Use of bacteria, bacterial products, and other immunomodulatory entities in combination with anti-CTLA-4 and / or anti-PD-1 antibodies to treat solid tumor malignancies

Abstract

To provide methods and kits for treating solid tumors in a subject by using a combination of anti-CTLA-4 and / or anti-PD-1 antibodies with at least one member of the group consisting of bacteria, bacterial products, and immunoregulatory entities.SOLUTION: In particular embodiments, the bacteria are toxin-depleted, anaerobic bacteria, such as Clostridium novyi-NT. In one aspect, the invention provides a method for treating a solid tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody in combination with at least one member of the group consisting of bacteria, bacterial products, and immunoregulatory entities, thereby treating the solid tumor.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 972,633, filed March 31, 2014, and U.S. Provisional Application No. 62 / 035,291, filed August 8, 2014, which are incorporated by reference in their entireties. [Background technology]

[0002] Patients presenting with advanced cancer of the pancreas, colon, lung, breast, ovary, brain or prostate have a poor prognosis. This tragic situation has spurred a surge of research that has led to a revolution in our understanding of cancer pathogenesis, a marked increase in the application of traditional chemotherapy drugs, and several promising new agents. Unfortunately, these innovations have yet to have a significant impact on the treatment of common solid tumors. Many believe that the greatest hope for future therapeutic outcomes lies in combining novel approaches, such as spores of the anaerobic bacterial strain Clostridium novyi (C. novyi), with more traditional drugs.

[0003] The rationale for using anaerobic bacteria is the unique vascularization conditions present within tumors. Solid tumors require angiogenesis to grow, and as they grow, parts of the tumor become poorly vascularized. These avascular regions tend to have low concentrations of therapeutic agents. Furthermore, drug molecules that do reach avascular regions typically rely on both oxygen and actively replicating cells for full efficacy.

[0004] It has previously been shown that solid tumor malignancies can be treated by using several species of anaerobic bacteria. C. novyi is a gram-positive endospore-forming obligately anaerobic bacterium. Clostridium novyi-NT (C. novyi-NT) is an attenuated form of C. novyi that lacks the major toxin. The use of C. novyi-NT for the treatment of cancer has been reported previously (Agrawal et al., (2004) Proc. Natl. Acad. Sci. USA 101(42):15172-15177; Bettegowda et al., (2003) Proc. Natl. Acad. Sci. USA 100(25):15083-15088; Bettegowda et al., (2006) Nat. Biotechnol. 24(12):1573-1580; Cheong et al., (2006) Science 314(5803):1308-1311; Dang et al., (2004) Cancer Biol. Ther. 3:326-337; Dang et al., (2004) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; Diaz et al. (2005) Toxicol. Sci. 88(2):562-575; Krick et al. (2012) Am. J. Vet. Re. 73(1):112-118). Immunotherapy is also a promising approach to eradicate metastatic cancer. Recent clinical trials of neutralizing antibodies targeting CTLA-4 and PD-1, two key checkpoints for T cell-mediated immunity, have shown clinical responses in patients with solid tumor malignancies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Agrawal et al., Proc. Natl. Acad. Sci. USA (2004) 101(42):15172-15177 [Non-Patent Document 2] Bettegowda et al., Proc. Natl. Acad. Sci. USA (2003) 100(25):15083-15088 [Non-Patent Document 3] Bettegowda et al., Nat. Biotechnol. (2006) 24(12):1573-1580 Summary of the Invention [Means for solving the problem]

[0006] In one embodiment, the presently disclosed subject matter provides a method for treating a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody in combination with at least one member of the group consisting of a bacterium, a bacterial product, and an immunomodulatory entity to treat the solid tumor. In a particular embodiment, the bacterium is a lethal toxin-depleted anaerobic bacterium. In another particular embodiment, the bacterial product is a bacterial component, such as a bacterial membrane component.

[0007] In certain embodiments, the presently disclosed subject matter provides a kit for treating a solid tumor, the kit comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and at least one member of the group consisting of a bacterium, a bacterial product, and an immunomodulatory entity.

[0008] In other aspects, the presently disclosed subject matter provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody to treat the cancer.

[0009] While certain aspects of the presently disclosed subject matter have been described above and are addressed in whole or in part by the presently disclosed subject matter, other aspects will become apparent as the description proceeds when considered in conjunction with the accompanying examples and figures, as best described herein below.

[0010] Having described the presently disclosed subject matter in these general terms, reference is now made to the accompanying drawings, which are not necessarily drawn to scale. The present invention provides, for example, the following items. (Item 1) 1. A method for treating a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody, in combination with at least one member of the group consisting of a bacteria, a bacterial product, and an immunomodulatory entity, to treat the solid tumor. (Item 2) 2. The method according to item 1, wherein the bacterium is an anaerobic bacterium. (Item 3) The method according to item 2, wherein the anaerobic bacterium is Clostridium novyi. 3. The method of claim 2, wherein the bacterium or the bacterial product is a toxin-depleted anaerobic bacterium. (Item 5) 5. The method of claim 4, wherein the toxin-depleted anaerobic bacterium is Clostridium novyi-NT. (Item 6) 5. The method of claim 4, wherein some or all of the toxin genes of the wild-type toxin-depleted anaerobic bacterium are deleted. (Item 7) 5. The method of claim 4, wherein the toxicity of the toxin-depleted anaerobic bacteria is reduced by at least 2-fold compared to the corresponding wild-type bacteria. (Item 8) 2. The method of claim 1, wherein the bacterial product is at least one bacterial membrane component. (Item 9) 2. The method of claim 1, wherein the at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities is administered intravenously or intratumorally. (Item 10) 2. The method of claim 1, wherein the at least one antibody is administered by at least one method selected from the group consisting of intravenous, intramuscular, subcutaneous, or intratumoral. (Item 11) 2. The method of claim 1, wherein the solid tumor is malignant. (Item 12) 2. The method of claim 1, wherein the solid tumor regresses or the growth of the solid tumor is slowed or stopped after the solid tumor is treated. (Item 13) 2. The method of claim 1, wherein the subject is a human. (Item 14) 2. The method of claim 1, wherein the subject is a non-human animal. (Item 15) 1. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody to treat the cancer. (Item 16) 16. The method of item 15, wherein the combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody is administered by at least one method selected from the group consisting of intravenous, intramuscular, subcutaneous, and intratumoral. (Item 17) 16. The method of claim 15, wherein the subject is a human. (Item 18) 2. The method of claim 1, wherein the subject is a non-human animal. (Item 19) 1. A kit for treating a solid tumor, comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and at least one member of the group consisting of a bacterium, a bacterial product, and an immunomodulatory entity. (Item 20) 20. The kit according to item 19, wherein the bacterium is an anaerobic bacterium. (Item 21) 21. The kit according to item 20, wherein the anaerobic bacterium is Clostridium novyi. (Item 22) 21. The kit according to item 20, wherein the anaerobic bacterium is a toxin-depleted anaerobic bacterium. (Item 23) 23. The kit according to item 22, wherein the anaerobic bacterium is Clostridium novyi-NT. (Item 24) 23. The kit according to item 22, wherein some or all of the toxin genes of the wild-type toxin-depleted anaerobic bacterium are deleted. (Item 25) 23. The kit of item 22, wherein the toxicity of the toxin-depleted anaerobic bacteria is reduced by at least 2-fold compared to the corresponding wild-type bacteria. (Item 26) 20. The kit of item 19, wherein the bacterial product is at least one bacterial membrane component. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing data from BALB / c mice bearing subcutaneous CT26 tumors treated with anti-CTLA-4 and / or anti-PD-1 antibodies with or without C. novyi-NT spores. [Diagram 2] 2A and 2B are graphs showing data from BALB / c mice bearing subcutaneous 4T1 tumors treated with anti-CTLA-4 and / or anti-PD-1 antibodies with or without C. novyi-NT spores: A) tumor growth; and B) survival data. [Diagram 3]Figures 3A-C are graphs and images showing the response to intratumoral C. novyi-NT treatment in a rat orthotopic brain tumor model. (A) Kaplan-Meier curves showing survival of F344 Fischer rats after orthotopic implantation of a syngeneic glioma cell line (F98). Red lines are C. novyi-NT spores injected intratumorally 12-15 days after tumor implantation. Black lines are controls. (B) Bioluminescence (Xenogen imaging system) in three representative F344 Fischer rats after orthotopic implantation of the F98 glioma cell line. Images obtained on day 0 (pretreatment-day of C. novyi-NT spore injection), day 1 after intratumoral injection of C. novyi-NT spores, and day 2 after intratumoral injection of C. novyi-NT spores; and (C) Luciferase activity (in millions) on day 0 (pretreatment), day 1 after intratumoral injection of C. novyi-NT spores, and day 2 after intratumoral injection of C. novyi-NT spores. [Figure 4] Figures 4A-D are images showing C. novyi-NT bacteria germinating within microscopic rat brain tumor lesions. Gram staining showed vegetative C. novyi-NT bacteria (yellow arrows) localized to tumors (T) and stellate microinvasions (S) in F344 Fischer rats, but not in normal brain tissue (Br): (A) tumor-normal brain interface, scale bar 30 μm; (B) tumor-normal brain interface, scale bar 10 μm; (C) normal brain, tumor, and stellate microinvasion interface of neoplastic tissue, scale bar 30 μm; and (D) C. novyi-NT germination evident in stellate microinvasion lesions, scale bar 10 μm. [Diagram 5]Figures 5A-F are images showing photographic and CT images from dog 11-R01 demonstrating a partial response to C. novyi-NT therapy. Images range from pretreatment to 70 days after the first intratumoral dose of C. novyi-NT spores: (A) pretreatment image of a peripheral nerve sheath tumor; (B) abscess formation on study day 3, limited in extent to the tumor; (C) spontaneous abscess rupture and secretion of necrotic and purulent material followed by medical debridement allowed healing by secondary intention; (D) the wound was completely healed by study day 70, with a 77.6% reduction in the maximum diameter of the tumor; (E) a pretreatment CT image taken 4 days prior to the first treatment showed tumor extension (yellow circle) at the intersection of the pinna and skull; and (F) a posttreatment CT image on study day 10 showing near complete debulking of the tumor. [Figure 6] 6A-6F are images showing photographic and CT images from dog 04-R03 showing a complete response to C. novyi-NT therapy. Images range from pretreatment to 60 days after the first intratumoral dose of C. novyi-NT spores: (A) pretreatment image of soft tissue sarcoma; (B) tumor-localized abscess formed 1 day after the third dose of C. novyi-NT spores, on study day 15; (C) tumor debulking was complete by study day 27 with healthy granulation tissue; (D) wound was completely healed by study day 60 with no residual tumor (complete response); (E) pretreatment CT image taken 5 days before the first treatment showing tumor spread (yellow circle) on the forearm; and (F) posttreatment CT image on study day 62 showing complete disappearance of tumor mass. [Figure 7] Figure 7 is a graph showing survival analysis of dogs treated with intratumoral injection of C. novyi-NT. Kaplan-Meier curves showing time to progression for dogs that showed either a complete or partial response to intratumoral injected C. novyi-NT. Dogs are followed if they have no progression at the last known evaluation. [Figure 8]8A-8D are images showing CT and MRI images from a human patient: (A) post-treatment CT with contrast on day 3 showing evidence of intramedullary and extramedullary air collection; (B) pre-treatment MRI (T1 with gadolinium contrast) of the right upper humerus showing a contrast enhancing mass involving soft tissue and possibly adjacent bone; (C) post-treatment MRI on day 4 showing reduced contrast enhancement in the tumor burden compared to baseline; and (D) post-treatment MRI on day 29 showing a homogenous non-enhancing mass consistent with ongoing necrosis. The tumor is highlighted by the yellow arrow. [Figure 9] Figures 9A-D are images showing extensive tumor necrosis in a human patient treated with C. novyi-NT spores: (A, B) pre-treatment tumor biopsy showing viable tumor (leiomyosarcoma) cells, scale bars are 100 and 30 μm, respectively; and (C, D) post-treatment tumor biopsy 4 days after intratumoral injection of C. novyi-NT spores showing extensive necrosis of tumor cells, scale bars are 100 and 30 μm, respectively. [Figure 10-1] FIG. 10 is a table showing summary data of the sequenced samples. [Figure 10-2] FIG. 10 is a table showing summary data of the sequenced samples. [Figure 10-3] FIG. 10 is a table showing summary data of the sequenced samples. [Figure 10-4] FIG. 10 is a table showing summary data of the sequenced samples. [Figure 11-1] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-2] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-3] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-4] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-5] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-6] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-7]FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-8] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-9] FIG. 11 is a table showing somatic alterations in canine sarcoma. [Figure 11-10] FIG. 11 is a table showing somatic alterations in canine sarcoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The subject matter disclosed herein will now be described more fully below with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Like numbers refer to like elements throughout. The subject matter disclosed herein can be embodied in many different forms, and should not be construed as being limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the subject matter disclosed herein described will come to mind to those skilled in the art to which the subject matter disclosed herein pertains, with the benefit of the teachings presented in the foregoing description and the accompanying drawings. Therefore, it is to be understood that the subject matter disclosed herein is not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0013] The subject matter disclosed herein provides methods and kits for treating tumors. It was hypothesized that abrogation of the negative regulation mediated through the PD-1 and CTLA-4 pathways could enhance the anti-cancer immune response induced by intratumoral bacterial infection, thus curing metastatic tumors. It has been shown herein below that the therapeutic effect of anti-tumor bacteria is significantly enhanced by combining with anti-CTLA-4 and / or anti-PD-1 antibodies. In a subcutaneous mouse tumor model, essentially 100% of the tumors were eradicated by this approach. In a metastatic tumor model, the number of metastases was significantly reduced, resulting in a significant survival benefit. Furthermore, in both tumor models, the combination of anti-CTLA-4 and anti-PD-1 antibodies produced better results than the use of either antibody alone.

[0014] Thus, the methods and kits disclosed herein use anti-CTLA-4 and / or anti-PD-1 antibodies in combination with bacteria, bacterial products, or other immunomodulatory entities to antagonize the negative regulatory mechanisms of anti-tumor immune responses induced by the immunomodulatory entities. Additionally, the methods and kits disclosed herein can be used to treat cancer by combining anti-CTLA-4 and anti-PD-1 antibodies.

[0015] I. Methods for Treating Cancer In some embodiments, the presently disclosed subject matter provides a method for treating a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody in combination with at least one member of the group consisting of a bacterium, a bacterial product, and an immunomodulatory entity to treat the solid tumor. Examples of antibodies that can be used in the presently disclosed methods include, but are not limited to, ipilimumab and tremelimumab against CTLA-4 and nivolumab against PD-1.

[0016] CTLA-4 (cytotoxic T lymphocyte antigen 4; e.g., GenBank Accession No. AAD00698.1), also known as CD152 (cluster of differentiation 152), is a T cell surface molecule that is a negative regulator of T cell activation. CTLA-4 was initially identified by differential screening of a mouse cytolytic T cell cDNA library (Brunet et al., (1987) Nature 328:267-270). CTLA-4 is also a member of the immunoglobulin (Ig) superfamily and contains a single extracellular Ig domain. CTLA-4 transcripts have been found in T cell populations with cytotoxic activity, suggesting that CTLA-4 may function in the cytolytic response (Brunet et al., (1987) Nature 328:267-270; Brunet et al., (1988) Immunol. Rev. 103:21-36). Researchers have reported the cloning and mapping of the gene for the human counterpart of CTLA-4 (Dariavach et al., (1988) Eur. J. Immunol. 18:1901-1905) to the same chromosomal region (2q33-34) as CD28 (Lafage-Pochitaloff et al., (1990) Immunogenetics 31:198-201). Sequence comparison between this human CTLA-4 DNA and the DNA encoding the CD28 protein revealed significant sequence homology, with the greatest degree of homology in the juxtamembrane and cytoplasmic regions (Brunet et al., (1987) Nature 328:267-270; Dariavach et al., (1988) Eur. J. Immunol. 18:1901-1905). Some studies have suggested that CTLA-4 has a similar function as a secondary costimulatory molecule (Linsley et al., (1992) J. Exp. Med. 176:1595-1604; Wu et al., (1997) J. Exp. Med. 185:1327-1335; U.S. Patent Nos. 5,977,318, 5,968,510, 5,885,796, and 5,885,579). However, others have reported that CTLA-4 has an opposing role as a buffer of T cell activation (Krurnmel (1995) J. Exp. Med. 182:459-465; Krurnmel et al. (1996) Int'l. Immunol. 8:519-523; Chambers et al. (1997) Immunity 7:885-895). CTLA-4-deficient mice have been reported to suffer from massive lymphoproliferation (Chambers et al. (1997) Immunity 7:885-895). Blockade of CTLA-4 has been reported to increase T cell responses in vitro (Walunas et al. (1994) Immunity 1:405-413) and in vivo (Kearney (1995) J. Immunol. 155:1032-1036), exacerbate antitumor immunity (Leach (1996) Science 271:1734-1736), and enhance induced autoimmune disease (Luhder (1998) J. Exp. Med. 187:427-432). CTLA-4 has also been reported to have alternative or additional effects on the initial features of T cell immune responses (Chambers (1997) Curr. Opin. Immunol. 9:396-404; Bluestone (1997) J. Immunol. 158:1989-1993; Thompson (1997) Immunity 7:445-450).

[0017] PD-1 (programmed cell death protein 1; e.g., GenBank Accession No. NP_005009.2), also known as CD279 (cluster of differentiation 279), is a cell surface membrane protein expressed primarily on a subset of activated T lymphocytes and encoded in humans by the PDCD1 gene (Entrez Gene GeneID: 5133; Ishida et al., (1992) EMBO J. 11:3887; Shinohara et al., (1994) Genomics 23:704; see also U.S. Pat. No. 5,698,520). PD-1 is a member of the immunoglobulin gene superfamily, with an extracellular region containing immunoglobulin superfamily domains, a transmembrane domain, and an intracellular region containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs; Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704). These features define a relatively large family of polypeptides called immunoinhibitory receptors, which also include gp49B, PIR-B, and killer cell inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). It is often assumed that the tyrosyl-phosphorylated ITIM motifs of these receptors interact with SH2 domain-containing phosphatases to generate inhibitory signals. A subset of these immunoinhibitory receptors bind MHC polypeptides, e.g., KIR and CTLA-4 bind B7-1 and B7-2. A phylogenetic relationship between the MHC and B7 genes has been proposed (Henry et al., (1999) Immunol. Today 20(6):285-8). Like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al., (1996) Int. Immunol. 8:765). In contrast to CTLA-4, however, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata et al., (1996) Int. Immunol. 8:765; Nishimura et al., (1996) Int. Immunol. 8:773).

[0018] Two human PD-1 ligands, PDL1 and PDL2, have been identified. PD-1 ligands contain a signal sequence, as well as IgV and IgC domains, a transmembrane domain, and a short cytoplasmic tail. Both PDL1 (NCBI Reference Sequence: NP_001254635.1; Freeman et al., (2000) J. Exp. Med. 192:1027) and PDL2 (NCBI Reference Sequence: NP_079515.2; Latchman et al., (2001) Nat. Immunol. 2:261) are members of the B7 family of polypeptides. Both PDL1 and PDL2 are expressed in the placenta, spleen, lymph nodes, thymus, and heart. Only PDL2 is expressed in the pancreas, lung, and liver, and only PDL1 is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells. The fact that PD-1 binds to PDL1 and PDL2 maps it, along with CTLA-4, to a family of inhibitory receptors.

[0019] A "functional variant" of CTLA-4 or PD-1 includes a functional fragment, a functional mutein, and / or a functional fusion protein. A functional variant of a selected polypeptide is an isolated and / or recombinant protein or polypeptide that has at least one property, activity, and / or functional characteristic of a selected polypeptide (e.g., CTLA-4 or PD-1). As used herein, the term "activity" when used in reference to a polypeptide, e.g., CTLA-4 or PD-1, includes an activity inherent to the structure of the wild-type protein.

[0020] For example, with respect to CTLA-4 or PD-1, the term "activity" includes the ability of CTLA-4 or PD-1 to regulate inhibitory signals in activated immune cells, for example, by binding natural CTLA-4 or PD-1 ligands on antigen-presenting cells. PD-1 transmits inhibitory signals to immune cells in a manner similar to CTLA-4. Regulation of inhibitory signals in immune cells regulates immune cell proliferation and / or cytokine secretion by immune cells. Thus, the term "CTLA-4 activity" or "PD-1 activity" includes the ability of CTLA-4 or PD-1 to bind to its natural ligand(s), regulate immune cell co-stimulatory or inhibitory signals, and regulate immune responses.

[0021] As used herein, the term "co-stimulate" includes the ability of a co-stimulatory polypeptide to provide a second, non-activating receptor-mediated signal ("co-stimulatory signal") that induces proliferation or effector function when used in relation to an activated immune cell. For example, a co-stimulatory signal can result in cytokine secretion, for example, in a T cell that has received a T cell receptor-mediated signal. For example, an immune cell that has received a cell receptor-mediated signal via an activating receptor is referred to herein as an "activated immune cell." As used herein, the term "co-stimulatory receptor" includes a receptor that transmits a co-stimulatory signal to an immune cell. As used herein, the term "inhibitory receptor" includes a receptor that transmits a negative signal to an immune cell (e.g., CTLA-4 or PD-1). The inhibitory signal transmitted by the inhibitory receptor can occur even if a costimulatory receptor (such as CD28) is not present on the immune cell, and therefore is not simply a function of competition between the inhibitory receptor and the costimulatory receptor for binding of the costimulatory polypeptide (Fallarino et al., (1998) J. Exp. Med. 188:205). The transmission of an inhibitory signal to an immune cell can result in unresponsiveness or anergy or programmed cell death in the immune cell. Preferably, the transmission of an inhibitory signal operates through a mechanism that does not involve apoptosis. As used herein, the term "apoptosis" includes programmed cell death that can be characterized using techniques known in the art. Apoptotic cell death can be characterized, for example, by cell shrinkage, membrane blebbing, and chromatin condensation resulting in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of internucleosomal DNA breaks.

[0022] In general, fragments or portions of CTLA-4 or PD-1 encompassed by the presently disclosed subject matter include fragments or portions of CTLA-4 or PD-1 that have a deletion (i.e., one or more deletions) of an amino acid (i.e., one or more amino acids) compared to wild-type CTLA-4 or PD-1 (such as an N-terminal, C-terminal or internal deletion). Fragments or portions that have only contiguous amino acids deleted or that have non-contiguous amino acids deleted compared to wild-type CTLA-4 or PD-1 are also contemplated. In general, mutants or derivatives of CTLA-4 or PD-1 encompassed by the presently disclosed subject matter include naturally occurring or artificial variants that differ by the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues, or modified polypeptides in which one or more residues have been altered, as well as mutants that include one or more altered residues. Preferred mutants are naturally occurring or artificial variants of CTLA-4 or PD-1 that differ by the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues.

[0023] Generally, a functional variant of CTLA-4 or PD-1 has an amino acid sequence that is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the wild-type amino acid sequence of CTLA-4 or PD-1 over the length of the variant.

[0024] "Sequence identity" or "identity" in the context of proteins or polypeptides refers to the amino acid residues of two amino acid sequences that are the same when aligned for maximum correspondence over a particular comparison window.

[0025] Thus, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where a portion of the amino acid sequence within the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (which contains no additions or deletions) of the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical amino acid residues are present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Useful examples of percent sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage between 50% and 100%. These identities can be determined using any of the programs described herein.

[0026] Sequence alignment and percent identity or similarity calculations can be determined using a variety of comparison methods designed to detect homologous sequences, including, but not limited to, the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Within the context of this application, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" shall mean any set of values ​​or parameters that originally came with the software when first initialized. The "Clustal V alignment method" is designated Clustal V (described by Higgins and Sharp (1989) CABIOS 5:151-153; Higgins et al. (1992) Comput. Appl. Biosci. 8:189-191) and corresponds to the alignment method found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).

[0027] It is well understood by those of skill in the art that many levels of sequence identity are useful for identifying proteins or polypeptides (e.g., from other species) that have the same or similar function or activity. Useful examples of percent identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage between 50% and 100%. Indeed, any integer amino acid identity between 50% and 100%, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% may be useful in describing the subject matter disclosed herein.

[0028] The term "antibody", also known as immunoglobulin (Ig), is a large Y-shaped protein produced by B cells that is used by the immune system to identify and neutralize foreign substances such as bacteria and viruses by recognizing unique portions (epitopes) of foreign targets called antigens. As used herein, the term "antibody" also includes the "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1 or CTLA-4). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR). Additionally, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, the VL and VH domains may be paired together to form a monovalent polypeptide (known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 341:544-546). 242:423-426; Huston et al., (1988) Proc. Natl. Acad Sci. USA 85:5879-5883; and Osbourn et al., (1998) Nature Biotechnology 16:778). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to create an expression vector encoding a complete IgG polypeptide or other isotype. The VH and VL can also be used to create Fab, Fv, or other fragments of immunoglobulins using protein chemistry or recombinant DNA techniques. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, forcing the domains to pair with the complementary domains on another chain and generating two antigen-binding sites (e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., (1994) Structure 2:1121-1123).

[0029] Additionally, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesin polypeptide formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesin polypeptides include the use of streptavidin core regions to generate tetrameric scFv polypeptides (Kipriyanov et al., (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine tags to generate bivalent biotinylated scFv polypeptides (Kipriyanov et al., (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Additionally, antibodies, antibody portions and immunoadhesin polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0030] The antibodies may be polyclonal or monoclonal, xenogeneic, allogeneic or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). The antibodies may be fully human. Preferably, the antibodies of the presently disclosed subject matter bind specifically or substantially specifically to PD-1 or CTLA-4 or functional variants thereof. The terms "monoclonal antibody" and "monoclonal antibody composition," as used herein, refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for a particular antigen with which it immunoreacts.

