WO2013043591A1 - Combination therapy for cancer - Google Patents

Abstract

A combination therapy to treat a cancer or a tumor is provided. A method of delaying resistance to an anti-cancer/anti-tumor therapy is also provided.

Description

COMBINATION THERAPY FOR CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 61/537,250, filed September 21, 2011, the contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Throughout this application various publications are referred to by number in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of each of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0003] The development of new agents to treat cancer requires a careful balance between efficacy and toxicity. Because patients with advanced-stage disease often have numerous sites of involvement, systemic delivery is the most practical way to treat all of those sites. However, systemically-delivered therapies often have significant unwanted side effects due to their indiscriminant activity on both neoplastic and normal tissue. Whether the target is a specific tissue type or pathway, targeted therapy is therefore indicated as a strategy for cancer treatment because it theoretically delivers the anti-cancer agent only to the sites required. Previously, this laboratory has shown that targeted gene therapy delivers anti-tumor agents directly to the tumor, produces sustained expression of the therapeutic gene product, and maximizes efficacy and minimizes toxicity (1-2).

[0004] Recently, this laboratory has also developed hybrid bacteriophages (phage), based on prokaryotic viruses that represent a promising vector strategy for the delivery of therapeutic transgenes (3-5). Systemic administration of bacteriophage has been shown to be an effective therapy for antibiotic-resistant septicemia in patients (6) and can be safely used in immunocompromised patients without significant toxicity (7). Bacteriophage has no natural tropism for eukaryotic cells. A new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis elements of recombinant adeno-associated virus (AAV) and bacteriophage (termed AAVP), has therefore been developed to express and deliver transgenes such as TNF-a, a potent cytotoxic and anti-vascular cytokine (1-2, 8-10).

[0005] The AAVP vector used in the studies disclosed herein was engineered to target tumor-associated vasculature selectively by its expression of an RGD (Arginine-Glycine- Aspartic acid) amino-acid motif (termed RGD-4C) on its surface (8-10), which can bind to the ligand ανβ3, an integrin that is over-expressed on tumor vascular endothelium (8-10). Recently, this laboratory demonstrated that the use of an AAVP vector delivering an anti- vascular agent capable of directed cell transduction in the targeted tumor vasculature resulted in the sustained expression of TNF-a without systemic toxicity (9-10). This is of significance because TNF-a is a cytokine that affects tumor cells and the tumor microenvironment. The mechanism of TNF-based anti-cancer therapy consists of three parameters: 1) an increase in vascular permeability leading to improved anti-cancer drug penetration within the tumor tissue (11-12); 2) induction of apoptosis in tumor cells; and 3) targeting of tumor vessels with a selective killing of angiogenic endothelial cells that results in the destruction of the tumor microenvironment (13-15).

[0006] Several clinical trials have been performed to investigate the efficacy of TNF-a in treating patients with cancer (16-23). Systemic administration of TNF-a as an anticancer agent at therapeutic doses is limited by the severe systemic side effects, such as hypotension and organ failure (24). To address this shortcoming, this laboratory developed a novel therapeutic agent, RGD-AA VP -TNF-a (16, 25), that can be administered systemically but delivers its gene product specifically to tumor vasculature via the av integrin ligand (RGD- 4C) motif (1-2)_. After intravenous administration, RGD-AA VP -TNF-a was found in tumor- associated blood vessels, but not in healthy tissues. Furthermore, expression of TNF-a was detected only in tumor tissue; the exposure of normal tissue beds during a therapeutic response was therefore minimized (1-2). An important aspect of TNF-a activity is its induction of apoptosis in both tumor and endothelial cells. However, it is unknown if this apoptotic effect can be improved upon.

[0007] The present invention address the need for targeted anti-cancer treatments and improved anti-apoptotic therapies.

SUMMARY OF THE INVENTION

[0008] A method is provided of treating a cancer or a tumor in a subject comprising administering to the subject an amount of an inducer of apoptosis and an amount of an inhibitor of Inhibitor of Apoptosis Protein (IAP), in amounts effective to treat a cancer or a tumor in a subject.

[0009] An inducer of apoptosis is provided for use with an inhibitor of Inhibitor of Apoptosis Protein (IAP) for treating a cancer or tumor in a subject.

[0010] An inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) are provided for treating a cancer or tumor in a subject.

[0011] An inducer of apoptosis for treating a cancer or a tumor in a subject, wherein the inducer of apoptosis is administered concurrently, separately or sequentially with an inhibitor of Inhibitor of Apoptosis Protein (IAP).

[0012] An inducer of apoptosis and an inhibitor of Apoptosis Protein (IAP) as a combined preparation for treating a cancer or a tumor in a subject.

[0013] A method is provided of delaying or preventing resistance of a cancer or a tumor in a subject to an anti-cancer or anti-tumor therapy, respectively, comprising administering to the subject an inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) in amounts effective to prevent resistance of the cancer or tumor to the anti-cancer or anti -tumor therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A-1E: Inhibition of M21 human melanoma xenografts in nude mice treated with targeted AAVP-TNF-a and/or LCL161. (A): Photographs of representative tumors in mice treated with AAVP-TNF-α plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-α plus NaAC Buffer at the beginning of the first treatment cycle (day 0). These pictures show that the initial tumor volumes for all mice from each group were the same at 115 mm³. (B): Photographs of representative tumors of mice treated with AAVP- TNF-a plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-α plus NaAC Buffer at the end of the first treatment cycle (day 27); (C): Photographs of representative tumors of mice treated with AAVP-TNF-α plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-α plus NaAC Buffer at the beginning of the second treatment cycle (day 62); (D) Photographs of representative tumors of mice treated with AAVP-TNF-α plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-a plus NaAC Buffer at the end of the second treatment cycle (day 90); (E) Tumor growth inhibition in all four groups during the first treatment cycle. [0015] Figure 2A-2B: Survival time of mice treated with AAVP-TNF-a and LCL161. (A) Kaplan-Meier survival curve. The mice were treated for two cycles with AAVP-TNF-a plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-α plus NaAC Buffer and monitored over time for their survival; (B) Tumor-free curve for two cycles of treatment with AAVP-TNF-α plus LCL161, PBS plus LCL161, PBS plus NaAC Buffer, and AAVP-TNF-α plus NaAC Buffer. Each day represents the number of mice that were tumor- free at the indicated time point.

[0016] Figure 3: Synergistic effect of AAVP-TNF-α and LCL161 in M21 xenograft nude mice. The curve shows the Fraction Effect-Combination Indices plot for the combination of AAVP-TNF-α and LCL161 with the CalcuSyn Software. These results indicated that AAVP- TNF-a and LCL161 were synergistic with CI values <1. Fractional effect: % CK (tumor cell killing) = (Volume Control-Volume Treat) / Volume Control; CI: Combination Index; CK 1 : Synergism is indicated.

[0017] Figure 4A-4C: RGD- AAVP-TNF-α trafficking resulted in selective expression of human TNF-a mRNA in tumor tissue. Levels of human TNF-a mRNA from tumor and normal tissues (heart, spleen, kidney, liver, and muscle) from all groups were analyzed by TaqMan assay. The Y-axis represents the relative human TNF-a mRNA levels after normalization to GAPDH mRNA. All data are shown as mean ± SD. (A) mRNA levels of human TNF-a in tumor tissues were significantly increased in targeted AAVP-TNF-α treated groups, in comparison with non-targeted AAVP-TNF-α groups, on day 7 and day 21 after injection (P<0.01 or PO.001, respectively). (B, C) mRNA levels of human TNF-a were detected only in tumors and absent in normal tissues from targeted AAVP-TNF-a-treated groups (AAVP-TNF-α alone and AAVP-TNF-α plus LCL161) on day 7 (PO.0001) (B) and day 21 after injection (PO.0001) (C). (H: heart; L: liver; K: kidney; S: spleen; M: muscle; T: tumor).

[0018] Figure 5A-5C: RGD- AAVP-TNF-α trafficking resulted in selective human TNF- α expression in tumor tissue. The levels of human TNF-a protein from tumor, normal tissues (heart, spleen, kidney, liver, and muscle), and peripheral blood of all groups, were evaluated by ELISA. The Y-axis represents human TNF-a in 100 μg of lysate or 50 μΐ of peripheral blood. All data are shown as mean ± SD. (A) The level of human TNF-a in tumor tissues was significantly increased in targeted AAVP-TNF-a-treated groups compared with non- targeted AAVP-TNF-α groups on day 7 and day 21 after injection (PO.001). (B) The expression of human TNF-a was detected only in tumors and absent in normal tissues from targeted AAVP-TNF-a treated groups (AAVP-TNF-a alone and AAVP-TNF-a plus LCL161) on days 7 and 21 after injection (PO.0001). (C) Human TNF-a was absent in peripheral blood from all four groups at pre-treatment and on days 7 and 21 post-treatment with targeted AAVP-TNF-α injected systemically.

[0019] Figure 6A-6B: Apoptosis was induced and enhanced in tumor and tumor vasculature by the combination of AAVP-TNF-α and LCL161. Apoptotic cell nuclei in tumor tissues from all groups were detected on day 7 and day 21 after treatment. Apoptotic cells were stained red by TUNEL assay and nuclei were stained blue with DAPI. The apoptotic index was defined as the average number of apoptotic cells per high power view counted from 6 sections from 3 mice at each time point. All data are shown as mean + SD. The number of apoptotic cells in tumor tissues was increased in treated groups compared with the control group on day 7 after treatment. The combination of targeted AAVP-TNF-α plus LCL161 exhibited the greatest number of apoptotic cells compared with either AAVP-TNF-a alone or LCL161 alone. The number of apoptotic cells in tumor tissues was increased in the treated groups compared with the control on day 21 after treatment. The combination of AAVP-TNF-α and LCL161 exhibited the greatest number of apoptotic cells compared with either AAVP-TNF-a alone or LCL161 alone. (A and B) The number of apoptotic cells in the tumor tissues was quantified by apoptotic index. The apoptotic index in tumor tissues was increased significantly in the treatment groups compared with the control group on days 7 and 21 after treatment (P<0.05). The combination of AAVP-TNF-α and LCL161 resulted in the highest apoptotic index, in comparison with either AAVP-TNF-α alone or LCL161 alone on days 7 and 21 after treatment (PO.001).

