1 -1.3 2,4 EFNA3 Efrin-AZ -1.3 -1.3 LASSI Longevity homolog LAG1 1 -1.5 1 EFNA4 Efrin-A4 -1.8 1,3 PDGFD DNA damage-inducing protein 1 -1.8 1, 8 IL10 Interleukin 10 -1.9 1, 6 GDF5 Growth Differentiation Factor 5 -1.9 1,3 EFNA2 Efrin-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 BMP 10 Bone morphogenetic protein 10 -2.4 -4, 6 CXCL12 Chemokine (C-X-C motif), ligand 12 -2.5 4 I ENG Interferon, gamma

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-2, 6 1,2 ERO Erythropoietin -2.7 -2.1 GAS 6 Specific growth inhibitory factor 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 No data X 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 CCL2 6 Chemokine (C-C motif), ligand 26 -8 9, 7 CCL19 Chemokine (C-C motif), ligand 27 No data No data X BMP 15 Bone morphogenetic protein 15 No data No data X CCL15 Chemokine (C-C motif), ligand 14 No data No data CCL16 Chemokine (C-C motif), ligand 16 No data No data X CCL18 Chemokine (C-C motif), ligand 18 No data No data X CCL23 Chemokine (C-C motif), ligand 24 No data No data CD40LG CD40 ligand (TNF superfamily)

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X No data No data X CSF3 Colony stimulating factor 3 (granulocyte) No data No data X CXCL5 Chemokine (C-X-C motif), ligand 5 No data No data FGF4 Fibroblast growth factor 4 No data No data X FGF6 Fibroblast growth factor 6 No data No data GH2 Growth hormone 2 No data No data X IL2 Interleukin 2 No data No data IL21 Interleukin 21 No data No data X IL28A Interleukin 28A (interferon, lambda 2) No data No data INHBA Inhibin, beta A No data No data X NRG3 Neuregulin 3 No data No data X TNFSF11 Tumor necrosis factor superfamily, member 11 (ligands) No No TNFSF13 Superfamily of factors (ligands) data data X tumor necrosis, member 13 No data 6, 5 NRG4 Neuregulin 4 No data 6, 1 IL3 Interleukin 3 (colony stimulating factor, multiple) No data 1,8 TNFSF9 Tumor necrosis factor superfamily, member 9 (ligands)

Oncolytic VSV potentiates SMC therapy in preclinical animal models of malignancy.

To evaluate SMC and VSV co-oncolytic therapy in vivo, EMT6 breast carcinoma was used as a syngeneic orthotopic model. Preliminary safety and

- 76 046706 Pharmacodynamic experiments showed that 50 mg/kg LCL161 given orally via gavage was well tolerated and induced cIAP1/2 knockdown in tumors for at least 24 h and up to 48-72 h in some cases (Figs. 19A, 19B, and 24G). When tumor volumes reached ~100 mm3, we began treating mice twice weekly with SMC and VSVA51, systemically. As single agents, SMC therapy resulted in decreased tumor growth rate and modest prolongation of survival, whereas VSVA51 treatment had no effect on tumor size or survival (Figs. 4A and 4B). In stark contrast, combined treatment with SMC and VSVA51 induced dramatic tumor regression and resulted in durable cure in 40% of treated mice. Consistent with the elucidated mechanism of nonspecific killing in vitro, immunofluorescence analysis revealed that VSVA51 infectivity was transient and limited to small intratumor foci (Fig. 4C), whereas caspase-3 activation was widespread in SMC- and VSVA51-cotreated tumors (Fig. 4D). Furthermore, immunoblots with tumor lysates showed caspase-8 and -3 activation in doubly treated tumors (Figs. 4E, 24B, and 24G). Although animals in the combination treatment cohort experienced weight loss, mice fully recovered after the final treatment (Fig. 20A).

To confirm these in vivo data in another model system, the human colorectal adenocarcinoma xenograft model HT-29 was tested in nude mice. HT29 is a cell line that is highly sensitive to killing by SMC and VSVA51 in vitro (Figs. 21A and 21B). Similar to the results obtained in the EMT6 model system, combination therapy with SMC and VSVA51 induced tumor regression and significantly increased mouse survival (Fig. 21C). In contrast, monotherapy had no effect on HT-29 tumors. Furthermore, there was no additional weight loss in double-treated mice compared with SMC-treated mice (Fig. 21D). These results indicate that the synergistic effect is highly effective in the refractory xenograft model and that the adaptive immune response does not play a major role in the efficacy of SMC and OV co-therapy.

The role of innate antiviral responses and immune effectors in the synergistic effect of combined treatment.

We then determined whether oncolytic VSV infection combined with SMC treatment resulted in TNFa- or IFNe-mediated cell death in vivo. We examined whether blocking TNFa signaling with neutralizing antibodies affected SMC and VSVA51 proliferation in the EMT6 tumor model. Compared with isotype-matched control antibodies, treatment with TNF neutralizing antibodies abolished tumor regression and reduced survival to values similar to control and monotherapy groups (Figs. 4F and 4G). This demonstrates that TNFa is required in vivo for the efficacy of the antitumor combination of SMC and oncolytic VSV.

To investigate the role of IFNe signaling in the SMC and OV combination paradigm, EMT6 tumor-bearing Balb/c mice were treated with IFNAR1 blocking antibodies. Mice treated with IFNAR1 blocking antibodies died from viremia within 24-48 h post-infection. Prior to death, tumors were harvested 18-20 h post-infection and tumors were analyzed for caspase activity. Although these animals with defective type I IFN signaling were sick due to high viral load, excised tumors showed no evidence of caspase-8 activity and minimal evidence of caspase-3 activity (Fig. 22), in contrast to controls, which showed the expected caspase activation in the tumor (Fig. 22). These results support the hypothesis that intact type I IFN signaling is required to mediate the antitumor 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, EMT6 tumor treatment was first undertaken in immunodeficient NOD-scid or NSG (NOD-scid-IL2Rγ ^noΛь ) mice. However, as in the IFNAR1 depletion studies, these mice also rapidly died due to viremia. Therefore, the contribution of innate immune cells was addressed using an ex vivo splenocyte culture system as a surrogate model. Innate immune cell populations with TNFa-producing capacity were positively selected and further sorted from naïve splenocytes. Macrophages (CD11b+ F4/8+), neutrophils (CD11b+ Gr1+), NK cells (CD11bCD49b+), and the myeloid-negative (lymphoid) population (CD11b-CD49-) were stimulated with VSVA51 and the conditioned medium was transferred to EMT6 cells to measure cytotoxicity in the presence of SMC. These results indicate that VSVAS1-stimulated macrophages and neutrophils, but not NK cells, are able to produce factors that lead to malignant cell death in the presence of SMC (Fig. 23A). Primary bone marrow-derived macrophages were also isolated, and these macrophages also responded to oncolytic VSV infection in a dose-dependent manner by producing factors that killed EMT6 cells (Fig. 23B). Overall, these data indicate that multiple populations of innate immune response cells can respond to and mediate the observed antitumor effects and that macrophages are the most likely effectors of this response.

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

We next examined whether synthetic TLR agonists, which are known to induce an innate proinflammatory response, would have synergistic activity with SMC therapy. EMT6 cells were cocultured with mouse splenocytes in a tranwell insert system, and the splenocytes were treated with SMC and TLR 3, 4, 7, or 9 agonists. All TLR agonists tested were found to induce nonspecific cell death in SMC-treated EMT6 cells (Fig. 5A). The TLR4, 7, and 9 agonists LPS, imiquimod, and CpG, respectively, were required by splenocytes for nonspecific cell death in EMT6 cells, presumably because their target TLR receptors are not expressed on EMT6 cells. However, the TLR3 agonist poly(I:C) resulted in cell death in EMT6 cells directly in the presence of SMC. Poly(I:C) and CpG were then tested in combination with SMC therapy in vivo. These agonists are selected to be safe for humans and are currently in numerous early-stage clinical trials for the treatment of malignancies. EMT6 tumors were generated and treated as described above. Although poly(I:C) treatment did not affect tumor growth when given alone, the combination with SMC resulted in significant tumor regression and, when administered intraperitoneally, resulted in durable cure in 60% of treated mice (Figs. 5B and 5C). Similarly, CpG monotherapy did not affect tumor size or survival, but when given in combination with SMC therapy, it resulted in tumor regression and durable cure in 8.8% of treated mice (Figs. 5D and 5E). Importantly, the combination therapy was well tolerated by the mice, with their weights returning to pre-treatment levels shortly after cessation of therapy (Figs. 20D and 20C). These data, taken together with the oncolytic VSV results, demonstrate that a series of actively investigated innate immune adjuvants potently and safely interact with SMC therapy in vivo to induce tumor regression and durable cure in several mouse models of refractory aggressive malignancies.

Example 2: Inactivated viral particles, cancer vaccines and stimulatory cytokines synergistically interact with SMC to kill tumors.

The use of modern anti-cancer immunotherapeutics such as BCG (Bacillus Calmette-Guerin), recombinant interferon (e.g., IFNa), and recombinant tumor necrosis factor (e.g., TNFa used in isolated limb perfusion), and the recent clinical use of biologics (e.g., blocking antibodies) together with immune checkpoint inhibitors that overcome tumor-mediated immune suppression (e.g., anti-CTLA-4 and anti-PD-1 or PDL-1 monoclonal antibodies) indicate the potential of cancer immunotherapy as an effective treatment. As shown in Example 1, we demonstrated the robust potential of non-viral immune stimulators to synergize with SMC (Fig. 5). To extend these studies, we also investigated the interaction of SMC with non-replicating rhabdovirus 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 whether NRRPs could directly interact with SMCs, we co-treated different cancer cell lines, EMT6, DBT, and CT-2A, with SMCs and assessed cell viability using Alamar blue. We observed that NRRPs synergized with SMCs in these cancer cell lines (Fig. 25A). To assess whether NRRPs can induce a strong proinflammatory response, we treated fractionated mouse splenocytes with NRRP (or the synthetic CpG ODN 2216 as a positive control), transferred cell culture supernatants into EMT6 cells in culture in a dose-response manner, and treated the cells with vehicle or SMC. It was noted that the immunogenicity of NRRP was at a level similar to that of CpG, as there was a significant proinflammatory response that resulted in a high degree of cell death of EMT6 cells in the presence of SMC (Fig. 25B). Since treatment with CpG and SMC in the EMT6 tumor model resulted in an 88% cure rate (Fig. 5D), these data indicate that the combination of SMC and NRRP may have a strong synergistic effect in vivo.

The authors' success is attributed to the detection of synergistic activity between SMC and live or inactivated oncolytic single-stranded RNA rhabdoviruses (e.g., VSVA51, Maraba-MG1, and NRRP), suggesting that a clinically approved attenuated vaccine may have synergistic activity with SMC. To test this possibility, the authors assessed the synergistic ability of the tuberculosis mycobacterium vaccine, BCG, which is commonly used to treat bladder cancer in situ due to its high local TNFa production, with SMC. Indeed, the combination of SMC and BCG exhibited significant synergistic interactions in killing EMT6 cells in vitro (Fig. 26A). These results were similarly assessed in vivo; The authors observed significant tumor regression with combined treatment with oral SMC and BCG administered locally or systemically (i.e., either intratumorally or intraperitoneally, respectively) (Fig. 26B). These data suggest the applicability

- 78,046,706 approved vaccines in combination cancer immunotherapy with SMC.

Type I IFN has synergistic activity with SMC in vivo

The effects of viruses and possibly other TLR agonists and vaccines appear to be mediated, in part, by type I IFN production, which is controlled by a variety of signaling mechanisms including mRNA translation. Our data demonstrate the clear potential of combining SMC therapy with existing immunotherapies such as recombinant IFN as an effective approach to the treatment of malignancy. To investigate the potential of this combination, we administered two SMC treatment regimens and either intraperitoneal or intratumoral injections of recombinant IFNa in a syngeneic orthotopic EMT6 breast carcinoma model. Although IFNa treatment did not affect EMT6 tumor growth or overall survival, SMC therapy slightly increased mouse survival and had a cure rate of 17% (Fig. 27). However, combination therapy with SMC and intraperitoneal or intratumoral injections of IFNa significantly delayed tumor growth and increased survival of tumor-bearing mice, resulting in cure rates of 57 and 86%, respectively (Fig. 27). These results support the hypothesis that direct stimulation of type I IFN may have synergistic activity with SMC to kill tumors in vivo.

Evaluation of additional oncolytic rhabdoviruses for potential synergistic interactions with SMC.

Although VSVA51 is a preclinical candidate, the oncolytic rhabdoviruses VSV-IFNe and Maraba-MG1 are currently in clinical trials in patients with cancer. As shown in Example 1, we showed that Maraba-MG1 synergized with SMC in vitro (Fig. 9). We also confirmed that SMC synergized with the clinical candidates, VSV-IFNe and VSV-NIS-IFNP (i.e., carrying the imaging gene, NIS, sodium iodide symporter) in EMT6 cells (Fig. 28). To assess whether these viruses could induce a proinflammatory state in vivo, we treated infected mice i.v. 5x10 ⁸ PFU of VSVA51, VSV-IFNe and Maraba-MG1 and measured serum TNFa levels in infected mice. In all cases, a transient but robust increase in TNFa was observed by oncolytic virus infection at 12 h post infection, which was barely detectable at 24 h (Fig. 29). This is logical, since the infection is self-limited in immunocompetent hosts. These results suggest that clinical candidate oncolytic rhabdoviruses may synergize with SMC in a similar manner to VSVA51.

As shown in Example 1, we documented that a form of VSVA51 that was engineered to express full-length TNFa could enhance oncolytic virus-induced killing in the presence of SMC (Fig. 15). To confirm these results, we also engineered VSVA51 to express TNFa in a form in which its intracellular and transmembrane components were replaced with the secretory signal fragment of human serum albumin (VSVA51-solTNFa). Compared to full-length TNFa (memTNFa), solTNFa is constitutively secreted by host cells, while the memTNFa form can be anchored to the plasma membrane (but retains the ability to induce cell death in a juxtacrine manner) or released via endogenous processing by metalloproteases (e.g., ADAM17) to kill cells in a paracrine manner. We assessed whether both forms of TNFa from oncolytic VSV-infected cells would synergize with SMC in an orthotopic syngeneic EMT6 breast cancer model. As expected, SMC treatment slightly delayed the rate of EMT6 tumor growth and slightly increased the survival of tumor-bearing mice, while the combination of vehicle and VSV-51memTNFa or VSVA51-solTNFa had no effect on overall survival or tumor growth rates (Figs. 30A and 30B). On the other hand, virally expressed TNFa significantly delayed tumor growth rates and resulted in a 30% and 70% increase in survival, respectively. Notably, the 40% tumor cure rate with combined SMC and VSVA51 treatment (Fig. 4A) required four treatments and a dose of ^5x108 PFU VSVA51. However, the combination of oncolytic VSV expressing TNFalpha and SMC resulted in increased cure rates and two treatment regimens at a viral dose of 1 x 10 ⁸ PFU. To assess whether this treatment strategy could be applied to other refractory syngeneic models, we assessed whether VSVA51-solTNFa synergized with SMC in a subcutaneous CT-26 colon carcinoma cell line model. As expected, we observed no effect on tumor growth rate or survival with VSVA51solTNFa and observed a modest decrease in tumor growth rate and a small increase in survival (Fig. 30C). However, we were able to enhance tumor growth delay and increase survival of tumor-bearing mice with combined treatment with SMC and VSVA51-solTNFa.

Therefore, the inclusion of a TNFa transgene in an oncolytic virus is a significant advantage for combination with SMC. One could easily envisage the inclusion of other death ligand transgenes such as TRAIL, FasL or lymphotoxin in viruses for synergistic activity.

- 79 046706 sti with SMC.

Exploring the potential of SMCs to kill brain tumors.

The combination of SMC with immune stimulatory agents may be useful in many different types of malignancies, including brain malignancies, for which effective therapies are lacking and immunotherapy is a promising treatment option. As a first step, we determined whether SMC could cross the blood-brain barrier (BBB) in a mouse brain tumor model, as the BBB is a significant barrier to brain invasion. We observed SMC-induced degradation of cIAP1/2 proteins in CT-2A intracranial tumors within hours of drug administration, indicating that SMC are able to cross the BBB and have antagonist activity against cIAP1/2 and possibly XIAP in brain tumors (Fig. 31A). The authors also showed that direct injection of SMC (10 μl of a 100 μM solution) by the intracranial route could result in potent inhibition of both cIAP1/2 and XIAP proteins (Fig. 31B), which could be a direct consequence of SMC-induced autoubiquitination of IAP proteins or a result of induction of tumor cell death in the case of XIAP depletion. Second, the authors wanted to determine whether systemic stimulation of immune stimulators could lead to a proinflammatory response in the brain of naïve mice. Indeed, the authors observed a marked upregulation of brain TNFa levels in mice that were injected intraperitoneally with the TLR3 agonist, poly(I:C), a viral mimetic (Fig. 32A). Based on these data, we extracted crude protein lysates from the brains of mice treated with poly(I:C) or the clinical candidates for oncolytic rhabdoviruses VSVA51, VSV-IFNe, or Maraba-MG1 and then applied these lysates to CT-2A or K1580 glioblastoma cells in the presence of SMC. We observed that stimulation of the innate immune response with these non-viral synthetic or biological viral agents resulted in increased cell death in the presence of SMC in these two glioblastoma cell lines (Fig. 32B). As a third step, we also confirmed that poly(I:C) can be administered directly via the intracranial route without obvious toxic effects, which may provide even greater cytokine induction at the tumor site (Fig. 32C). Finally, we assessed whether direct stimulation of the immune system in the brain or systemic stimulation would result in durable cure in SMC-treated mouse brain tumor models. The combination of oral SMC and poly(I:C), intracranial route, or VSVA51, i.v., resulted in near-complete survival of CT-2A glioma-bearing mice (Figs. 32D and 32E) with expected survival rates of 86 and 100%, respectively. As a consequence of the observed synergy between SMC and intracranial poly(I:C) treatment, we also assessed the possibility of treating CT-2A glioma with direct, simultaneous intracranial injections of SMC and recombinant human IFNa (B/D). Indeed, we observed a marked benefit in mouse survival with the combination treatment, with a cure rate of 50% (Fig. 33). Importantly, single or combined treatment with SMC or IFNa did not result in any overt neurotoxicity in tumor-bearing mice. Overall, these results indicate that various SMC treatments may have synergistic activity with a variety of locally or systemically administered innate immune stimulators to kill malignant cells in vitro and eradicate tumors in animal models of malignancy. METHODS LCL161 reagents from Novartis (Houghton, P. J. et al. Initial testing (stage 1) of LCL161, a SMAC mimetic, by the Pediatric Preclinical Testing Program. Pediatr Blood Cancer 58: 636-639 (2012); 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 (2012). SM-122 and SM-164 from 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 BIR3 domains in XIAP. J Am Chem Soc 129: 1527915294 (2007)). AEG40730 (Bertrand, M. J. et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell 30: 689–700 (2008)) was synthesized by Vibrant Pharma Inc (Brantford, Canada). OICR720 was synthesized by Ontario Institute for Cancer Research (Toronto, Canada) (Enwere, E. K. et al. TWEAK and cIAP1 regulate myoblast fusion through the noncanonical NF-kappaB signalling pathway. Sci Signal 5: ra75 (2013)). IFNa, IFNe, IL28 and IL29 were obtained from PBL Interferonsource (Piscataway, USA). All dsRNAs were obtained from Dharmacon (Ottawa, Canada; ON TARGETplus SMARTpool). CpGODN 2216 was synthesized by IDT (5'-gggGGACGATCGTCgggggg-3' (SEQ ID NO: 1), lowercase indicates phosphorothioate linkages between the indicated nucleotides and italics indicate three CpG motifs with phosphodiester linkages). Imiquimod was purchased from BioVision Inc. (Milpitas, USA). Poly(:C) was obtained from InvivoGen (San Diego, USA). LPS was from Sigma (Oakville, Canada). Cell culture

Cells were maintained at 37°C and 5% _CO2 in DMEM supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and 1% essential amino acids (Invitrogen, Burlington, USA). All cell lines were obtained from ATCC except the following: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Cell lines were routinely tested for mycoplasma contamination. For dsRNA transfections,

- 80 046706 cells were reverse transfected with RNAiMAX Lipofectamine (Invitrogen) or DharmaFECT I (Dharmacon) for 48 h according to the manufacturer's protocol.

Viruses

The Indiana VSVA51 serotype (Stojdl, DF 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 expanded in Vero cells. VSVA51-GFP is a recombinant derivative of VSVA51 expressing jellyfish green fluorescent protein. VSVA51-Fluc expresses firefly luciferase. VSVA51 with a deletion of the gene encoding the glycoprotein (VSVA51AG) was expanded in HEK293T cells transfected with pMD2-G using Lipofectamine2000 (Invitrogen). To generate the VSVA51-TNFa construct, the full-length TNFa gene was inserted between the viral G and L genes. All VSVA51 viruses were purified on a sucrose cushion. Maraba-MG1, WDD-B18R-, reovirus, and HSV1 ICP34.5 were generated as described previously (Brun, J. et al. Identification of genetically modified Maraba virus as an oncolytic rhabdovirus. Mol Ther 18, 1440-1449 (2010); Le Boeuf, F. et al. Synergistic interaction between oncolytic viruses enhances cell death. Mol Ther 18, 888-895 (2011); 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, 1927-1936 (2010)). Generation of adenoviral vectors expressing GFP or expressing GFP together with dominant negative IKKe was performed as described previously 16.

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 μM LCL161 and infected with the indicated MOI of OV or treated with 250 U/ml IFNe, 500 U/ml IFNa, 500 U/ml IFNy, 10 ng/ml IL28, or 10 ng/ml IL29 for 48 h. Cell viability was determined with Alamar blue (resazurin sodium salt (Sigma)) and data were normalized to vehicle treatment. The sample size chosen is consistent with previous reports that used a similar assay for viability assays. For combination indices, cells were seeded overnight, treated with serial dilutions of a fixed mixture of VSVA51 and LCL161 (5000:1, 1000:1, and 400:1 PFU VSVA51: mM LCL161) for 48 h, and cell viability was assessed with Alamar blue. Combination indices (CIs) were calculated according to the method of Chou and Talaly 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, 64386442 (1977)). Statistical parameters (mean with standard deviation or standard error) were determined using n=3 biological replicates.

Distribution analysis

A confluent monolayer of 786-0 cells was overlaid with 0.7% agarose in complete medium. A small hole was made in the agarose layer in the middle of the well with a pipette and 5x103 PFU VSVA51-GFP was added. Medium containing vehicle or 5 μM LCL161 was overlaid, the cells were incubated for 4 days, fluorescence imaging was performed, and the cells were stained with crystal violet.

Co-culture of splenocytes

EMT6 cells were cultured in multiwell plates and cell culture inserts containing unfractionated splenocytes were placed on top. Briefly, single-cell suspensions were generated by passaging mouse spleens through 70 μm nylon mesh and red blood cells were lysed with ASA lysis buffer. Splenocytes were treated for 24 h with either 0.1 MOI VSVA51AG, 1 μg/ml poly(I:C), 1 μg/ml LPS, 2 μM imiquimod or prior to 0.25 μM CpG in the presence of 1 μM LCL161. EMT6 cell viability was determined by crystal violet staining. Statistical parameters (mean, standard deviation) were determined using n=3 biological replicates.

Bioassay for cytokine responses

Cells were infected with the indicated MOI of VSVA51 for 24 h and the cell culture supernatant was UV-irradiated for 1 h to inactivate VSVA51 particles. The UV-inactivated supernatant was then applied to naïve cells in the presence of 5 μM LCL161 for 48 h. Cell viability was assessed using Alamar blue. Statistical parameters (mean, standard deviation) were determined using n=3 biological replicates.

Microscopy

To measure caspase-3/7 activation, 5 μM LCL161, the indicated MOI of VSVA51, and 5 μM CellPlayer Apoptosis Caspase-3/7 Reagent (Essen Bioscience, Ann Arbor, USA) were added to the cells. The cells were placed in an incubator equipped with an IncuCyte Zoom microscope with a 10X objective, and phase contrast and fluorescence images were acquired for 48 h. Alternatively, the cells were treated with 5 μM LCL161 and 0.1 MOI of VSV-51-GFP and SMC for 36 h and labeled using the Magic Red Caspase-3/7 Assay Kit (ImmunoChemsitry Technologies, Bloomington, USA). To measure the proportion of apoptotic cells, 1 μg/ml Appin V-CF594 (Biotium, Hayward, USA) and 0.2 μM YOYO-1 (Invitrogen) were added to SMC- and VSYA51-treated cells. Images were acquired for 24 h after treatment.

- 81 046706 using IncuCyte Zoom. Fluorescence signals were numbered using the counting algorithm integrated into the IncuCyte Zoom software. Statistical parameters (mean, standard deviation) were determined using n=2 (caspase-3/7) or n=9 (Annexin V, YOYO-1) biological replicates.

Multistage growth curves

Cells were treated with vehicle or 5 μM LCL161 for 2 h and then infected at the indicated MOI with VSVA51 for 1 h. Cells were washed with PBS and replenished with vehicle or 5 μM LCL161 and incubated at 37°C. Aliquots were prepared at the indicated time points and virus titers were assessed by standard plaque assay using African green monkey VERO cells.

Western immunoblotting

Cells were scraped, collected by centrifugation and lysed in RIPA lysis buffer containing 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: pSTAT1 (9171), caspase-3 (9661), caspase-8 (9746), caspase-9 (9508), DR5 (3696), TNF-R1 (3736) cFLIP (3210) and PARP (9541) from Cell Signaling Technology (Danvers, USA); caspase-8 (1612) from Enzo Life Sciences (Farmingdale, USA); IFNAR1 (EP899) and TNF-R1 (19139) were from Abcam (Cambridge, USA); cappase-8 (AHZ0502) from Invitrogen; cFLIP (clone NF6) from Alexis Biochemicals (Lausen, Switzerland); RIP1 (clone 38) from BD Biosciences (Franklin Lakes, USA); and E7 from the Developmental Studies Hybridoma Bank (Iowa City, USA). Rabbit anti-rat polyclonal antibodies, IAP1 and IAP3, were used to detect human and mouse cIAP1/2 and XIAP, respectively. AlexaFluor680 (Invitrogen) or IRDye800 (Li-Cor, Lincoln, USA) were used to detect primary antibodies, and infrared fluorescence signals were detected with an 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 III (Invitrogen) and SsoAdvanced SYBR Green supermix (BioRad, Mississauga, Canada) on a Mastercycler ep realplex (Eppendorf, Mississauga, Canada). All primers were obtained from realtimeprimers.com. Statistical parameters (mean, standard deviation) were determined using n=3 biological replicates.

ELISA

Cells were infected with virus at the indicated MOI or treated with IFNe for 24 h, and clarified cell culture supernatants were concentrated using Amicon Ultra filter units. Cytokines were measured using the Quantikine high sensitivity TNFa assay kits, TNFa DuoSet, TRAIL DuoSet (R & D Systems, Minneapolis, USA), and VeriKine IFNe (PBL Interferonsource). Statistical analysis used n=3 biological replicates.

EMT6 breast tumor model

Mammary tumors were generated by injecting 1x105 native EMT6 or firefly luciferase-labeled EMT6 (EMT6-F1uc) cells into the mammary fat pad of 6-week-old female BALB/c mice. Mice bearing palpable tumors (~100 ^mm3 ) were treated concomitantly with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc, pH 4.63) or 50 mg/kg LCL161 per os, and either i.v. injection of either PBS or 5x108 PFU VSVA51 twice weekly for two weeks. For poly(:C) 25 and SMC treatment, animals were injected with LCL161 twice weekly and either BSA (i.v.), 20 μg poly(I:C), or 2.5 mg/kg poly(:C) i.p. four times weekly. The SMC and CpG group was injected with 2 mg/kg CpG (i.p.) followed by CpG and SMC the following day. CpG and SMC were repeated 4 days later. Treatment groups were assigned per cell, and each group had a min n=4-8 for statistical measurements (mean, standard error, Kaplan-Meier log-rank analysis). The sample size is consistent with previous reports examining tumor growth and survival in mice after cancer treatment. Blinding was not possible. Animals were euthanized when the tumor metastasized intraperitoneally or when the tumor burden exceeded 2000 ^mm3 . Tumor volume was calculated using (π)(W) ² (L)/4, where W=tumor width and L=tumor length. Tumor bioluminescence was visualized using a Xenogen 2000 IVIS CCD system (Caliper Life Sciences Massachusetts, USA) after injection of 4 mg luciferin (Gold Biotechnology, St. Louis, USA).

Subcutaneous tumor model NT-29

Subcutaneous tumors were generated by injecting 3x106 HT-29 into the right phalanx of 6-week-old female CD-1 nude mice. Palpable tumors (~200 ^mm3 ) were treated with five intrauterine injections of PBS or 1x108 PFU VSVA51. Four hours later, mice were treated with vehicle or 50 mg/kg LCL161 per os. Treatment groups were assigned per cage, and each group had a min n=5-7 for statistical measurements (mean, standard error, Kaplan-Meier analysis with log-rank). The sample size is consistent with previous reports examining tumor growth and survival in mice after cancer treatment. Blinding was not possible. Animals were euthanized when tumor burden exceeded 2000 ^mm3 . Tumor volume was calculated using (π)(W) ² (L)/4, where

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W=tumor width and L=tumor length.

All animal experiments were performed with the approval of the University of Ottawa Animal Care and Veterinary Service in accordance with accepted guidelines approved by the Canadian Council on Animal Care.

Antibody-mediated cytokine neutralization

To neutralize TNFa signaling in vitro, EMT6 cells were treated with 25 μg/ml α-TNFa (XT3.11) or isotype control (HRPN) for 1 h prior to co-treatment with LCL161 and VSVA51 or IFNe for 48 h. Viability was assessed using Alamar blue. To neutralize TNFa in the EMT6-F1uc tumor model, 0.5 mg α-TNFa or α-HRPN were administered at 8, 10, and 12 days post-implantation. Mice were treated with 50 mg/kg LCL161 (p.o.) at 8, 10, and 12 days post-implantation and infected with 5 x 10 ⁸ PFU VSVA51 i.v. at days 9, 11, and 13. To neutralize the type I IFN signaling pathway, EMT6 tumor-bearing mice were injected with 2.5 mg of a-IFNAR1 (MAR1-5A3) or isotype control (MORS-21) and treated with 50 mg/kg LCL161 (p.o.) for 20 h. Mice were infected with 5x10 ⁸ PFU VSVA51 (i.v.) for 18-20 h, and tumors were analyzed by Western blotting. All antibodies were from BioXCell (West Lebanon, USA).

Flow cytometry and sorting

EMT6 cells were treated simultaneously with 0.1 MOI VSVA51-GFP and 5 μM LCL161 for 20 h. Cells were trypsinized, permeabilized with 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 were analyzed with FlowJo (Tree Star, Ashland, USA).

Splenocytes were enriched for CD11b using the EasySep CD11b Positive Selection Kit (StemCell Technologies, Vancouver, Canada). CD49+ cells were enriched using the EasySep CD49b Positive Selection Kit (StemCell Technologies) from the CD11b- fraction. CD11b+ cells were stained with F4/80PE-Cy5 (BM8, eBioscience) and Gr1-FITC (RB6-8C5, BD Biosciences) and then sorted using MoFlo Astrios (Beckman Coulter). Flow cytometry data were analyzed using Kaluza (Beckman Coulter). Isolated cells were infected with VSVA51 for 24 h, and purified cell culture supernatants were added to EMT6 cells for 24 h in the presence of 5 μM LCL161.

Bone marrow derived macrophages

Mouse femurs and radii were obtained and washed to remove bone marrow. Cells were cultured in RPMI with 8% FBS and 5 ng/ml M-CSF for 7 days. Flow cytometry was used to confirm the purity of macrophages (F4/80+ CD11b+).

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 12 μm. Sections were permeabilized with 0.1% Triton X100 in blocking solution (50 mM Tris-HCl, pH 7.4, 100 mM L-lysine, 145 mM NaCl, and 1% BSA, 10% goat serum). α-cleaved caspase 3 (C92-605, BD Pharmingen, Mississauga, Canada) and VSV polyclonal antiserum (Dr. Earl Brown, University of Ottawa, Canada) were incubated overnight and then reincubated with AlexaFluor-coupled secondary antibodies (Invitrogen).

Statistical analysis

Comparison of Kaplan-Meier survival plots by log-rank analysis and subsequent pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot, San Jose, USA). _EC50 values were calculated in GraphPad Prism using normalized nonlinear regression analysis. The _EC50 shift was calculated by subtracting the _{log10 EC50} of SMC-treated VSVA51-infected cells from the _{log10 EC50} of vehicle-treated VSVA51-infected cells. To normalize the degree of SMC synergy, the _EC50 value was normalized to 100% to compensate for cell death caused by SMC treatment alone.

Example 3: SMC-containing immunotherapy showed anti-myeloma activity.

Immune checkpoint blockade has synergistic activity with SMC treatment and slows disease progression in MM.

MPC-11 cells stably expressing the luciferase transgene were implanted by intravenous injection into BALB/c mice. This in vivo model of MM effectively mimics the human disease and subsequent prognosis of disease progression. MPC-11 cells were derived from mouse plasmacytoma. After two rounds of treatment with SMC and monoclonal antibodies against PD-1 or CTLA-4, anti-PD-1 monotherapy showed a response in terms of delayed disease progression. Mice treated with the combination of anti-PD-1 and SMC showed a better response, with virtually no tumor burden as determined by the luminescence signal (Fig. 35). This combination also significantly prolonged mouse survival compared to controls (p=0.01) and was comparable to PD-1 monotherapy (p=0.0163).

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Type 1 interferons have synergistic activity with SMC and induce MM cell death.

In an in vitro study analyzing the effects of various cytokines in combination with SMC, attention was focused on type 1 IFN. In particular, IFNa and IFNe showed very strong synergistic activity in killing MM cells together with SMC in most of the cell lines tested (Fig. 36A). Using the same MPC-11 mouse model, mice were treated with recombinant IFNa and SMC at three different time points (Fig. 37).

Oncolytic viruses have synergistic activity with SMC and cause death of MM cells.

The vesicular somatic virus-derived oncolytic virus VSVA51 has synergistic activity with SMC in vitro and induces cell death in MPC-11 cells (Figs. 36B and 36C). SMC-containing combinations were also tested in a syngeneic MPC-11 mouse model. Combination treatment did not reduce tumor burden as effectively as VSVA51 and was poorly tolerated by the mice. However, VSVA51 monotherapy delayed disease progression and the improvement in survival was significant compared with the untreated control group (p=0.0379, log-rank analysis) (Fig. 38).

SMC has synergistic activity with standard therapies in MM.

In vitro viability assays showed synergistic killing of MM cells in the SMC-based combination with the synthetic glucocorticoid dexamethasone (Dex) (Fig. 39B). When SMC was combined with the glucocorticoid receptor antagonist RU486, cell death levels were comparable, suggesting that the synergistic activity may not be due to GCR activation but to its inhibitory effect on NF-κB.

SMC-based combination treatment activates NF-κB signaling and induces MM cell apoptosis.

SMC treatment effectively induced rapid degradation of cIAP1 and cIAP2 (Fig. 40A). When given alone, SMC treatment increased NF-κB signaling; starting with a small transient increase in the classical pathway, as evidenced by a higher ratio of phosphorylated-p65 to p65, and then decreasing (Fig. 40B). As classical pathway activation was attenuated, the alternative pathway became highly activated, as evidenced by an increase in the ratio of p52 to p100 (Fig. 40C). Apoptosis in cells was confirmed by the presence of cleaved poly(ADP-ribose) polymerase (PARP). PARP cleavage is often used as an apoptotic marker because it is a substrate for caspases in the early stages of apoptosis.

The combination of SMC with either IFNe (Fig. 41) or VSVA5 or VSVmIFN (containing an integrated murine IFNe gene) (Fig. 42) shared many of the same characteristics as SMC monotherapy. For instance, the classical pathway was eventually down-regulated and the alternative pathway was upregulated. Apoptosis was also present, as evidenced by PARP cleavage and caspase 8 cleavage. The IFN receptor, IFNAR1, was also inhibited by IFN treatment, which is of particular interest since it is required for a sustained response. With VSV treatment, RIP1 was almost completely degraded at late time points; this is another signal for apoptosis, as it is degraded by caspase 8 after ripoptosome formation.

SMC sensitivity in MM1R and MM1S is associated with glucocorticoid receptor expression.

The response to SMC-mediated cell death varies greatly between the human MM1R and MM1S cell lines, which are derived from the same parental line and differ only in GCR expression. MM1R, which lacks detectable GCR expression (Fig. 39A), is highly sensitive to SMC (Fig. 39C), whereas MM1S, which has high levels of GCR expression, is resistant. MM1S can be made sensitive to SMC treatment by treatment with either Dex or the GCR antagonist RU486 (Fig. 39B).

Stimulators of the innate immune response activate inhibitors of the adaptive immune response.

Human MM cell lines, U266, MM1R, and MM1S, robustly upregulated PD-L1 in response to IFNe treatment. A comparable upregulation was also seen for the combination of SMC and IFNe. Another ligand for PD-1, PD-L2, was similarly upregulated by IFNe-based treatment. This effect was seen at both early and late time points for both proteins (Fig. 43). This suggests that any immune stimulants that upregulate type 1 IFNs will result in upregulation of T cell coinhibitory molecules.

The combination of SMC and immunomodulatory agents results in the death of malignant cells, which also include CD8+ T cells.

Figures 44A and 44B are graphs showing data from an experiment in which mice cured after double treatment were re-injected intramammary fat pad with EMT6 cells (180 days after the initial postimplantation date) or intracranially injected with CT-2A cells (190 days after the initial postimplantation date).

Fig. 44C shows a graph that shows the data from an experiment in which

- 84 046706 CT-2A glioma cells or EMT6 breast cancer cells were trypsinized, surface-stained with isotype control-conjugated IgG or anti-PD-L1, and subjected to flow cytometry.

Figure 44D is a graph showing data from an experiment in which CD8+ T cells were isolated from splenocytes (from naïve mice or mice previously cured of EMT6 tumor) using a CD8 T cell positive magnetic selection kit and subjected to an ELISpot assay for IFNy and Granzyme B. CD8+ T cells were cocultured with medium or cancer cells (12:1 cancer to CD8+ T cell ratio) and 10 mg control IgG or anti-PD-1 for 48 h. Three mice were used as independent biological replicates (previously cured of EMT6 tumor). 4T1 cells served as a negative control since 4T1 and EMT6 cells share the same MHC antigens.

SMC and immune checkpoint inhibitors are synergistic in orthotopic mouse models of cancer.

Fig. 45A is a graph showing data from an experiment in which mice bearing EMT6 mammary tumors were injected intramuscularly with PBS or 1 x ¹⁰⁸ PFU VSVD51 once and five days later the mice were injected with combinations of vehicle or 50 mg/kg LCL161 (SMC) orally and 250 mg anti-PD intraperitoneally (i.p.). Figs. 45B and 45C are graphs showing data from an experiment in which mice bearing CT-2A or GL261 intracranial tumors were injected four times with vehicle or 75 mg/kg LCL161 (orally) and 250 mg (i.p.) control IgG, anti-PD-1, or anti-CTLA4.

Fig. 45D is a graph showing data from an experiment in which athymic CD-1 nude mice bearing CT-2A intracranial tumors were treated with 75 mg/kg LCL161 (po) and 250 mg (ip) anti-PD-1.

Example 4. Smac mimetics have synergistic activity with immune checkpoint inhibitors and promote tumor immunity.

Cell culture

Cell lines RPMI-8226, U266, MM1R, MM1S, MPC-11 were purchased from ATCC. MPC-11 was cultured in DMEM (Hyclone) with 10% FBS (Hyclone), U266 was cultured in RPMI-1640 (Hyclone) with 15% FBS, all other lines were cultured in RPMI-1640 with 10% FBS.

Cells were maintained at 37°C and 5% _CO2 in DMEM supplemented with 10% heat-inactivated fetal calf serum and 1% essential amino acids (Invitrogen). All cell lines were obtained from ATCC except the following: SNB75 (Dr. D. Stojdl, Children's Hospital of Eastern Ontario Research Institute) and SF539 (UCSF Brain Tumor Bank). Primary NF1-/+p53-/+ cells were obtained from C57B1/6J p53+/-/NF1+/- mice. Cell lines were routinely tested for mycoplasma contamination. BTIC were cultured in serum-free culture medium supplemented with EGF and FGF-250. For dsRNA transfection, cells were reverse transfected with RNAiMAX Lipofectamine (Invitrogen) or DharmaFECT I (Dharmacon) for 48 h according to the manufacturer's protocol. Cell lines were continuously tested for mycoplasma contamination. BTIC were cultured in serum-free culture medium supplemented with EGF and FGF-2. For dsRNA transfection, cells were reverse transfected with RNAiMAX Lipofectamine (Invitrogen) or DharmaFECT I (Dharmacon) for 48 h according to the manufacturer's protocol. Antibodies and reagents In vivo: LCL161 was a generous gift from Novartis. Anti-PD-1 (clone J43) was purchased from BioXcell. Poly(:C) (HMW vaccine grade, Invivogen). IFNa (for in vivo use) was a generous gift from Dr. Peter Staeheli, Germany. Tetralogic Pharmaceuticals received Birinapant.

In vitro: IFN was obtained from PBL assay; Dexamethasone and RU486 were purchased from Sigma Aldrich.

Antibodies used include RIAP1 (available from stock), PD-L1 (Abcam), PD-L2 (R&D Systems), GCR (Snata Cruz), P100 (Cell Signaling), P65 (Cell signaling), p-P65 (Cell signaling), IFNAR1 (Abcam), PARP (Cell Signaling), tubulin (Developmental Studies Hybridoma Bank), RIP1 (R&D Systems), caspase 8 (R&D Systems).

AT-406, GDC-0917 and AZD-5582 were purchased from Active Biochem.

TNF-α was purchased from Enzo. TNF-α was obtained from PBL Assay Science. Broad host range IFNaB/D was produced in yeast and purified by affinity immunochromatography. Non-targeting dsRNA or csRNA targeting cFLIP was obtained from Dharmacon (ON-TARGETplus SMARTpool). High molecular weight poly(:C) was obtained from Invivogen.

Working with animals

Four- to five-week-old BALB/c mice were purchased from Charles River and injected i.v. with 1 x 10 ⁶ MPC-II Fluc cells stably expressing firefly luciferase (Fluc) transgenes. Treatment included 50 mg/kg LCL161, 250 μg anti-PD-1, 250 μg anti-CTLA4, 25 μg poly(:C), 5 x 10 ⁸ pfu VSVA51, and 1 μg IFNa. Mice were imaged using the IVIS in vivo imaging system after i.v. injection of 200 μl luciferin to measure luminescence.

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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 amplified in Vero cells and purified on an OptiPrep gradient. VSVA51 with a deleted gene encoding the glycoprotein (VSVA51AG) was amplified in HEK293T cells transfected with pMD2-G using Lipofectamine2000 (Invitrogen) and purified on a sucrose cushion. NRRP was generated by treating VSV-EGFP with ultraviolet light (250 mJ/cm ² ) using the 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 IFN-aB/D, 0.1 ηγ μλ ^-1 TNF-α, or the indicated amount of NRRP for 48 h. Cell viability was determined with Alamar blue (resazurin sodium salt (Sigma)), and data were normalized to vehicle treatment. The sample size chosen is consistent with previous reports that used a similar assay for viability assays but did not employ statistical methods to determine the size.

Western blotting

Cells were scraped, collected by centrifugation, and lysed in RIPA lysis buffer containing protease inhibitor cocktail (Roche). Tumors were dissected, 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 cFLIP (7F10, 1:500, Alexis Biochemicals) and β-tubulin (1:1000, E7 from Hybridoma Bank Developmental Studies). Rabbit anti-rat polyclonal antibodies, IAP1 and IAP3, were used to detect human and mouse cIAP1/2 and XIAP, respectively (1:5000; Cyclex Co.). AlexaFluor680 (Invitrogen) or IRDye800 (Li-Cor) (1:2500) was used to detect primary antibodies, and infrared fluorescent signals were detected using an Odyssey infrared imaging system (Li-Cor). Full-length blots are shown in Fig. 68A-68D.

ELISA

For in vivo TNF-α detection, mice were treated with 50 μg poly(:C) intraperitoneally (i.p.) or 5x108 PFU VSVA51 intravenously (i.v.). Brains were homogenized in 20 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 10% glycerol, and 1 mM MgCl2, supplemented with EDTA-free protease inhibitor cocktail (Roche). NP-40 was added to a final concentration of 0.1% and clarified by centrifugation. Equal amounts were processed for TNF-a detection using QuantKine TNF-α 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 and age-matched control (naïve) C57BL/6 females were injected subcutaneously with 1x106 CT-2A cells. Seven days later, splenocytes were isolated and co-cultured with CT-2A cells for 48 h (spleen to tumor cell ratio 20:1) in the presence of vehicle or 5 μM SMC or 20 μg ml ^-1 of the indicated antibodies. Secretion of IFN-γ, GrzB, TNF-α, IL-17, IL-6 and IL-10 was determined by ELISA (R&D Systems kits). CT-2A and GL261 Brain Tumor Models Five-week-old female C57BL/6 or CD-1 nude mice were anesthetized with isofluorane and the surgical site was shaved and treated with 70% ethanol. 5x104 cells were stereotaxically injected in a volume of 10 μl into the left striatum for 1 min at the following coordinates: 0.5 mm anterior, 2 mm lateral to the parietal bone and 3.5 mm deep. The skin was closed with surgical glue. Mice were treated with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc, pH 4.63) or 75 mg kg ^-1 LCL161 orally and intratumorally (i.v.), in 10 μl with 50 μg poly(:C) intravenously (i.v.), with 5x108 VSVA51, or intraperitoneally (i.p.) with 250 μg anti-CD4 (GK1.5), anti-CD8 (YTS169.4), anti-PD1 (J43), or CTLA-4 (9H10).

For birinpatine administration, mice were treated with vehicle (12.5% Captisol) or 30 mg kg ^-1 birinpatine (i.p.). In some cases, animals were treated with anti-IFNAR1 (MAR1-5A3), anti-IFN-γ (R4-6A2), or anti-TNF-α (XT3.11). Isotype IgG antibody controls were used as follows: BE0091, BE0087, BP0090, MORS-21, or HPRN. All neutralizing and control antibodies were purchased from BioXCell. For intracranial administration of SMC and type I IFN, mice were treated with 10 μl of a combination of vehicle (0.5% DMSO), 100 μM LCL161, 0.01% BSA, or 1 μg IFN-aB/D. Alternatively, mice were orally treated with vehicle or 75 mg kg ^-1 LCL161 and 1 μg IFN-α B/D (i.p.). Animals were euthanized when they showed predefined signs of neurological deficit (inability to ambulate, weight loss >20% of body weight, lethargy, hunched posture). Treatment groups were assigned by cage, and each group had 5 to 9 mice for statistical measurements (Kaplan-Meier analysis with log-rank test). There was no randomization, and the principal investigator was blinded to group assignment.

The sample size is consistent with previous reports that examined tumor growth and survival in mice following cancer treatment, but to determine the sample size

- 86 046706 statistical analyses were not performed.

MRI

MRI of the living mouse brain was performed at the University of Ottawa Preclinical Imaging Facility using a 7 Tesla GE/Agilent MR 901. Mice were anesthetized for MRI using isoflurane. A dual fast echo (FSE) pulse sequence was used to acquire a 2D image with the following parameters: 15 slices, slice thickness = 0.7 mm, distance = 0 mm, field of view = 2 cm, matrix = 256 χ 256, echo time = 25 ms, repetition time = 3000 ms, echo length = 8, bandwidth = 16 kHz, 1 mean and fat saturation. The FSE sequence was acquired in both the transverse and coronal planes, with a total imaging time of approximately 5 min.

EMT6 breast tumor model

Mammary tumors were generated by injecting 1x105 EMT6 cells into the mammary fat pad of 5-week-old female BALB/c mice. Mice bearing palpable tumors (~100 ^mm3 ) were co-administered with either vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc, pH 4.63) or 50 mg/kg LCL161 orally and either i.p. injection of 5x108 PFU VSVA51 or i.p. injections of control IgG (BE0091) or anti-PD-1 (O43). Animals were euthanized when tumors metastasized intraperitoneally or when tumor burden exceeded 2000 ^mm3 . Tumor volume was calculated using (π)(W) ² (L)/4, where W=tumor width and L=tumor length. Treatment groups were assigned by cage, and each group had 4 to 5 mice for statistical measurements (mean, standard error, Kaplan-Meier log-rank analysis). There was no randomization, and the principal investigator was blinded to group assignment.

Multiple myeloma model MRS-11

A mouse model of multiple myeloma and plasmacytoma was established by injecting 1x106 MPC luciferase-labeled MPC-11 cells (i.v.) into 4-5-week-old female BALB/c mice. Mice were treated with vehicle (30% 0.1 M HCl, 70% 0.1 M NaOAc pH 4.63) or 75 mg kg ^-1 LCL161 orally and 250 μg control IgG or a-PD-1 antibodies (i.p.). Bioluminescence was visualized using a Xenogen 2000 IVIS CCD system (Caliper Life Sciences) after injection of 4 mg luciferin (Gold Biotechnology). Treatment groups were assigned by cage, and each group had 3 to 4 mice for statistical measurements (Kaplan-Meier analysis with log-rank test). There was no randomization, and the principal investigator was blinded to group assignment.

Recurrent tumor burden

Age-matched naïve female C57BL/6 mice or mice previously cured of CT2A intracranial tumors with SMC-based combination therapy and immunostimulants (at least 180 days post-implantation) were reinjected with CT-2A cells i.v. as described above or with 5x105 cells subcutaneously. Naïve BALB/c mice or mice previously cured of luciferase-labeled EMT6 mammary tumors with SMC and VSVA51 combination treatment (90 to 120 days post-implantation) were reinjected with 5x105 unlabeled EMT6 cells intrafat. Animals were euthanized as described above. Blinding or randomization was not possible.

All animal experiments were performed with the approval of the University of Ottawa Animal Care and Veterinary Service in accordance with accepted guidelines approved by the Canadian Council on Animal Care.

Flow cytometry

For in vitro analysis, cells were treated with vehicle (0.01% DMSO) or 5 μM LCL161 and 0.01% BSA, 1 npm mL- ¹ TNF-α, 250 U mL-1 IFN-β, or 0.1 MOI VSVA51 for 24 h. Cells were detached from plates with enzyme-free dissociation buffer (Gibco) and stained with Zombie Green and the indicated antibodies. For analysis of immune infiltrates within tumors, CT-2A intracranial tumors were mechanically dissociated, red blood cells were lysed in ASA lysis buffer, and stained with Zombie Green and the indicated antibodies. In some cases, cells were stimulated with 5 μg/ml PMA and 500 ng/ml ionimicin in the presence of Brefeldin A for 5 h, and intracellular antigens were processed using the BD Cytofix/Cytoperm kit. Antibodies included Fc block (101319, 1:500), PD-L1 (10F.9G2, 1:250), PD-L2 (TY25, 1:100), IA/IE (M5/114.15.2, 1:200) and H-2Kd/H-2Dd- (34-1-2S, 1:200), CD45 (30-F11, 1:300), CD3 (17A2, 1:500), CD4 (GK1.5, 1:500), CD8 (53-6.7,1:500), PD-1 (29.1A12, 1:200), CD25 (PC61, 1:150), Gr1 (RB6-AC5, 1:200), F4/80 (BM8, 1:200), GrzB (GB11, 1:150), and IFN-γ (XMG1.2, 1:200). All antibodies were from BioLegend except TNF-α (MP6-XT22, 1:200) and CD11b (M1/70, 1:100), which were from BD Biosciences. Cells were analyzed on Cyan ADP 9 (Beckman Coulter) or BD Fortessa (BD Biosciences), and data were analyzed using FlowJo (Tree Star).

Microscopy

Detection of mKate2-CT-2A cells was performed in an incubator equipped with an Incucyte Zoom microscope equipped with a 10X objective. Fluorescence signals from the Incucyte Zoom were numbered using a counting algorithm integrated into the IncuCyte Zoom software.

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Multiplex ELISA

Detection of serum proteins after treatment with a combination of SMC and anti-PD-1 was analyzed using a flow cytometry-based multiplex kit (LEGENDplex 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 performed using PD-L1 and PD-L2 primers (Qiagen) and SIBR green reagent (Bio-Rad). Relative expression was calculated as AACt using RPL13A as a control.

A library panel of cytokine and chemokine genes was obtained from realtimeprimers.com. Each run was n=4 and data were normalized to eight different reference genes and compared to each sample and the IgG sample. Data were analyzed by hierarchical analysis using Morpheus.

ELISpot

CD8+ T cells were obtained from splenocytes of age-matched naïve female mice or mice previously cured of CT-2A intracranial tumor (180 days post-implantation) or EMT6 mammary tumors (120 days post-implantation) using a CD8 Magnetic Field Assay Kit (Stemcell Technologies). CD8+ cells were co-cultured with malignant cells (1:20 for CT-2A, LLC and 1:12.5 for EMT6 or 4T1 cells) and with 10 μg ml-1 IgG (BE0091) or anti-PD-I (J43) for 48 h using IFN-γ or Granzyme B ELISpot kits (R&D Systems).

Statistics

For all animal studies, survival was calculated as days after MM cell implantation and plotted as Kaplan-Meier curves. From these, the log-rank test was used to determine significance. For in vitro viability assays, the standard deviation is shown. Post hoc pairwise multiple comparisons were performed using the Holm-Sidak method (SigmaPlot). Comparisons between multiple treatment groups were analyzed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test with adjustments for multiple comparisons (GraphPad). Estimates of variance were analyzed using GraphPad. Comparisons between treatment pairs were analyzed using two-tailed t-tests (GraphPad).

Example 5. Combination of immunostimulating agents for the treatment of glioblastoma.

The authors showed that cultured primary glioblastoma cell lines were killed by SMC in combination with exogenous TNF-α, the oncolytic virus VSVA51, or by infection with the non-replicating VSVA51AG (Figs. 46A and 46B). The authors confirmed that the synergistic effects of SMC, LCL161, and TNF-α are a general phenomenon in this class of drugs, as the authors observed cell death of glioblastoma cells in combination with TNF-α and various SMCs (Fig. 47). In addition, the authors also observed enhanced efficacy of SMC with the oncolytic rhabdoviruses VSVA51 or Maraba-MG1 against human tumor-initiating cells (BTICs) (Fig. 46C). Non-replicating rhabdovirus particles (NRRPs), which retain their infectious and immunostimulatory properties but are unable to replicate, were similarly found to interact with SMC and induce cell death in glioblastoma cells. Notably, only approximately 50% of defined cancer cell lines were sensitized to the combination of SMC and TNF-α or TNF-related apoptosis-inducing ligand (TRAIL), resulting in cell death; the majority of resistant cell lines were additionally sensitized to inhibition of the caspase-8 inhibitor, cFLIP (cellular FLICE-like inhibitory protein), resulting in cell death. Consistent with previous results, two glioblastoma cell lines that were not refractory to combination treatment with SMC and TNF-α or VSVA51AG were killed by cFLIP silencing (Figs. 48A and 48B). In contrast, normal human diploid fibroblasts were not sensitized to cFLIP inhibition and combination treatment, resulting in cell death. These data suggest that IFN and/or cytokine responses, rather than direct virus-induced cytolysis, are responsible for SMC-induced glioblastoma cell death.

Because VSVA51 is neurotoxic and because challenges remain in the immune-protected brain microenvironment and in drug penetration across the blood-brain barrier (BBB), we decided to test the effects of systemic delivery and the intracranial route of administration of the immunotherapeutic agent. After generating CT-2A intracranial tumors (Figs. 49A and 49B), we tested whether systemic administration via oral gavage of SMC, LCL161, could induce transient degradation of the primary target proteins, cIAP1 and CIAP2, within mouse intracranial tumors. However, we did not observe cIAP inhibition in adjacent non-tumor tissues.

- 88 046706 brain, as well as in the cortex or cerebellum of tumor-free mice (Fig. 51). Thus, SMC are able to infiltrate brain tumors with defective BBB. Systemic administration of immunostimulatory agents such as the synthetic TLR3 agonist poly(:C) injected intraperitoneally (i.p.) or the oncolytic virus VSVA51 administered intravenously (i.v.) induced TNF-α cytokine production in the serum and brain of tumor-free mice.

When mice bearing intracranial CT-2A glioblastoma were given a single dose of SMC (oral gavage), VSVA51 (i.v.), or poly(I:C) (intracranial route, i.v.), the survival benefit was minimal in mice with aggressive malignancies (17% survival rate) (Fig. 55C). However, the combination of systemic SMC and the immune-stimulatory trigger, VSVA51 or poly(I:C), significantly prolonged survival and resulted in durable cure in 71% or 86% of mice, respectively. Tumors (which were not labeled with foreign protein to enhance immunity) were imaged at 40 days postimplantation using MRI to confirm the observed treatment effects.

Virus-induced immune effects are mediated in part by type I IFN. We showed that CT-2A cells were partially sensitive to combined SMC and recombinant IFN-α in vitro (Fig. 50A). We found that intracranial administration of SMC resulted in even greater degradation of IAP proteins in CT-2A brain tumors (Fig. 33). For in vivo studies, we used a form of recombinant IFN-α that consists of a hybrid of human IFN-α B and IFN-α D isoforms that exhibits potent antiviral activity against a broad range of species. A single coadministration of SMC and IFN-α significantly increased mouse survival and resulted in a 50% cure rate. Survivors showed no overt physical or behavioral defects to single or combined intracranial administration of SMC, poly(:C), or IFN-α (Fig. 54). Furthermore, because we observed a transient increase in intracranial TNF-α in the brain upon systemic VSVA51 infection or upon poly(:C) treatment, we sought to determine whether systemic administration of recombinant IFN-α along with SMC treatment would be effective in the CT-2A glioblastoma model. Similar to the combination of SMC and VSVA51, the combination of i.p. IFNα along with oral gavage of SMC resulted in durable cure in 55% of the mice (Fig. 50B). These results indicate that the presence of a transient inflammatory environment in the brain is permissible and suggest the feasibility of indirect and other direct (intracranial) routes of combination therapy.

Example 6. Obtaining long-term immunity to a tumor in cured mice.

The innate immune system plays a key role in SMC-mediated tumor cell killing. However, fundamental questions remain regarding the role of the adaptive immune system in this combined SMC approach. Furthermore, a potential pitfall of the proposed use of oncolytic viruses or other immunostimulatory agents in combination with SMC treatment may be the upregulation of cancer checkpoint ligand inhibitor expression, thereby nullifying the tumor-mediated CTL attack. Flow cytometric analysis revealed that treatment of glioma cells with recombinant type I IFN or VSVA51 infection, but not TNF-α treatment, resulted in increased surface expression of PD-L1 and major histocompatibility complex (MHC) I markers. Furthermore, tumor surface expression of these molecules was not affected by SMC treatment (Figs. 25A and ​and5B).

Interestingly, mice previously cured of EMT6 orthotopic mammary carcinoma with SMC combination therapy were completely resistant to tumor engraftment when rechallenged with EMT6 tumor cells (Fig. 52B). However, another syngeneic cell line, 4T1, which also utilizes MHC proteins, was not rejected by these cured mice. We found that mice cured of CT-2A intracranial tumors were also resistant to engraftment of CT-2A tumor cells injected either subcutaneously or intracranially (Fig. 52C). We then assessed the cytotoxic potential of CD8 T cells from cured mice using an ELISpot assay. Stimulation of CD8+ T cells from cured mice, but not from naïve mice, with CT-2A cells revealed the presence of specific reactive T cells, as demonstrated by increased IFN-γ and Granzyme B (GrzB) production (Fig. 54A). Treatment with anti-PD-1 blocking antibodies further increased IFN-γ and GrzB expression. Similar results were observed in mice cured of EMT6 tumors (Fig. 44D). Collectively, these results suggest the generation of robust and tumor-specific long-term immunity using SMC combination therapy.

Example 7. Immune checkpoint inhibitors have synergistic activity with IAP antagonists.

The authors then investigated whether an existing class of cancer immunotherapies known as immune checkpoint inhibitors, or ICIs, could enhance the efficacy of SMCs. ICI treatment of glioblastoma in mice was recently reported to result in at least partial survival. The authors first tried

- 89 046706 aimed to determine whether SMC treatment affects PD-1 expression in a subset of infiltrating immune cells in CT-2A brain tumors. Although there was no statistical difference between the levels of infiltrating CD3+ or CD3+CD8+ cells within intracranial CT-2A tumors, we did observe a robust increase in CD3+ and CD3+CD8+ cells expressing the immune checkpoint protein, PD-1 (Figs. 54B and 55). Despite an overall increase in PD-L1 expression in CD25− cells, which are predominantly CT-2A cells, this trend did not reach statistical significance (Fig. 55C).

To determine whether elevated levels of PD-1+ CD8 T cells could negatively modulate the efficacy of SMC, we evaluated blockade of PD-1 as well as CTLA-4 in combination with SMC using two murine glioblastoma models. Systemic administration of anti-PD-1 or anti-CTLA4 antibodies showed no activity on their own (Figures 54D and 54E). In contrast, the combination of anti-PD-1 and SMC significantly prolonged survival and, respectively, provided 71 and 33% durable cure rates in the CT-2A and GL261 models. Furthermore, when combined with SMC, the bioactivity of anti-PD-1 was superior to that of anti-CTLA-4 in the CT-2A model (71% vs. 43%, Figure 54D).

There are two structural classes of SMC: monomers and dimers. Monomeric SMCs are composed of a single chemical molecule that binds to the BIR domains of IAPs, whereas dimeric SMCs are composed of two SMC molecules joined by a linker, allowing for cooperative binding and/or attachment to IAPs. An advanced SMC, LCL161, is the focus of much of the authors’ research and is a potent monomer. The authors then sought to evaluate whether another advanced dimeric SMC similarly interacts with ICIs for the treatment of glioblastoma. The authors noted a significant increase in survival in mice bearing CT-2A intracranial tumors when treated with the anti-PD-1 and SMC dimer, birinapant (Fig. 54F). Because combined blockade of PD-1 or CTLA-4 is effective in patients with melanoma, we sought to determine whether the combination of PD-1 and CTLA-4 would also significantly improve SMC therapy. The combination of antibodies targeting PD-1 and CTLA-4 was effective in inducing a durable therapeutic effect in mouse models of malignancy, with the authors observing an overall survival of 67% (Fig. 54G). Strikingly, administering SMC therapy with both anti-PD-1 and anti-CTLA-4 together resulted in a robust cure rate of 100%.

The synergistic effect of SMC and ICI is not limited to brain tumors. The authors also observed a significant increase in survival in a mouse model of highly aggressive and refractory multiple myeloma using MPC-11 cells (Figs. 56A and 56B). A robust cure rate of 75% was also obtained in mice bearing EMT6 mammary tumors, which was further increased to 100% by administration of the immune response stimulator (Figs. 57A-57C).

Example 8: CD8+ T cells are required for the efficacy of SMC and ICI.

To gain insight into the underlying cellular mechanism of action, we modulated immune factor production from CT-2A cells co-cultured with splenocytes obtained from mice cured of CT-2A intracranial tumors using combined SMC and anti-PD-1 therapy. We observed significant increases in IFN-γ and GrzB production from CT-2A cells co-cultured with splenocytes obtained from surviving mice (Figures 58A and 59A). Notably, there was an increase in interleukin 17 (IL17) production. We also observed a decrease in the expression of the proinflammatory cytokines IL-6 and TNF-α, which was unexpected given that IL-17 has previously been shown to stimulate the NFκB pathway.

However, IFN-γ and IL-17 expression from splenocytes isolated from cured mice was significantly increased by anti-PD-1 or PD-L1 treatment, suggesting that T cell-based immune responses can be enhanced by inhibition of PD-1 signaling axis checkpoints. We next sought to determine whether this gene response was affected by SMC treatment. Among the cytokines previously analyzed, inclusion of SMC in these cocultures along with anti-PD-1 blockade increased the secretion of IFN-γ, GrzB, IL-17, and TNF-α (Figures ​(Figures59B and ​and9C). 59C). Notably, IL-6 levels in the supernatant were not affected by SMC treatment. Furthermore, the immunosuppressive cytokine IL-10 had a general trend for decreased secretion with combined SMC and anti-PD-L1 treatment.

Given the elevated levels of GrzB, a cytotoxic factor that is partially blocked by XIAP31-33 and TNF-α, we next assessed whether co-culture of glioblastoma cells and splenocytes in naïve mice or mice previously cured of CT-2A intracranial tumors resulted in CT-2A cell death. Using various patterned SMCs, we observed a statistically significant increase in CT-2A cell death in the presence of SMCs, and this response was enhanced by the inclusion of anti-PD-1 antibodies (Fig. 59C).

Taken together, these results indicate that robust effector T cell responses are induced by combined ICI and SMC treatment. To further elucidate the cellular mechanism, the authors

- 90 046 706 depleted immune cells using specific CD4 or CD8-targeting antibodies. The 71% cure rate with the combination therapy was found to be completely reversed by depletion of CD8+ T cells (Fig. 59D). Interestingly, 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 indicate that depletion of CD4+ immunosuppressive cells (such as regulatory T cells) facilitates the induction of tumor regression and that CD4+ cells are not required for the efficacy of the combination treatment approach. In a second approach, CD1 nude mice lacking functional T cells developed intracranial CT-2A tumors and were then treated with a combination of anti-PD-1 antibody and vehicle or SMC. The survival advantage conferred by the combination of SMC and αanti-PD-1 was lost in these T cell-deficient mice (Fig. 59E). Overall, the synergistic effect of SMC and anti-PD-1 depends 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 infiltration by immune cells.

To understand the immune cellular response aspect of SMC and ICI synergism, we assessed the profiles of infiltrating CD45+ immune cells in glioblastoma-bearing mice. In these studies, we assessed infiltrating immune cells in advanced glioblastoma following combination therapy with PD-1 and SMC (Fig. 61A). Flow cytometric analysis of tumor-infiltrating T cells showed a statistically nonsignificant trend in the proportion of CD4+ and CD8+ T cells between the vehicle and IgG control groups and all single- and double-treated mice (Fig. 66B). However, analysis of CD4+ and CD25+ T cells, indicative of the regulatory T cell (Treg) population, revealed a significant reduction in this cell population with SMC alone or the combination of SMC and ICI (Fig. 61C).

We then characterized the surface presentation of PD-1 on T cells following treatment with SMC alone or the combination. We noted a significant increase in CD8+ T cells expressing PD-1 in mice treated with SMC alone, and anti-PD-1 treatment or combined SMC and anti-PD-1 treatment resulted in less detectable cellular presentation of PD-1 (Figure 61D). We also observed a trend toward decreased PD-1 presentation on CD4+ T cells in the SMC- or anti-PD-1-treated groups. However, detectable surface PD-1 was abolished by combined SMC and anti-PD-1 treatment (Figure 61E).

In addition to the observed T cell infiltration of intracranial glioblastoma tumors, we also characterized the presence of myeloid-derived suppressor cells (MDSCs) and astrocytes/microglia. In contrast to the previous report, we found no differences in the MDSC population (CD11b+ Gr1+) in any treatment cohorts (Fig. 61F). However, we did find that the astrocyte/microglia population was significantly reduced in the treatment cohorts that included anti-PD-1 (Fig. 61G). Overall, these results indicate that combination treatment results in a reduction in the immunosuppressive CD4− T cell population with a concomitant reduction in PD-1 presentation on T cells and a reduction in the number of astrocytes and/or microglia.

Example 10. Synergistic activity of SMC and ICI is dependent on TNF-α.

We then characterized tumor cell cytokine and chemokine profiles in mice bearing intracranial glioblastomas treated with combinations of SMC and anti-PD-1. Flow cytometric analysis showed an increase in GrzB-expressing CD8+ cells upon administration of anti-PD-1 antibodies. The ratio of cytotoxic CD8+ (Figure 62A) to CD4+ Tregs was also increased in the anti-PD-1 and SMC and anti-PD-1 treatment cohorts (Figure 62B). In addition to assessing GrzB expression, we analyzed IFN-γ and TNF-α levels in T cells. Surprisingly, we found a decrease in the proportion of CD4+ cells expressing IFN-γ upon SMC treatment (even with the addition of PD1-targeting antibodies), but no change in IFN-γ expression levels in any CD8+ cell treatment cohort (Fig. 62C). We then analyzed the expression levels of TNF-α in T cells. In this context, we noted a significant increase in CD4+ and CD8+ T cells expressing TNF-α (Fig. 69D), indicating that these T cells can directly induce SMC-mediated tumor cell death.

The authors also assessed the effect of combination therapy with SMC and anti-PD-1 blockade on serum concentrations and gene expression levels of cytokines and chemokines in a CT-2A intracranial glioblastoma model. The authors found statistically significant increases in proinflammatory cytokines IFN-, IL-1-α, IL-1e, and IL-17 and multifactorial cytokines IFN-γ, IL-27, and GM-CSF (Figures 6O and 62E). Notably, there was no difference in the presence of anti-inflammatory cytokines such as IL-10. Similarly, analysis of cytokine and chemokine expression profiles within CT2A intracranial tumors after combination treatment with SMC and ICI showed clustering of proinflammatory cytokines and chemokines (Figures 62F and 63). In addition to SMC candidates or SMC and ICI combination therapy, proinflammatory cytokines IFN-β, IL-1e, IL-17, Osm, and TNF-α, chemokines Ccl2 (also known as MCP-1), Ccl5, Ccl7, Ccl22, Cxcl9, Cscl10, and Cxcl11, and multifactorial factors such as

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FasL, IL-2, IL-12 and IFN-γ.

Since we observed a consistent increase in IFN-β and IFN-γ levels, we sought to characterize the functional role of these signaling molecules using blocking/neutralizing antibodies in mice bearing CT-2A intracranial tumors treated with SMC and anti-PD-1. Blockade of the type I IFN signaling pathway using an antibody that blocks the IFNAR1 receptor abolished the synergistic effects that increased survival in mice bearing CT-2A intracranial tumors after combined treatment with SMC and anti-PD1 (Fig. 62G).

In contrast, antagonism of IFN-γ function using an anti-IFN-γ antibody partially inhibited the synergistic effect of combination therapy with SMC and ICI. Overall, these results indicate that each therapeutic agent, including the combination, results in the production of a different set of genes and proteins, but overall depends on an intact type I IFN signaling pathway.

Overall, the results showed that the synergistic effects of SMC and ICI may be mainly due to the enhancement of CTL-mediated attack on glioblastoma cells, and this is associated with a proinflammatory response that includes type I IFN. Co-culture of CT-2A cells and CD8+ T cells isolated from mice previously cured of intracranial tumors resulted in an increase in GrzB-positive CD8+ T cells that were not increased by SMC treatment alone (Fig. 65A). However, there was a slight decrease in the number of viable CT-2A cells when co-incubated with the same CTLs, even when the PD-1/PD-L1 axis was neutralized (Fig. 65B).

As previously noted, that type I IFN response also results in TNF-α production, we examined the ability of T cells to produce TNF-α after SMC treatment in the presence of glioblastoma cells. Accordingly, TNF-α production was then assessed.

Inclusion of SMC significantly increased the proportion of TNF-α-expressing CD8+ T cells, independent of the administration of PD-1-targeted antibodies (Fig. 65A). Consistent with the increased expression of TNF-α from CD8 T cells, we observed a significant reduction in CT-2A cells in a coculture system using CT-2A cells and CD8 T cells from cured mice (Fig. 65B). Notably, SMC-mediated effects on CT-2A cell death induction were mainly dependent on TNF-α (the major mediator of SMC-induced cell killing). We next assessed whether SMC therapy improved T cell proliferation. Indeed, the authors observed a significant reduction in CFSE-loaded CD8+ T cells along with the emergence of a new population of weakly CFSE-labeled cells after co-incubation of CT-2A cells, and this effect was pronounced with SMC and anti-PD-1 administration (Fig. 64).

These results indicate that cytotoxic T cells in response to SMC and anti-PD-1 treatment may result in enhanced tumor cell killing due to increased production of GrzB and TNF-α, death profactors that induce tumor cell death through IAP antagonism. We functionally characterized the role of TNF-α using blocking antibodies targeting TNF-α. With systemic TNF-α blockade, there was a near-complete reversal of the efficacy of combined SMC and ICI treatment (Fig. 65C), highlighting the importance of TNF-α for the synergistic effects of these disparate agents.

The immunomodulatory anti-cancer effects of SMC are multimodal (Figs. 66 and 67). SMC can polarize macrophages from the immunosuppressive M2 type toward the inflammatory TNF-α-producing M1 phenotype. Moreover, the anti-tumor effects of SMC are strongly potentiated by pro-inflammatory cytokines, and the presence of these cytokines, such as TNF-α or TRAIL, in the tumor microenvironment results in tumor cell death. In particular, SMC-mediated cIAP depletion converts the TNF-α-mediated survival response into a cell death pathway in cancer cells.

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

Thus, the authors showed for the first time that SMC can enhance ICI activity in mouse tumor models.

Furthermore, this combinatorial effect depends on the presence of CD8+ T cells with concomitant reduction of immunosuppressive CD4+ T cells and IFN type I and II and TNF-α signaling pathways, strongly implying a role for adaptive immunity for SMC-mediated therapy in mice. Thus, SMC-mediated T cell co-stimulatory signals provide the impetus for adaptive immune responses that develop against the tumor, and this is fully realized if the brakes imposed on ICI by coinhibitory signals such as PD-1 or PD-L1 are released.

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Claims (10)

Other embodiments of the invention All publications, patent applications and patents mentioned in this specification are incorporated herein by reference. Although the invention has been described by way of example with specific embodiments, it should be understood that it can be further modified. Accordingly, the present application covers any variations, applications or modifications of the invention that generally follow from the principles of the invention, including departures from the present disclosure that are obvious in known or customary practice within the scope of the prior art. CLAUSE OF INVENTION 1. An antitumor combination containing a monovalent Smac mimetic compound (SMC) and Atezolizumab. 2. The combination of claim 1, wherein said monovalent SMC is AT406/SM406/Debio1143/D1143. 3. Use of a combination according to any of paragraphs 1-2 for treating a patient with a malignant neoplasm. 4. The use according to paragraph 3, where the malignant neoplasm is resistant to treatment with only the Smac mimetic. 5. The use according to any one of claims 3 or 4, wherein said malignant neoplasm 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, digestive system cancer, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasm, renal cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cavity cancer oral cancer, ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, respiratory cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and urinary tract cancer. 6. A method for treating a malignant neoplasm, wherein said method comprises administering to a patient an effective amount of a combination according to any one of claims 1-2, wherein SMC and Atezolizumab are administered simultaneously or at an interval of 28 days from each other in amounts that are together effective for treating said malignant neoplasm. 7. The method of claim 6, wherein the SMC and Atezolizumab are administered within 14 days of each other, or within 10 days of each other, or within 5 days of each other, or within 24 hours of each other, or within 6 hours of each other, or substantially simultaneously. 8. The method according to claim 6 or 7, wherein the malignant neoplasm is resistant to treatment with only the Smac mimetic. 9. The method according to any one of claims 6 to 8, wherein said method also comprises administering a therapeutic agent comprising an interferon, wherein said interferon is preferably type 1 interferon. 10. The method according to any one of claims 6 to 9, wherein said malignant neoplasm 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, digestive system cancer, endometrial cancer, epipharyngeal carcinoma, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hepatocellular carcinoma, intraepithelial neoplasm, renal cancer, laryngeal cancer, leukemia, liver cancer, liver metastases, lung cancer, lymphoma, melanoma, myeloma, multiple myeloma, neuroblastoma, mesothelioma, neuroglioma, myelodysplastic syndrome, multiple myeloma, oral cavity cancer oral cancer, ovarian cancer, childhood cancer, pancreatic cancer, pancreatic endocrine tumors, penile cancer, plasma cell tumors, pituitary adenoma, thymoma, prostate cancer, renal cell carcinoma, respiratory cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small bowel cancer, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and urinary tract cancer. -