[0031] The term "humanized antibody", as used herein, is intended to include antibodies made by non-human cells having variable and constant regions that have been altered to more closely resemble antibodies made by human cells, e.g., by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies of the presently disclosed subject matter can, for example, include amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0032] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CTLA-4 or PD-1 is substantially free of antibodies that specifically bind to antigens other than CTLA-4 or PD-1). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0033] When isolated CTLA-4 or PD-1 or functional variants thereof (or nucleic acids encoding such polypeptides) are used as immunogens, it is possible to generate antibodies that bind to the respective CTLA-4 or PD-1 or functional variants thereof using standard techniques for polyclonal and monoclonal antibody preparation. Full-length CTLA-4 or PD-1 can be used, or alternatively, the subject matter disclosed herein relates to antigenic peptide fragments of CTLA-4 or PD-1 ligands or functional variants thereof for use as immunogens. The antigenic peptides of CTLA-4 or PD-1 or functional variants thereof contain at least 8 amino acid residues and encompass epitopes present in the respective full-length molecules such that antibodies raised against the peptides form specific immune complexes with the respective full-length molecules. Preferably, the antigenic peptides comprise at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptides are regions of CTLA-4 or PD-1 or functional variants thereof that are located on the surface of the protein, e.g., hydrophilic regions. Standard hydrophobicity analysis of the polypeptide molecule can be performed to identify hydrophilic regions. Highly preferred epitopes encompassed by antigenic peptides are regions of the polypeptide molecule that are in the extracellular domain and thus involved in binding. In one embodiment, such epitopes can be specific to a given polypeptide molecule from a species, such as mouse or human (i.e., antigenic peptides spanning regions of the polypeptide molecule that are not conserved across species are used as immunogens, and such non-conserved residues can be determined using alignments such as those provided herein).

[0034] An immunogen comprising CTLA-4 or PD-1 or a functional variant thereof is typically used to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal) with the immunogen. A suitable immunogen preparation can contain, for example, a recombinantly expressed or chemically synthesized molecule or fragment thereof that will generate an immune response. The preparation can further contain an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant. Immunization of a suitable subject with the immunogen preparation induces a polyclonal antibody response against the antigenic peptide contained therein.

[0035] Polyclonal antibodies can be prepared by immunizing a suitable subject with a polypeptide immunogen as described above. Polypeptide antibody titers in the immunized subject can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, antibodies directed against the antigen can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain an IgG fraction. At an appropriate time after immunization, for example when antibody titers are highest, antibody-producing cells are obtained from the subject and used to generate antibodies using the hybridoma technique first described by Kohler and Milstein (1975) Nature 256:495-497; Brown et al., (1981) J. Immunol. 127:539-46; Brown et al., (1980) J. Biol. Chem. 255:4980-83; Yeh et al., (1976) Proc. Natl. Acad. Sci. 76:2927-31; and Yeh et al., (1982) Int. J. Cancer 29:269-75), human B cell hybridoma technique (Kozbor et al., (1983) Immunol. Monoclonal antibodies can be prepared by standard techniques such as the EBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or the trioma technique. Techniques for producing monoclonal antibody hybridomas are well known (see generally, Kenneth, RH Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, NY (1980); Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefter et al., (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused with lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the resulting hybridoma cell culture supernatants are screened to identify hybridomas producing monoclonal antibodies that bind, preferably specifically, to the polypeptide antigen.

[0036] Any of the many well-known protocols used to fuse lymphocytes with immortalized cell lines can be used to generate anti-PD-1 ligand monoclonal antibodies (e.g., Galfre, G. et al. (1977) Nature 266:55052; Kenneth, RH Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, NY (1980); Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Moreover, one of skill in the art will recognize that there are many variations of such methods that would also be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be made by fusing lymphocytes from mice immunized with the immunogen preparations of the present subject matter disclosed herein with an immortalized mouse cell line. A preferred immortal cell line is a mouse myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of several myeloma cell lines can be used as fusion partners according to standard techniques, e.g., P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and non-productively fused myeloma cells (unfused splenocytes are not transformed and will die after a few days).Hybridoma cells producing a monoclonal antibody of the presently disclosed subject matter are detected by screening the hybridoma culture supernatants for antibodies that bind a given polypeptide, e.g., using a standard ELISA assay.

[0037] As an alternative to preparing monoclonal antibody-secreting hybridomas, monoclonals specific for one of the above polypeptide antibodies can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the appropriate polypeptide, thereby isolating immunoglobulin library members that bind to the polypeptide. Kits for generating and screening phage displays are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01 and the Stratagene SurfZAP™ Phage Display Kit, catalog number 240612).Additionally, examples of particularly acceptable methods and reagents for use in generating and screening antibody display libraries can be found in, e.g., U.S. Pat. No. 5,223,409, PCT Patent Application Publication No. 92 / 18619, PCT Patent Application Publication No. 91 / 17271, PCT Patent Application Publication No. 92 / 20791, PCT Patent Application Publication No. 92 / 15679, PCT Patent Application Publication No. 93 / 01288, PCT Patent Application Publication No. 92 / 01047, PCT Patent Application Publication No. 92 / 09690, PCT Patent Application Publication No. 90 / 02809, Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

[0038] Additionally, recombinant anti-CTLA-4 or anti-PD-1 antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the presently disclosed subject matter. Such chimeric and humanized monoclonal antibodies are described, for example, in PCT Patent Application Publication No. PCT / US86 / 02269, European Patent Application No. 184,187, European Patent Application No. 171,496, European Patent Application No. 173,494, PCT Application Publication No. WO 86 / 01533, U.S. Pat. No. 4,816,567, European Patent Application No. 125,023, Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison, SL (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060, by recombinant DNA techniques known in the art.

[0039] Additionally, humanized antibodies can be made according to standard protocols, such as those disclosed in U.S. Patent No. 5,565,332. In another embodiment, the antibody chain or specific binding pair member can be produced by recombination between a vector comprising a nucleic acid molecule encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and a vector containing a nucleic acid molecule encoding a second polypeptide chain of a single binding pair member, using techniques known in the art, for example, as described in U.S. Patent Nos. 5,565,332, 5,871,907, or 5,733,743. The use of intracellular antibodies to inhibit protein function within a cell is also known in the art (e.g., Carlson (1988) Mol. Cell. Biol. 8:2638-2646; Biocca et al. (1990) EMBO J. 9:101-108; Werge et al. (1990) FEBS Lett. 274:193-198; Carlson (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca et al. (1994) Biotechnology (NY) 12:396-399; Chen et al. (1994) Hum. Gene Ther. 5:595-601; Duan et al. (1994) Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli et al. (1994) J. Biol. Chem. 269:23931-23936; Beerli et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672; Mhashilkar et al. (1995) EMBO J. 14:1542-1551; Richardson et al. (1995) Proc. Natl. Acad. Sci. USA 92:3137-3141; PCT Publication No. 94 / 02610, and PCT Publication No. 95 / 03832).

[0040] Additionally, fully human antibodies against CTLA-4 or PD-1 or functional variants thereof can be made. Fully human antibodies can be made in mice transgenic for human immunoglobulin genes, for example, according to Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual," Cold Spring Harbor Laboratory. Briefly, transgenic mice are immunized with purified CTLA-4 or PD-1 or functional variants thereof. Spleen cells are harvested and fused to myeloma cells to generate hybridomas. Hybridomas are selected based on their ability to produce antibodies that bind to CTLA-4 or PD-1 or functional variants thereof. Fully human antibodies are believed to reduce the immunogenicity of such antibodies in humans.

[0041] In one embodiment, the antibodies for use in the presently disclosed subject matter are bispecific antibodies. Bispecific antibodies have binding sites for two different antigens within a single antibody polypeptide. Antigen binding can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines capable of secreting bispecific antibodies. Examples of bispecific antibodies produced by hybrid hybridomas or triomas are disclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628 and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA 83:1453 and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent No. 5,959,084. Bispecific antibody fragments are described in U.S. Patent No. 5,798,229.

[0042] Bispecific agents can also be made by fusing hybridomas or other cells making different antibodies to create heterologous hybridomas, followed by identifying clones producing or co-assembling both antibodies. Bispecific agents can also be made by chemically or genetically conjugating complete immunoglobulin chains or portions thereof, such as Fab and Fv sequences. The antibody components are capable of binding to CTLA-4 or PD-1 or a functional variant thereof. In one embodiment, the bispecific antibody is capable of specifically binding to both a PD-1 ligand or a functional variant thereof and a PD-1 polypeptide or a functional variant thereof.

[0043] Yet another aspect of the presently disclosed subject matter relates to an antibody obtainable by a process comprising immunizing an animal with an immunogenic CTLA-4 or PD-1, or a functional variant thereof, or an immunogenic portion thereof that is unique to CTLA-4 or PD-1, and then isolating from the animal antibodies that specifically bind to the polypeptide.

[0044] In some embodiments, the presently disclosed subject matter provides methods for treating solid tumors using bacteria, bacterial products, and / or other immunomodulatory entities. In other embodiments, the bacteria or bacterial products are anaerobic bacteria or bacterial products. Suitable genera include, but are not limited to, Bifidobacteria, Lactobacilli, and Clostridia, such as Clostridium novyi or Clostridium sordellii (C. sordellii). In yet other embodiments, the bacteria or bacterial products are obligate anaerobic bacteria or bacterial products. As used herein, "anaerobic bacteria" are bacteria that do not require oxygen to grow. As used herein, "obligate anaerobic bacteria" are bacteria that not only do not require oxygen to grow, but are also harmed by normal levels of atmospheric oxygen. In further embodiments, the anaerobic bacteria or bacterial products are Clostridium novyi or bacterial products.

[0045] In some embodiments, the bacterium or bacterial product thereof is a toxin-depleted anaerobic bacterium or bacterial product thereof. In other embodiments, the toxin-depleted anaerobic bacterium or bacterial product thereof is Clostridium novyi-NT or a bacterial product thereof.

[0046] It is desirable to reduce the natural production of toxins for therapeutic use of bacteria. Toxin-depleted strains do not need to be completely non-toxic, but it is desirable that at least one of the toxin genes is mutated, deleted, or otherwise inactivated to make the bacteria less harmful to the subject. As used herein, the term "toxic" means to act as a toxin or have the effect of a toxin. Preferably, toxicity is reduced by at least 2, 5, 10, 50, 100, 1000-fold, or more. If the toxin gene is episomal or on a bacteriophage, episomal or bacteriophage curing can be used to delete the toxin gene. Techniques for mutagenesis, curing, and screening of mutants are well known in the art.

[0047] In some embodiments, some or all of the toxin genes of the wild-type form of the toxin-depleted anaerobic bacterium or its bacterial product are deleted to produce a "toxin-depleted" bacterium or its bacterial product. For example, the lethal alpha-toxin gene is deleted in C. novyi-NT. In other embodiments, the virulence of the toxin-depleted anaerobic bacterium is at least two-fold reduced compared to the corresponding wild-type bacterium. In yet other embodiments, the virulence of the toxin-depleted Clostridium novyi is at least two-fold reduced compared to the corresponding Clostridium novyi. As used herein, the term "wild-type" refers to the normal, non-mutated version of a bacterium or gene, etc. As used herein, the term "deletion" refers to a change in the nucleotide sequence in which one or more nucleotides are removed.

[0048] In some embodiments, the bacterial product is at least one bacterial membrane component.The bacterial membrane component may include, for example, a bacterial membrane protein that is bound to or associated with the membrane of Clostridium novyi, suitably a protein that has a domain that is exposed on the outside of the bacteria upon infection and thus is considered to be visible to the human immune system. Reference herein to a bacterial membrane protein includes variants of naturally occurring bacterial membrane proteins such as proteins having an amino acid sequence that is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the wild-type amino acid sequence for a given bacterial membrane over the length of a deletion, insertion, and substitution mutation or suitably immunogenic variant of a given bacterial membrane protein.

[0049] In some embodiments, other immunomodulatory entities can be combined with antibodies against CTLA-4 and / or PD-1. Such immunomodulatory entities can include, for example, immune stimulatory cytokines such as GM-CSF, interleukin-12 (IL-12), and IL-15. Additional examples of bacterial products used for immune stimulatory purposes include inactivated bacteria or bacterial components such as Freund's complete adjuvant and Coley's toxin.

[0050] In some embodiments, at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities is administered intravenously or intratumorally. In other embodiments, at least one antibody is administered by at least one method selected from the group consisting of intravenous, intramuscular, subcutaneous, and intratumor.

[0051] "Cancer" in a subject refers to the presence of cells with typical characteristics of cancer-causing cells, such as uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cell markers. In some circumstances, cancer cells become tumor-shaped, and such cells may be present locally in an animal or circulate in the bloodstream as independent cells, e.g., leukemia cells. Cancers may include, but are not limited to, head, neck, lung, breast, prostate, colorectal, esophageal, gastric, leukemia / lymphoma, uterine, skin, endocrine, urological, pancreatic, gastrointestinal, ovarian, cervical, and adenoma. "Tumor," as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and any precancerous and cancerous cells and tissues. A "solid tumor," as used herein, is an abnormal mass of tissue that generally does not contain cysts or liquid areas. Solid tumors may be present in, by way of non-limiting example, the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovaries, cervix, stomach, colon, rectum, bladder, uterus, testes, and pancreas. In some embodiments, after a solid tumor is treated with the methods disclosed herein, the solid tumor regresses or the growth of the solid tumor is slowed or stopped. In other embodiments, the solid tumor is malignant.

[0052] In some embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody to treat the cancer. It has been found that the combination of anti-CTLA-4 antibody and anti-PD-1 antibody for treating cancer provides better results than administration of the antibodies separately. In other embodiments, the combination of anti-CTLA-4 antibody and anti-PD-1 antibody is administered by at least one method selected from the group consisting of intravenous, intramuscular, subcutaneous, and intratumoral.

[0053] As used herein, the term "treating" can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition.

[0054] Although the subjects treated by the methods disclosed herein in many embodiments thereof are desirably human subjects, it should be understood that the methods described herein are effective with respect to any vertebrate species, and all vertebrate species are intended to be encompassed by the term "subject." Thus, a "subject" can include a human subject for medical purposes, such as for treatment of an existing condition or disease or for prophylactic treatment to prevent the onset of a condition or disease, or an animal subject for medical, veterinary, or development purposes. Suitable animal subjects include mammals, including, but not limited to, primates, such as humans, monkeys, apes, and the like; bovines, such as cattle, bulls, and the like; ovines, such as sheep, and the like; caprines, such as goats, and the like; porcines, such as pigs, boars, and the like; equines, such as horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. The animal may be a transgenic animal. In some embodiments, the subject is a human, including fetal, neonatal, infant, juvenile, and adult subjects. Additionally, a "subject" can include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein.

[0055] More specifically, the compositions disclosed herein comprising at least one antibody selected from the group consisting of anti-CTLA-4 antibody and anti-PD-1 antibody combined with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities as described herein can be administered to a subject for treatment by any suitable route of administration, including orally, intranasally, mucosally, ophthalmically, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary, and intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, intracisternally, including buccal and sublingually, topically by powder, ointment, or drops (including eye drops), transdermally, via inhalation spray, or other delivery modes known in the art. The compositions disclosed herein can also be administered intratumorally, such as by injection or other means, such that the compositions are administered directly to a solid tumor.

[0056] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of a composition comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities, i.e., compounds, drugs, or other substances, other than directly into the central nervous system, e.g., subcutaneously, so that the antibody enters the patient's system and thus undergoes metabolic and other similar processes.

[0057] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0058] The pharmaceutical compositions disclosed herein can be manufactured in a manner known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0059] In some embodiments, the pharmaceutical compositions disclosed herein can be administered by a rechargeable or biodegradable device. For example, various sustained release polymeric devices have been developed and tested in vivo for controlled delivery of drugs, including protein biologics. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., (1981) J. Biomed. Mater. Res. 15:167; Langer (1982), Chem. Tech. 12:98), ethylene vinyl acetate (Langer et al., (1981) J. Biomed. Mater. Res. 15:167), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A). The sustained release compositions also include liposomally entrapped compositions comprising at least one antibody selected from the group consisting of anti-CTLA-4 and anti-PD-1 antibodies in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities, which can be prepared by methods known per se (Epstein et al., (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., (1980) Proc. Natl. Acad. Sci. USA 77:4030; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A). Typically, the liposomes are small (about 200-800 angstroms) unilamellar type with a lipid content of more than about 30 mol % cholesterol, the selected ratio being adjusted for optimal therapy. Such materials can include, for example, in some embodiments, implants for sustained release of the compositions disclosed herein that can be implanted at specific, predetermined target sites, such as solid tumors.

[0060] In another embodiment, the pharmaceutical compositions disclosed herein can include a pegylated therapeutic agent (e.g., a pegylated antibody or bacterial product). Pegylation is a well-established and validated approach for the modification of a range of antibodies, proteins, and peptides, and involves the attachment of polyethylene glycol (PEG) to specific sites on antibodies, proteins, and peptides (Chapman (2002) Adv. Drug Deliv. Rev. 54:531-545). Some of the effects of PEGylation include: (a) significantly improved circulating half-life in vivo by avoidance of renal clearance as a result of the polymer increasing the apparent size of the molecule beyond the glomerular filtration limit and / or through avoidance of cellular clearance mechanisms; (b) improved pharmacokinetics; (c) improved solubility, PEG has been shown to be soluble in many different solvents ranging from water to many organic solvents such as toluene, methylene chloride, ethanol and acetone; (d) PEGylated antibody fragments can be concentrated up to 200 mg / ml, which opens up a range of formulation and administration options, such as subcutaneous administration of high protein doses, in contrast to many other therapeutic antibodies, which are typically administered intravenously; (e) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et al., (1992) J. Immunol. Meth. 152:177-190); (f) improved bioavailability due to reduced losses at the subcutaneous injection site; (g) reduced toxicity has been observed; for drugs where toxicity is related to peak plasma levels, the flatter pharmacokinetic profile achieved by subcutaneous administration of PEGylated proteins is advantageous; proteins that elicit immune responses with toxic consequences can also benefit as a result of PEGylation; and (h) improved thermal and mechanical stability of PEGylated molecules.

[0061] Pharmaceutical compositions for parenteral administration include aqueous solutions of compositions comprising at least one antibody selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities. For injection, the pharmaceutical compositions disclosed herein can be formulated in aqueous solutions, for example, in some embodiments, in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. In addition, suspensions of compositions comprising at least one antibody selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities or vehicles can include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the composition comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities, so as to allow for the preparation of highly concentrated solutions.

[0062] For nasal or transmucosal administration, penetrants appropriate to the particular barrier to be permeated are generally used in the formulation, and such penetrants are generally known in the art.

[0063] For inhalation delivery, the agents of the present disclosure may be formulated by methods known to those of skill in the art and may include, for example but not limited to, solubilizing, diluting or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption enhancers, and fluorocarbons.

[0064] Additional ingredients can be added to the composition for topical administration, as long as such ingredients are pharma- ceutically acceptable and not harmful to epithelial cells or their functions.Furthermore, such additional ingredients should not adversely affect the epithelial penetration efficiency of the composition, and should not cause deterioration of the stability of the composition.For example, fragrances, opacifiers, antioxidants, gelling agents, stabilizers, surfactants, emollients, colorants, preservatives, buffers, etc. can be present.The pH of the topical compositions disclosed herein can be adjusted to a physiologically acceptable range of about 6.0 to about 9.0 by adding a buffer thereto, so that the composition is physiologically compatible with the skin of a subject.

[0065] Regardless of the route of administration selected, the compositions disclosed herein comprising at least one antibody selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities are formulated into a pharma- ceutically acceptable dosage form as described herein or by other conventional methods known to those of skill in the art.

[0066] In general, an "effective amount" or "therapeutically effective amount" of an active agent or drug delivery device is the amount necessary to induce a desired biological response. As one skilled in the art will recognize, the effective amount of an agent or device may vary depending on factors such as the desired biological endpoint, the agent being delivered, the composition of the encapsulation matrix, the target tissue, and the like.

[0067] The term "combination" is used in its broadest sense and means that a subject is administered at least two agents, more specifically, at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody, in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities. More specifically, the term "in combination" refers to the simultaneous administration of two (or more) active agents, for example, for the treatment of a single disease condition. As used herein, the active agents may be combined and administered in a single dosage form, may be administered simultaneously in separate dosage forms, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., where it is desirable to vary the amount of one dosage form but not the other). The single dosage form may contain an additional active agent for the treatment of a disease condition.

[0068] Additionally, the compositions disclosed herein can be administered alone or in combination with adjuvants that enhance the stability of the agent, in certain embodiments facilitate administration of pharmaceutical compositions containing the agent, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of conventional therapeutic agents, thus avoiding possible toxic and adverse side effects incurred when those agents are used as monotherapies.

[0069] The timing of administration of the compound of anti-CTLA-4 antibody and / or anti-PD-1 antibody in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents can be varied, so long as the beneficial effects of the combination of these agents are achieved. Thus, the phrase "in combination" refers to simultaneous, sequential, or combination administration of anti-CTLA-4 and / or anti-PD-1 antibody in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents. Thus, a subject administered a combination of anti-CTLA-4 and / or anti-PD-1 antibody in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents can receive anti-CTLA-4 and / or anti-PD-1 antibody in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effects of the combination of all agents are achieved in the subject.

[0070] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or more of each other. In other embodiments, agents administered sequentially can be administered within 1, 2, 3, 4, 5, 10, 15, 20 or more days of each other. When the compounds of anti-CTLA-4 and / or anti-PD-1 antibodies in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents are administered simultaneously, the compounds can be administered to the subject as separate pharmaceutical compositions, each comprising either anti-CTLA-4 and / or anti-PD-1 antibodies in combination with bacteria, bacterial products, or other immunomodulatory entities and optionally additional agents, or can be administered to the subject as a single pharmaceutical composition comprising all of the agents.

[0071] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing for a reduction in the dosage of one or more agents compared to the dosage required when the agent is administered as a single agent. The effect of the multiple agents may be, but need not be, additive or synergistic. The agents may be administered multiple times.

[0072] In some embodiments, two or more agents can have a synergistic effect when administered in combination. As used herein, the terms "synergy," "synergistic," "synergistically," and derivatives thereof, such as "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of a combination of agents, e.g., an anti-CTLA-4 and / or anti-PD-1 antibody in combination with a bacterium, bacterial product, or other immunomodulatory entity, and at least one additional therapeutic agent, is greater than the sum of the biological activities of each agent when administered individually.

[0073] Synergy can be expressed in terms of the Synergy Index (SI), which is generally defined as: Q a Q A +Q b Q B = Synergy Index (SI) from the ratio determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), Q A is the concentration of component A acting alone that produced the endpoint for component A, and Q a is the concentration of component A in the mixture that gave rise to the endpoint, Q B is the concentration of component B acting alone that produced the endpoint for component B, and Q b is the concentration of component B in the mixture that gave rise to the endpoint.

[0074] In general, Q a / Q A and Q b / Q B If the sum is greater than 1, antagonism is indicated. If the sum is equal to 1, additivity is indicated. If the sum is less than 1, synergy is indicated. The lower the SI, the greater the synergy exhibited by that particular mixture. Thus, a "synergistic combination" has greater activity than can be predicted based on the observed activity of the individual components when used alone. Furthermore, a "synergistically effective amount" of a component is, for example, the amount of the component required to induce a synergistic effect in another therapeutic agent present in the composition.

[0075] As used herein, the term "reduce" or "inhibit" and its grammatical derivatives refer to the ability of an agent to block, partially block, impede, reduce, diminish or deactivate a pathway or mechanism of action. Thus, those skilled in the art will recognize that the term "reduce" encompasses complete and / or partial loss of activity, for example, at least 10% loss of activity, and in some embodiments, at least 20%, 30%, 50%, 75%, 95%, 98% and up to 100% loss of activity.

[0076] In another aspect, the presently disclosed subject matter provides pharmaceutical compositions comprising the bacteria, bacterial products, or other immunomodulatory entities, and, optionally, anti-CTLA-4 and / or anti-PD-1 antibodies in combination with an additional agent, alone or in combination with one or more additional therapeutic agents, in admixture with a pharma- ceutical acceptable excipient.

[0077] More specifically, the presently disclosed subject matter provides pharmaceutical compositions comprising a bacterium, a bacterial product, or other immunomodulatory entity, and, optionally, an anti-CTLA-4 and / or anti-PD-1 antibody in combination with an additional agent, and a pharma- ceutically acceptable carrier.

[0078] For therapeutic and / or diagnostic applications, the compounds of the present disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams and Wilkins (2000).

[0079] It is within the scope of the present disclosure to use pharmaceutically acceptable inert carriers to formulate the compounds disclosed herein into dosages suitable for systemic administration to carry out the present disclosure.By proper selection of carriers and appropriate manufacturing practices, the compositions of the present disclosure, particularly those formulated as solutions, can be administered parenterally, such as by intravenous injection.The compounds can be easily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art.Such carriers allow the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject (e.g., patient) to be treated.

[0080] For nasal or inhalation delivery, the agents of the present disclosure may be formulated by methods known to those skilled in the art and may include, but are not limited to, examples of solubilizing, diluting or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption enhancers, and fluorocarbons.

[0081] Pharmaceutical compositions suitable for use in the present disclosure include compositions in which the active ingredient is contained in an effective amount to achieve its intended purpose. The determination of effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, compounds according to the present disclosure are effective over a wide range of dosages. For example, in the treatment of adult humans, dosages of 0.01 to 1000 mg per day, 0.5 to 100 mg, 1 to 50 mg, and 5 to 40 mg per day are examples of dosages that can be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the weight of the subject being treated, and the preference and experience of the attending physician.

[0082] In addition to the active ingredient, these pharmaceutical compositions can contain suitable pharma- ceutical acceptable carriers, including excipients and auxiliary substances that facilitate the processing of the active compound into pharma- ceutical usable preparations. Preparations formulated for oral administration can be in the form of tablets, dragees, capsules, or solutions.

[0083] II. Kits for Treating Cancer The presently disclosed subject matter also relates to kits for carrying out the methods of the presently disclosed subject matter. In general, the presently disclosed kits contain some or all of the components, reagents, supplies, etc., for carrying out the methods according to the presently disclosed subject matter. In some embodiments, the term "kit" refers to any intended article of manufacture (e.g., package or container) that includes at least one antibody selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies combined with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities, and a set of specific instructions for carrying out the methods of the presently disclosed subject matter. The kits can be packaged in divided or undivided containers such as cartons, bottles, ampoules, tubes, etc. The compositions disclosed herein can be packaged in dry, lyophilized, or liquid form. Additional components provided can include vehicles for reconstitution of the dry components. Preferably, all such vehicles are sterile and non-pyrogenic, so as to be suitable for injection into a subject without causing adverse reactions.

[0084] In some embodiments, the presently disclosed subject matter provides a kit for treating a solid tumor, the kit comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, and at least one member of the group consisting of a bacterium, a bacterial product, and an immunomodulatory entity. In other embodiments, the kit comprises a bacterium or bacterial product thereof and at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody. In yet other embodiments, the bacterium or bacterial product is an anaerobic bacterium or bacterial product thereof. In further embodiments, the anaerobic bacterium or bacterial product thereof is Clostridium novyi or a bacterial product thereof.

[0085] In some embodiments, the anaerobic bacterium or bacterial product is a toxin-depleted anaerobic bacterium or bacterial product. In other embodiments, the anaerobic bacterium or bacterial product is Clostridium novyi-NT or bacterial product. In some other embodiments, some or all of the toxin genes of the wild-type form of the toxin-depleted anaerobic bacterium or bacterial product are deleted. In further embodiments, the toxicity of the toxin-depleted anaerobic bacterium is at least 2-fold reduced compared to the corresponding wild-type bacterium. In further embodiments, the bacterial product is at least one spore.

[0086] In some embodiments, a kit containing a combination of an anti-CTLA-4 antibody and an anti-PD-1 antibody is used to treat cancer.

[0087] Following long-standing patent law practice, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to "a subject" includes a plurality of subjects, and so forth, unless the context clearly indicates to the contrary (e.g., a plurality of subjects).

[0088] Throughout this specification and the claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term "include" and grammatical variations thereof are intended to be open-ended, such that the enumeration of items in a list is not to the exclusion of other similar items that may be substituted for or added to the listed items.

[0089] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, contents, characteristics and other numerical values ​​used in the specification and claims are to be understood as being modified in all instances by the term "about," even if the term "about" is not expressly stated in conjunction with the value, amount or range. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are not and need not be precise, but may be approximated and / or larger or smaller as desired, reflecting tolerances, conversion rates, rounding, measurement errors and the like, as well as other factors known to those of ordinary skill in the art depending upon the desired properties sought to be obtained by the subject matter disclosed herein. For example, the term "about" when referring to a value can be intended to encompass variations from the particular amount of, in some embodiments, ±100%, in some embodiments, ±50%, in some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, ±0.5%, and in some embodiments, ±0.1%, as such variations are suitable for practicing the disclosed methods or using the disclosed compositions.

[0090] Furthermore, the term "about," when used in reference to one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers within the range, and to modify the range by extending its boundaries above and below the numerical values ​​set forth. Recitation of numerical ranges by endpoints includes all numbers, e.g., all integers, including fractions thereof contained within the range (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, and 5 as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. EXAMPLES

[0091] The following examples are included to provide guidance to those skilled in the art for carrying out representative embodiments of the subject matter disclosed herein. In light of this disclosure and the general level of skill in the art, those skilled in the art will recognize that the following examples are intended to be illustrative only, and that numerous changes, modifications, and variations can be used without departing from the scope of the subject matter disclosed herein. The synthetic descriptions and specific examples that follow are intended for illustrative purposes only, and should not be construed in any way as limiting the making of the compounds of the present disclosure in other ways.

[0092] Example 1 Combination treatment in tumor models Two mouse tumor models, the CT26 tumor model and the 4T1 tumor model, were used to determine the effects of CTLA-4 and PD-1 antibodies on tumors with or without administration of C. novyi-NT.

[0093] Using the CT26 tumor model, BALB / c mice bearing subcutaneous CT26 tumors were treated with C. novyi-NT spores by intravenous injection and / or with the indicated antibodies by intraperitoneal injection. Animals were followed for >3 weeks and tumor volumes were determined. In combination, CTLA-4 and PD-1 antibodies were able to eradicate immunogenic CT26 tumors with or without administration of spores of C. novyi-NT, a tumor-targeting bacterial strain currently in clinical development (Figure 1).

[0094] Using the 4T1 tumor model, BALB / c mice bearing subcutaneous 4T1 tumors were treated with C. novyi-NT spores by intravenous injection and / or with the indicated antibodies by intraperitoneal injection. Animals were followed for approximately >70 days. Both tumor volume (Figure 2A) and animal survival (Figure 2B) are shown.

[0095] In contrast to the CT26 tumor model, 4T1 tumors are minimally immunogenic and naturally metastatic, making them a good model of human disease. 4T1 tumor-bearing mice show increased tumor size, especially when tumors reach 200 mm3 For tumors larger than 100 mm, cures were rarely reported. Here, we show that neither individual antibodies nor antibody combinations were sufficient to eradicate large primary 4T1 tumors (Figure 2A), even though survival rates were significantly increased with antibody combinations (Figure 2B), likely due to improved immunological control of micrometastases. Importantly, when spores of an attenuated anaerobic tumor-targeting bacterial strain, C. novyi-NT, were administered intravenously (IV) in addition to the combined antibodies, a significant proportion of large primary tumors were eradicated (Figure 2A), resulting not only in extended survival but also in cures, which are extremely rare in this tumor model (Figure 2B).

[0096] These results suggest that aggressive immune stimulation (e.g., intratumoral bacterial infection) combined with the absence of immunological checkpoints (e.g., PD-1 and CTLA-4 antibodies) can predict significant clinical benefit even within minimally immunogenic tumors.

[0097] Example 2 Clostridium novyi-NT induces antitumor responses summary Clostridium species are known for their ability to lyse tumor cells growing in hypoxic environments. Here, an attenuated strain of Clostridium novyi (C. novyi-NT) is shown to induce a microscopically well-defined tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. However, it is well known that experimental models often do not reliably predict the response of human patients to therapeutic agents. Naturally occurring canine tumors were therefore used as a bridge to human clinical trials. Canine tumors are more similar to human tumors as they arise in animals with heterogeneous genetic backgrounds, are of host origin, and result from spontaneous mutations rather than engineered mutations. Intratumoral injection of C. novyi-NT spores was found to be well tolerated in companion animal dogs with naturally occurring solid tumors, with the most frequent toxicities being the expected symptoms associated with bacterial infection. Objective responses were observed in 6 of 16 dogs (37.5%), with 3 complete responses and 3 partial responses. Based on these promising results, human patients with aggressive leiomyosarcoma were treated with intratumoral injections of C. novyi-NT spores. This treatment produced a dramatic response, significantly reducing the tumor in and around the bone. Collectively, these results demonstrate that C. novyi-NT can act as a controlled tool to precisely eradicate neoplastic tissue and suggest that further clinical trials of this agent in selected patients are justified.

[0098] introduction Therapies that specifically target and destroy cancer must recognize the difference between normal and malignant tissue (Krause and Van Etten (2005) New Engl. J. Med. 353:172-187; Imai and Takaoka (2006) Nat. Rev. Cancer 6:714-727; Sosman et al. (2012) New Engl. J. Med. 366:707-714; Wilson and Hay (2011) Nat. Rev. Cancer 11:393-410). These differences include genetic alterations as well as pathophysiological changes that result in heterogeneous tumor masses with areas of hypoxia and necrosis (Wilson and Hay (2011) Nat. Rev. Cancer 11:393-410; Hanahan and Weinberg (2011) Cell 144:646-674; Kerbel (2008) New Engl. J. Med. 358:2039-2049; Chung and Ferrara (2011) Annu. Rev. Cell Dev. Bio. 27:563-584; Baish et al. (2011) Proc. Natl. Acad. Sci. USA 108:1799-1803). Systemically delivered anticancer drugs rely on the tumor vasculature for delivery and are therefore less effective in poorly vascularized, hypoxic tumor regions (Wilson and Hay (2011) Nat. Rev. Cancer 11:393-410). Furthermore, radiation therapy cannot kill hypoxic cells because oxygen is a necessary effector of radiation-induced cell death (Horsman et al. (2012) Nat. Rev. Clin. Oncol. 9:674-687. For these important reasons, unresectable locally advanced tumors are particularly difficult to treat with conventional therapies.

[0099] Tumors are composed of necrotic, hypoxic, and well-oxygenated regions. Hypoxic tumor regions are more resistant to systemic anticancer drugs and radiation therapy. However, hypoxic tumor regions provide fertile soil for the growth of anaerobic bacteria. Thus, hypoxic regions of tumors provide a perfect niche for the growth of anaerobic bacteria. In principle, this provides an opportunity for the targeted eradication of advanced local tumors, sparing the surrounding well-vascularized normoxic tissue. Since Corey's first work treating cancer patients with Strepococcus pyogenes over 100 years ago, various anaerobic bacteria have been explored for this purpose (Coley (1910) Proc. Roy. Soc. Med. 3:1-48; Coley (1991) Clin. Orthop. Relat. Res. 3-11). This early work failed to produce viable anticancer drugs, in part due to poor reproducibility and unacceptable toxicity. More recent studies have involved Salmonella typhimurium and other attenuated strains (Forbes (2010) Nat. Rev. Cancer 10:785-794; Wei et al. (2008) Cancer Lett. 259:16-27). However, Phase I clinical trials of S. typhimurium in both dogs and human patients have demonstrated that the bacteria can be safely administered and targeted to tumors, although the observed efficacy was limited (Toso et al. (2002) J. Clin. Oncol. 20:142-152; Thamm et al. (2005) Clin. Cancer Res. 11:4827-4834). In an attempt to enhance the efficacy of S. typhimurium treatments, genetically modified strains incorporating cytosine deaminase that converts systemically administered 5-flurocytosine to 5-flurouracil have been developed and evaluated in patients (Nemunaitis et al. (2003) Cancer Gene Ther. 10:737-744).

[0100] However, one particularly promising bacterium is Clostridium novyi (Dang et al., (2001) Proc. Natl. Acad. Sci. USA 98:15155-15160). C. novyi is a highly mobile, spore-forming bacterium that is exquisitely sensitive to oxygen. A derivative of the wild-type strain, called C. novyi-NT, was created through removal of the alpha-toxin gene (Dang et al., (2004) Cancer Bio. Ther. 3:326-337; Dang et al. (2001) Proc. Natl. Acad. Sci. USA 98:15155-15160). A single dose of intravenously injected C. novyi-NT spores into mice and rabbits bearing transplanted syngeneic tumors resulted in localized tumor necrosis, a robust inflammatory response, and complete responses in 25-30% of treated animals (Agrawal et al. (2004) Proc. Natl. Acad. Sci. USA 101:15172-15177). Based on these data, intravenously injected C. novyi-NT spores were evaluated in naturally occurring canine tumors (Krick et al. (2012) Amer. J. Vet. Res. 73:112-118). However, no complete responses were observed at doses that showed acceptable toxicity.

[0101] Given the remarkable ability of intravenously injected C. novyi-NT spores to localize, germinate within, and destroy mouse tumors while leaving the surrounding normal tissues untreated, it was hypothesized that direct intratumoral injection of spores into solid tumors may be advantageous over administration by the intravenous route. One problem encountered with systemic injection of spores is that a low percentage of spores are actually delivered to the tumor (Diaz et al. (2005) Toxicol. Sci. 88:562-575). This problem is compounded in larger animals and human patients, who have relatively large blood volumes and relatively small tumors compared to mice. With intratumoral injection, it is possible to overcome this problem by depositing orders of magnitude more spores directly within the target tumor. Furthermore, intratumoral injection of spores may also be advantageous over other conventional local treatments such as surgery and radiation therapy. In theory, C. novyi-NT therapy could allow for precise and fine ablation of neoplastic cells from tumors without the need to ablate a margin of normal tissue. Intratumoral injection of C. novyi-NT spores could induce a strong and localized inflammatory response as well as an adaptive immune response against tumor cells (Agrawal et al., (2004) Proc. Natl. Acad. Sci. USA 101:15172-15177). Based on such reasoning, the safety and efficacy of intratumorally injected C. novyi-NT spores were investigated in preclinical animal models and in controlled studies of dogs with naturally occurring cancer. We also report first human data from patients treated with intratumorally injected C. novyi-NT spores.

[0102] material and method Study Design: A preclinical proof-of-concept study was performed using a rat orthotopic F98 glioma model to demonstrate C. novyi-NT-induced infection specifically and precisely localized to tumor lesions. Luciferase activity and Kaplan-Meier survival curves were used to evaluate treatment efficacy. A comparative study in companion animal dogs with spontaneous solid tumors was used to bridge the gap between preclinical and human trials. The experimental unit was one study dog, with each dog receiving up to four cycles of treatment. No placebo control, blinding, or randomization was used in this study. No formal a priori statistical hypotheses were planned for this comparative study. Descriptive summary statistics and analyses were provided post hoc. The human clinical trial is an ongoing open-label, non-randomized, multicenter Phase I study, using standard "3+3" dose escalation. The study was designed to (i) determine the safety profile, dose-limiting toxicity, and maximum tolerated dose of C. novyi-NT spores in humans with treatment-resistant solid tumor malignancies when administered as a single intratumoral injection, (ii) describe preliminary antitumor activity in both injected tumors and overall responses, (iii) examine the disposition of circulating C. novyi-NT spores, and (iv) measure host immune and inflammatory responses associated with C. novyi-NT treatment.

[0103] Cell lines and tissue culture: Rat F98 glioma cell lines transfected with luciferase constructs via lentivirus were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin.

[0104] Rat orthotopic brain tumor model: All animal experiments involving rats were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Six-week-old female F344 Fischer rats (body weight 100–150 grams) were purchased from the National Cancer Institute. For the implantation procedure, female F344 Fischer rats were administered 100 mg / kg or 100 mg / mL ketamine HCl (Abbot Laboratories), 100 mg / mL xylazine (7.5 mg / kg or 100 mg / mL Xyla-ject; Phoenix) in a sterile NaCl (0.9%) solution. Each mouse was anesthetized with an intraperitoneal injection of 100 mM NaCl (Chemical Pharmaceutical, Burlingame, CA) and ethanol (14.25%). F98 glioma cells (2 × 10 4) were stereotactically implanted into the right frontal lobe, 3 mm lateral and 2 mm anterior to bregma, through a burr hole as previously described (Bai et al., (2011) Neuro-oncology 13:974-982). Tumor size was assessed by intraperitoneal injection of 8 mg / rat D-luciferin potassium salt 12 days after tumor cell implantation using a Xenogen instrument. Subsequently, 3 million C. novyi-NT spores, generated as previously described (Dang et al., (2004) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; Bettegowda et al., (2006) Nat. Biotechnol. 24:1573-1580), were stereotactically injected into the intracranial tumor using the same coordinates as above. Rats were treated with 10 mg / kg / day intraperitoneal dexamethasone for the first 2 days to minimize the risk of postoperative edema, closely mimicking the standard clinical protocol used in human patients after brain tumor surgery and biopsy. Control rats were stereotactically injected with the same volume of PBS and treated with 10 mg / kg / day intraperitoneal dexamethasone for the first 2 days. Animals were observed daily for any signs of deterioration, lethargy, neurotoxicity, or pain in accordance with the Johns Hopkins Animal Care Guidelines. In the event of symptoms of distress, hydration and supportive treatment with doxycycline (15 mg / kg intraperitoneal loading dose followed by 10 mg / kg every 12 hours as maintenance) was initiated and continued for 7 days. If symptoms persisted and / or declined, moribund animals were euthanized. The efficacy of intratumorally injected C. novyi-NT spores was assessed by Kaplan-Meier survival curves, as well as the remaining tumor burden in the brain sections. In the latter, brains were collected postmortem for further pathological examination, placed in formaldehyde, and embedded in paraffin. Gram-stained slides, counterstained with safranin, and H&E slides were obtained according to standard procedure guidelines.

[0105] Statistical Analysis: Kaplan-Meier survival curves and luciferase counting graphs were generated and analyzed with the Mantel-Cox test and the Mann-Whitney test, respectively, using GraphPad Prism v.5.00 (GraphPad Software, San Diego, Calif.).

[0106] Genomic DNA isolation for sequencing: Genomic DNA from dogs participating in a comparative study of intratumorally injected C. novyi-NT spores was extracted from peripheral blood lymphocytes (PBLs) and formalin-fixed, paraffin-embedded tumor tissues using a QIAamp DNA mini kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol.

[0107] Sequencing and bioinformatic analysis: Genomic purification, library construction, exome capture, next-generation sequencing, and bioinformatic analysis of tumor and normal samples were performed at Personal Genome Diagnostics (PGDx, Baltimore, MD). Briefly, genomic DNA from tumor and normal samples was fragmented and used for Illumina TruSeq library construction (Illumina, San Diego, CA). Exome regions were captured in solution using the Agilent Canine All Exon kit according to the manufacturer's instructions (Agilent, Santa Clara, CA). Paired-end sequencing, yielding 100 bases from each end of the fragments, was performed using a HiSeq 2000 Genome Analyzer (Illumina, San Diego, CA). Tags were aligned to the canine reference sequence (CanFam2.0) using the Eland algorithm of CASAVA 1.7 software (Illumina, San Diego, CA). Sequence reads were selected for subsequent analysis using the pure filter in Illumina's BaseCall software. Then, the ELAND algorithm in CASAVA 1.7 software (Illumina, San Diego, CA) was applied to identify point mutations and small insertions and deletions. Known polymorphisms recorded in dbSNP131 (CanFam2.0) were removed from the analysis. Potential somatic mutations were filtered and visually inspected as previously described (Jones et al., (2010) Science 330:228-231).

[0108] Preparation and intratumoral injection of C. novyi-NT spores in spontaneous canine tumors: C. novyi-NT spores for use in comparative canine studies were generated as previously described (Dang et al., (2004) Proc. Natl. Acad. Sci. USA 98(26):15155-15160; Bettegowda et al., (2006) Nat. Biotechnol. 24:1573-1580). Briefly, bacteria were cultured in sporulation medium for at least 2 weeks to ensure maximum yield of mature spores. Mature spores were purified through two successive uninterrupted Percoll gradients followed by four washes and resuspension in PBS. Sterility testing of the final product was performed according to FDA 21CFR610.12 guidelines (Nelson Laboratories, Salt Lake City, CA). Germination efficiency assays were performed by culturing the products in soybean casein digest medium and thioglycollate medium according to the method described in the literature (Lake City, UT). Germination efficiency assays were performed under anaerobic conditions on Brucella agar containing 5% horse blood to ensure that the spores met pre-established viability criteria. Spores were cultured at a volume of 1000 μL and a concentration of 1 × 10 9 Spores / mL were packaged into sterile 1.8 mL cryovials (Simport, Beloeil, Canada) with O-ring sealed screw caps. C. novyi-NT cryovials were stored at 2–8 °C. For dosing, 0.4 mL aliquots of the stock spore solution were packaged into 0.5 mL cryovials. After dosing, the cryovials and unused C. novyi-NT spores were discarded in accordance with applicable regulations for disposal of biosafety level 2 materials.

[0109] Prior to intratumoral injection, spores were resuspended by vortexing while mixing at maximum speed for 10 seconds, a total of three times, before being drawn into a 1 mL syringe. The injection site was prepared aseptically. Necrotic areas of the tumor were identified using ultrasound or computed tomography (CT), if available. If no necrotic areas were identified, the injection was directed toward the center of the tumor. The needle was inserted once into the predefined area, and 100 μL of the spore suspension (1 × 10 8C. novyi-NT spores were dispensed with equal pressure, the needle was slowly removed, and the injection site was sterilized.

[0110] Design and Conduct of the Comparative Dog Study: All animal studies involving dogs were conducted in accordance with applicable local, state, national, and international animal welfare regulations and adhered to the highest standards of animal care and use. Written informed consent was obtained from the owner prior to enrollment of each dog. The study protocol and informed consent were approved by the Animal Care and Use Committee of Animal Clinical Investigation (ACI, Washington, DC) to ensure ethical care of the dogs enrolled in the study.

[0111] Client-owned dogs with spontaneous tumors received up to four cycles of intratumoral C. novyi-NT spores. A cycle consisted of 1 × 10 8 The study consisted of a single intratumoral injection of C. novyi-NT spores (in 100 μL PBS). Cycles of intratumoral C. novyi-NT spores were typically spaced 1 week apart. No placebo control or masking was used. Dogs were followed for 90 days, with extended follow-up for disease progression and survival guaranteed when available. Early withdrawal from the study was permitted for toxicity or progressive disease.

[0112] Dogs were enrolled at multiple facilities participating in the Animal Clinical Investigation Oncology Network (ACI, Washington, DC). Treatment, management, and study evaluations were overseen by board-certified veterinary oncologists. Enrollment was offered to client-owned dogs with spontaneous solid tumors, with priority given to dogs with soft tissue sarcomas that had failed standard treatment or whose owner(s) declined such treatment. Participation was restricted to tumor-bearing dogs with target lesions measuring 1 to 7 cm in longest diameter. Dogs whose tumors were located in areas where abscess development was considered catastrophic (e.g., nasal tumors that had spread into the brain or significant pulmonary metastatic disease) were excluded from the study. Dogs with evidence of active bacterial infection requiring systemic antibiotic treatment within 7 days or cancer treatment (chemotherapy, radiation therapy, and immunotherapy) within 21 days of C. novyi-NT spore treatment were ineligible. Dogs were required to have a performance score of 0 or 1 (Table 1) and to be available for the entire study period of enrollment. Concurrent use of anticancer drugs and participation in other clinical trials were prohibited.

[0113] Dogs were hospitalized for observation for 4 days after the first intratumoral injection of C. novyi-NT and for 24-48 hours after subsequent intratumoral injections, at the investigator's discretion. Intravenous fluid treatment was administered for 2 hours after each intratumoral injection of C. novyi-NT spores at a rate of 4 mL / kg / h. Subcutaneous fluid treatment was administered for 4 days after each intratumoral injection of C. novyi-NT spores at a rate of 20 mL / kg / day. Dogs were closely monitored for 6 hours after each intratumoral injection of C. novyi-NT spores.

[0114] Study evaluations were performed as described in Table 2. Prescreening evaluations were performed 1–14 days before the first cycle of intratumoral C. novyi-NT spores. Dogs were monitored regularly during the study on both an inpatient and outpatient basis. Laboratory samples were collected as specified in Table 2 and included complete blood count, serum biochemistry, prothrombin time, partial thromboplastin time, and urinalysis. Imaging was performed at screening and included focal CT, chest radiography, and abdominal ultrasound. Additional imaging was performed during the study at the investigator's discretion.

[0115] Whenever possible, the Veterinary Dictionary for Drug Related Affairs) (VeDDRA) rev.4 (European Medicines Agency (2012)) Combined VeDDRA list of clinical terms for reporting Adverse events were assessed using the Veterinary Co-operative Oncology Group Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.0 (Veterinary Co-operative Oncology Group, 2004, Vet. Comp. Oncol. 2:195-213), using terminology from the Veterinary Co-operative Oncology Group (VCOG-CTCAE) Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.0 (Veterinary Co-operative Oncology Group, 2004, Vet. Comp. Oncol. 2:195-213). Terminology for adverse events (target lesion reactions) related to C. novyi-NT germination is defined in Table 3. Clinical findings without an appropriate VeDDRA or target lesion reaction term were classified separately as uncoded signs (Table 4). Relationship to C. novyi-NT therapy was determined by the reporting investigator.

[0116] Longest diameter tumor measurements of target (injected) lesions were performed on days 0, 7, 14, 21, 60, and 90 after treatment (Table 2). Non-target and new lesions were recorded but not measured. Best overall target response was assessed at or after the day 21 study visit, with complete response (CR) defined as complete disappearance of the target lesion, partial response (PR) defined as at least a 30% decrease in the longest diameter of the target lesion, and progressive target disease (PD) defined as at least a 20% increase in the longest diameter of the target lesion or the appearance of a new non-target lesion. Stable disease (SD) was defined as an insufficient decrease or increase in the longest diameter of the target lesion to qualify as CR, PR, or PD. In cases of C. novyi-NT-associated abscesses, medical or surgical debridement of necrotic tissue was at the discretion of the investigator.

[0117] Evaluation of surgical specimens and necropsies was performed by a board-certified veterinary pathologist. Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. H&E stained and / or Gram stained slides were prepared for evaluation according to standard procedure guidelines. For immunohistochemistry (IHC), formalin-fixed, paraffin-embedded tumor tissues were sectioned at 5 μm, deparaffinized in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed by heating slides for 10 minutes in unmasking solution (catalog number H-3300, Vector Laboratories, Burlingame, CA). All slides were then incubated in 10 percent blocking serum from the animal species from which secondary antibodies were made in PBS for 10 minutes at room temperature. Primary antibodies S100 (Cat. No. Z0311, DAKO, Carpinteria, CA) and anti-smooth muscle actin (Cat. No. M0851, DAKO, Carpinteria, CA) were used at 1:100 for 60 min at room temperature (Duke et al., (2014) Vet. Pathol.; Zarfoss et al., (2007) Vet. Pathol. 44:276-284). Secondary antibodies labeled with DAB (Cat. Nos. BA-1000 and BA-2000, Vector Laboratories, Burlingame, CA) were used at 1:500 for 30 min at room temperature. Sections were incubated with ABC reagent (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. Tumor grade was assigned independently based on published criteria (Dennis et al., (2011) Vet. Pathol. 48:73-84; Patnaik et al., (1984) Vet. Pathol. 21:469-474; Smedley et al., (2011) Vet. Pathol. 48:54-72; Sabattini et al., (2014) Vet. Pathol.).

[0118] Phase I human clinical trial of intratumorally injected C. novyi-NT spores: An open-label, non-randomized, multicenter, phase I safety study of a single intratumoral injection of C. novyi-NT spores is currently underway in patients with treatment-resistant solid tumors. The clinical trial protocol was reviewed and approved by the Institutional Review Board (IRB) of each participating center, and all regulatory steps were performed under the guidance of the Food and Drug Administration (FDA) (http: / / www.clinicaltrials.gov; NCT01924689). All patients were asked to sign a written informed consent form (ICF) before enrollment in the study.

[0119] The primary objective of this phase I study was to determine the safety profile, dose-limiting toxicity, and maximum tolerated dose of intratumorally injected C. novyi-NT. Additionally, we investigated the antitumor activity of intratumoral C. novyi-NT.

[0120] Preparation and intratumoral injection of C. novyi-NT spores in a Phase I study: C. novyi-NT spores were manufactured and formulated by Omnia Biologics, Inc. (Rockville, MD). The clinical supply of C. novyi-NT spores consisted of 8.52 × 10 spores suspended in 1.0 mL of sterile phosphate-buffered saline (PBS) in single-use 2 mL sterile pyrogen-free type I borosilicate glass vials with rubber stoppers and aluminum seals with tamper-evident caps. 8 The vials were packed at a concentration of spores / mL. The vials were stored at 2–8°C in a controlled temperature environment with constant temperature monitoring.

[0121] After the patient was enrolled in the study, one vial was shipped to the study site. Further preparation of C. novyi-NT was required and was performed on the same day as the intratumoral injection. Dilution of the concentrated spore suspension was performed in a designated biological safety cabinet using appropriately sized sterile saline (0.9%) infusion bags to achieve the required dose based on the assigned cohort. The injection volume (3 mL) was then drawn from the saline bag and injected under radiographic guidance. C. novyi-NT spores were injected with an 18-gauge multi-pronged needle (Quadra-Fuse®, Rex-Medical, Conshohocken, PA).

[0122] Human Clinical Trial Design and Execution: The study was conducted using a standard 3+3 dose escalation design. To be enrolled in the study, patients had to have been diagnosed with advanced solid tumor malignancies, target tumors that were palpable and clearly identifiable under ultrasound or radiographic guidance. Furthermore, target lesions had to be ≥1 cm in longest dimension, measurable as defined by RECIST 1.1 criteria, and amenable to percutaneous injection of C. novyi-NT spores.

[0123] Eligibility criteria included a history of treatment-resistant solid tumor malignancy; age at least 18 years; Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2; ability to stay within 45 minutes of an emergency room and have a caregiver present for 28 days after intratumoral injection. Exclusion criteria included pregnancy; primary cerebral malignancy or brain metastases; clinically significant ascites or clinical evidence or history of portosystemic hypertension or cirrhosis; Glasgow coma score (GCS) < 15; serum creatinine level > 1.5 × upper limit of normal (ULN) or chronic renal failure requiring hemodialysis or peritoneal dialysis; oxygen saturation (Sp02) < 95% (room air); mean arterial blood pressure (BP) < 70 mmHg; platelet count ≤ 100,000 / mm3; hemoglobin < 9.0 g / dL; absolute neutrophil count (ANC) < 1,000 / mm3; clinically significant pleural effusion, pericardial effusion, circumferential pericardial effusion, or any effusion > 1.0 cm in any location around the heart; the need for ongoing treatment with immunosuppressants; history of solid organ transplantation; and systemic or localized infection.

[0124] Eligible patients were entered into a dosing cohort and enrolled. Patients remained hospitalized after C. novyi-NT spore injection and were observed for 8 days. Patients returned to the clinical site for routinely scheduled follow-up visits, during which time safety and efficacy assessments were performed.

[0125] Clinical response and progression were assessed using RECIST version 1.1. Objective responses were measured by serial CT or MRI scans of injected tumors and distant metastases (up to 5 lesions).

[0126] Public Health Implications of C. novyi-NT Therapy: C. novyi is a spore-forming, Gram-positive, obligately anaerobic bacterium commonly present in soil (Nishida and Nakagawara (1964) J. Bacteriol. 88:1636-1640). C. novyi-NT was derived from a strain of C. novyi by deleting the toxin genes required for systemic virulence (Dang et al. (2004) Proc. Natl. Acad. Sci. USA 98(26):15155-15160). Extensive preclinical evaluation of C. novyi-NT failed to demonstrate germination of C. novyi-NT spores in non-tumor tissues (Diaz et al. (2005) Toxicol. Sci. 88:562-575). Furthermore, C. novyi-NT spores are resistant to oxygen, whereas vegetative C. novyi-NT is highly sensitive to oxygen (Diaz et al., (2005) Toxicol. Sci. 88:562-575). Thus, vegetative C. novyi-NT cannot survive outside the hypoxic tumor microenvironment. Although the risk to public health with C. novyi-NT therapy is thought to be minimal, caution has been encouraged regarding the handling of C. novyi-NT and the disposal of C. novyi-NT contaminated materials. In the dog-controlled study, protective gloves were worn when handling feces, urine, saliva, or tumor secretions from treated dogs; feces were placed in a sealed plastic bag and disposed of with general household waste; and items soiled with urine, feces, or tumor secretions were washed separately from other laundry. In human clinical trials, standard protective clothing and gloves were required for health care providers.

[0127] result Intratumorally injected C. novyi-NT spores specifically target tumor tissue and extend rat survival: High-grade gliomas exhibit significant histopathological variability with widespread areas of hypoxia and necrosis. Although this tumor type does not commonly metastasize, its complexity along with its protected location within the central nervous system has made this cancer one of the most difficult to treat. Complete surgical resection is almost always impossible due to anatomical constraints and an infiltrative growth pattern that inexorably results in tumor recurrence. Thus, gliomas appeared to represent a tumor type in which local injection of C. novyi-NT spores may be therapeutically useful. To evaluate this possibility, F98 rat glioma cells engineered to express luciferase were orthotopically implanted into 6-week-old F344 Fischer rats, resulting in locally invasive tumors that became rapidly lethal (Figure 3A). Stereotactic intratumoral injection of C. novyi-NT spores into the tumors of these rats resulted in germination within 24 hours and a rapid decline in luciferase activity, an indicator of tumor burden, within 48 hours (Figures 3B and 3C). C. novyi-NT germination was indicated by the appearance of vegetative forms of the bacteria. Strikingly, C. novyi-NT localized precisely to the tumor, sparing adjacent normal cells only a few microns away (Figures 4A and 4B). Moreover, these vegetative bacteria could be seen to grow specifically within islands of microinvasive tumor cells embedded within normal brain parenchyma, and at the same time destroy the islands (Figures 4C and 4D). This bacterial treatment conferred a significant survival advantage in this highly aggressive rat model (Figure 3A, P value < 0.0001). Cerebral edema as a result of C. novyi-NT germination was common and was medically managed. Brain abscess formation was not evident in the syngeneic rat model with appropriate antibiotic use. However, abscess formation is a potential side effect of this treatment that may develop in human patients and would require neurosurgical abscess resection and drainage, a routine clinical procedure. Nevertheless, given the dismal prognosis of high-grade gliomas, the benefits of C. novyi-NT treatment may outweigh the associated potential risks.

[0128] Canine soft tissue sarcomas resemble human tumors: preclinical animal testing of anticancer drugs often does not reproduce the effects observed in humans. However, in companion dogs, clinically used therapeutic agents induce similar toxicities and effects as seen in humans (Paoloni and Khanna (2008) Nat. Rev. Cancer 8:147-156). Testing of investigational therapeutics in companion dogs can represent a vital bridge between preclinical animal testing and human clinical trials. In particular, canine soft tissue sarcomas are an excellent model because they are common in many breeds of dogs and have clinical and histopathological features that are strikingly similar to human soft tissue sarcomas (Paoloni and Khanna (2008) Nat. Rev. Cancer 8:147-156; Vail and MacEwen (2000) Cancer Invest. 18:781-792). Furthermore, the superficial location of many soft tissue sarcomas allows for rapid evaluation and management of treatment-related abscess formation.

[0129] Recent advances in genomics have expanded our knowledge of cancer genetics in humans, recently providing evidence of an association between mutational burden, tumor immunogenicity and response to immunotherapy such as anti-PD-1 and anti-PD-L1 antibodies (Champiat et al., (2014) Oncoimmunology 3:e27817). However, relatively little is known about the genetic landscape of canine cancer. As C. novyi-NT has been shown to induce potent anti-tumor immune responses (Agrawal et al., (2004) Proc. Natl. Acad. Sci. US,A 101(42):15172-15177), it was sought to determine whether canine soft tissue tumor sarcomas are genetically similar to human soft tissue tumor sarcomas and therefore would be suitable comparative models. Therefore, tumor exomes were sequenced and matched with normal DNA from 10 dogs with soft tissue sarcomas (7 with peripheral nerve sheath tumor, 1 with fibrosarcoma, 1 with myxosarcoma, and 1 with synovial cell sarcoma) participating in a comparative study (Figure 10). The analysis included matching of 30,194 nominal genes comprising 32.9 megabases (Mb) of DNA. On average, 16.2 gigabases (Gb) (range: 8.1-23.3 Gb) of generated sequence were mapped to the genome, with 92.2% of bases in the targeted regions covered by at least 10 unique reads in tumor DNA. Similarly, on average, 16.2 Gb (range: 14.6-19.7 Gb) of sequence were mapped to the genome in normal DNA, with 93.6% of bases in the targeted regions covered by at least 10 unique reads. The average coverage per targeted base in tumors was 158-fold (range: 73-227-fold) and 151-fold (range: 130-178-fold) in matched normal samples.

[0130] Using strict analytical criteria, 156 somatic mutations and 28 somatic copy number alterations among the 10 soft tissue sarcomas were identified (Figure 11 and Table 5). Somatic mutations ranged from 0 to 95, with an average of 16 per tumor. Mutation prevalence in soft tissue sarcomas was low, averaging 0.47 per Mb (range: 0.00-2.89 per Mb). Exclusion of one sample outlier with 95 somatic alterations resulted in an average prevalence of 0.21 mutations per Mb (range: 0.00-0.61 per Mb) (Figure 10), similar to estimates of mutation rates in human pediatric rhabdoid tumors (Lee et al., (2012) J. Clin. Invest. 122:2983-2988) and other soft tissue sarcomas (Joseph et al., (2014) Gene Chromosome Canc. 53:15-24). The most common type of somatic alteration was a missense mutation, predominantly C to T (45.5%) and G to A transitions (34.0%; Tables 6 and 7). Amplifications and deletions were less common, averaging 3 per tumor (range: 0 to 17) (Figure 10). Seven of the 10 canine soft tissue sarcomas had no amplifications or deletions.

[0131] Single base substitutions were identified in three tumor suppressor genes (NF1, MLL3, and PTCH1) that are frequently mutated in human tumors. In addition, MDM4, an oncogene shown to be amplified but not point mutated in human cancers, was found to be amplified (but not point mutated) in one canine tumor (Lee et al., (2012) J. Clin. Invest. 122:2983-2988; Barretina et al., (2010) Nat. Genet. 42:715-721; Chmielecki et al., (2013) Nat. Genet. 45:131-132; Vogelstein et al., (2013) Science 339:1546-1558). The only genes mutated in more than one tumor were ATP7B (missense mutation in two tumors) and AIG1 (amplified in two tumors). Interestingly, mutations in ATP7B were also found in human liposarcoma (Joseph et al. (2014) Gene Chromosome Canc. 53:15-24). Of the 184 somatic alterations in the canine tumors, 22 occurred in genes previously shown to be mutated in human soft tissue sarcomas (Table 8). Because the analysis encompassed several soft tissue sarcoma histiotypes, further extensive testing of soft tissue sarcomas in both species will be required to determine whether these represent driver mutations indicative of important, conserved tumorigenic pathways. Nevertheless, the genetic landscape of the canine tumors was similar to that of human tumors in terms of the number of genetic alterations and the range of mutations. Specifically, this excludes the possibility that canine tumors harbor a very large number of mutations that may make them more likely to mount an immune response than similar tumor types in humans.

[0132] Intratumoral injection of C. novyi-NT spores in spontaneous canine tumors: To investigate the safety and efficacy of intratumoral injection of C. novyi-NT spores, a comparative study of 16 dogs with naturally occurring solid tumors was conducted (Table 9). Each dog received 1 × 10 spores in one target tumor. 8Dogs received at least one cycle of C. novyi-NT spore treatment, defined as a single intratumoral injection of C. novyi-NT spores. Dogs received up to four cycles of treatment with a one-week interval between cycles. Treated dogs were followed for at least 90 days after the first intratumoral injection.

[0133] Nine neutered males, six neutered females, and one intact male were enrolled in the study (Table 2). The mean weight of the dogs was 29.4 kg (range 8.1–44.3 kg) and their mean age was 10.9 years (range 7.2–14.3 years). Thirteen dogs had a histomorphic diagnosis of soft tissue sarcoma (8 peripheral nerve sheath tumors, 1 fibrosarcoma, 1 myxosarcoma, 1 rhabdomyosarcoma, and 1 synovial cell sarcoma) as well as one each of osteosarcoma, malignant melanoma, and mast cell tumor. Of the 13 soft tissue sarcomas, six peripheral nerve sheath tumors were available for immunohistochemistry (IHC). All six were positive for S100 and negative for smooth muscle actin, confirming the histomorphic diagnosis. Seven of the tumours were grade I, five were grade II and four were grade III. Eight dogs had previously been surgically treated for their cancer.

[0134] All dogs received at least one cycle of treatment, with 53 cycles administered out of a planned maximum of 64. The majority of dogs, 10 of 16, received the intended four cycles. In dogs that showed early tumor response, toxicity, or progressive disease after the first cycle, subsequent cycles were stopped (Table 9). In general, adverse events were mild in severity (>90% grade I or grade II) and consistent with local infection at the C. novyi-NT spore injection site, including fever (17 incidents), tumor inflammation (12 incidents), tumor abscess (10 incidents), anorexia (9 incidents), and lethargy (6 incidents) (Table 10). Clinical signs of an inflammatory response at the site of the injected target lesion were observed in 14 of 16 dogs (87.5%), including tumor inflammation (12 / 14), tumor abscess (7 / 14), tumor pain (5 / 14), and tumor secretion (4 / 14) (Table 11).

[0135] Dogs were evaluated for best response on or after day 21 of the study. Two of the 16 dogs, 04-R04 and 04-R08, could not be evaluated for response because the injected tumor was surgically removed before day 21. Dog 04-R04 had a humerus osteosarcoma that developed robust germination 2 days after the first intratumoral injection of C. novyi-NT, and due to the deep location of the tumor, limb amputation was performed on day 21 for abscess management. Dog 04-R08 had a peripheral nerve sheath tumor on the medial aspect of the hind leg and received three cycles of treatment before limb amputation on day 15 for management of progressive disease. Fourteen of the 16 dogs were evaluated for response to treatment. Three had a complete response (CR) to treatment, three had a partial response (PR), five had stable disease (SD), and three had progressive disease (PD). The response rate for treatment was 37.5% (6 of 16 dogs; 95% confidence interval: 15.2-64.6%). Tumor abscesses and responses occurred after 1 to 4 cycles of treatment. Dog 11-R01 experienced a PR after a single cycle, 04-R03 had a CR after 3 cycles, dogs 04-R02 and 04-R05 had a PR after 4 cycles, and 04-R01 and 04-R06 had a CR after 4 cycles. Figures 5 and 6 show representative changes in dogs with partial (11-R01) and complete responses (04-R03), respectively. In 3 of the 6 dogs that experienced an objective response, resolution of the abscess occurred with operative management. In these cases, debridement was performed an average of 22 days after the first cycle of treatment. In dog 04-R02, tumor response was evaluated before the owner elected limb amputation for wound management. In dogs 04-R03 and 11-R02, tumor response was evaluated after wound debridement. Dissected tissue was available for histopathological analysis in dogs 04-R02 and 04-R03, which showed extensive tumor necrosis and inflammation with numerous Gram-positive bacilli morphologically consistent with Clostridium spp. In dog 04-R02, there were no viable tumor cells at the tumor margins. In dog 04-R03, rare scattered tumor cells were observed.However, given the aggressive nature of C. novyi-NT-associated abscess formation and subsequent immune infiltration and wound healing, it is difficult to speculate on its ultimate fate in the absence of debridement. Independent of debridement, wound healing was uneventful and complete after 2 to 4 weeks. In addition to surgical management, three of the six dogs with objective responses received antibiotics (ampicillin, amoxicillin, and metronidazole) and analgesics (opioids, tramadol, and nonsteroidal anti-inflammatory drugs) during the course of the study. However, overt abscess formation was not always observed before the objective response. Dogs 04-R01 and 04-R06 received four cycles of treatment and tumor inflammation but no abscess formation was observed at the study visit on day 21. Complete responses were observed in these two dogs at study visits on days 42 (unscheduled visit) and 60, respectively. Three of the six dogs that experienced either CR or PR had long-term responses (Figure 7). In the remaining three dogs, the mean progression-free interval was 106 days (range: 60-169).

[0136] C. novyi-NT causes rapid local tumor destruction in the first human patient: The promising results and favorable risk / benefit profile of C. novyi-NT treatment in a comparative dog study, together with the results observed in rats, provided a rationale for the attempt of this treatment in humans. Thus, a phase I investigational study in human patients with solid tumors that are resistant to standard treatment or have no available standard treatment was initiated (NCT01924689). Herein, we report the first patient enrolled in this study, a 53-year-old woman diagnosed with retroperitoneal leiomyosarcoma in August 2006. The patient had undergone several surgical resections and multiple chemotherapy and radiotherapy treatments. However, the patient's disease progressed and metastatic lesions were present in the liver, lung, peritoneum, and soft tissues in the right shoulder and adjacent right humerus.

[0137] Planned starting dose 1 x 10 4Treatment was performed by injecting C. novyi-NT spores with an 18-gauge multi-pronged needle into the patient's metastatic right shoulder tumor (day 0). On day 1, the patient had mild right shoulder pain radiating to the scapula, which responded to tramadol and acetaminophen. On day 2, the patient's pain required intravenous patient-controlled analgesia with hydromorphone, the white blood cell count increased to 18,300 per μL, and the patient was febrile with a maximum temperature of 39.2°C. On day 3, the patient's right shoulder and scapula pain was difficult to control. The maximum temperature was 37.8°C. A CT scan of the right upper extremity showed extensive tumor destruction with gas in the soft tissue and bony components of the tumor (Figure 8A). The gas permeation pattern was consistent with extensive necrosis of the proximal humerus. CT-guided aspirate of the patient's tumor revealed C. novyi-NT growth under anaerobic culture conditions. The patient was then started on antibiotics (piperacillin / tazobactam, metronidazole, and vancomycin), and the patient's fever abated shortly thereafter. On day 4, magnetic resonance imaging (MRI) of the right upper extremity showed a marked decrease in enhancement that was confined to the tumor mass compared to baseline (Figure 8B and 8C). A biopsy from the tumor showed numerous gram-positive bacteria and the absence of viable tumor cells (Figure 9). At the time of biopsy, a percutaneous drain was placed within the tumor abscess to drain fluid and debris. The patient remained afebrile, and his white blood cell count gradually normalized. The patient continued on antibiotics and was placed in the hospital for intravenous analgesia until day 20, when he was transitioned to oral analgesics. The patient was discharged on metronidazole and doxycycline administered orally by protocol. On day 29, a follow-up MRI showed a progressive decrease in tumor enhancement (Figure 8D). On day 55, the patient presented with localized pain as a result of a patient-effort-induced pathologic fracture of the necrotic right proximal humerus. Subsequent partial resection of the humerus, debridement, and internal fixation with an intramedullary nail and cement spacer markedly improved pain and increased range of motion. Intraoperative cultures revealed C. novyi-NT growth under anaerobic culture conditions. Histopathology showed extensive tumor necrosis and small foci of residual tumor cells.The patient continues to be monitored and currently has an Eastern Cooperative Oncology Group scale (ECOG) performance status of 1 with no clinical signs of infection.

[0138] Consideration Most conventional anticancer treatments target the well-vascularized components of tumors. However, to cure the disease, all neoplastic cells must be destroyed, and any remaining cancer cells are capable of regenerating tumors. This principle has been dramatically illustrated in recent studies using targeted anticancer drugs. Although it is possible to induce significant remissions, tumors almost always recur within a few months due to a small percentage (<0.0001%) of cells that harbor resistance mutations prior to treatment (Sharma et al., (2007) Nat. Rev. Cancer 7:169-181; Chapman et al., (2011) New Engl. J. Med 364:2507-2516; Kwak et al. (2010) New Engl. J. Med 363:1693-1703).

[0139] Treatment with C. novyi-NT spores injected intratumorally offers a method to precisely eradicate neoplastic cells, in principle, independent of tumor-specific genetic alterations. In addition to directly killing tumor cells in its hypoxic environment, C. novyi-NT has been shown to induce potent antitumor immune responses, both innate and acquired, in preclinical models (Agrawal et al., (2004) Proc. Natl. Acad. Sci. USA vol. 101(no. 42):15172-15177). Although there was no clear evidence to demonstrate an acquired antitumor immune response in either human patients or companion dogs, the striking inflammatory response induced by intratumoral injection of C. novyi-NT spores provides clear evidence of an innate immune response. Because C. novyi-NT is acutely sensitive to oxygen and has not been shown to germinate in normoxic areas of tumors, it is plausible that immunity (either innate or acquired) played a role in the dogs achieving durable complete responses. Furthermore, the first human experience with intratumorally injected C. novyi-NT spores produced a rapid and robust local antitumor response. In this case, the proximity of the underlying bone may have contributed to the pathological fracture that ultimately required surgery. However, patient selection may minimize the risk of similar complications in the future. It is important to point out that this result was produced with only 10,000 spores, a small fraction of the dose used to treat dogs or rats. As Phase I trials progress, it will be interesting to see whether higher doses will affect distant metastases, either directly through the spread of spores released from local sites into the circulation or through host-mediated immunity.

[0140] Comparative studies of dogs with spontaneous tumors should be incorporated into discussions of the translational potential of studies in experimental animal models of cancer (Vail and MacEwen (2000) Cancer Invest. 18:781-792). Demonstration of therapeutic efficacy in spontaneous tumors in dogs can be a powerful complement to studies of transplanted or genetically induced tumors in preclinical animal models. This complementarity is reinforced by the genetic similarities between human and canine tumors described herein. Taken together, such similarities can provide a compelling rationale for directing studies in humans, which is particularly pertinent for new forms of therapy associated with significantly more potent toxicity, such as those using C. novyi-NT and other biological agents.

[0141] The next steps in this line of research are clear. First, it will be important to further characterize the safety and efficacy of intratumoral C. novyi-NT spore treatment. The efficacy of C. novyi-NT spores, at least when administered systemically, is dramatically enhanced by combining them with carefully selected chemotherapeutic agents or radiation therapy (Dang et al., (2004) Cancer Bio. Ther. 3:326-337; Cheong et al., (2006) Science 314(5803):1308-1311; Bettegowda et al., (2003) Proc. Natl. Acad. Sci. USA 100(25):15083-15088). A multiple model approach seems particularly attractive because the mechanism by which C. novyi-NT kills tumor cells does not overlap with the mechanism of action of other forms of therapy (Dang et al., (2004) Cancer Bio. Ther. 3:326-337). Finally, it will be of great interest to determine whether immune checkpoint blockade can enhance the antitumor immunity expected from intratumoral C. novyi-NT spore treatment (Agrawal et al. (2004) Proc. Natl. Acad. Sci. USA 101(42):15172-15177).

[0142] In some embodiments, the presently disclosed subject matter uses an attenuated strain of the anaerobic spore-forming bacterium Clostridium novyi (C. novyi-NT) and demonstrates precise, robust and reproducible antitumor responses when C. novyi-NT spores are injected into tumors in rats, domestic dogs, and humans. These results indicate that intratumoral C. novyi-NT spores can be used as a therapeutic agent for patients with locally advanced unresectable cancer. [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 4] [Table 5-1] [Table 5-2] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10-1] [Table 10-2] [Table 11]

[0143] References All publications, patent applications, patents, and other references mentioned in this specification are indicative of the level of ordinary skill in the art to which the subject matter disclosed herein pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although several patent applications, patents, and other references are mentioned in this specification, it will be understood that such mention does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0144] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A combination for treating a solid breast tumor in a subject, the combination comprising a therapeutically effective amount of an anti-CTLA-4 antibody in combination with Clostridium novyi bacteria.

2. A composition for treating a solid breast tumor in a subject, comprising a therapeutically effective amount of an anti-CTLA-4 antibody, said composition being administered in combination with Clostridium novyi bacteria.

3. A composition for treating a solid breast tumor in a subject, comprising Clostridium novyi bacteria, said composition being administered in combination with a therapeutically effective amount of an anti-CTLA-4 antibody.

4. 4. The combination of claim 1 or the composition of claim 2 or 3, wherein the Clostridium novyi bacterium is a toxin-depleted anaerobic bacterium.

5. The combination or composition of claim 4, wherein the Clostridium novyi bacterium is Clostridium novyi-NT.

6. 5. The combination or composition of claim 4, wherein some or all of the toxin genes of the wild-type Clostridium novyi bacterium have been deleted.

7. 5. The combination or composition of claim 4, wherein the virulence of the Clostridium novyi bacteria is reduced by at least 2-fold compared to the corresponding wild-type Clostridium novyi bacteria.

8. 4. The combination of claim 1 or the composition of claim 2 or 3, characterized in that the Clostridium novyi bacteria are administered intravenously or intratumorally.

9. The combination of claim 1 or the composition of claim 2 or 3, characterized in that the anti-CTLA-4 antibody is administered by at least one method selected from the group consisting of intravenous, intramuscular, subcutaneous, or intratumoral.

10. 4. The combination of claim 1 or the composition of claim 2 or 3, wherein the solid breast tumor is malignant.

11. 4. The combination of claim 1 or the composition of claim 2 or 3, wherein after the solid breast tumor is treated, the solid breast tumor regresses or the growth of the solid breast tumor is slowed or stopped.

12. The combination of claim 1 or the composition of claim 2 or 3, wherein the subject is a human.

13. The combination of claim 1 or the composition of claim 2 or 3, wherein the subject is a non-human animal.

14. A kit for treating a solid breast tumor, comprising Clostridium novyi bacteria and an anti-CTLA-4 antibody.

15. 15. The kit of claim 14, wherein the Clostridium novyi bacterium is a toxin-depleted anaerobic bacterium.

16. The kit of claim 15, wherein the Clostridium novyi bacterium is Clostridium novyi-NT.

17. 16. The kit of claim 15, wherein some or all of the toxin genes of wild-type Clostridium novyi bacteria have been deleted.

18. 16. The kit of claim 15, wherein the virulence of the Clostridium novyi bacteria is reduced by at least 2-fold compared to corresponding wild-type Clostridium novyi bacteria.

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