[0020] Figure 7A-7B: (A, B) The expression of caspase 3 in tumor tissues was quantified and was increased significantly in the treated groups compared with the control group on day 7 (A) and day 21 (B) after treatment (P< 0.05 or PO.001, respectively). The levels of caspase 3 was the highest in the group receiving the combination of targeted AAVP-TNF-a and LCL161, in comparison with groups treated with either AAVP-TNF-α alone or LCL161 alone on day 7 (A) and day 21 (B) after treatment (PO.001).

[0021] Figure 8A-8B: (A, B) caspase 9 in tumor tissues was quantified. Levels were increased significantly in the treated groups compared with the control group on day 7 (A) and day 21 (B) after treatment (P< 0.05 or P< 0.001, respectively). Caspase 9 was highest after combined AAVP-TNF-α and LCL161 administration, in comparison with that of either AAVP-TNF-α alone or LCL161 alone on day 7 (A) and day 21 (B) after treatment (PO.001).

[0022] Figure 9A-9D: Treatment of M21 xenografted nude mice. Twenty-six mice were assigned to four groups for tumor growth inhibition and survival studies: (A) Control group (N=8), received PBS by tail vein injection weekly (Monday: M) and received NaAc Buffer control by gavage daily (Monday -Friday: M-F). (B) AAVP-TNF-a group (N=4), received AAVP-TNF-a by tail vein weekly (M) and was gavaged with NaAc Buffer daily (M-F). (C) LCL161 group (N=7), received PBS by tail vein weekly (M) and received LCL161 daily (M- F) by gavage. (D) Combination AAVP-TNF-α plus LCL161 group (N=7), received AAVP- TNF-a weekly (M) by tail vein and LCL161 daily (M-F) by gavage. All mice were treated for two cycles. In each cycle, 1 x 10¹¹ AAVP particles (or PBS control) were administered intravenously into the tail vein weekly and 100 mg/kg LCL161 (or NaAc Buffer control) was administered by gavage daily.

[0023] Figure 10A-10B: Proliferating cell nuclear antigen (PCNA) was analyzed by IF in tumor sections from the groups on day 7 and day 21 after treatment. PCNA appears red (Alexa Fluor 647), blood vessels are stained green by anti-CD31 antibody (Alexa Fluor 488), and nuclei are stained blue with DAPI. Relative levels of PCNA in tumor tissue appeared similar in all groups on day 7 after treatment. Relative levels of PCNA in tumor tissue appeared similar in all groups on day 21 after treatment. (A, B): The number of proliferative cells in the tumor tissues was quantified by PCNA detection, The levels of PCNA in tumor tissues are no significant change in the treatment groups compared with the control group on days 7 and 21 after treatment (P>0.05).

DETAILED DESCRIPTION OF THE INVENTION

[0024] A method is provided of treating a cancer or a tumor in a subject comprising administering to the subject an amount of an inducer of apoptosis and an amount of an inhibitor of Inhibitor of Apoptosis Protein (IAP), in amounts effective to treat a cancer or a tumor in a subject.

[0025] In an embodiment, the inducer of apoptosis and the inhibitor of IAP are administered concurrently. In an embodiment, the inducer of apoptosis and the inhibitor of IAP are administered sequentially. In an embodiment, the amount of the inducer of apoptosis and the amount of the inhibitor of IAP combined elicit a synergistic effect in treating the cancer or tumor. In an embodiment, the synergistic effect comprises enhanced apoptosis of tumor cells. In an embodiment, the synergistic effect comprises enhanced apoptosis of tumor- associated vasculature cells. IN an embodiment the CI is < 1.0. In an embodiment, the CI is from 0.5 to 0.95. In an embodiment the CI is from 0.5 to 0.85.

[0026] In an embodiment, the inhibitor of IAP is a Smac mimetic (Smac is second mitochondrial-derived activator of caspases). Examples of Smac inhibitors are known in the art, for example birinapant (N,N'-[(6,6'-difluoro[2,2'-bi-lH-indole]-3,3'- diyl)bis[methylene[(2R,4S)-4-hydroxy-2, l-pyrrolidinediyl][(l S)-l-ethyl-2-oxo-2, l- ethanediyl]]]bis[2-(methylamino)-,(2S,2'S)-propanamide; (2S,2'S)-N,N'-[(6,6'-difluoro- lH, l'H-2,2'-biindolyl-3,3'-diyl)bis {methylene[(2R,4S)-4- hydroxypyrrolidine-2,l-diyl][(l S)- l-ethyl-2-oxoethylene]}]bis[2- (methylamino)propanamide), or a compound having the following structure:

[0027] In an embodiment, the inhibitor of IAP is a small molecule of 2000 daltons or less or 1500 daltons or less. In an embodiment the small molecule is a small organic molecule. In an embodiment, the inhibitor of IAP is a peptide or an antigen-binding fragment of an antibody. In an embodiment, the inducer of apoptosis is tumor necrosis factor (TNF) or wherein the inducer of apoptosis elicits production in the subject of TNF. In an embodiment, the inducer of apoptosis TNF is TNFa.

[0028] In an embodiment, the inducer of apoptosis is administered locally into the cancer or locally into the tumor. In an embodiment, the inducer of apoptosis is administered in a manner which targets it to and/or selectively delivers it to the cancer or tumor. In an embodiment, the inducer of apoptosis is administered in a manner which targets it to and/or selectively delivers it to the cancer or tumor by administering it using a peptide, protein, aptamer or antibody targeting ligand. In an embodiment, the inducer of apoptosis is administered using a vector comprising a nucleic acid encoding the inducer under conditions permitting expression therefrom of the inducer. In an embodiment, the inducer of apoptosis is recombinant TNFa. In an embodiment, the TNFa is conjugated to a nanoparticle. In an embodiment, the nanoparticle is predominantly spherical. In an embodiment, the nanoparticle has a diameter of less than 100 nm. In an embodiment, the nanoparticle comprises gold.

[0029] In an embodiment, the inhibitor of IAP is administered systemically. In an embodiment, the inhibitor of IAP is administered orally. In an embodiment, the IAP is administered locally into the cancer or tumor. In an embodiment, the IAP is administered in the form of a vector encoding the IAP. In an embodiment, the vector is a hybrid prokaryotic- eukaryotic vector. In an embodiment, the hybrid prokaryotic-eukaryotic vector comprises a genetic cis element of a recombinant adeno-associated virus. In an embodiment, the hybrid prokaryotic-eukaryotic vector comprises a phage vector. In an embodiment, the phage is an Ml 3 -derived phage. In an embodiment, the hybrid prokaryotic-eukaryotic vector comprises a bacteriophage expressing the amino acid motif arginine-glycine-aspartic acid on its surface.

[0030] In an embodiment, the inhibitor of IAP inhibits one or more of XIAP, CIAPi and CIAP₂.

[0031] In an embodiment, the treatment effects a decrease in tumor volume or effects tumor regression. In an embodiment, the treatment effects a decrease in tumor growth.

[0032] In an embodiment, the cancer or tumor is a cancer or tumor of the breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid or skin. In an embodiment, the cancer or tumor is a cancer or tumor of the skin. In an embodiment, the cancer or tumor is a melanoma.

[0033] An inducer of apoptosis is provided for treating a cancer or a tumor in a subject, wherein the inducer of apoptosis is administered concurrently, separately or sequentially with an inhibitor of Inhibitor of Apoptosis Protein (IAP). An inducer of apoptosis and an inhibitor of Apoptosis Protein (IAP) are provided as a combined preparation for treating a cancer or a tumor in a subject. An inducer of apoptosis is provided for use with an inhibitor of Inhibitor of Apoptosis Protein (IAP) for treating a cancer or tumor in a subject. An inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) are provided for treating a cancer or tumor in a subject.

[0034] In an embodiment, inducer of apoptosis and the inhibitor of IAP are formulated for use concurrently. In an embodiment, the inducer of apoptosis and inhibitor of IAP are formulated for use sequentially. In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is a Smac mimetic. In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is a small molecule of 2000 daltons or less. In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is a small molecule of 1500 daltons or less. In an embodiment, the small molecule is a small organic molecule. In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is an antibody or an antigen-binding fragment of an antibody. In an embodiment, the inducer of apoptosis is tumor necrosis factor (TNF) or elicits production in the subject of TNF. In an embodiment, the inducer of apoptosis is TNFa. In an embodiment, the inducer of apoptosis is formulated as an expressible vector comprising a nucleic acid encoding the inducer.

[0035] A method is provided of delaying or preventing resistance of a cancer or a tumor in a subject to an anti-cancer or anti-tumor therapy, respectively, comprising administering to the subject an inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) in amounts effective to prevent resistance of the cancer or tumor to the anti-cancer or anti -tumor therapy.

[0036] In an embodiment, the inducer of apoptosis is TNFa. In an embodiment, the inducer of apoptosis and the inhibitor of IAP are administered concurrently. In an embodiment, the inducer of apoptosis and the inhibitor of IAP are administered sequentially. In an embodiment, the cancer or tumor is a cancer or tumor of the breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid or skin.

[0037] As used herein, a "cancer" is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication. In an embodiment, the cancer is a tumorous cancer. In an embodiment, the cancer is a non-tumorous cancer. As used herein a "tumor" is a detectable malignant tumor usually presenting as a lesion or lump located in an organ or tissue in a subject.

[0038] In an embodiment, the cancer or tumor is a cancer or tumor of the skin, breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate or thyroid.

[0039] As used herein, "treating" a cancer or tumor means that one or more symptoms of the disease, such as the cancer or tumor itself, metastasis thereof, vascularization of the tumor, or other parameters by which the disease is characterized, are reduced, ameliorated, prevented, placed in a state of remission, or maintained in a state of remission. "Treating" a tumor or cancer also means that one or more hallmarks of the tumor or cancer may be eliminated, reduced or prevented by the treatment.

[0040] Inhibitors of Apoptosis Proteins (IAPs) are well-known in the art and, in non- limiting examples, include X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein- 1 and 2 (CIAPl and CIAP2, respectively). Herein, "Inhibitor of Apoptosis Proteins" and "Inhibitor of Apoptosis Protein" are used interchangeably unless context dictates otherwise.

[0041] As used herein, an "inhibitor" of IAP or IAPs includes Smac mimetics, small molecules, aptamers, antibodies, RNAi-based inhibitors (e.g. siRNA and shRNA), peptides, and fragments of antibodies able to act intracellular. In an embodiment the inhibitor of IAP inhibits one type of IAP only (for example, XIAP, CIAPl or CIAP2). In an embodiment, the inhibitor of IAP inhibits more than one type of IAP (for example, any two or more of XIAP, CIAPl and CIAP2). XIAP inhibitors are known in the art, e.g. embelin, embelin-6g. Also see Oost et al, J. Med. Chem., (2004), 47 (18), pp 4417-4426, hereby incorporated by reference. Inhibitors of CIAP 1 and CIAP2 are known in the art, for example, LB W242 which has the structure:

and MeBS ((-)-N-[(2S, 3R)-3-amino-2-hydroxy-

4-phenyl-butyryl]-L-leucine methyl ester. Selective CIAP2 inhibitos include SmacN7 (H- AVPIAQK-OH).

[0042] In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is a nucleic acid. In an embodiment, the inhibitor of Inhibitor of Apoptosis Protein is RNAi, in non-limiting examples, an siRNA or shRNA.

[0043] In an embodiment, the siRNA (small interfering RNA) as used in the methods or compositions described herein comprises a portion which is complementary to an mRNA encoding an Inhibitor of Apoptosis Protein, and the siRNA is effective to inhibit expression of the Inhibitor of Apoptosis Protein. In an embodiment, the siRNA comprises a double- stranded portion (duplex). In an embodiment, the siRNA is 20-25 nucleotides in length. In an embodiment the siRNA comprises a 19-21 core RNA duplex with a one or 2 nucleotide 3 ' overhang on, independently, either one or both strands. The siRNA can be 5' phosphorylated or not and may be modified with any of the known modifications in the art to improve efficacy and/or resistance to nuclease degradation. In an embodiment the siRNA can be administered such that it is transfected into one or more cells.

[0044] In one embodiment, a siRNA of the invention comprises a double-stranded RNA wherein one strand of the double-stranded RNA is 80, 85, 90, 95 or 100% complementary to a portion of an RNA transcript of a gene encoding an Inhibitor of Apoptosis Protein. In another embodiment, a siRNA of the invention comprises a double-stranded RNA wherein one strand of the RNA comprises a portion having a sequence the same as a portion of 18-25 consecutive nucleotides of an RNA transcript of a gene encoding an Inhibitor of Apoptosis Protein. In yet another embodiment, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker. Alternately, a siRNA of the invention comprises a double-stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.

[0045] In one embodiment, a single strand component of a siRNA of the invention is from 14 to 50 nucleotides in length. In another embodiment, a single strand component of a siRNA of the invention is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 21 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 22 nucleotides in length. In yet another embodiment, a single strand component of a siRNA of the invention is 23 nucleotides in length. In one embodiment, a siRNA of the invention is from 28 to 56 nucleotides in length. In another embodiment, a siRNA of the invention is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides in length. In yet another embodiment, a siRNA of the invention is 46 nucleotides in length.

[0046] In another embodiment, an siRNA of the invention comprises at least one 2'-sugar modification. In another embodiment, an siRNA of the invention comprises at least one nucleic acid base modification. In another embodiment, an siRNA of the invention comprises at least one phosphate backbone modification.

[0047] In one embodiment, RNAi inhibition of the Inhibitor of Apoptosis Protein is effected by a short hairpin RNA ("shRNA"). The shRNA is introduced into the cell by transduction with a vector. In an embodiment, the vector is a lentiviral vector. In an embodiment, the vector comprises a promoter. In an embodiment, the promoter is a U6 or HI promoter. In an embodiment the shRNA encoded by the vector is a first nucleotide sequence ranging from 19-29 nucleotides complementary to the target gene, in the present case, a gene encoding an Inhibitor of Apoptosis Protein. In an embodiment the shRNA encoded by the vector also comprises a short spacer of 4-15 nucleotides (a loop, which does not hybridize) and a 19-29 nucleotide sequence that is a reverse complement of the first nucleotide sequence. In an embodiment the siRNA resulting from intracellular processing of the shRNA has overhangs of 1 or 2 nucleotides. In an embodiment the siRNA resulting from intracellular processing of the shRNA overhangs has two 3 ' overhangs. In an embodiment the overhangs are UU.

[0048] In an embodiment the inhibitor of Inhibitor of Apoptosis Protein is an antibody or a fragment of an antibody which is able to act intracellularly. As used herein, the term "antibody" refers to complete, intact antibodies. As used herein "antibody fragment" refers to Fab, Fab', F(ab)2, and other antibody fragments, which fragments (like the complete, intact antibodies) bind the antigen of interest, in this case an Inhibitor of Apoptosis Protein. Complete, intact antibodies include, but are not limited to, monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies. Various anti-IAP antibodies are known in the art, including those available from commercial suppliers.

[0049] Various forms of antibodies may be produced using standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-99, 1991). For example, "chimeric" antibodies may be constructed, in which the antigen binding domain from an animal antibody is linked to a human constant domain (an antibody derived initially from a nonhuman mammal in which recombinant DNA technology has been used to replace all or part of the hinge and constant regions of the heavy chain and/or the constant region of the light chain, with corresponding regions from a human immunoglobulin light chain or heavy chain) (see, e.g., Cabilly et al, U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. 81 : 6851- 55, 1984). Chimeric antibodies reduce the immunogenic responses elicited by animal antibodies when used in human clinical treatments. In addition, recombinant "humanized" antibodies may be synthesized. Humanized antibodies are antibodies initially derived from a nonhuman mammal in which recombinant DNA technology has been used to substitute some or all of the amino acids not required for antigen binding with amino acids from corresponding regions of a human immunoglobulin light or heavy chain. That is, they are chimeras comprising mostly human immunoglobulin sequences into which the regions responsible for specific antigen-binding have been inserted (see, e.g., PCT patent application WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated and the portion of the variable region sequences responsible for specific antigen binding are removed. The animal-derived antigen binding regions are then cloned into the appropriate position of the human antibody genes in which the antigen binding regions have been deleted. Humanized antibodies minimize the use of heterologous (inter-species) sequences in antibodies for use in human therapies, and are less likely to elicit unwanted immune responses. Primatized antibodies can be produced similarly.

[0050] Another embodiment of the antibodies employed in the compositions and methods of the invention is a human antibody, which can be produced in nonhuman animals, such as transgenic animals harboring one or more human immunoglobulin transgenes. Such animals may be used as a source for splenocytes for producing hybridomas, as is described in U.S. Pat. No. 5,569,825.

[0051] Antibody fragments and univalent antibodies may also be used in the methods and compositions of this invention wherein they can be delivered so as to act intracellularly. Univalent antibodies comprise a heavy chain/light chain dimer bound to the Fc (or stem) region of a second heavy chain. "Fab region" refers to those portions of the chains which are roughly equivalent, or analogous, to the sequences which comprise the Y branch portions of the heavy chain and to the light chain in its entirety, and which collectively (in aggregates) have been shown to exhibit antibody activity. A Fab protein includes aggregates of one heavy and one light chain (commonly known as Fab'), as well as tetramers which correspond to the two branch segments of the antibody Y, (commonly known as F(ab)₂), whether any of the above are covalently or non-covalently aggregated, so long as the aggregation is capable of specifically reacting with a particular antigen or antigen family.

[0052] The antibody can be, e.g., any of an IgA, IgD, IgE, IgG, or IgM antibody. In an embodiment the antibody is an immunoglobulin G. In an embodiment the antibody fragment is a fragment of an immunoglobulin G. In an embodiment the antibody is an IgGl, IgG2, IgG2a, IgG2b, IgG3 or IgG4. The IgA antibody can be, e.g., an IgAl or an IgA2 antibody. A combination of any of these antibodies subtypes can also be used. One consideration in selecting the type of antibody to be used is the desired serum half-life of the antibody. IgG has a serum half-life of 23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days. (Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology, 4th edition, W.B. Saunders Co., Philadelphia, 2000). Another consideration is the size of the antibody. For example, the size of IgG is smaller than that of IgM allowing for greater penetration of IgG into tumors.

[0053] As used herein, the term "bind", or grammatical equivalent, means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof, including the interaction between an antibody and a protein. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.

[0054] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences. The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0055] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0056] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0057] As used herein, an "inducer of apoptosis" is an agent which induces apoptosis of cells in vivo and includes, in non-limiting examples, including TNF-alpha, Fas-associated death domain (FADD), endothelial monocyte-activating polypeptide II (EMAP II), TNF- related apoptosis-inducing ligand (TRAIL) and those agents known in the art which activate cysteine proteases resulting in apoptosis. The inducer can be delivered by any means known in the art including by hybrid prokaryotic-eukaryotic phage vector, (e.g. see Ref. 9), bound to nanoparticles, such as gold (e.g. see Ref. 39), locally injected.

[0058] The inhibitor of IAP and/or the induce of apoptosis may, independently be, peptides.

[0059] As used herein a "small molecule" refers to an organic compound characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 2000 daltons. In an embodiment, the small molecule is less than 1500 daltons.

[0060] The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered by any means known in the art. Preferably, the inducer of apoptosis is targeted to the tumor or cancer, i.e. it is administered in a manner so as to selectively deliver it to the tumor or cancer or to deliver the majority of the inducer to the cancer or tumor in preference to other areas, organs and tissues of the subject's body, including up to 95% or more of the administered inducer being delivered to the tumor or cancer. The inhibitor of Inhibitor of Apoptosis Protein can be delivered systemically or locally into the tumor or cancer.

[0061] The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered parentally, enterally or topically. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered subcutaneously. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered intravenously. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered orally. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered topically. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered via an osmotic pump. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered inhalationally. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered directly into the site of the disease, e.g. cannulation into or injection into a cancer, tumor or blood vessel thereof.

[0062] The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein referred to herein can, independently, be administered to the subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein can be administered in a single composition or administered in separate compositions. The pharmaceutically acceptable carrier used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, oral administration, parenteral administration, subcutaneous administration, intravenous administration, transdermal administration, intranasal administration, and administration through an osmotic mini-pump. The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein can be applied to the skin, for example, in compositions formulated as skin creams, e.g. to treat a skin cancer, or as sustained release formulations or patches.

[0063] The inducer of apoptosis and inhibitor of Inhibitor of Apoptosis Protein can be administered as a sequential therapy where the patient is treated first with one agent and then the other. For example, the inducer of apoptosis can be administered first and the inhibitor of Inhibitor of Apoptosis Protein be administered second. Alternatively, the inducer of apoptosis can be administered after administration of the inhibitor of Inhibitor of Apoptosis Protein. In an embodiment, the inducer of apoptosis is administered concurrently with the inhibitor of Inhibitor of Apoptosis Protein. In an embodiment, the inducer of apoptosis is administered before or after the inhibitor of Inhibitor of Apoptosis Protein, but there is a period of overlap of administration of both agents. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein can be administered independently by the same route or by two or more different routes of administration.

[0064] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, a suspending vehicle, for delivering the instant agents to the animal or human subject. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known in the art, and include, but are not limited to, additive solution-3 (AS-3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate acid dextrose solution.

[0065] Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al. , 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol. 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol. 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0066] The methods disclosed herein can be used with any mammalian subject. Preferably, the mammal is a human.

[0067] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0068] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

Introduction

[0069] It is unknown whether the TNF-a response described in the background can be augmented by agents that enhance apoptosis. The strategy employed herein was to combine targeted expression of TNF-a with an agent that could potentially enhance pro-apoptotic effects. The target chosen was proteins that inhibit apoptosis (Inhibitor of Apoptosis Proteins, "IAPs"), a class of proteins that inhibit caspases and control the levels of a network of signaling molecules relevant to cancer (30-31). IAPs can be inhibited by second mitochondria-derived activator of caspases (Smac), a mitochondrial protein (32-33), and activation of Smac leads to increased apoptosis via inhibition of IAPs.

[0070] An inhibitor of IAPs used was LCL161 ( ovartis Institute for BioMedical Research, Cambridge, MA), a novel, orally-bioavailable mimetic of Smac that binds to IAPs with high affinity, initiates the destruction of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein- 1 and 2 (CIAPi and CIAP₂), and induces apoptosis via the activation of caspases. Recently, XIAP, CIAPi, and CIAP₂ were reported to be elevated in many cancers, with subsequent resistance to the induction of apoptotic pathways by TNF-a (34-35). Whether the combination of a Smac-mimetic, LCL161, with the tumor- targeted TNF-a biotherapeutic agent would lead to enhanced apoptosis in tumor cells and tumor associated vasculature was investigated. The responses seen with the tumor-targeted TNF-a biotherapeutic agent were found to be augmented. In this study, it was also determined whether the combination of RGD-AAVP-TNF-a and LCL161 resulted in enhanced efficacy, reduced toxicity, and the delay/prevention of the development of resistance in a mouse xenograft model of human melanoma. The effects of this combination on cellular apoptosis and proliferation were also examined.

Materials and Methods

[0071] Cell Culture: M21 human melanoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown at 37 °C as a monolayer culture with RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2mM glutamine, 100 U/mL penicillin G, 100 μg/mL streptomycin S04, 100 μg/mL gentamicin, and 250 μg/mL fungizone.

[0072] M21 xenograft mouse experiments: All animal experiments were conducted according to protocols approved by the Animal Institute of Albert Einstein College of Medicine. Female 6-week-old nude mice with a body weight of approximately 20 grams (g) were purchased from Jackson Laboratories and were housed in the animal facility of Albert Einstein College of Medicine. M21 human melanoma cells (4xl0⁶) were implanted subcutaneously into the right flank. Two weeks after implantation, tumor volumes (mm³) were measured in 3 dimensions and were calculated as length x width x height x 0.52. When tumor volumes reached approximately 100-150 mm³, the mice were assigned to groups such that the average tumor size was equivalent in each group. [0073] Treatment of M21 xenografts with AAVP-TNF-a and LCL161 : RGD-AAVP- TNF-a was constructed, purified, and quantified as described in published protocols (1-2). See ref. (9) for design and construction of AAVP vectors for mammalian cell transduction. LCL161 was provided as part of a collaboration with NOVARTIS Pharmaceutical Corporation. Two weeks after tumor cell implantation, mice were treated according to two protocols.

[0074] The first protocol evaluated the effects of therapy on the inhibition of tumor growth and on animal survival: Twenty-six M21 xenografted nude mice were assigned to four groups: 1) Control group (N=8), received phosphate buffered saline (PBS) by tail vein injection weekly (Monday: M) and received sodium acetate-acetic acid (NaAc) Buffer control by gavage daily (Monday -Friday: M-F); 2) AAVP-TNF-α group (N=4), received AAVP-TNF-a by tail vein injection weekly (M) and was gavaged with NaAc Buffer daily (M-F); 3) LCL161 group (N=7), received PBS by tail vein injection weekly (M) and received LCL161 daily (M-F) by gavage; 4) Combination AAVP-TNF-α plus LCL161 group (N=7), received AAVP-TNF-a weekly (M) by tail vein injection and LCL161 daily (M-F) by gavage. All mice were treated for two cycles (Table 1 and Figure 9). In each cycle, 1 x 10¹¹ AAVP particles (or PBS control) were administered intravenously into the tail vein weekly, and 100 mg/kg LCL161 (or NaAc Buffer control) was administered by gavage daily for four weeks (Table 1 and Figure 9). In the LCL161 treatment groups, LCL161 was maintained at a dose of 100 mg/kg daily (M-F) (Table 1 and Figure 9).

[0075] Table 1 : The First Therapy Protocol for Tumor Growth Inhibition and Survival Analysis

* In the second cycle, all mice from LCL161 alone and control groups were dead with larger tumors

[0076] The second protocol was designed to examine vector trafficking, gene expression, and activation of apoptotic pathways: Twenty- four M21 xenografted nude mice were assigned to four groups: 1) Control group (n=6), received PBS weekly by tail vein injection (M) and NaAc Buffer daily (M-F) for 7 days (n=3) or 21 days (n=3); 2) AAVP-TNF-a group (n=6), received AAVP-TNF-a by tail vein weekly (M) and NaAc Buffer daily (M-F) by gavage for 7 days (n=3) or 21 days (n=3); 3) LCL161 group (n=6), received PBS weekly by tail vein injection (M) and LCL161 daily (M-F) by gavage for 7 days (N=3) or 21 days (n=3); 4) Combination AAVP-TNF-α plus LCL161 group (n=6), received AAVP-TNF weekly (M) by tail vein injection and LCL161 daily (M-F) by gavage for 7 days (n=3) or 21 days (n=3). Animals were sacrificed at the established time intervals (Table 2). Resected tumor tissues and normal tissues (liver, kidney, heart, spleen, and skeletal muscle) were flash- frozen and stored at -80°C. Formalin-fixed tissue was paraffin-embedded for further analysis. Peripheral blood was also collected from all animals at days 0, 7, and 21.

[0077] Table 2: The Second Therapy Protocol for Trafficking, Gene Expression, and Apoptotic Pathway Detection

[0078] Tumor size and survival analysis: Tumor growth was defined as an increase in tumor volume, measured in a blinded fashion for average diameter before, during, and after treatment. Tumor volume was calculated according to the equation: tumor size = length x width x height x 0.52 (1-2). The treatment resistance response was analyzed after a total of two cycles. Survival time was followed in each group during and after treatment, and the average tumor-specific survival rate and tumor-free survival rate calculated for each group. Photographs of all the mice in each group were taken every week.

[0079] Toxicity assay: Toxicity was assessed in each group in vivo by analysis of body mass, feeding status, and mobility. All mice were weighed once per week.

[0080] Analysis of drug combined effects: The unexpected drug synergy was analyzed and quantified by the drug combination-index methods by the use of CalcuSyn software (Biosoft, Ferguson, MO) (36-37). The combination-index (CI) method is a mathematical and quantitative representation of a two-drug pharmacologic interaction (36-37). Using the CalcuSyn software, CI values over a range of Fraction levels (Fa) from 0.05 - 0.90 (5% - 90% growth inhibition) were generated. A CI of 1 indicates an additive effect between AAVP-TNF-a and LCL161, whereas a CI < 1 indicates the presence of synergistic activity.

[0081] In Vivo AAVP trafficking detection by immmunofluorescence assay (IF) with anti-filamentous ss-DNA (fd) bacteriophage: For detection of AAVP, 5^M-thick paraffin sections from the resected tumor tissues and normal tissues (liver, kidney, heart, spleen, and skeletal muscle) were stained by dual IF 1-2. The sections were treated with blocking buffer (5% goat serum and 2.5 % bovine serum albumin in PBS) for 1 hour at room temperature. The sections were incubated overnight at 4°C in a 1 : 1000 dilution of rabbit anti-fd bacteriophage antibody (Sigma Chemical Company) and a concentration of 10 ng/μΐ of antigen affinity -purified rat anti-mouse CD31 antibody (BD Biosciences, San Jose, Calif)(l- 2). Slides were next incubated with the secondary antibodies (1 :200 dilutions each of goat anti-rabbit Alexa Fluor 647 and goat anti-rat Alexa Fluor 488) (Invitrogen Corp) for 45 min in the dark (1-2). The slides were mounted in Vectashield mounting medium with 4'6- diamidino-2-phenylinodole (DAPI) (Vector Laboratories, Burlingame, Calif). Images were taken using on fluorescence microscope with camera.

[0082] In Vivo AA VP-mediated TNF-a transcription detection by Real-Time polymerase chain reaction (PCR): Human TNF-a mRNA was measured by RT-PCR with primer-probe sequences unique to human TNF-a inserted into RGD-A-TNF-a2. Total RNA was extracted from frozen tumor and normal tissues (liver, kidney, heart, spleen, and skeletal muscle) with Trizon (Invitrogen Corp) and RNeasy total RNA kit (Qiagen Corp). First-strand cDNAs were generated from the total RNA, and quantitative RT-PCR was performed with a Gene Amp 7500 Sequence Detector (Applied Biosystems). PCR products were measured as fluorescent signal intensity after standardization with a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) internal control. The following sense (S) and antisense (AS) primers and probes for human TNF-a were used: Sense primer: 5'- TTCAGCTCTGCATCGTTTTG-3 ' (SEQ ID NO: l); Antisense primer: 5'- CTCAGCTTGAGGGTTTGCTACA-3 ' (SEQ ID NO:2); and Probe 5'-FAM- TTCTCTTGGCGTCA GATCATCTTCTCGAAC-TAMARA-3 ' (2) (SEQ ID NO:3).

[0083] In Vivo AA VP-mediated TNF-a expression by an enzyme-linked immunosorbent assay (ELISA): Levels of human TNF-a were assessed by ELISA (1-2). Total cell lysates from peripheral blood, frozen tumor tissues, and frozen normal tissues (liver, kidney, heart, spleen, and skeletal muscle) were prepared in lysis buffer (50 mM Tris-HCl, pH 7.4; 140 mM NaCl; 0.1% sodium dodecyl sulfate; 1% NP40, and 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Roche, Branchburg, NJ) (1). The lysates were cleared by centrifugation at 13,000 rpm for 10 min. The amount of protein was quantified by the use of a protein assay reagent (BioRad, Hercules, Calif). 100 micrograms ^g) of total protein was assayed for human TNF-a by ELISA (Biosource, Camarillo, Calif) (1-2).

[0084] Measurement of apoptotic cells in tumor tissues by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay: Apoptotic status in tumor tissues was assessed from control and treated mice at day 7 and day 21 by TUNEL assay. For quantification of apoptosis, the TUNEL assay was performed according to the manufacturer on paraffin-embedded sections with an In Situ cell Death Detection Kit (Roche Diagnostic). The tissue sections were deparaffinized and treated with proteinase K (10 μg/ml) for 20 min. The sections were next washed twice with PBS, labeled and stained with the TUNEL reaction mixture (label plus enzyme solutions) for 60 min at 37 °C, and washed twice with PBS. The slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). The apoptotic fluorescent cells were counted under a fluorescent microscope, and the numbers were expressed as the percentage of total cells ± standard deviation (SD). A negative control without enzyme treatment and a positive control with DNase I treatment were also performed.

[0085] Measurement of the activity of Caspase 3 and Caspase 9 by IF: The activity of pro-apoptotic pathway components (caspase 3 and caspase 9) was measured in the tumor tissues from control and treated mice at day 7 and day 21. The paraffin-embedded sections were deparaffinized and were incubated with a concentration of 10 ng/μΐ of rat anti-mouse CD31 (BD Biosciences) and a concentration of 2.5 ng/μΐ of rabbit anti-active Caspase 3 (BD Biosciences) or Caspase 9 (BD Biosciences) antibodies, respectively, overnight at 4°C. Sections were washed with PBS and were incubated with a 1 :200 dilution of anti-rabbit Alexa Fluor 647 and goat anti-rat Alexa Fluor 488 secondary antibodies for 45 min in the dark. The slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent cells were counted under a fluorescence microscope, and the numbers were expressed as the percentage of total cells ± SD. A negative control without primary antibody was also performed.

[0086] Assessment of cell proliferation by IF: Cell proliferation was measured in tumor tissues from control and treated mice by the use of proliferating cell nuclear antigen (PCNA). Staining for PCNA on sections was performed in the same manner used to detect caspase 3 and caspase 9. Rabbit anti-PCNA antibody source (BD Biosciences) was incubated at a concentration of 4 ng/μΐ overnight at 4°C. Sections were also washed with PBS and were incubated with a 1 :200 dilution of anti-rabbit Alexa Fluor 647 secondary antibody for 45 min in the dark. The slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent cells were counted under a fluorescence microscope, and the numbers were expressed as the percentage of total cells ± SD. A negative control without primary antibody was also performed.

[0087] Power calculation and statistical analysis: An SD of 200 mm and a mean difference between control and treatment groups of 400 mm was used for tumor volume for the power calculation based on the two-sided, two-sample Student's t test (nQuery 5.0). Using P < 0.05, it was observed that n = 3 mice per group were adequate to achieve a power (l-β) of 90%. The statistical analysis was also conducted to determine significant differences among groups under study, with respect to tumor-specific survival rate, tumor-free survival rate, tumor size, TNF-a expression, and apoptotic status and its components. All groups were compared by the use of analyses of variance and Tukey comparison post-tests (GraphPad Instat software, San Diego CA). P values <0.05 were considered statistically significant.

Results

[0088] AAVP-TNF-a plus an IAPs inhibitor such as LCL161 enhances the inhibition of tumor growth and delays development of treatment resistance. The tumor volume in each group under study was measured and calculated for average diameter before, during, and after treatment. Serial blinded measurements (during and after treatment) of tumor volumes were taken for 120 days (Figure 1 and Table 3). The initial tumor volumes (day 0) for all mice from each group were approximately 115 ± 24 mm³. The reduction in tumor volume in the group receiving AAVP-TNF-α plus LCL161 was statistically significant by day 10 after treatment (P=0.0001). The AAVP-TNF-α plus LCL161 treatment group had a mean (+ SD) tumor volume of 235.31+97.00 mm³; the AAVP-TNF-α treatment group had a mean tumor volume of 388.85+188.76 mm³; the LCL161 treatment group had a mean tumor volume of 673.94+118.20mm³, and the control group had a mean tumor volume of 812.98+324.58 mm³ (Figure IF and Table 3) (day 10). Tumor growth in the AAVP-TNF-α plus LCL161 treatment group was rapidly inhibited, in comparison with that of the AAVP-TNF-a treatment group, the LCL161 treatment group, and control group (Figure 1 and Table 3). Antitumor activity was also seen in mice receiving either AAVP-TNF-α alone or LCL161 alone; however, the magnitude of the effect was significantly less than that associated with the combination of the agents (Figure 1 and Table 3). Mice in the group receiving combination therapy showed regression of tumors from their baseline measurements and in 3 mice, a complete tumor- free response was seen (Figure 1). Although the mice receiving either AAVP-TNF-α alone or LCL161 alone exhibited a decrease in the rate of tumor growth, neither group showed evidence of tumor regression (Figure 1).

[0089] Table 3 : Tumor Growth Inhibition With the Combination of AAVP-TNF-α and LCL161 Treatment

* The tumor volume can't be measured because all mice were dead in these groups

[0090] In this study, the development of treatment resistance was analyzed after two cycles of treatment. Tumor growth in the mice receiving the combination of AAVP-TNF-a and LCL161 continued to be inhibited at a <71% reduction (from 175.96 mm³ + 173.48 mm³ to 51.18 mm³ + 66.05 mm³) in the final tumor volumes in the second cycle (Table 3). In contrast, tumor growth in animals receiving AAVP-TNF-α alone was not inhibited (from 1306.42 mm³ to 1699.34 mm ) in the second cycle (Table 3). There were no live mice in the LCL161 alone treatment group or the control group after the first cycle. These results indicate that the combination of AAVP-TNF-α plus LCL161 can prevent or delay the development of resistance to treatment, because a repeat cycle again resulted in tumor responses (Figure 1 and Table 3).

[0091] Treatment with AAVP-TNF-a plus LCL161 prolonged survival in nude mice with human melanoma xenografts: Tumor-specific survival was compared with tumor progression-free survival on day 120 in each group. The tumor-specific survival rate was statistically significant among groups (PO.0001), 100%* for the AAVP-TNF-a plus LCL161 treatment group, 25% for the AAVP-TNF-α alone treatment group, 0% for the LCL161 alone treatment group, and 0% for the control group (Figure 2A) ("*": One mouse with a small tumor was sacrificed at day 82 in the combination treatment group for a skin infection often seen in nude mice. The necropsy confirmed the cause of death was "scaly skin" disease and not tumor progression). The median survival time was day 59 for the AAVP-TNF-α treatment group, day 45 for the LCL161 treatment group, and day 31 for the control group (Figure 2A). The combination group did not reach median survival.

[0092] The tumor-free complete response rate on day 120 was 50% for the AAVP-TNF- α plus LCL161 treatment group, and was 0% for the AAVP-TNF-α treatment group, the LCL161 treatment group, and the control group (PO.0001) (Figure 2B).

[0093] Doses of AAVP-TNF-α alone, LCL161 alone, and AAVP-TNF-α plus LCL161 did not increase systemic toxicity: Toxicity was assessed in each group by assessment of body mass, eating status, and mobility. The mice tolerated AAVP-TNF-α and/or LCL161 treatment as demonstrated by no changes in any of these parameters among the four groups.

[0094] Synergistic effect of AAVP-TNF-α and LCL161 in M21 xenografted nude mice: The additive activity of AAVP-TNF-α and LCL161 was evaluated and quantified with CalcuSyn Software. In fact, synergistic activity of AAVP-TNF-α and LCL161 was observed that resulted in combination indices under 0.8 at a fractional effect of 0.5 (50% tumor cell killing) and under 0.5 at a fractional effect of 0.75 (75% tumor cell killing) (Figure 3). Thus, AAVP-TNF-α and LCL161 were synergistic, with CI values less than 1 as determined with CalcuSyn Software.

[0095] AA VP-specific trafficking to tumor vasculature, but not to normal vasculature and normal tissues: 1 x 10¹¹ targeted AAVP particles were injected systemically into M21 xenografted nude mice. The tumor tissues and normal tissues (heart, spleen, kidney, liver, and muscle) from each group were removed on day 7 and day 21 after treatment. The presence of AAVP particles was determined by IF. On day 7 and day 21, AAVP particles were localized to the tumor-associated vasculature in mice treated with AAVP-TNF-a, but were absent from those animals that were not treated with AAVP-TNF-α. RGD-AAVP-TNF-a targeted specifically to tumor vasculature after systemic administration. Tumor and normal tissues (heart, spleen, kidney, liver, and muscle) from all groups were analyzed for AAVP particles by IF. AAVP particles were stained red with anti-bacteriophage antibody (Alexa Fluor 647), tumor vasculature was stained green with CD31 antibody (Alexa Fluor 488), and nuclei were stained blue with 4'6-diamidino-2-phenylindole (DAPI). AAVP-TNF-a selectively targeted tumor-associated vasculature in AAVP-TNF-α treated groups and was absent in non-targeted AAVP-TNF-α groups by day 7 and day 21 after injection. Furthermore, it was evaluated whether systemically administered AAVP particles were localized in normal vasculature of the heart, spleen, kidney, liver, and muscle. The imaging results showed that AAVP particles were not observed in any normal tissue vasculature in the mice that received AAVP-TNF-a. These data were consistent with previously reported results (1-2).

[0096] AA VP-mediated TNF-a expression in tumor tissues: The effect of AA VP- mediated TNF-a expression in tumor tissues was examined by real-time PCR. On day 7 and day 21, the levels of TNF-a mRNA were significantly increased in tumor tissues from groups treated with targeted AAVP-TNF-α, in comparison with groups lacking AAVP-TNF-a treatment (P<0.01 or 0.001) (Figure 4A). TNF-a mRNA levels were quantified in normal tissues after treatment with AAVP-TNF-α as shown in Figures 4B and 4C, TNF-a was not detected in normal tissues from mice receiving targeted AAVP-TNF-a.

[0097] Levels of TNF-a protein were measured in peripheral blood, tumor, and normal tissues by ELISA at day 7 and day 21. The mice receiving targeted AAVP-TNF-α exhibited significantly increased expression of TNF-a in tumor tissues, in comparison with those mice not receiving AAVP-TNF-α (P<0.001) (Figure 5A). The levels of TNF-a in peripheral blood were determined and normal tissues after treatment with targeted AAVP-TNF-α by ELISA. That TNF-a was not detected in these tissues of mice receiving targeted AAVP-TNF-a confirmed the tumor-selective delivery of TNF by this vector (Figure 5B and 5C).

[0098] Enhancement of tumor sensitivity to the induction of apoptosis with combined AAVP-TNF-α and LCL161 : It had been previously demonstrated by this laboratory that targeted AAVP-TNF-α induced the activation of caspase 3 in the tumor vasculature of mice with M21 tumor xenografts (9). In this study, it was investigated whether AAVP-TNF-α and LCL161 synergistically could enhance apoptosis in tumor cells and tumor vasculature. The combination of AAVP-TNF-α and LCL161 increases the expression of caspase 3 in tumor tissues. Caspase 3 was analyzed in tumor sections from the treatment groups on days 7 and 21 after treatment by IF. Caspase 3 was stained red by an anti-caspase 3 antibody (Alexa Fluor 647), blood vessels were stained green by an anti-CD31 antibody (Alexa Fluor 488), and nuclei were stained blue with DAPI. The expression of caspase 3 in tumor tissues was increased in the treatment groups compared with the control group on day 7 after treatment. The expression of caspase 3 was the highest in the group receiving the combination of targeted AAVP-TNF-a plus LCL161, in comparison with either AAVP-TNF-a alone or LCL161 alone. The expression of caspase 3 in tumor tissues was increased in the treated groups compared with the control group on day 21 after treatment. The combination of AAVP-TNF-α and LCL161 resulted in the highest level of caspase 3, relative to that seen in tissues treated with either AAVP-TNF-α alone or LCL161 alone. The combination of AAVP- TNF-a and LCL161was determined to result in an increased proportion of apoptotic cells in the tumor tissues, approximately 41% on day 7 (Fig. 6A) and 83% on day 21 (Fig. 6B). However, treatment with either AAVP-TNF-α alone or LCL161 alone resulted in only 10% on day 7 (Figure 7A and 7C) and 20% on day 21 (Figure 7B and 7D). These results indicate that the combination of AAVP-TNF-α and LCL161 induced apoptosis within tumor tissues to a greater degree than treatment with either AAVP-TNF-α or LCL161 alone (P<0.01) (Figure 6A and 6B). This observation supports the data shown for tumor response and survival data (Figure 2 and 3).

[0099] Activation of apoptotic pathways in response to expression of caspase 3 and caspase 9 after therapy with a combination of AAVP-TNF-α and LCL161 : To characterize further the activation of apoptotic pathways as a result of treatment with AAVP-TNF-α and LCL161, a series of IF experiments was performed. Tumors treated with the combination of targeted AAVP- TNF-a and LCL161 exhibited the highest levels of caspase 3, relative to either AAVP-TNF-α alone or LCL161 alone, as well as controls on day 7 (Figure 7A) and day 21 (Figure 7B).

[00100] The high-affinity binding of LCL161 to XIAP results in the destruction of XIAP, a reaction that precipitates increased sensitivity to TNF-a and further induction of caspase 9. Expression of caspase 9 was analyzed by IF in tumor sections from the treated and control groups on day 7 and day 21 after treatment. Caspase 9 was stained red by an anti-caspase 9 antibody (Alexa Fluor 647), blood vessels were stained green by an anti-CD31 antibody (Alexa Fluor 488), and nuclei were stained blue with DAPI. Levels of caspase 9 in tumor tissues were increased in treated groups compared with the control on day 7 after treatment. The highest expression of caspase 9 in tumor tissues was seen in the group receiving combination targeted AAVP-TNF-α plus LCL161, in comparison either AAVP-TNF-a alone or LCL161 alone. The expression of caspase 9 in tumor tissues was increased in the treated groups compared with the control group on day 21 after treatment. Likewise, caspase 9 in tumor tissues was highest in the targeted AAVP-TNF-a plus LCL161 treated group, relative to groups treated with either AAVP-TNF-α alone or LCL161 alone. It was observed that the activity of caspase 9 in the tumors increased significantly after treatment with the combination of targeted AAVP-TNF-α and LCL161, relative to either AAVP-TNF-α alone or LCL161 alone or to control groups on day 7 (Figure 8 A) and day 21 (Figure 8B). These results support the conclusion that the combination of AAVP-TNF-α and LCL161 enhance tumor-specific apoptosis through the activation of pathways in which caspase 3 and 9 participate.

[00101] Effect of the combination of targeted AAVP-TNF-α plus LCL161 on cell proliferation: To identify alternative mechanisms causing the antitumor effect seen with the combination of targeted AAVP-TNF-α and LCL161, cell proliferation by IF with anti-PCNA antibody was also examined. No differences in cell proliferation were found in any of the treated groups compared to controls and no evidence of inhibition of cell proliferation (P>0.05) (Figure 9A-9D). This observation further shows an apoptotic mechanism rather than an inhibition of proliferation for the effects on tumor growth observed.

Discussion

[00102] To enhance efficacy and reduce (or eliminate) toxicity remains the major challenge in anticancer therapy. Recently, this laboratory has developed a targeted hybrid vector that can be used for sustained expression and delivery of a transgene, such as the potent cytotoxic and anti- vascular cytokine TNF-a, to the tumor vasculature such that expression of the anti-tumor agent is restricted to the tumor microenvironment (1-2, 9-10). In this study, whether the combination of RGD-AA VP -TNF-a plus LCL161 could enhance tumor growth inhibition yet augment the pro- apoptotic activity of TNF-a was investigated. The results showed that the former was significantly enhanced by the combination of AAVP-TNF-a plus LCL161, in comparison with either compound alone (P<0.0001) (Figure 1 and Table 3). In fact, the combination resulted in tumor regression as well as tumor-free complete response and prolonged survival.

[00103] The reduction in tumor volume in the combined treatment group was statistically significant by day 10 (P=0.0001), (Figure 1 and Table 3). Tumor-specific survival rate was significantly increased with the combination of AAVP-TNF-a and LCL161, relative to AAVP- TNF-a alone or LCL161 alone (PO.0001), 100% for the AAVP-TNF-α plus LCL161 treatment groups, 25% for the AAVP-TNF-a alone treatment group, 0% for LCL161 alone treatment group, and 0% for the control group on day 120 after treatment (Figure 2A). Most importantly, complete tumor regression was seen in three mice in the AAVP-TNF-α plus LCL161 -treated group on day 23, day 62, and day 85. Moreover, there was an overall 50%> tumor-free survival rate for the AAVP-TNF-a plus LCL161 combination, with no tumor- free survival seen in any of the other groups (P<0.0001) (Figure 2B). An analysis of the combinatorial effect demonstrated that the combination of targeted AAVP-TNF-a and LCL161 can significantly enhance antitumor activity and prolong survival time by a synergistic mechanism (Figure 3).

[00104] In the current study, the systemic toxicity as well as the tissue selective trafficking of AAVP particles in animal's treatment with both AAVP-TNF-a and LCL161 was assessed. The mice tolerated AAVP-TNF-a and/or LCL161 as demonstrated by no change in body mass, eating status, and mobility among the four groups. Furthermore, it was identified that targeted RGD-AA VP particles localized selectively to the tumor vasculature of the M21 xenografts after systemic administration, data consistent with our previous observations (1-2). No evidence of vector or gene product expression was found in normal tissues, such as heart, liver, kidney, spleen, and muscle by day 7 and day 21. It was further demonstrated that RGD-AA VP -TNF-a mediated gene transcription of human TNF-a occurred specifically in the tumor vasculature and not in the normal tissues of mice on day 7 and day 21 (Figure 4). Human TNF-a protein was also observed selectively in tumor tissue and not in normal tissues and peripheral blood at all time points evaluated (Figure 5).

[00105] Smac is a mitochondrial protein and was found in the cytosol when a cell is primed for apoptosis by caspase activation. Smac was identified as an inhibitor of IAP-binding protein and it moderates caspase inhibition of IAP (32-33). LCL161 is a next generation SMAC- mimetic that can bind to many IAPs with high affinity and initiates the destruction of CIAPi_, CIAP₂, and XIAP, events associated with the induction of apoptosis. Previous studies have reported that CIAPi, CIAP₂, and XIAP are highly expressed in many tumors and function in the resistance to apoptotic pathways induced by TNF-a^34"35. A combination of a drug targeting XIAP and CIAPs, together with a tumor-targeted vector expressing TNF-a, could possibly result in the increased induction of apoptosis and an enhanced anti-tumor effect. The present study demonstrates that the combination of tumor-targeted AAVP-TNF-a and LCL161 led to increased apoptosis in the tumor and the tumor vasculature, in comparison with either targeted AAVP-TNF-a alone or LCL161 alone on days 7 (Figure 6A) and 21 (Figure 6B). This increased apoptotic activity was due to enhanced activity of enzymes in the apoptotic cascade. In particular, the activity of caspase 3 was significantly increased in the tumor tissues after combination therapy by day 7 (P<0.001) (Figure 7A) and day 21 (P<0.001) (Figure 7B), in comparison with tissues from animals treated with single agent or control. The activity of caspase 9 was also significantly increased following combination therapy by day 7 (P<0.001) (Figure 8A) and day 21 (P<0.001) (Figure 8B). Moreover, significant synergy was observed.

[00106] The combination of AAVP-TNF-α and LCL161 inhibits tumor growth through the induction of apoptosis rather than an inhibition of proliferation, as evaluated by PCNA, a marker of proliferation. Tumor cell proliferation was not inhibited in any of our treatment groups (P>0.05) (Figure 10). The combination of RGD-AA VP-TNF-a and LCL161 synergistically enhanced apoptotic effects via activation of caspases 3 and caspase 9.

[00107] In summary, these results demonstrate that the combination of targeted gene therapy with AAVP-TNF-a and oral administration of the Smac mimetic, LCL-161, has synergistic antitumor activity. This activity results from the induction of apoptosis in the tumor cells and the tumor-associated vasculature. Mice receiving this combination were cured of their tumors, with complete tumor regression in 50% of the treated animals. Survival was prolonged significantly in all animals treated with the combination. It is concluded that combinatorial therapy with targeted AAVP-TNF-a and LCL-161 can enhance efficacy, and reduce toxicity, as well as prevent and/or delay the development of resistance. This novel strategy of targeted gene therapy with AAVP-TNF-α and targeting IAPs (XIAP, CIAPl, and CIAP2) with inhibitors, exemplified herein by LCL161, will be of great benefit to patients with cancer.

REFERENCES

1. Tandle A, Hanna E, Lorang D, Hajitou A, Moya CA, Pasqualini R, Arap W, Adem A, Starker E, Hewitt S, Libutti SK. Tumor vasculature-targeted delivery of tumor necrosis factor-alpha. Cancer. 2009 Jan 1; 115(1): 128-39.

2. Paoloni MC, Tandle A, Mazcko C, Hanna E, Kachala S, Leblanc A, Newman S, Vail D, Henry C, Thamm D, Sorenmo K, Hajitou A, Pasqualini R, Arap W, Khanna C, Libutti SK. Launching a novel preclinical infrastructure: comparative oncology trials consortium directed therapeutic targeting of TNFalpha to cancer vasculature. PLoS One. 2009; 4(3):e4972.

3. Larocca D, Kassner PD, Witte A, Ladner RC, Pierce GF, Baird A. Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. FASEB J 1999; 13:727-34.

4. Barrow PA, Soothill JS. Bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential. Trends Microbiol 1997; 5:268-71.

5. Ivanenkov V, Felici F, Menon AG. Uptake and intracellular fate of phage display vectors in mammalian cells. Biochim Biophys Acta 1999; 1448:450-62.

6. Weber-Dabrowska B, Mulczyk M, Gorski A. Bacteriophages as an efficient therapy for antibiotic-resistant septicemia in man. Transplant Proc 2003; 35: 1385-6.

7. Borysowski J, Gorski A. Is phage therapy acceptable in the immunocompromised host? Int J Infect Dis 2008; 12:466-71.

8. Hajitou A, Trepel M, Lilley CE, et al. A hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell 2006; 125:385-98. Hajitou A, Rangel R, Trepel M, et al. Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat Protoc 2007; 2:523-31.

Pasqualini R, Koivunen E, Ruoslahti E. A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J Cell Biol 1995; 130: 1189-96.

Friedl J, Puhlmann M, Bartlett DL, et al. Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF. Blood 2002; 100: 1334-9.

Kerkar S, Williams M, Blocksom JM, Wilson RF, Tyburski JG, Steffes CP. TNF-alpha and IL-lbeta increase pericyte/endothelial cell co-culture permeability. J Surg Res 2006; 132:40-5.

Lienard D, Ewalenko P, Delmotte JJ, Renard N, Lejeune FJ. High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 1992; 10:52-60. Olieman AF, van Ginkel RJ, Hoekstra HJ, Mooyaart EL, Molenaar WM, Koops HS. Angiographic response of locally advanced soft-tissue sarcoma following hyperthermic isolated limb perfusion with tumor necrosis factor. Ann Surg Oncol 1997; 4:64-9.

Van Horssen R, Ten Hagen TL, Eggermont AM. TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist 2006; 11 :397-408. Bartlett DL, Buell JF, Libutti SK, et al. A phase I trial of continuous hyperthermic peritoneal perfusion with tumor necrosis factor and cisplatin in the treatment of peritoneal carcinomatosis. Cancer 1998; 83: 1251-61.

Bartlett DL, Libutti SK, Figg WD, Fraker DL, Alexander HR. Isolated hepatic perfusion for unresectable hepatic metastases from colorectal cancer. Surgery 2001; 129: 176-87. Alexander HR, Jr., Bartlett DL, Libutti SK, Fraker DL, Moser T, Rosenberg SA. Isolated hepatic perfusion with tumor necrosis factor and melphalan for unresectable cancers confined to the liver. J Clin Oncol 1998; 16: 1479-89.

Alexander HR, Jr., Bartlett DL, Libutti SK. Current status of isolated hepatic perfusion with or without tumor necrosis factor for the treatment of unresectable cancers confined to liver. Oncologist 2000; 5:416-24. Alexander HR, Libutti SK, Bartlett DL, Puhlmann M, Fraker DL, Bachenheimer LC. A phase I-II study of isolated hepatic perfusion using melphalan with or without tumor necrosis factor for patients with ocular melanoma metastatic to liver. Clin Cancer Res 2000;6:3062-70.

Alexander HR, Jr., Libutti SK, Bartlett DL, et al. Hepatic vascular isolation and perfusion for patients with progressive unresectable liver metastases from colorectal carcinoma refractory to previous systemic and regional chemotherapy. Cancer 2002; 95:730-6.

Libutti SK, Barlett DL, Fraker DL, Alexander HR. Technique and results of hyperthermic isolated hepatic perfusion with tumor necrosis factor and melphalan for the treatment of unresectable hepatic malignancies. J Am Coll Surg 2000;191 :519-30

Lans TE, Bartlett DL, Libutti SK, et al. Role of tumor necrosis factor on toxicity and cytokine production after isolated hepatic perfusion. Clin Cancer Res 2001;7:784-90. Lejeune FJ, Lienard D, Matter M, Ruegg C. Efficiency of recombinant human TNF in human cancer therapy. Cancer Immun 2006;6:6.

Rivera F, Lopez -Tarruella S, Vega-Villegas ME, Salcedo M. Treatment of advanced pancreatic cancer: from gemcitabine single agent to combinations and targeted therapy. Cancer Treat Rev 2009;35:335-9.

