WO2016014899A1 - Flagellin derivatives and uses - Google Patents

Abstract

The present invention relates, in part, to compositions comprising improved flagellin derived constructs and methods of using the same in the treatment of various diseases and conditions.

Description

FLAGELLIN DERIVATIVES AND USES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/029,193, filed July 25, 2014, the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to, inter alia, methods and compositions that are useful for the treatment, prevention, and/or diagnosis, of various diseases, including cancer.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: CLE_015PC_SeqList_ST25.txt; date recorded: July 21 , 2015; file size: 27.2 KB).

BACKGROUND

[0004] Toll-like receptors (TLRs) are type I membrane glycoproteins that are key receptors in innate immunity. The ten TLRs known in humans recognize different microbial antigens, and when activated by ligand binding, mediate rapid production of cytokines and chemokines. In addition to their role in host defense, TLRs play a role in cancer progression and development as well as in cell protection.

[0005] TLR5 binds flagellin, a globular protein that arranges itself in a hollow cylinder to form the filament in bacterial flagella. Binding of flagellin to TLR5 initiates a cascade of pro-inflammatory molecules, notably NF-KB and its targets. TLR5 agonists derived from flagellin have been developed as therapies for various diseases. However, these molecules may suffer from specific limitations, including for example, unsatisfactory binding and signaling. Additionally, many possible hosts already produce anti-flagellin antibodies that also target the TLR5 agonist derivatives, thereby clearing the therapeutics from the body and limiting their efficacy. Moreover, as intrinsically immunogenic bacterial proteins, flagellin derivatives may possess disadvantageous antigenicity and immunogenicity, and therefore warrant improvement.

SUMMARY OF THE INVENTION

[0006] Accordingly, the present invention provides flagellin derivatives that overcome limitations observed among this group of biologies.

[0007] The present invention is based, in part, on the discovery that minimized constructs of flagellin can exhibit reduced immunogenicity and improved pharmacokinetics while still retaining the ability to activate TLR5 signaling. In various embodiments, the flagellin construct is derived from an extremophile. In an embodiment, the extremophile is a thermophile, such as a hyperthermophile. In various embodiments, the flagellin construct may be derived from organisms not found among, and not closely related to, human pathogens or normal microflora, for example, Firmicutes, Thermotogae, and Aquificae. In another embodiment, the flagellin construct is derived from a microorganism that is well tolerated by humans. For example, the microorganism is from the human microflora, such as Lactobacillus.

[0008] In various embodiments, the flagellin derivative comprises various modifications compared to the native flagellin from which it is derived. In some embodiments, the flagellin derivative comprises a truncation in one or more domains. In an embodiment, the flagellin derivative comprises a deletion in the N-terminal domain. In a further embodiment, the flagellin derivative comprises a deletion in the NDO domain. In yet a further embodiment, the flagellin derivative comprises a deletion of the entire NDO domain. In a further embodiment, the flagellin derivative comprises a deletion in a C-terminal domain. In yet another embodiment, the flagellin derivative comprises a deletion in the CDO domain. In yet another embodiment, the flagellin derivative comprises a deletion of the entire CDO domain. In other embodiments, the flagellin derivative lacks both the NDO and CDO domains. In still further embodiments, the flagellin derivative comprises a ND1 and CD1 domain. In other embodiments, the flagellin derivative comprises a NDO, ND1 , CDO, and CD1 domain.

[0009] In one aspect, the invention provides a flagellin derivative that retains the ability to activate TLR5 signaling. In a further embodiment, the flagellin derivative comprises mutations that decrease the antigenicity and immunogenicity of the construct. In a further embodiment, the flagellin derivative is not recognized by flagellin (FliC) neutralizing antibodies. In yet a further embodiment, the flagellin derivative activates TLR5 signaling at a level that is the same as or similar to that of a full-length flagellin and/or CBLB502. In a further embodiment, the flagellin derivative demonstrates improved pharmacokinetics compared with a full length flagellin and/or CBLB502. In yet a further embodiment, the flagellin derivative demonstrates increased retention in the host.

[0010] In some embodiments, the flagellin derivative comprises a tag. In yet a further embodiment, the tag is attached to the N-terminus of the flagellin derivative. In yet another embodiment, the tag is attached to the C- terminus of the flagellin derivative.

[0011] In some embodiments, the flagellin derivative comprises a flexible linker. In a further embodiment, the flexible linker comprises SEQ ID NO: 16. In yet a further embodiment, the flexible linker comprises SEQ ID NO: 17.

[0012] In some embodiments, the flagellin derivative comprises any one of the polypeptides having the sequence of SEQ ID NOs: 18-27.

[0013] In some embodiments, the flagellin derivative activates TLR5 signaling. In a further embodiment, the flagellin derivative induces expression of NF-κΒ. In yet a further embodiment, the minimized flagellin derivative induces expression of one or more of cytokines. In yet a further embodiment, the cytokines are selected from IL- 6, IL-12, IL-8, keratinocyte chemoattractant (KC), IL-10, G-CSF, MCP-1 , TNF-a, MIG, and MIP-2.

[0014] In one aspect, the invention provides a pharmaceutical composition comprising the flagellin derivative of the invention with a pharmaceutically accepted carrier. [0015] In one aspect, the invention provides a method of stimulating TLR5 signaling comprising administering a flagellin derivative of the invention to a subject in need thereof. In some embodiments, the subject has cancer. In a further embodiment, the tumor expresses TLR5. In a further embodiment, the tumor does not express TLR5. In yet a further embodiment, the cancer is selected from breast cancer, lung cancer, colon cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, promyelocytic leukemia, astrocytoma, neuroblastoma, glioma, schwannomas, fibrosarcoma, rhabdomyoscarcoma, osteosarcoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, and cancers of the gastrointestinal tract or the abdominopelvic cavity.

[0016] In some embodiments, the subject suffers from radiation-induced damage. In a further embodiment, the subject has been subjected to a lethal dose of radiation. In yet a further embodiment, the subject is undergoing radiation treatment. In another embodiment, the flagellin derivative is administered prior to exposure to radiation. In yet another embodiment, the flagellin derivative is administered during exposure to radiation. In yet another embodiment, the flagellin derivative is administered after exposure to radiation.

[0017] In some embodiments, the subject suffers from reperfusion injury. In a further embodiment, the reperfusion is caused by an injury. In a further embodiment, the injury is ischemia or hypoxia. In a further embodiment, the flagellin derivative is administered prior to the influx of oxygen. In a further embodiment, the flagellin derivative is administered during the influx of oxygen. In a further embodiment, the flagellin derivative is administered after the influx of oxygen.

[0018] In various embodiments, the flagellin derivative is administered in conjunction with other therapeutics and/or treatments. In a further embodiment, the flagellin derivative is administered in conjunction with chemotherapy. In a further embodiment, the flagellin derivative is administered with radiation treatment. In a further embodiment, the flagellin derivative is administered in conjunction with an antioxidant. In a further embodiment, the flagellin derivative is administered in conjunction with amifostine and/or vitamin E. In some embodiments, the flagellin derivative is administered prior to administration of other therapeutics and/or treatments. In further embodiments, the flagellin derivative is administered at the same time as other therapeutics and/or treatments. In yet further embodiments, the flagellin derivative is administered after administration of other therapeutics and/or treatments.

[0019] In one aspect, the invention provides a method of treating or preventing cancer comprising administering a flagellin derivative of the invention to a subject in need thereof.

[0020] In one aspect, the invention provides a method of treating or preventing radiation-induced damage comprising administering a flagellin derivative of the invention to a subject in need thereof. In one aspect, the invention provides a method of treating or preventing acute radiation syndrome (ARS) comprising administering a flagellin derivative of the invention to a subject in need thereof.

[0021] In one aspect, the invention provides a method of treating or preventing reperfusion injury comprising administering a flagellin derivative of the invention to a subject in need thereof.

[0022] The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

BRIEF DESCRIPTION OF THE FIGURES

[0023] Figures 1A and 1 B show exemplary flagellin derivatives of the present invention. Figure 1A shows a schematic of Tpe275 derived from Thermotoga petrophiia (Tpe), Chy270 derived from Carboxydothermus hydrogenoformans (Chy), and Lru283 derived from Lactobacillus ruminis (Lru), all of which includes the ND0, ND1 , CD0, and CD1 domains. Figure 1 B shows a schematic of Chy162w and Tpe159w, which include the ND1 and CD1 domains.

[0024] Figure 2 shows a SDS-PAGE gel of various flagellin derivatives purified by metal affinity chromatography using either the Pierce HisPur Co++ column or the Sigma His-Select plate.

[0025] Figures 3A-3D show the activity of various flagellin derivatives in activating a NF-κΒ regulated lacZ reporter gene in vitro. The assays are carried out in the presence of either a non-neutralizing anti-CBLB502 antibody (Figure 3A), a neutralizing anti-CBLB502 mAb (Figure 3B), or neutralizing human sera #012 (Figure 3C) or sera #004 (Figure 3D).

[0026] Figures 4A-4E show the activity of various flagellin derivatives in activating a NF-κΒ regulated lacZ reporter gene in vitro in the presence of CBLB502 boosted human sera.

[0027] Figures 5A-5E show the activity of various flagellin derivatives in activating a NF-κΒ regulated lacZ reporter gene in vitro in the presence of pre-immune normal human sera.

[0028] Figures 6A-6D show exemplary Chy162w variants which include the ND1 and CD1 domains.

[0029] Figure 7 shows a SDS-PAGE gel of various flagellin derivatives purified by metal affinity chromatography using Sigma His-Select iLAP-5 column.

[0030] Figures 8A-8F show the activity of various flagellin derivatives in activating a NF-κΒ regulated lacZ reporter gene in vitro.

[0031] Figures 9A-9B show SDS-PAGE gel of different fractions of purified flagellin derivatives. [0032] Figures 10A-10D show the activity of different fractions of purified flagellin derivatives in activating a NF-KB regulated lacZ reporter gene in vitro.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based, in part, on the surprising discovery that minimized flagellin constructs derived from microorganisms such as, for example, thermophiles, exhibit improved pharmacologically relevant properties. By way of non-limiting examples, these flagellin constructs show altered antigenicity and immunogenicity relative to the native flagellin from which the constructs are derived from as well as from CBLB502, a flagellin construct derived from Salmonella. CBLB502 is described, for example, in U.S. Patent No. 8,287,882, which is incorporated herein in its entirety. The flagellin constructs also retain the ability to active TLR5 signaling at levels the same as, or similar to, for example, CBLB502.

Flagellin Derivatives

[0034] Flagellin and previously described variants (e.g. flagellin or "FliC") suffer from high antigenicity and immunogenicity in large part, without wishing to be bound by theory, because they are intrinsically immunogenic bacterial proteins. A practical limitation in preexisting flagellin constructs is that many subjects have high titers of pre-existing antibodies capable of neutralizing the TLR5-stimulating activity of these constructs. These individuals would be desensitized (or completely resistant) to flagellin-derived treatment, sometimes even in case of single-injections and, without wishing to be bound by theory, more likely upon recurrent treatment. Moreover, the titer of such pre-existing antibodies, even if initially present at lower levels, may be rapidly boosted by a single flagellin-derived injection thereby compromising even a larger group of individuals for the purpose of multi- dose regimen as projected for medical applications. The widespread preexistence of anti-FliC antibodies (including neutralizing Abs) in a population likely reflects humanity's life-long exposure to numerous species of flagellated enterobacteria (e.g. Salmonella spp., E. coli) colonizing (and infecting) the human body.

[0035] The present invention is based, in part, on the discovery that flagellin constructs derived from microorganisms which have either none or very little contact with human exhibit reduced immunogenicity while still retaining the ability to active TLR5 signaling. These flagellin derivatives are capable of activating TLR5 signaling at levels that is the same as, or similar to, for example, CBLB502. Alternatively and in addition, it was discovered that flagellin constructs derived from microorganisms that are well tolerated by humans, such as those microorganisms which are part of the human microflora, also exhibit improved immunogenicity and TLR5 activation activity. The reduced immunogenicity allows the construct to persist in the host as long as or even longer than, for example, CBLB502.

[0036] Accordingly, in various embodiments, the present invention provides flagellin derivatives and compositions comprising the same. In some embodiments, the present invention provides for flagellin constructs that have (1) improved pharmacological properties, including reduced antigenicity and immunogenicity, which, for example, allow for use in wide variety of disease states and patient types and/or (2) improved functional properties which, for example, allow for improved medical effects. [0037] The flagellin construct may be derived from microorganisms that have little or no contact with humans. For example, the flagellin construct may be derived from a microorganism other than a bacteria from the phylum Proteobacteria. In one embodiment, the flagellin construct is derived from a microorganism other than an enterobacteria such as Escherichia or Salmonella (e.g., S. typhimurium and S. Dublin).

[0038] In some embodiments, the flagellin construct is derived from an extremophile. Exemplary extremophile includes, for example, acidophile, alkaliphile, anaerobe, cryptoendolith, halophile, thermophile, hyperthermophile, hypolith, lithoautotroph, metallotolerant microorganisms, oligotroph, osmophile, piezophile, polyextremophile, psychrophile, radioresistant microorganisms, thermoacidophile, and xerophile.

[0039] In an embodiment, the flagellin construct is derived from a thermophilic or hyperthemophilic bacteria, including but not limited to members of Thermotogae, such as Thermotoga petrophila, Thermotoga elfeii, Thermotoga hypogea, Thermotoga maritima, Thermotoga neapolitana, Thermotoga subterranean, and Thermotoga thermarum. In one embodiment, the thermophilic bacterium is Thermotoga petrophila. Other thermophilic bacteria such as Aquificae (e.g., Aquifex aeoiicus) or Fimicutes (e.g., those from the classes of Bacilli, Clostridia, and Mollicutes) are also contemplated. In one embodiment, the flagellin construct is derived from Carboxydothermus hydrogenoformans.

[0040] In other embodiments, the flagellin construct is derived from an obligate anaerobe. For example, the obligate anaerobe may be a chemotroph.

[0041] In yet other embodiments, the microorganism may be part of the microflora tolerated by humans, such as Lactobacillus. Exemplary Lactobacillus species include, but are not limited to, L. acetotolerans, L. acidifarinae, L. acidipiscism L. acidophilusm L. agiiis, L. aigidus, L. aiimentarius, L. amyioiyticus, L. amyiophiius ,L. amylotrophicus, L. amylovorusm L. animalis, L. antri, L. apodemi, L. aviariusm L. bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L. catenaformis, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatusmm L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. dextrinicusm, L. diolivorans, L. equi, L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. graminis, L. hammesii, L. hamsteri, L. harbinensis, L. hayakitensis, L. helveticus, L. hilgardii, L. homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchii, L. kitasatonis, L. kunkeei,, L. leichmannii, L. lindneri, L. malefermentans, L. mali, L. manihotivorans, L. mindensis, L. mucosae, L. murinus, L. nagelii, L. namurensis, L. nantensis, L. oligofermentans, L. oris, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei , L. paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L. protectus, L. psittaci, L. rennini, L. reuteri, L. rhamnosus, L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L. sharpeae, L. siliginis, L. spicheri, L. suebicus, L. thailandensis, L. ultunensis, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. Mini, L. vitulinus, L. zeae, and L. zymae. In one embodiment, the microorganism is Lactobacillus ruminis. [0042] The flagellin construct may be a fragment, variant, analog, homolog, or derivative of wild type flagellin from which it is derived. A fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of flagellin and the conserved structure recognized by TLR5. For example, it was surprisingly discovered that a flagellin construct lacking at least the NDO domain, the CDO domain, or both the NDO and CDO domains still retains most of the molecule's ability to activate TLR5 signaling. This is in sharp contrast to prior studies by, for example, Eaves-Pyles 2003, Murthy 2004, and DiDonato 2005, which indicate that deletions removing more than approximately 55 amino acids from either the N-terminus or C-terminus of flagellin would render the protein inactive.

[0043] The flagellin derivative may be composed of one, or two, or three, or four, or five, or six, or seven domains or fragments thereof (see, e.g. Figure 10 of US Patent 8,324,163, the contents of which are incorporated herein by reference in their entirety). The domains may be selected from NDO, ND1, ND2, D3, CD2, CD1 , and CDO. Domains 0 (DO), 1 (D1), and 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure. The amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable. The non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes. The potent proinflammatory activity of flagellin may reside in the highly conserved ND1 and CD1 regions. In one embodiment, the flagellin derivative comprises or consists of the NDO, ND1 , CDO, and CD1 domains. In another embodiment, the flagellin derivative comprises or consists of the ND1 and CD1 domains.

[0044] In some embodiments, the flagellin derivative may comprise insertions, deletions, and other amino acid changes to any one of the DO, D1 , D2, D3 domains, as well as the hairpin structure connecting the discontinuous D1 and D2 domains. For example, the D3 domain may be substituted in part, or in whole, with a hinge or linker polypeptide that allows the D1 and D2 domains to properly fold such that the variant stimulates TLR5 activity. In an embodiment, the hairpin structure connecting the discontinuous D1 or D2 domains is partially or completely deleted. In another embodiment, the ND1 domain is truncated.

[0045] In some embodiments, the flagellin derivative comprises mutations in epitopes recognized by neutralizing anti-CBLB502 antibodies. The flagellin derivative may comprise one or more mutations in the epitopes recognized by neutralizing anti-CBLB502 antibodies which inhibit or abrogate the ability of the antibodies to neutralize the composition. In yet a further embodiment, the flagellin derivative comprises a truncation and mutations in one or more epitopes. In a further embodiment, the mutations comprise replacement of the epitope residues with alanine. In various embodiments, the flagellin derivative comprises mutations that eliminate all T-cell and B-cell epitopes.

[0046] In some embodiments, the flagellin derivative comprises a tag. In yet a further embodiment, the tag is attached to the N-terminus of the flagellin derivative. In yet another embodiment, the tag is attached to the C- terminus of the flagellin derivative. In one embodiment, the tag is a His tag. In some embodiments, the tag is may be, but not limited to, epitope tags, affinity tags, reporters, or combinations thereof.

[0047] In some embodiments, the tag is an epitope tag. The epitope tag may comprise a random amino acid sequence, or a known amino acid sequence. A known amino acid sequence may have, for example, antibodies generated against it, or there may be no known antibodies generated against the sequence. The epitope tag may be an antibody epitope tag for which commercial antibodies are available. Non-limiting examples of suitable antibody epitope tags include but are not limited to myc, AcV5, AU1 , AU5, E, ECS, E2, FLAG, HA, Maltose binding protein, nus, Softag 1 , Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, 51 , T7, V5, VSV-G, 6*His, BCCP, and calmodulin.

[0048] In some embodiments, the tag is a reporter. Non-limiting examples of reporters include affinity tags, visual reporters or selectable-marker reporters. