Use of bacteria, bacterial products and other immunoregulatory 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 Mar. 31, 2014, and U.S. Provisional Application No. 62 / 035,291, filed Aug. 8, 2014, which are hereby incorporated by reference in their entirety.

Background Art

[0002] The prognosis for patients presenting with advanced cancer of the pancreas, colon, lung, breast, ovary, brain or prostate is poor. As a result of intensive research from this tragic situation, there have been innovations in the understanding of cancer etiology, a marked increase in the application of conventional chemotherapeutic agents, and the availability of some promising new agents. Unfortunately, these innovations have not yet had a major impact on the treatment of common solid tumors. More than anything else, what is desired for future therapeutic effects is to combine more new approaches, such as spores of the anaerobic bacterium Clostridium novyi (C. novyi), with a greater number of conventional agents, as many people think.

[0003] The theoretical basis for using anaerobic bacteria lies in the unique angiogenesis state present within tumors. Solid tumors require angiogenesis to grow, and as they grow, some of the tumor vasculature becomes insufficient. These avascular regions tend to have low therapeutic drug concentrations. Furthermore, drug molecules that actually reach the avascular regions usually rely on both oxygen and actively replicating cells for full efficacy.

[0004] Solid tumor malignancies have previously been shown to be treatable by using several species of anaerobic bacteria. C. novyi is a Gram-positive, endospore-forming, obligate anaerobic bacterium. Clostridium novyi-NT (C. novyi-NT) is an attenuated form of C. novyi that lacks the major toxins. The use of C. novyi-NT for the treatment of cancer has been previously reported (Agrawal et al., (2004) Proc. Natl. Acad. Sci. U.S.A. 101(42):15172-15177; Bettegowda et al., (2003) Proc. Natl. Acad. Sci. U.S.A. 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. U.S.A. 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 eradicating metastatic cancer. Recent clinical trials of neutralizing antibodies targeting CTLA-4 and PD-1, two important checkpoints for T cell-mediated immunity, have shown clinical responses in patients with solid tumor malignancies.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] In one aspect, the subject matter disclosed herein is 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 a bacterium, a bacterial product, and an immunomodulatory entity to treat the solid tumor. In certain embodiments, the bacterium is a lethal toxin-depleted anaerobic bacterium. In another specific embodiment, the bacterial product is a component of the bacterium, such as a bacterial membrane component.

[0007] In certain embodiments, the subject matter disclosed herein is 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 and 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 subject matter disclosed herein is a method for treating cancer in a subject, the method 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] Certain embodiments of the subject matter disclosed herein have been described above, and while the subject matter disclosed herein is wholly or partially addressed, other embodiments will become apparent as the description proceeds in connection with the accompanying examples and figures, as best described below.

[0010] The subject matter disclosed herein has been described in such general terms that reference is made here to the accompanying drawings, which are not necessarily drawn to scale. The present invention provides, for example, the following items. (Item 1) 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 immunomodulatory entities to treat the solid tumor. (Item 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. (Item 4) The method according to item 2, wherein the bacterium or the bacterial product is a toxin-depleted anaerobic bacterium. (Item 5) The method according to item 4, wherein the toxin-depleted anaerobic bacterium is Clostridium novyi-NT. (Item 6) The method according to item 4, wherein part or all of the toxin gene of the wild-type toxin-depleted anaerobic bacterium is deleted. (Item 7) The method according to item 4, wherein the toxicity of the toxin-depleted anaerobic bacterium is reduced by at most one-half compared to the corresponding wild-type bacterium. (Item 8) The method according to item 1, wherein the bacterial product is at least one bacterial membrane component. (Item 9) The method according to item 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) The method according to item 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) The method according to item 1, wherein the solid tumor is malignant. (Item 12) The method according to item 1, wherein after the solid tumor is treated, the solid tumor regresses, or the growth of the solid tumor is delayed or stopped. (Item 13) The method according to item 1, wherein the subject is a human. (Item 14) The method according to item 1, wherein the subject is a non-human animal. (Item 15) A method for 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) The method according to 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) The method according to item 15, wherein the subject is a human. (Item 18) The method according to item 1, wherein the subject is a non-human animal. (Item 19) A kit for treating a solid tumor, comprising at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody, and at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities. (Item 20) The kit according to item 19, wherein the bacteria are anaerobic bacteria. (Item 21) The kit according to item 20, wherein the anaerobic bacteria are Clostridium novyi. (Item 22) The kit according to item 20, wherein the anaerobic bacteria are toxin-depleted anaerobic bacteria. (Item 23) The kit according to item 22, wherein the anaerobic bacterium is Clostridium novyi-NT. (Item 24) The kit according to item 22, wherein part or all of the toxin gene of the wild-type toxin-depleted anaerobic bacterium is deleted. (Item 25) The kit according to item 22, wherein the toxicity of the toxin-depleted anaerobic bacterium is reduced by at most one-half compared to the corresponding wild-type bacterium. (Item 26) The kit according to item 19, wherein the bacterial product is at least one bacterial membrane component.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0012] The subject matter disclosed herein will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Throughout the drawings, like numerals refer to like elements. The subject matter disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. 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 will come to mind to one skilled in the art to which the subject matter disclosed herein pertains, and the benefits of the teachings are set forth in the foregoing description and the accompanying drawings. Accordingly, the subject matter disclosed herein should not be limited to the particular embodiments disclosed, but it is intended that modifications and other embodiments be included within the scope of the appended claims.

[0013] The subject matter disclosed herein provides methods and kits for treating tumors. Ablation of negative regulation mediated through the PD-1 and CTLA-4 pathways has been hypothesized to enhance the anti-cancer immune response induced by intratumoral bacterial infection, and thus to cure metastatic tumors. It is shown herein that the therapeutic effect of anti-tumor bacteria is significantly enhanced by combination with anti-CTLA-4 antibody and / or anti-PD-1 antibody. 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, combining anti-CTLA-4 antibody and anti-PD-1 antibody gave better results than using either antibody alone.

[0014] Accordingly, 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 the anti-tumor immune response induced by the immunomodulatory entity. Furthermore, it is possible to treat cancer by combining anti-CTLA-4 antibody and anti-PD-1 antibody using the methods and kits disclosed herein.

[0015] I. Method for Treating Cancer In some embodiments, the subject matter disclosed herein is 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 anti-CTLA-4 antibody and anti-PD-1 antibody in combination with at least one member of the group consisting of bacteria, bacterial products, and immunomodulatory entities to treat the solid tumor. Examples of antibodies that can be used in the methods disclosed herein 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 number AAD00698.1), also known as CD152 (surface antigen classification 152), is a T cell surface molecule that is a negative regulator of T cell activation. CTLA-4 was first identified by differential screening of a mouse cytotoxic 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 cytotoxic 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 homology in the region near the membrane and the cytoplasmic region (Brunet et al., (1987) Nature 328:267-270; Dariavach et al., (1988) Eur. J. Immunol. 18:1901-1905). Several 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, other researchers have reported that CTLA-4 has an opposing role as a buffer for T cell activation (Krurnmel (1995) J. (Exp. Med. 182: 459 - 465; Krummel 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 lymphocyte proliferation (Chambers et al., (1997) Immunity 7: 885 - 895). Blockade of CTLA-4 has been reported to enhance T cell responses in vitro (Walunas et al., (1994) Immunity 1: 405 - 413) and in vivo (Kearney (1995) J. Immunol. 155: 1032 - 1036), to exacerbate antitumor immunity (Leach (1996) Science 271: 1734 - 1736), and to enhance induced autoimmune diseases (Luhder (1998) J. Exp. Med. 187: 427 - 432). CTLA-4 has also been reported to have another or additional effect on the initial characteristics of the T cell immune response (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 number NP_005009.2), also known as CD279 (cluster of differentiation 279), is a cell surface membrane protein that is expressed primarily on subsets of activated T lymphocytes and is 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. Patent No. 5,698,520). PD-1 is a member of the immunoglobulin gene superfamily and has an extracellular region containing an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region containing an immunoreceptor tyrosine-based inhibitory motif (ITIM; Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704). These features define a relatively large family of polypeptides, also called immune inhibitory receptors, that also includes gp49B, PIR-B, and killer cell inhibitory receptors (KIR) (Vivier and Daeron (1997) Immunol. Today 18:286). The tyrosine phosphorylated ITIM motifs of these receptors are thought to often interact with phosphatases containing SH2 domains to generate inhibitory signals. A subset of these immune inhibitory receptors binds MHC polypeptides; for example, KIR and CTLA-4 bind B7-1 and B7-2. A phylogenetic relationship has been proposed between the MHC and B7 genes (Henry et al. (1999) Immunol. Today Volume 20 (Issue 6): pp. 285-288. Similar to 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). However, in contrast to CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed on subsets 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 an IgV domain, an IgC domain, a transmembrane domain, and a short cytoplasmic tail. 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 placenta, spleen, lymph nodes, thymus, and heart. Only PDL2 is expressed in pancreas, lung and liver, and only PDL1 is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells. Due to the fact that PD-1 binds to PDL1 and PDL2, together with CTLA-4, PD-1 maps to the family of inhibitory receptors.

[0019] "Functional variants" of CTLA-4 or PD-1 include functional fragments, functional mutant proteins, and / or functional fusion proteins. A functional variant of a selected polypeptide is an isolated and / or recombinant protein or polypeptide having at least one characteristic, activity, and / or functional feature of the selected polypeptide (e.g., CTLA-4 or PD-1). As used herein, the term "activity," when used with respect to a polypeptide, e.g., CTLA-4 or PD-1, includes activities inherent to the structure of the wild-type protein.

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

[0021] As used herein, the term "co-stimulate," when used in connection with an activated immune cell, 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. For example, a co-stimulatory signal can result in cytokine secretion 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 an inhibitory receptor can occur even if a co-stimulatory receptor (such as CD28) is not present on the immune cell, and thus, this is not simply a function of competition between the inhibitory receptor and the co-stimulatory receptor with respect to binding of the co-stimulatory polypeptide (Fallarino et al., (1998) J. Exp. Med. 188:205). Transmission of the inhibitory signal to an immune cell can result in unresponsiveness or anergy or programmed cell death in the immune cell. Preferably, transmission of the inhibitory signal operates through a mechanism that does not include 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 that results in fragmentation of the cell. Cells undergoing apoptosis also exhibit a characteristic pattern of DNA fragmentation between nucleosomes.

[0022] Generally, fragments or portions of CTLA-4 or PD-1 encompassed by the subject matter disclosed herein include fragments or portions of CTLA-4 or PD-1 having deletions of amino acids (i.e., one or more amino acids) (such as N-terminal, C-terminal, or internal deletions) compared to wild-type CTLA-4 or PD-1. Fragments or portions are also envisioned in which only contiguous amino acids are deleted or non-contiguous amino acids are deleted compared to wild-type CTLA-4 or PD-1. Generally, mutants or derivatives of CTLA-4 or PD-1 encompassed by the subject matter disclosed herein include natural or artificial mutants 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 modified, as well as mutants that include one or more modified residues. Preferred mutants are natural or artificial mutants 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 mutant 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 mutant.

[0024] "Sequence identity" or "identity" in the context of a protein or polypeptide refers to amino acid residues of two amino acid sequences that are the same when aligned to maximize matches over a specified comparison window.

[0025] Thus, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the amino acid sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence of the optimal alignment of the two sequences (excluding additions and deletions). The percent is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences, obtaining 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 percent 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 percent from 50% to 100%. These identities can be determined using any of the programs described herein.

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

[0027] Many levels of sequence identity are well understood by those skilled in the art to be useful for identifying proteins or polypeptides having the same or similar functions or activities (e.g., from other species). 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 from 50% to 100%. In fact, any integer amino acid identity from 50% to 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 for 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 used by the immune system to identify and neutralize foreign substances such as bacteria and viruses by recognizing unique parts (epitopes) of foreign targets called antigens. As used herein, the term "antibody" also includes the "antigen-binding portion" (or simply "antibody portion") of the antibody. 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 carried out by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains, (ii) F(ab’)2 fragment, a divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, (iii) Fd fragment consisting of VH and CH1 domains, (iv) Fv fragment consisting of VL and VH domains of a single arm of an antibody, (v) dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546), and (vi) isolated complementarity-determining regions (CDRs). Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but the VL and VH regions pair to form a monovalent polypeptide (known as single-chain Fv (scFv); e.g., Bird et al., (1988) Science The two domains can be joined using recombinant methods with a synthetic linker that allows them to be made as a single protein chain that forms (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Osbourn et al., Nature Biotechnology 16:778 (1998), Vol. 242: pages 423-426). Such single-chain antibodies are also intended to be included within the antibody term "antigen-binding portion". To create an expression vector encoding a full IgG polypeptide or other isotype, any VH and VL sequences of a particular scFv can be ligated to human immunoglobulin constant region cDNA or genomic sequences. VH and VL can also be used in the production of 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 included. 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, thereby forcing the domains to pair with complementary domains on another chain, resulting in two antigen-binding sites (e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994)).

[0029] Furthermore, the antibody or antigen-binding portion thereof may be part of a larger immunoadhesion polypeptide formed by covalent or non-covalent attachment of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the use of streptavidin core regions to create tetrameric scFv polypeptides (Kipriyanov et al., (1995) Human Antibodies and Hybridomas 6:93-101) as well as the use of cysteine residues, marker peptides and C-terminal polyhistidine tags to create divalent 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 of the whole antibody. Furthermore, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0030] The antibody can be polyclonal or monoclonal, heterologous, homologous or syngeneic, or a modified form thereof (e.g., humanized, chimeric, etc.). The antibody can be fully human. Preferably, the antibodies of the subject matter disclosed herein specifically or substantially specifically bind to PD-1 or CTLA-4 or a functional variant thereof. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a population of antibody polypeptides containing only one antigen-binding site capable of immunoreacting with a particular epitope of an antigen, while the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a population of antibody polypeptides containing multiple antigen-binding sites capable of interacting with a particular antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the particular antigen to which they immunoreact.

[0031] As used herein, the term "humanized antibody" is intended to include antibodies made by non-human cells that have variable and constant regions that have been altered to more closely resemble antibodies made by human cells. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the subject matter disclosed herein can, for example, include in the CDRs amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed 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, are grafted onto human framework sequences.

[0032] As used herein, "isolated antibody" is intended to refer to an antibody that substantially lacks other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CTLA-4 or PD-1 substantially lacks antibodies that specifically bind to antigens other than CTLA-4 or PD-1). In addition, an isolated antibody may substantially lack other cellular materials and / or chemical substances.

[0033] An isolated CTLA-4 or PD-1 or a functional variant thereof (or a nucleic acid encoding such a polypeptide) can be used as an immunogen to generate antibodies that bind to the respective CTLA-4 or PD-1 or a functional variant thereof using standard techniques for the preparation of polyclonal and monoclonal antibodies. It is possible to use full-length CTLA-4 or PD-1, 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. 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 contain at least 10 amino acid residues, more preferably at least 15 amino acid residues, still 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 located on the surface of the protein, e.g., hydrophilic regions, among CTLA-4 or PD-1 or functional variants thereof. Hydrophilic regions can be identified by performing standard hydrophobicity analysis of the polypeptide molecule. Highly preferred epitopes encompassed by the antigenic peptides are those in the extracellular domain of the polypeptide molecule and thus involved in binding. In one embodiment, such epitopes can be specific for a given polypeptide molecule from a species such as mouse or human (i.e., antigenic peptides that span 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] Immunogens containing CTLA-4 or PD-1 or functional variants thereof are typically used to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal) with the immunogen. Suitable immunogen preparations can contain, for example, recombinantly expressed or chemically synthesized molecules or fragments thereof that will elicit 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 peptides contained therein.

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

[0036] Any of a number of well-known protocols used to fuse lymphocytes with immortalized cell lines can be used for the purpose of generating anti-PD-1 ligand monoclonal antibodies (e.g., Galfre, G. et al., (1977) Nature 266:550-52; Kenneth, R. H. Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner (1981) Yale J. Biol. Med. 54:387-402; Gefter et al., (1977) Somatic Cell Genet. 3:231-36). Furthermore, one of ordinary skill in the art will recognize that there are many variations of such methods that are also considered useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, a mouse hybridoma can be made by fusing lymphocytes from a mouse immunized with the immunogen preparation of the subject matter disclosed herein with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to a culture medium containing hypoxanthine, aminopterin, and thymidine ("HAT medium"). Any of several myeloma cell lines, such as the P3-NS1 / 1-Ag4-1, P3-X63-Ag8.653, or Sp2 / O-Ag14 myeloma lines, can be used as fusion partners according to standard techniques. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse spleen cells using polyethylene glycol ("PEG"). Next, the hybridoma cells resulting from the fusion are selected using HAT medium, which kills unfused myeloma cells and non-productively fused myeloma cells (unfused spleen cells die after a few days because they are not transformed).Hybridoma cells that produce monoclonal antibodies of the subject matter disclosed herein are detected, for example, by screening the hybridoma culture supernatant for antibodies that bind to a given polypeptide using a standard ELISA assay.

[0037] As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal specific to 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 an appropriate polypeptide, thereby isolating immunoglobulin library members that bind to the polypeptide. Kits for creating and screening phage displays are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01 and Stratagene SurfZAP™ Phage Display Kit, catalog number 240612).Furthermore, examples of methods and reagents that are particularly acceptable for creating and screening antibody display libraries can be found, for example, in U.S. Patent 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] Furthermore, recombinant anti-CTLA-4 antibodies or anti-PD-1 antibodies, such as chimeric and humanized monoclonal antibodies that contain both human and non-human portions and that can be made using standard recombinant DNA techniques, are within the scope of the subject matter disclosed herein. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art using, for example, the methods described 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 International Publication No. 86 / 01533, U.S. Patent 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. 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, S. L. (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.

[0039] Furthermore, the humanized antibody can be made according to standard protocols such as the protocol disclosed in U.S. Patent No. 5,565,332. In another embodiment, the antibody chain or specific binding pair member can be prepared using techniques known in the art, as described, for example, in U.S. Patent No. 5,565,332, No. 5,871,907, or No. 5,733,743, by recombination between a vector containing 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. The use of intracellular antibodies to inhibit protein function within cells 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 Volume 90: pages 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] Furthermore, a fully human antibody against CTLA - 4 or PD - 1 or a functional variant thereof can be made. A fully human antibody can be made, for example, in mice transgenic for human immunoglobulin genes 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 a functional variant thereof. Spleen cells are harvested and fused to myeloma cells to generate hybridomas. The hybridomas are selected based on their ability to produce an antibody that binds to CTLA - 4 or PD - 1 or a functional variant thereof. A fully human antibody is thought to reduce the immunogenicity of such an antibody in humans.

[0041] In one embodiment, an antibody for use in the subject matter disclosed herein is a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may 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. Patent 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. Fragments of bispecific antibodies are described in U.S. Patent No. 5,798,229.

[0042] Bispecific agents can also be made by fusing hybridomas or other cells that make different antibodies to create heterohybridomas, and then identifying clones that produce or co-assemble both antibodies. Bispecific agents can also be made by chemically or genetically conjugating intact 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 functional variants thereof. In one embodiment, the bispecific antibody can specifically bind 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 subject matter disclosed herein relates to antibodies obtainable by a process that includes 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 an antibody that specifically binds to the polypeptide from the animal.

[0044] In some embodiments, the subject matter disclosed herein provides methods for treating solid tumors using bacteria, bacterial products, and / or other immunomodulatory entities. In other embodiments, the bacteria or bacterial products thereof are anaerobic bacteria or bacterial products thereof. 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 thereof are obligate anaerobic bacteria or bacterial products thereof. As used herein, an “anaerobic bacterium” is a bacterium that does not require oxygen for growth. As used herein, an “obligate anaerobic bacterium” is a bacterium that not only does not require oxygen for growth but is harmed by normal levels of atmospheric oxygen. In further embodiments, the anaerobic bacterium or bacterial product thereof is Clostridium novyi or a bacterial product thereof.

[0045] In some embodiments, the bacteria or bacterial products thereof are toxin-depleted anaerobic bacteria or bacterial products thereof. In other embodiments, the toxin-depleted anaerobic bacteria or bacterial products thereof are Clostridium novyi-NT or bacterial products thereof.

[0046] For therapeutic use of bacteria, it is desirable to reduce the natural production of toxins. A toxin-depleted strain of bacteria need not be completely non-toxic, but it is desirable that at least one of the toxin genes is mutated, deleted, or otherwise inactivated such that the bacteria are less harmful to the subject. As used herein, the term "toxic" means acting as a poison or having the effects of a poison. It is preferred that the 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, curing of the episome or bacteriophage 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 bacteria or their bacterial products are deleted to produce the "toxin-depleted" bacteria or their bacterial products. For example, the lethal alpha toxin gene is deleted in C. novyi-NT. In other embodiments, the toxicity of the toxin-depleted anaerobic bacteria is reduced by at most one-half compared to the corresponding wild-type bacteria. In yet other embodiments, the toxicity of the toxin-depleted Clostridium novyi is reduced by at most one-half 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 a nucleotide sequence in which one or more nucleotides have been removed.

[0048] In some embodiments, the bacterial product is at least one bacterial membrane component. Bacterial membrane components can include, for example, bacterial membrane proteins that are bound to or associated with the membrane of Clostridium novyi, and suitably proteins having domains that are exposed to the outside of the bacteria when the bacteria are infected and are thus thought to be visible to the human immune system. References herein to bacterial membrane proteins include deletions, insertions, and substitution mutations of a given bacterial membrane protein or variants that are suitably immunogenic and are 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 the variant, including variants of naturally occurring bacterial membrane proteins such as proteins having such amino acid sequences.

[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, immunostimulatory cytokines such as GM-CSF, interleukin-12 (IL-12), and IL-15. Additional examples of bacterial products used for immunostimulatory 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 intratumoral.

[0051] "Cancer" in a subject refers to the presence of cells having characteristics typical of cancer - causing cells, such as uncontrolled proliferation, loss of specialized function, immortality, significant metastatic ability, a marked increase in anti - apoptotic activity, rapid growth and proliferation rates, and certain characteristic morphologies and cell markers. In some situations, cancer cells form tumors, and such cells may be present locally within an animal or may circulate in the bloodstream as independent cells, such as leukemia cells. Cancer includes, but is not limited to, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, and adenoma. "Tumor" as used herein refers to any neoplastic cell growth and proliferation, and any pre - cancerous and cancerous cells and tissues, whether malignant or benign. "Solid tumor" as used herein is generally an abnormal mass of tissue that does not contain cysts or liquid regions. Solid tumors may be present, by way of non - limiting example, in the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovary, cervix, stomach, colon, rectum, bladder, uterus, testis, and pancreas. In some embodiments, after a solid tumor is treated by the methods disclosed herein, the solid tumor regresses, or the growth of the solid tumor is delayed or stopped. In other embodiments, the solid tumor is malignant.

[0052] In some embodiments, the subject matter disclosed herein is a method of treating cancer in a subject, the method 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 combinations of anti-CTLA-4 and anti-PD-1 antibodies for treating cancer provide better results than when the antibodies are administered 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 a disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition.

[0054] In many of its embodiments, the subject to be treated by the methods disclosed herein is desirably a human subject, although it should be understood that the methods described herein are effective for any vertebrate species, and any vertebrate species is intended to be included within the term "subject". Thus, "subject" can include a human subject for medical purposes such as the 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 developmental purposes. Suitable animal subjects include, but are not limited to, mammals such as primates, e.g., humans, monkeys, apes, etc.; cattle, e.g., dairy cows, bulls, etc.; ovine, e.g., sheep, etc.; caprine, e.g., goats, etc.; porcine, e.g., pigs, boars, etc.; equine, e.g., horses, donkeys, zebras, etc.; feline, including wild and domestic cats; canine, including dogs; lagomorphs, including rabbits, wild rabbits, etc.; and rodents, including mice, rats, etc. The animal may be a transgenic animal. In some embodiments, the subject is a human including fetal, neonatal, infant, juvenile, and adult subjects. Further, "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, as described herein, 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 can be administered to a subject for treatment by any suitable route of administration, including orally, nasally, transmucosally, to the eyes, rectally, intravaginally, by intramuscular injection, subcutaneous injection, intramedullary injection, as well as by intracerebroventricular injection, direct intraventricular injection, intravenous injection, intra-articular injection, intrasternal injection, intrasynovial injection, intrahepatic injection, intralesional injection, intracranial injection, intraperitoneal injection, intranasal injection, or intraocular injection, parenterally, into the tank, locally by powder, ointment or droplet (including eye drops), transdermally, through an inhalation spray, or by other delivery modes known in the art. The compositions disclosed herein can also be administered into a solid tumor so that the composition is administered directly into the tumor, such as by injection or other means.

[0056] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" mean that the antibody enters the patient's system and thus undergoes metabolism and other similar processes, other than entering directly into the central nervous system, for example, by subcutaneous administration, of a composition 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, i.e., compounds, drugs or other substances.

[0057] As used herein, the phrases "parenteral administration" and "parenterally administered" mean a mode of administration other than enteral and topical administration, typically by injection, including, without limitation, intravenous, intramuscular, intra-arterial, intracerebroventricular, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subdural, intraspinal, and intrasternal injection and infusion.

[0058] The pharmaceutical compositions disclosed herein can be manufactured in a manner known in the art, for example, by conventional mixing, dissolving, granulating, tablet coating, levitating, emulsifying, encapsulating, entrapping or lyophilization processes.

[0059] In some embodiments, the pharmaceutical compositions disclosed herein can be administered by a refillable or biodegradable device. For example, various sustained release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein biopharmaceuticals. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, for example, films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Patent 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, vol. 98, p. 98), ethylene vinyl acetate (Langer et al., (1981) J. Biomed. Mater. Res. 15, vol. 167, p. 167), or poly-D-(-)-3-hydroxybutyric acid (EP133,988A). The sustained release composition 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 that can be prepared by methods known per se, encapsulated in liposomes (Epstein et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82, vol. 3688, p. 3688; Hwang et al., (1980) Proc. Natl. Acad. Sci. U.S.A. 77, vol. 4030, p. 4030; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP102,324A). Usually, the liposomes are small (about 200 - 800 angstroms) unilamellar with a lipid content of more than about 30 mol% cholesterol, and the selected ratio is adjusted for optimal treatment. Such substances can, for example, in some embodiments, include implants for the 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 the antibody, protein, and peptide (Chapman (2002) Adv. Drug Deliv. Rev. 54:531-545). Some of the effects of pegylation include: (a) a marked improvement in the in vivo circulation half-life 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 found 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 allows for an expansion of formulation and administration options such as subcutaneous administration of high protein doses, which is in contrast to many other therapeutic antibodies that 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 loss at the subcutaneous injection site; (g) for agents in which toxicity is related to peak plasma levels and a reduction in toxicity has been observed, the more flattened pharmacokinetic profile achieved by subcutaneous administration of the pegylated protein is advantageous, proteins that induce an immune response with toxic consequences can also benefit as a result of pegylation; and (h) improved thermal and mechanical stability of the pegylated molecule.

[0061] For parenteral administration, pharmaceutical compositions 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 an aqueous solution, for example, in some embodiments, in a physiologically compatible buffer 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. Further, 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 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 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 to enable the preparation of highly concentrated solutions.

[0062] For nasal or transmucosal administration, generally, penetration enhancers suitable for the particular barriers to be permeated are used in the formulation. Such penetration enhancers are generally known in the art.

[0063] For inhalation delivery, the agents of the present disclosure can also be formulated by methods known to those skilled in the art and can include, but are not limited to, solubilizing, diluting, or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption promoters, and examples of fluorocarbons.

[0064] Additional components can be added to the composition for topical administration as long as such components are pharmaceutically acceptable and not harmful to the epithelial cells or their functions. Further, such additional components should not adversely affect the epithelial permeation 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. may be present. The pH of the topical composition disclosed herein can be adjusted to a physiologically acceptable range from about 6.0 to about 9.0 by adding a buffer thereto so that the composition is physiologically compatible with the skin of the subject.

[0065] Regardless of the administration route selected, the compositions disclosed herein that comprise 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 pharmaceutically acceptable dosage form as described herein or by other conventional methods known to those of skill in the art.

[0066] Generally, an “effective amount” or “therapeutically effective amount” of an active agent or drug delivery device is the amount necessary to induce the desired biological response. As will be recognized by those of skill in the art, the effective amount of an agent or device can vary depending on factors such as the desired biological endpoint, the agent being delivered, the composition of the encapsulating matrix, the target tissue, etc.

[0067] The term "combination" is used in its broadest sense and means that a subject is administered 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 at least two agents, more specifically bacteria, bacterial products, and immunomodulatory entities. More specifically, the term "in combination" means, for example, the simultaneous administration of two (or more) active agents for the treatment of a single disease state. As used herein, the active agents may be combined and administered in a single dosage form, simultaneously in separate dosage forms, or administered as separate dosage forms alternately or sequentially on the same day or on separate days. In one embodiment of the subject matter disclosed herein, 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., in which case it may be desirable to vary the amount of one dosage form but not the amount of the other dosage form). A single dosage form may contain additional active agents for the treatment of the disease state.

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

[0069] The timing of administration of a compound of an anti-CTLA-4 antibody and / or an anti-PD-1 antibody in combination with a bacterium, bacterial product, or other immunomodulatory entity and optionally an additional agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Thus, the phrase "in combination" refers to the administration, simultaneously, sequentially, or a combination thereof, of an anti-CTLA-4 and / or anti-PD-1 antibody in combination with a bacterium, bacterial product, or other immunomodulatory entity and optionally an additional agent. Thus, a subject administered a combination of an anti-CTLA-4 and / or anti-PD-1 antibody in combination with a bacterium, bacterial product, or other immunomodulatory entity and optionally an additional agent can receive the anti-CTLA-4 and / or anti-PD-1 antibody in combination with a bacterium, bacterial product, or other immunomodulatory entity and optionally an additional agent 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 any combination of the agents are achieved in the subject.

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

[0071] When administered in combination, the effective concentration of each agent that induces a specific biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more agents compared to the dose required when the agent is administered as a single agent. The effects of the plurality of agents may be additive or synergistic, but need not be. The agents can be administered multiple times.

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

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

[0074] Generally, Q a / Q A and Q b / Q B If the sum of is greater than 1, an antagonistic effect is shown. If the sum is equal to 1, an additive effect is shown. If the sum is less than 1, a synergistic effect is shown. As the SI decreases, the synergistic effect shown by that particular mixture increases. Thus, a "synergistic combination" has an activity higher than that which can be predicted based on the observed activity of the individual components when used alone. Further, the "synergistically effective amount" of a component is, for example, the amount of the component necessary to induce a synergistic effect in another therapeutic agent present in the composition.

[0075] As used herein, the terms "reduce" or "inhibit", and their grammatical derivatives, refer to the ability of an agent to block, partially block, interfere with, reduce, diminish or inactivate an action pathway or mechanism of action. Thus, one of ordinary skill in the art would recognize that the term "diminish" encompasses a complete and / or partial loss of activity, e.g., a loss of at least 10% of the activity, and in some embodiments, losses of at least 20%, 30%, 50%, 75%, 95%, 98%, and up to a maximum of 100% of the activity.

[0076] In another aspect, the subject matter disclosed herein provides a pharmaceutical composition comprising a bacterium, a bacterial product, or another immunomodulatory entity, and, optionally, an anti - CTLA - 4 and / or anti - PD - 1 antibody, alone or in combination with one or more additional therapeutic agents, admixed with a pharmaceutically acceptable excipient.

[0077] More specifically, the subject matter disclosed herein provides a pharmaceutical composition comprising a bacterium, a bacterial product, or another immunomodulatory entity, and, optionally, an anti - CTLA - 4 and / or anti - PD - 1 antibody in combination with an additional agent, and a pharmaceutically 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 local administration. Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy (20th Edition) 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 in the present disclosure into dosages suitable for systemic administration. By the correct 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 enable the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a subject (e.g., a patient) to be treated.

[0080] For nasal or inhalation delivery, the agents of the present disclosure can also be formulated by methods known to those skilled in the art, including, but not limited to, solubilizing, diluting, or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption promoters, and examples of 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. Determination of the effective amount is well within the ability of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein. Generally, the 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, 0.5 to 100 mg, 1 to 50 mg, and 5 to 40 mg per day 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, as well as the preference and experience of the attending physician.

[0082] In addition to the active ingredient, these pharmaceutical compositions can contain a suitable pharmaceutically acceptable carrier that includes excipients and auxiliary substances that facilitate the processing of the active compound into a pharmaceutically usable preparation. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0083] II. Kit for Treating Cancer The subject matter disclosed herein also relates to kits for performing the methods of the subject matter disclosed herein. Generally, the kits disclosed herein contain some or all of the components, reagents, supplies, etc. for performing a method in accordance with the subject matter disclosed herein. In some embodiments, the term "kit" refers to any intended product (e.g., a package or container) containing 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, as well as a set of specific instructions for performing the methods of the subject matter disclosed herein. The kit can be packaged in a divided or undivided container such as a carton, bottle, ampule, tube, etc. The compositions disclosed herein can be packaged in a dry, lyophilized, or liquid form. The additional components provided can include a vehicle 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 an adverse reaction.

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

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

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

[0087] In accordance with longstanding patent law convention, the terms "a", "an", and "the" when used in this application, including the claims, mean "one or more". Thus, for example, a reference to "a subject" includes one or more subjects, such as multiple subjects, unless the context clearly dictates otherwise (e.g., multiple subjects).

[0088] Throughout this specification and the claims, the terms "comprise", "comprises", and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Similarly, the term "include" and its grammatical variations are intended to be non-limiting, such that a listing of items in a list is not to be construed as excluding other similar items that can be substituted for or added to the listed items.

[0089] For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, parameters, contents, characteristics and other numerical values used in this specification and the claims shall be understood to be modified in all instances by the term "about" even if the term "about" does not explicitly appear with the value, quantity 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 exact, but rather approximate and / or may be larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those of ordinary skill in the art in light of the desired characteristics of the subject matter disclosed herein. For example, the term "about", when referring to a value, can be intended to encompass variations 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% from a particular quantity. This is because such variations are suitable for practicing the disclosed methods or using the disclosed compositions.

[0090] Furthermore, when the term "about" is used with respect to one or more numbers or numerical ranges, it should be understood to refer to all numbers within and including such numbers, modifying the range by extending it above and below the numerical values recited for its boundaries. The recitation of numerical ranges by endpoints includes all numbers, e.g., all integers and their fractions included within that range (e.g., the recitation from 1 to 5 includes 1, 2, 3, 4, and 5 as well as their fractions, 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 the present 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 variations, modifications, and alterations can be used without departing from the scope of the subject matter disclosed herein. The following synthesis descriptions and specific examples are intended for illustrative purposes only and should not be construed as limiting the compounds of the present disclosure in any way to other methods of making them.

[0092] (Example 1) Combination Therapy 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 with subcutaneous CT26 tumors were treated with C. novyi-NT spores by intravenous injection and / or the indicated antibodies by intraperitoneal injection. The animals were followed for up to 3 weeks and tumor volumes were determined. The CTLA-4 antibody and the PD-1 antibody, when combined, 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 with subcutaneous 4T1 tumors were treated with C. novyi-NT spores by intravenous injection and / or the indicated antibodies by intraperitoneal injection. The animals were followed for over 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 and serve as a good model of human disease. In mice with 4T1 tumors, especially when the tumor is 200 mm3 When larger, cures were rarely reported. Here, even though survival was significantly increased by using the antibody combination, presumably due to improved immunological control of micrometastases (Figure 2B), it was shown that neither individual antibodies nor the antibody combination was sufficient to eradicate large primary 4T1 tumors (Figure 2A). Importantly, in addition to the combined antibodies, when spores of C. novyi-NT, an attenuated anaerobic tumor-targeting bacterial strain, were administered intravenously (IV), a significant proportion of large primary tumors were eradicated (Figure 2A), resulting not only in extended survival but also in the extremely rare cure in this tumor model (Figure 2B).

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

[0097] (Example 2) Clostridium novyi-NT induces an antitumor response Summary Species of the Clostridium bacterium are known for their ability to lyse tumor cells that grow in a hypoxic environment. Here, a attenuated strain of Clostridium novyi (C. novyi-NT) has been shown to induce a microscopically distinct tumor localization 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. Therefore, naturally occurring canine tumors have been used as a bridge to human trials. Canine tumors are more similar to human tumors because they occur in animals with a heterogeneous genetic background, are host-derived, and result from spontaneous rather than engineered mutations. Intratumoral injection of C. novyi-NT spores was well tolerated in companion animals of dogs with spontaneous solid tumors, and the most common toxicities were the expected symptoms associated with bacterial infection. An objective response was observed in 6 out of 16 dogs (37.5%), with 3 achieving complete response and 3 achieving partial response. Based on these promising results, human patients with progressive leiomyosarcoma were treated with intratumoral injection of C. novyi-NT spores. This treatment resulted in a dramatic response, significantly reducing the tumor both within and around the bone. Overall, these results demonstrate that C. novyi-NT can act as a controlled agent to precisely eradicate neoplastic tissue, suggesting that further clinical trials of this agent in selected patients are warranted.

[0098] Introduction Therapies that specifically target and destroy cancer must recognize the differences between normal and malignant tissues (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 changes and pathophysiological changes that result in heterogeneous tumors with regions 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 agents rely on tumor vasculature for delivery and are therefore less effective in hypoxic tumor regions with insufficient angiogenesis (Wilson and Hay (2011) Nat. Rev. Cancer 11: 393 - 410). Furthermore, since oxygen is a necessary effector of radiation-induced cell death, radiotherapy cannot kill hypoxic cells (Horsman et al., (2012) Nat. Rev. Clin. Oncol. 9: 674 - 687). For these important reasons, locally advanced tumors that are unresectable are particularly difficult to treat with conventional therapies.

[0099] Tumors are composed of necrotic, hypoxic, and fully oxygenated regions. Hypoxic tumor regions are resistant to systemic anticancer agents and radiotherapy. However, hypoxic tumor regions provide a fertile soil for the growth of anaerobic bacteria. Thus, the hypoxic regions of tumors provide a perfect niche for the growth of anaerobic bacteria. In principle, this offers an opportunity for the precise eradication of advanced local tumors while sparing the surrounding well-vascularized normoxic tissues. Since Coley's first study over 100 years ago of treating cancer patients with Streptococcus pyogenes, various anaerobic bacteria have been investigated for this purpose (Coley (1910) Proc. Roy. Soc. Med. 3: 1-48; Coley (1991) Clin. Orthop. Relat. Res. 3-11). This initial research was not able to produce a viable anticancer agent because, in part, it had low reproducibility and unacceptable toxicity. More recent studies have included Salmonella typhimurium, and other attenuated strains (Forbes (2010) Nat. Rev. Cancer 10: 785-794; Wei et al., (2008) Cancer Lett. 259: 16-27). However, in phase I clinical trials of S. typhimurium in both canine and human patients, it was demonstrated that the bacteria could be safely administered and target tumors, but 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 efficacy using S. typhimurium treatment, genetically modified strains incorporating cytosine deaminase that converts systemically administered 5-fluorocytosine to 5-fluorouracil 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 motile spore-forming bacterium that is exquisitely sensitive to oxygen. A derivative of the wild-type strain, called C. novyi-NT, was created through the 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 C. novyi-NT spores injected intravenously into mice and rabbits bearing transplanted syngeneic tumors resulted in localized tumor necrosis, a strong inflammatory response, and complete responses in 25-30% of the treated animals (Agrawal et al., (2004) Proc. Natl. Acad. Sci. USA 101:15172-15177). Based on these data, intravenous injection of C. novyi-NT spores was evaluated in naturally occurring canine tumors (Krick et al., (2012) Amer. J. Vet. Res. 73:112-118). However, complete responses were not observed at doses that showed acceptable toxicity.

[0101] The surrounding normal tissues were left untreated and localized to the mouse tumors. Considering the remarkable ability of C. novyi-NT spores injected into the veins that germinate and destroy within them, it was hypothesized that direct intratumoral injection of spores into solid tumors might be more advantageous than intravenous administration. One problem encountered with systemic injection of spores is the low percentage of spores actually delivered to the tumor (Diaz et al., (2005) Toxicol. Sci. 88:562-575). This problem is compounded in large animals and human patients with relatively large blood volumes and relatively small tumors compared to mice. Using intratumoral injection, it is possible to overcome this problem by directly accumulating many more spores, even by orders of magnitude, into the target tumor. Furthermore, intratumoral injection of spores may also be more advantageous than other conventional local treatments such as surgery and radiotherapy. Theoretically, with C. novyi-NT therapy, precise and delicate excision of neoplastic cells from the tumor would be possible without the need to excise the surrounding normal tissues. Intratumoral injection of C. novyi-NT spores would also be able to 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 C. novyi-NT spores injected into tumors were investigated in preclinical animal models as well as in comparative trials in dogs with naturally occurring cancer. The first human data from patients treated with C. novyi-NT spores injected into tumors are also reported.

[0102] Materials and Methods Study Design: To demonstrate C. novyi-NT-induced infection that is specifically and accurately localized to tumor lesions, a preclinical proof-of-concept study was conducted using a rat syngeneic F98 glioma model. The treatment effect was evaluated using luciferase activity and Kaplan-Meier survival curves. A comparative study in companion dogs with naturally occurring solid tumors was used to bridge the preclinical and human trials. The experimental unit was one study dog, and each dog received treatment up to 4 cycles. Placebo control, blinding, or randomization was not used in this study. A formal deductive statistical hypothesis was not planned for this comparative study. Descriptive summary statistics and analysis were provided post hoc. The human clinical trial is an ongoing, non-blinded, non-randomized, multi-site Phase I trial using the standard “3 + 3” dose escalation. The trial 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) characterize the preliminary antitumor activity of both the injected tumor and the overall response, (iii) examine the disposition of circulating C. novyi-NT spores, and (iv) measure the host immune and inflammatory responses associated with C. novyi-NT treatment.

[0103] Cell Lines and Tissue Culture: Rat F98 glioma cell lines transfected with a luciferase construct via lentivirus were maintained in Dulbecco's Modified Eagle 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 (weighing 100 - 150 grams) were purchased from the National Cancer Institute. In the transplantation procedure, female F344 Fischer rats were anesthetized by intraperitoneal injection of ketamine hydrochloride (75 mg / kg; 100 mg / mL ketamine HCl; Abbot Laboratories), xylazine (7.5 mg / kg; 100 mg / mL Xyla-ject; Phoenix Pharmaceutical, Burlingame, CA), and ethanol (14.25%). F98 glioma cells transfected with a luciferase construct via lentivirus (2×10 4) was stereotactically implanted into the right frontal lobe located 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 evaluated by intraperitoneal injection of 8 mg / rat D - luciferin potassium salt on day 12 after tumor cell implantation using a Xenogen instrument. Subsequently, three million C. novyi - NT spores generated as previously described (Dang et al., (2004) Proc. Natl. Acad. Sci. U.S.A. 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 of intraperitoneal dexamethasone for the first 2 days to minimize the risk of postoperative edema, which closely mimics 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 of intraperitoneal dexamethasone for the first 2 days. Animals were observed daily for any signs of deterioration, lethargy, neurotoxicity, or pain according to the Johns Hopkins Animal Care Guidelines. In case of signs of distress, supportive treatment with fluid replacement and doxycycline (15 mg / kg loading dose intraperitoneally followed by 10 mg / kg every 12 hours as maintenance) was initiated and continued for 7 days. If symptoms persisted and / or the animals became debilitated, moribund animals were euthanized. The efficacy of C. novyi - NT spores injected into the tumor was evaluated by Kaplan - Meier survival curves and the remaining tumor tissue mass (tumor burden) in the brain portion. For the latter, the brain was collected post - mortem 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 count graphs were generated and analyzed using the Mantel-Cox test and the Mann-Whitney test, respectively, with GraphPad Prism v.5.00 (GraphPad Software, San Diego, CA).

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

[0107] Array determination and bioinformatics analysis: Genome purification, library construction, exome capture, next-generation sequencing, and bioinformatics 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 an Agilent Canine All Exon kit according to the manufacturer's instructions (Agilent, Santa Clara, CA). Paired-end sequencing, which generates 100 bases from each end of the fragment, 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 native filters of Illumina's BaseCall software. Next, the ELAND algorithm of CASAVA 1.7 software (Illumina, San Diego, CA) was applied to identify point mutations as well as 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. U.S.A. 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 yields of mature spores. Mature spores were purified through two consecutive continuous Percoll gradients followed by four washes and resuspension in PBS. Sterility testing of the final product was performed by culturing the product in soybean casein digest medium and thioglycollate medium according to FDA 21 CFR610.12 guidelines (Nelson Laboratories, Salt Lake City, UT). The germination efficiency assay was performed on Brucella agar containing 5% horse blood under anaerobic conditions to ensure that the spores met pre-set viability criteria. The spores were filled into sterile 1.8 mL cryovials with O-ring sealed screw caps (Simport, Beloeil, Canada) at a volume of 1000 μL and a concentration of 1×10 9 spores / mL. The C. novyi-NT cryovials were stored at 2-8°C. For dosing, a 0.4 mL aliquot of the stored spore solution was filled into a 0.5 mL cryovial. After dosing, the cryovials and unused C. novyi-NT spores were discarded according to applicable regulations for the disposal of biosafety level 2 materials.

[0109] Prior to intratumoral injection, the spores were resuspended by vortexing three times for 10 seconds each at maximum speed while mixing to ensure thorough resuspension before drawing into a 1 mL syringe. The injection site was prepared aseptically. If available, ultrasound or computed tomography (CT) was used to identify necrotic regions of the tumor. If no necrotic regions were identified, the injection was directed towards the center of the tumor. The needle was inserted once into the pre-specified area and 100 μL of the spore suspension (1×10 8C. novyi-NT spores were dispensed at equal pressure. The injection needle was removed slowly and the injection site was sterilized.

[0110] Design and conduct of comparative dog studies: All animal studies involving dogs were conducted in accordance with applicable local, state, national, and international animal rights protection 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 Clinical Investigation (ACI, Washington, DC) Animal Experimentation Committee to ensure the ethical conduct of dogs enrolled in the study.

[0111] Client-owned dogs with spontaneous tumors received intratumoral C. novyi-NT spores up to 4 cycles. A cycle consisted of a single intratumoral injection of 1 × 10 8 C. novyi-NT spores (in 100 μL PBS). Cycles of intratumoral C. novyi-NT spores were typically at 1-week intervals. Neither placebo controls nor masking were used. Dogs were followed for 90 days, with extended follow-up for disease progression and survival guaranteed if 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 supervised by board-certified veterinary oncologists. Enrollment was offered to client-owned dogs with spontaneously occurring solid tumors, with preference given to dogs with soft tissue sarcomas that had failed standard therapy or whose owner(s) declined such therapy. Participation was restricted to dogs with solitary tumors having a longest diameter of 1 to 7 centimeters. Dogs with tumors located in areas where abscess development was considered to be terminal (e.g., nasal tumors that had spread intracranially or significant pulmonary metastatic disease) were excluded from the study. Dogs with evidence of active bacterial infection requiring systemic antibiotic therapy within 7 days of C. novyi-NT spore treatment or cancer therapy (chemotherapy, radiation therapy, and immunotherapy) within 21 days were ineligible. Dogs were required to have a performance score of 0 or 1 (Table 1) and to be available for the duration of the entire study enrollment period. Concurrent use of anticancer agents and participation in other clinical trials were prohibited.

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

[0114] The test evaluations were conducted as described in Table 2. The prescreening evaluations were conducted 1 to 14 days prior to the first cycle of intratumoral C. novyi-NT spores. Dogs were monitored regularly during the study, both on an inpatient and outpatient basis. Laboratory samples were collected as specified in Table 2 and included complete blood counts, serum biochemistry, prothrombin time, partial thromboplastin time, and urinalysis. Imaging was performed at screening and included local CT, chest radiography, and abdominal ultrasonography. Additional imaging was performed during the study at the discretion of the investigator.

[0115] If possible, using the terms of the Veterinary Dictionary for Drug Related Affairs (VeDDRA) rev. 4 (European Medicines Agency (2012) Combined VeDDRA list of clinical terms for reporting suspected adverse reactions in animals and humans to veterinary medicinal products), the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.0 (Veterinary co-operative oncology group (2004) Vet. Comp. Oncol. 2:195-213) was used to evaluate adverse events. The terms for adverse events related to C. novyi-NT germination (target lesion responses) are defined in Table 3. Clinical findings without appropriate VeDDRA or target lesion response terms were classified separately as un-coded signs (Table 4). The relationship to C. novyi-NT therapy was determined by the reporting investigator.

[0116] The longest diameter tumor measurements of the 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. The best overall target response was evaluated at the 21-day study visit or later, and complete response (CR) was defined as the complete disappearance of the target lesions, partial response (PR) was defined as at least a 30% decrease in the longest diameter of the target lesions, and progressive target disease (PD) was defined as at least a 20% increase in the longest diameter of the target lesions or the appearance of new non-target lesions. Stable disease (SD) was defined as an inadequate decrease or increase in the longest diameter of the target lesions and was designated as CR, PR, or PD. In the case of C. novyi-NT-related abscesses, medical or surgical debridement of necrotic tissue was left to the discretion of the investigator.

[0117] The evaluation of surgical samples and biopsies was performed by board-certified veterinary pathologists. Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Slides stained with H&E and / or Gram stain slides were prepared for evaluation according to standard procedure guidelines. For immunohistochemistry (IHC), formalin-fixed, paraffin-embedded tumor tissue was sectioned at 5 μm, deparaffinized in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed by heating the slides in unmasking solution for 10 minutes (catalog number H-3300, Vector Laboratories, Burlingame, CA). All slides were then incubated in 10% blocking serum from the animal species, and secondary antibodies were made in PBS at room temperature for 10 minutes. Primary antibodies S100 (catalog number Z0311, DAKO, Carpinteria, CA) and anti-smooth muscle actin (catalog number M0851, DAKO, Carpinteria, CA) were used at 1:100 for 60 minutes at room temperature (Duke et al., (2014) Vet. Pathol.; Zarfoss et al., (2007) Vet. Pathol. 44:276-284). Secondary antibodies labeled with DAB (catalog numbers BA-1000 and BA-2000, Vector Laboratories, Burlingame, CA) were used at 1:500 for 30 minutes at room temperature. Sections were incubated with ABC reagent (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. Tumor malignancy was assigned to each 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 C. novyi-NT Spores Injected into Tumors: An open-label, non-randomized, multi-center Phase I safety trial of a single intratumoral injection of C. novyi-NT spores is currently ongoing in patients with treatment-resistant solid tumors. The clinical trial protocol was reviewed and approved by the Institutional Review Board (IRB) of each participating facility, and all regulatory steps were carried out under the guidance of the Food and Drug Administration (FDA) (http: / / www.clinicaltrials.gov; NCT01924689). All patients were required to sign a written informed consent form (ICF) before being enrolled in the study.

[0119] The primary objectives of this Phase I trial were to determine the safety profile, dose-limiting toxicity, and maximum tolerated dose of C. novyi-NT injected into tumors. Additionally, the antitumor activity of intratumoral C. novyi-NT was investigated.

[0120] Preparation and Intratumoral Injection of C. novyi-NT Spores in the Phase I Trial: C. novyi-NT spores were manufactured and formulated by Omnia Biologics, Inc. (Rockville, MD). The clinical supply of C. novyi-NT spores was filled into single-use 2 mL sterile, pyrogen-free Type I borosilicate glass vials with rubber stoppers and tamper-evident caps, suspended in 1.0 mL of sterile phosphate-buffered saline (PBS) at a concentration of 8.52×10 8 spores / mL. The vials were stored at 2 - 8°C in a controlled temperature environment under constant temperature monitoring.

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

[0122] Design and conduct of human clinical trial: This trial was conducted using a standard 3 + 3 dose-escalation design. To enroll in the trial, patients must have been diagnosed with a target tumor that is a progressive solid tumor lesion, clearly identifiable by palpation and ultrasonography or fluoroscopically guided, and not amenable to percutaneous injection of C. novyi-NT spores. Furthermore, the target lesion must be at least 1 cm in its longest diameter, measurable as defined by RECIST 1.1 criteria, and suitable for intratumoral injection of C. novyi-NT spores.

[0123] Eligibility criteria included a history of treatment-resistant solid tumor malignancies; at least 18 years of age; Eastern Cooperative Oncology Group (ECOG) performance status ≤2; ability to stay within the emergency treatment room for 45 minutes or less and the presence of a caregiver for 28 days after intratumoral injection. Exclusion criteria included pregnancy; primary brain malignancies or brain metastases; clinically significant ascites or clinical evidence or history of portal systemic hypertension or cirrhosis; Glasgow Coma Scale (GCS) <15; serum creatinine level >1.5 × upper limit of normal (ULN), chronic renal insufficiency 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, endocardial fluid leakage, circumferential pericardial effusion, or any effusion exceeding 1.0 cm at any location around the heart; the need for ongoing treatment with immunosuppressive agents; history of solid organ transplantation; and systemic or local infection.

[0124] Eligible patients were placed in the dosing cohort and registered. Patients remained hospitalized after C. novyi-NT spore injection and were observed for 8 days. Patients returned to the clinical facility for scheduled follow-up visits during which safety and efficacy evaluations were performed.

[0125] Clinical response and progression were evaluated using RECIST version 1.1. Objective response was measured by serial CT or MRI scans of the injected tumor as well as distant metastases (up to 5 lesions).

[0126] Public health implications of C. novyi-NT therapy: C. novyi is a spore-forming Gram-positive obligate anaerobic bacterium commonly found 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 pathogenicity (Dang et al., (2004) Proc. Natl. Acad. Sci. U.S.A. 98(26):15155-15160). In extensive preclinical evaluations of C. novyi-NT, germination of C. novyi-NT spores in non-tumor tissues could not be demonstrated (Diaz et al., (2005) Toxicol. Sci. 88:562-575). Furthermore, C. novyi-NT spores are resistant to oxygen, while proliferative C. novyi-NT is highly sensitive to oxygen (Diaz et al., (2005) Toxicol. Sci. 88:562-575). Therefore, proliferative C. novyi-NT cannot survive outside the hypoxic tumor microenvironment. Although the public health risk associated with the use of C. novyi-NT therapy is considered minimal, caution has been raised regarding the handling of C. novyi-NT and the disposal of C. novyi-NT contaminants. In dog comparative studies, protective gloves were worn when handling feces, urine, saliva, or tumor secretions from treated dogs; feces were placed in sealed plastic bags and disposed of with regular household trash; 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 of healthcare providers.

[0127] Results Intratumoral injection of C. novyi-NT spores specifically targets tumor tissue and extends the survival of rats: High-grade gliomas exhibit marked histopathological variability with extensive areas of hypoxia and necrosis. This tumor type generally does not metastasize, but 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 the infiltrative growth pattern that relentlessly leads to tumor recurrence. Therefore, gliomas were thought to represent a tumor type in which local injection of C. novyi-NT spores might be therapeutically useful. To evaluate this possibility, orthotopic transplantation of luciferase-expressing F98 rat glioma cells into 6-week-old F344 Fischer rats resulted in locally invasive tumors that rapidly became lethal (Figure 3A). Stereotactic intratumoral injection of C. novyi-NT spores into the tumors of these rats germinated within 24 hours, and luciferase activity, an indicator of tumor tissue mass, rapidly decreased within 48 hours (Figures 3B and 3C). C. novyi-NT germination was indicated by the appearance of the proliferative form of the bacteria. Notably, C. novyi-NT was precisely localized to the tumor, sparing adjacent normal cells by only a few microns (Figures 4A and 4B). Furthermore, these proliferative bacteria were specifically seen to grow within and sometimes destroy islands of microinvasive tumor cells embedded within normal brain parenchyma (Figures 4C and 4D). This bacterial treatment resulted in a significant survival advantage in this highly invasive rat model (Figure 3A, P value < 0.0001). Brain edema as a result of C. novyi-NT germination was common and medically managed. Abscess formation within the brain was not clearly observed in a syngeneic rat model with appropriate antibiotic use. However, abscess formation is a potential side effect of this treatment and may occur in human patients, and is thought to require neurosurgical abscess resection and drainage, which are standard clinical procedures. Nevertheless, considering the gloomy 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: often, the effects observed in humans are not reproduced in preclinical animal trials of anticancer agents. However, in companion animal dogs, therapeutics used clinically induce toxicities and effects similar to those seen in humans (Paoloni and Khanna (2008) Nat. Rev. Cancer 8:147-156). Investigational treatment trials in companion animal dogs can represent a critically important bridge between preclinical animal trials and human clinical trials. In particular, canine soft tissue sarcomas are common in many breeds of dogs and are an excellent model because they have clinical and histopathological features that closely resemble those of human soft tissue sarcomas (Paoloni and Khanna (2008) Nat. Rev. Cancer 8:147-156; Vail and MacEwen (2000) Cancer Invest. 18:781-792). Furthermore, because many soft tissue sarcomas are located superficially, rapid assessment and management of treatment-related abscess formation is possible.

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

[0130] Using stringent analysis criteria, 156 somatic mutations and 28 somatic copy number alterations were identified among 10 soft tissue sarcomas (Figure 11 and Table 5). The range of somatic mutations was 0 to 95, with an average of 16 per tumor. The mutation prevalence in soft tissue sarcomas was low, averaging 0.47 per Mb (range: 0.00 to 2.89 per Mb). Excluding one outlier sample with 95 somatic changes, the average mutation prevalence was 0.21 per Mb (range: 0.00 to 0.61 per Mb) (Figure 10), similar to the estimated 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 change was missense mutation, with the predominant ones being transitions from C to T (45.5%) and from G to A (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 neither amplifications nor deletions.

[0131] A single nucleotide substitution was identified in three tumor suppressor genes (NF1, MLL3, and PTCH1) that are frequently mutated in human tumors. Furthermore, MDM4, a cancer gene that has been 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 mutations in two tumors) and AIG1 (amplified in two tumors). Interestingly, mutations in ATP7B have also been found in human liposarcomas (Joseph et al., (2014) Gene Chromosome Canc. 53:15-24). Twenty-two of the 184 somatic changes in canine tumors occurred in genes that have already been shown to be mutated in human soft tissue sarcomas (Table 8). Since the analysis included several soft tissue sarcoma histiotypes, larger studies of soft tissue sarcomas in both species will be needed to determine whether these represent driver mutations indicating important and conserved tumorigenic pathways. Nevertheless, the genetic landscape of canine tumors was similar to that of human tumors in terms of the number of genetic changes and the range of mutations. Specifically, this rules out the possibility that canine tumors have a very large number of mutations that could potentially initiate an immune response more readily 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 spontaneous solid tumors was conducted (Table 9). Each dog received 1 × 10 8Received at least 1 cycle of C. novyi-NT spore treatment defined as single intratumoral injection of C. novyi-NT spores. Dogs were treated up to 4 cycles at 1-week intervals between cycles. Treated dogs were followed for at least 90 days after the first intratumoral injection.

[0133] Nine castrated males, six castrated females and one intact male were enrolled in the study (Table 2). The mean body 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 received 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, 6 peripheral nerve sheath tumors were available for immunohistochemistry (IHC). All 6 were positive for S100 and negative for smooth muscle actin, confirming the histiomorphic diagnosis. Seven of the tumors were grade I, five were grade II, and four were grade III. Eight dogs had previously received surgical treatment for their cancer.

[0134] All dogs received at least 1 cycle of treatment and 53 of 64 planned cycles were administered. Ten of 16 dogs, a majority, received the intended 4 cycles. In dogs showing early tumor response, toxicity, or progressive disease after the first cycle, subsequent cycles were stopped (Table 9). Generally, adverse events were of mild severity (>90% grade I or grade II) and were consistent with local infections 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 the inflammatory response at the injected target lesion site were observed in 14 of 16 dogs (87.5%) including tumor inflammation (12 / 14), tumor abscess (7 / 14), tumor pain (5 / 14), and tumor discharge (4 / 14) (Table 11).

[0135] Dogs were evaluated for best response on or after day 21 of the trial. Two of the 16 dogs, 04-R04 and 04-R08, could not be evaluated for response because the injected tumors were surgically resected before day 21. Dog 04-R04 had a humeral osteosarcoma that developed firm sprouts 2 days after the first intratumoral injection of C. novyi-NT. Due to the deep location of the tumor, amputation was performed on day 21 for abscess management. Dog 04-R08 had a peripheral nerve sheath tumor on the medial side of the hind paw and received 3 cycles of treatment before amputation on day 15 for management of the 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 to 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 response (04-R03), respectively. Abscess resolution occurred with surgical management in 3 of the 6 dogs that experienced an objective response. In these cases, debridement was performed an average of 22 days after the first cycle of treatment. In dog 04-R02, the tumor response was evaluated before the owner chose amputation for wound management. In dogs 04-R03 and 11-R02, the tumor response was evaluated after wound debridement. The dissected tissue was available for histopathological analysis in dogs 04-R02 and 04-R03, and these dogs showed extensive tumor necrosis and inflammation, and numerous gram-positive bacilli morphologically consistent with Clostridium spp. In dog 04-R02, no viable tumor cells were present at the tumor margin. In dog 04-R03, rare scattered tumor cells were observed.However, considering the active nature of C. novyi-NT-related abscess formation and subsequent immune infiltration and wound healing, it is difficult to speculate on the ultimate fate if debridement was not performed. Regardless of debridement, wound healing was uneventful and complete by 2 to 4 weeks. In addition to surgical management, 3 out of 6 dogs with an objective response received antibiotics (ampicillin, amoxicillin, and metronidazole) and analgesics (opioid, tramadol, and non-steroidal anti-inflammatory drugs) during the course of the trial. However, overt abscess formation was not always observed prior to the objective response. Dogs 04-R01 and 04-R06 received 4 cycles of treatment, and tumor inflammation was observed at the 21-day study visit, but abscess formation was not observed. Complete responses were noted in these 2 dogs at the 42-day (unscheduled visit) and 60-day study visits, respectively. 3 out of 6 dogs that experienced either a CR or PR had a long-term response (Figure 7). In the remaining 3 dogs, the median progression-free period 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 / effect profile of C. novyi-NT treatment in a comparative dog trial, combined with the results observed in rats, provided a rationale for the attempt of this treatment in humans. Therefore, a Phase I trial was initiated in human patients with solid tumors that are resistant to standard treatment or for which no standard treatment is available (NCT01924689). Here, we report on the first patient enrolled in this trial, a 53-year-old woman diagnosed with retroperitoneal leiomyosarcoma in August 2006. The patient had undergone several surgical resections and received multiple chemotherapy and radiotherapy treatments. However, the patient's disease had progressed, and metastatic lesions were present in the liver, lungs, peritoneum, and soft tissue in the right shoulder and adjacent right upper arm bone.

[0137] Planned starting dose 1×10 4Treatment was performed by injecting C. novyi-NT spores into the patient's metastatic right shoulder tumor with an 18-gauge multipronged needle (day 0). On day 1, the patient developed mild right shoulder pain that spread 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, the patient had a fever, and the maximum body temperature was 39.2°C. On day 3, the pain in the patient's right shoulder and scapula was difficult to control. The maximum body temperature was 37.8°C. A CT scan of the right upper extremity showed extensive tumor destruction with gas in the soft tissue and osseous components of the tumor (Figure 8A). The gas permeation pattern was consistent with extensive necrosis of the proximal humerus. CT-guided aspiration fluid from the patient's tumor revealed C. novyi-NT growth under anaerobic culture conditions. Next, the patient started antibiotics (piperacillin / tazobactam, metronidazole, and vancomycin), and the patient's fever subsequently subsided. On day 4, magnetic resonance imaging (MRI) of the right upper extremity showed a marked decrease in enhancement limited to the tumor mass compared to baseline (Figures 8B and 8C). Tumor-derived biopsy showed the absence of many gram-positive bacteria and viable tumor cells (Figure 9). At the time of biopsy, a percutaneous drain was placed into the tumor abscess to drain fluid and debris. The patient remained afebrile and the white blood cell count gradually normalized. The patient continued antibiotics and was hospitalized for intravenous analgesia until day 20 when switched to oral analgesics. The patient was discharged on metronidazole and doxycycline administered orally according to protocol. On day 29, follow-up MRI showed ongoing decrease in tumor enhancement (Figure 8D). On day 55, the patient presented with local 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 significantly improved the pain and increased the range of motion. Intraoperative culture revealed C. novyi-NT growth under anaerobic culture conditions. Histopathology showed extensive tumor necrosis and small foci of residual tumor cells.The patient has been continuously monitored and currently has an Eastern Cooperative Oncology Group scale (ECOG) performance status of 1 and no clinical signs of infection.

[0138] Discussion Most conventional anticancer therapies target the well-vascularized components of tumors. However, to cure this disease, all neoplastic cells must be destroyed, and any remaining cancer cells can potentially regenerate the tumor. This principle has been dramatically illustrated in recent studies using targeted anticancer agents. Although it is possible to induce significant remission, tumors almost always recur within months due to a small percentage (<0.0001%) of cells that have resistant 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 into the tumor provides a means to precisely eradicate neoplastic cells, in principle independent of tumor-specific genetic changes. In addition to directly killing tumor cells in its hypoxic environment, C. novyi-NT has been shown to induce a potent antitumor immune response in preclinical models, whether innate or adaptive (Agrawal et al., (2004) Proc. Natl. Acad. Proc. Natl. Acad. Sci. U.S.A. 101(42):15172-15177. Although there was no clear evidence of an acquired antitumor immune response in either human patients or companion animal dogs, the marked inflammatory response induced by intratumoral injection of C. novyi-NT spores provided clear evidence of an innate immune response. C. novyi-NT is highly sensitive to oxygen and has not been shown to germinate in the normoxic regions of tumors, so it is reasonable that immunity (either innate or acquired) played a role in dogs that achieved durable complete responses. Furthermore, the first human experience with intratumoral injection of C. novyi-NT spores resulted in a rapid and robust local antitumor response. In this case, the proximity of underlying bone may have contributed to a pathologic fracture that ultimately required surgery. However, patient selection may minimize the risk of similar complications in the future. It is important to note that this result occurred with only a small fraction of the dose used to treat dogs or rats (10,000 spores). As the Phase I trial proceeds, it will be interesting to see whether higher doses affect distant metastases either through diffusion of spores that are released directly from the local site into the circulation or through host-mediated immunity.

[0140] Comparative studies of dogs with spontaneous tumors should be incorporated into discussions about the translatability of cancer research in experimental animal models (Vail and MacEwen (2000) Cancer Invest. 18:781-792). Demonstration of therapeutic efficacy in spontaneous canine tumors can strongly complement studies of transplanted or genetically induced tumors in preclinical animal models. This complementarity is enhanced by the genetic similarities between human and canine tumors described herein. Together, such similarities can provide a compelling theoretical basis for guiding research in humans, particularly for new forms of therapy associated with significant and potent toxicity, such as novel forms of therapy using C. novyi-NT and other biological agents.

[0141] The next steps in this series of studies are clear. First, it will be important to further characterize the safety and efficacy of intratumoral C. novyi-NT spore treatment. The effects of C. novyi-NT spores, at least when administered systemically, are dramatically enhanced by combination 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. U.S.A. 100(25):15083-15088). Since the mechanism by which C. novyi-NT kills tumor cells does not overlap with the mechanisms of action of other forms of therapy, a multi-model approach seems particularly attractive (Dang et al., (2004) Cancer Bio. Ther. 3:326-337). Finally, it will also be very interesting 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. U.S.A 101(42):15172-15177).

[0142] In some embodiments, the subject matter disclosed herein uses an attenuated strain of the anaerobic spore-forming bacterium Clostridium novyi (C. novyi-NT) and demonstrates a precise and robust reproducible antitumor response when C. novyi-NT spores are injected intratumorally in rats, pet dogs, and humans. These results indicate that intratumoral C. novyi-NT spores can be used as a therapeutic agent for patients with locally advanced inoperable 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 The publications, patent applications, patents, and other references mentioned in this specification are all indicative of the level of those skilled in the art to which the disclosed subject matter pertains. The publications, patent applications, patents, and other references are each incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. It will be understood that although several patent applications, patents, and other references are mentioned in this specification, such mention does not admit 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 appreciated by those skilled in the art that certain changes and modifications within the scope of the appended claims are practicable.

Claims

[Claim 1] The invention described in this specification.

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