Xie Y, Sheng W, Miao J, Xiang J, Yang J. Enhanced antitumor activity by combining an adenovirus harboring ING4 with cisplatin for hepatocarcinoma cells. Cancer Gene Ther. 2010 Nov 5.

Seoane S, Montero JC, Ocana A, Pandiella A. Effect of multikinase inhibitors on caspase- independent cell death and DNA damage in HER2-overexpressing breast cancer cells. J Natl Cancer Inst. 2010 Sep 22; 102(18): 1432-46.

Dai MH, Zamarin D, Gao SP, Chou TC, Gonzalez L, Lin SF, Fong Y. Synergistic action of oncolytic herpes simplex virus and radiotherapy in pancreatic cancer cell lines. Br J Surg. 2010 Sep; 97(9): 1385-94.

Malvicini M, Rizzo M, Alaniz L, Pinero F, Garcia M, Atorrasagasti C, Aquino JB, Rozados V, Scharovsky OG, Matar P, Mazzolini G. A novel synergistic combination of cyclophosphamide and gene transfer of interleukin-12 eradicates colorectal carcinoma in mice. Clin Cancer Res. 2009 Dec 1; 15(23):7256-65. Gyrd-Hansen M, Meier P. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation and cancer. Nat Rev Cancer. 2010 Aug; 10(8):561-74

Gray D, McKenzie MD, Nachbur U, Huang DC, Bouillet P, Thomas HE, Borner C, Silke J, Strasser A, Kaufmann T. Jost PJ, Grabow S, XIAP discriminates between type I and type II FAS-induced apoptosis. Nature. 2009 Aug 20; 460(7258): 1035-9.

Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y. Structural basis of IAP recognition by Smac/DIABLO. Nature. 2000 Dec 21-28; 408(6815): 1008-12.

Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, Lee RA, Robbins PD, Fernandes-Alnemri T, Shi Y, Alnemri ES. A conserved XIAP -interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature. 2001 Mar l; 410(6824): 112-6.

Ashkenazi A, Herbst RS (2008). To kill a tumor cell: the potential of proapoptotic receptor agonists. J Clin Invest

Newsom-Davis T, Prieske S, Walczak H (2009). Is TRAIL the holy grail of cancer therapy? Apoptosis 14: 607-623

Damaraju VL, Bouffard DY, Wong CK, Clarke ML, Mackey JR, Leblond L, Cass CE, Grey M, Gourdeau H. Synergistic activity of troxacitabine (Troxatyl) and gemcitabine in pancreatic cancer. BMC Cancer. 2007 Jul 3; 7: 121.

Thoennissen NH, Iwanski GB, Doan NB, Okamoto R, Lin P, Abbassi S, Song JH, Yin D, Toh M, Xie WD, Said JW, Koeffier HP. Cucurbitacin B induces apoptosis by inhibition of the JAK/STAT pathway and potentiates antiproliferative effects of gemcitabine on pancreatic cancer cells. Cancer Res. 2009 Jul 15; 69(14):5876-84.

Kurbanov B.M., Fecker L.F., Geilen C.C., Sterry W., and Eberle J. (2007). Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4. Oncogene 26, 3364-3377.

Libutti SK et al., Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine, Clin., Cancer Res. 2010 Dec 15;16(24):6139-49. Epub 2010 Sep 27.

Claims

What is claimed:

1. A method of treating a cancer or a tumor in a subject comprising administering to the subject an amount of an inducer of apoptosis and an amount of an inhibitor of Inhibitor of Apoptosis Protein (IAP), in amounts effective to treat a cancer or a tumor in a subject.

2. The method of Claim 1, wherein the inducer of apoptosis and the inhibitor of IAP are administered concurrently.

3. The method of Claim 1, wherein the inducer of apoptosis and the inhibitor of IAP are administered sequentially.

4. The method of Claim 1, 2 or 3, wherein the amount of inducer of apoptosis and the amount of the inhibitor of IAP combined elicit a synergistic effect in treating the cancer or tumor.

5. The method of Claim 4, wherein the synergistic effect comprises enhanced apoptosis of tumor cells.

6. The method of Claim 4 or 5, wherein the synergistic effect comprises enhanced apoptosis of tumor-associated vasculature cells.

7. The method of any of Claims 1-6, wherein the inhibitor of IAP is a Smac mimetic.

8. The method of any of Claims 1-7, wherein the inhibitor of IAP is a small molecule of 2000 daltons or less.

9. The method of any of Claims 1-6, wherein the inhibitor of IAP is a peptide, an antibody, or an antigen-binding fragment of an antibody.

10. The method of any of Claims 1-9, wherein the inducer of apoptosis is tumor necrosis factor (TNF) or wherein the inducer of apoptosis elicits production in the subject of TNF.

11. The method of Claim 10, wherein the TNF is TNFa.

12. The method of any of Claims 1-11, wherein the inducer of apoptosis is administered locally into the cancer or locally into the tumor.

13. The method of any of Claims 1-11, wherein the inducer of apoptosis is administered in a manner which targets it to and/or selectively delivers it to the cancer or tumor.

14. The method of Claim 13, wherein the inducer of apoptosis is administered in a manner which targets it to and/or selectively delivers it to the cancer or tumor by administering it using a peptide targeting-ligand, protein targeting-ligand, aptamer targeting-ligand or antibody targeting-ligand.

15. The method of any of Claims 1-13, wherein the inducer of apoptosis is administered using a vector comprising a nucleic acid encoding the inducer under conditions permitting expression therefrom of the inducer.

16. The method of any of Claims 12-15, wherein the inducer of apoptosis is recombinant TNFa.

17. The method of Claim 16, wherein the TNFa is conjugated to a nanoparticle.

18. The method of Claim 17, wherein the nanoparticle comprises gold.

19. The method of any of Claims 1-18, wherein the inhibitor of IAP is administered systemically.

20. The method of any of Claims 1-18, wherein the inhibitor of IAP is administered orally.

21. The method of any of Claims 1-18 or 20, wherein the IAP is administered locally into the cancer or tumor.

22. The method of Claim 15, wherein the vector is a hybrid prokaryotic-eukaryotic vector.

23. The method of Claim 22, wherein the hybrid prokaryotic-eukaryotic vector comprises a genetic cis element of a recombinant adeno-associated virus.

24. The method of Claim 22 or 23, wherein the hybrid prokaryotic-eukaryotic vector comprises a phage vector.

25. The method of Claim 24, wherein the phage is an M13-derived phage.

26. The method of any of Claims 22-25, wherein the hybrid prokaryotic-eukaryotic vector comprises a bacteriophage expressing the amino acid motif arginine-glycine-aspartic acid on its surface.

27. The method of any of Claims 1-26, wherein the inhibitor of IAP inhibits one or more of XIAP, CIAPi and CIAP₂.

28. The method of any of Claims 1-27, wherein the treatment effects a decrease in tumor volume or effects tumor regression.

29. The method of any of Claims 1-27, wherein the treatment effects a decrease in tumor growth.

30. The method of any of Claims 1-29, wherein the cancer or tumor is a cancer or tumor of the breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid or skin.

31. The method of any of Claims 1-30, wherein the cancer or tumor is a cancer or tumor of the skin.

32. The method of Claim 31 , wherein the cancer or tumor is a melanoma.

33. An inducer of apoptosis for treating a cancer or a tumor in a subject, wherein the inducer of apoptosis is administered concurrently, separately or sequentially with an inhibitor of Inhibitor of Apoptosis Protein (IAP).

35. An inducer of apoptosis and an inhibitor of Apoptosis Protein (IAP) as a combined preparation for treating a cancer or a tumor in a subject.

36. An inducer of apoptosis for use with an inhibitor of Inhibitor of Apoptosis Protein (IAP) for treating a cancer or tumor in a subject.

37. An inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) for treating a cancer or tumor in a subject.

38. The inducer of apoptosis and the inhibitor of IAP of any of Claims 33-37, wherein the inducer of apoptosis and inhibitor of IAP are formulated for use concurrently.

39. The inducer of apoptosis and the inhibitor of IAP of any of Claims 33-37, wherein the inducer of apoptosis and inhibitor of IAP are formulated for use sequentially.

40. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein of any of Claims 33-39, wherein the inhibitor of Inhibitor of Apoptosis Protein is a Smac mimetic.

41. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein of any of Claims 33-40, wherein the inhibitor of Inhibitor of Apoptosis Protein is a small molecule of 2000 daltons or less.

42. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein of any of Claims 33-41, wherein the inhibitor of Inhibitor of Apoptosis Protein is an antibody or an antigen-binding fragment of an antibody.

43. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein of any of Claims 33-42, wherein the inducer of apoptosis is tumor necrosis factor (TNF) or elicits production in the subject of TNF.

44. The inducer of apoptosis and the inhibitor of Inhibitor of Apoptosis Protein of Claim 43, wherein the TNF is TNFa.

45. The method of any of Claims 33-44, wherein the inducer of apoptosis is formulated as an expressible vector comprising a nucleic acid encoding the inducer.

46. A method of delaying or preventing resistance of a cancer or a tumor in a subject to an anti-cancer or anti-tumor therapy, respectively, comprising administering to the subject an inducer of apoptosis and an inhibitor of Inhibitor of Apoptosis Protein (IAP) in amounts effective to prevent resistance of the cancer or tumor to the anti-cancer or anti-tumor therapy.

47. The method of Claim 46, wherein the inducer of apoptosis is TNFa.

48. The method of Claim 46 or 47, wherein the inducer of apoptosis and the inhibitor of IAP are administered concurrently.

49. The method of Claim 46 or 47, wherein the inducer of apoptosis and the inhibitor of IAP are administered sequentially.

50. The method of any of Claims 46-49, wherein the cancer or tumor is a cancer or tumor of the breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus, testes, ovary, uterus, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid or skin.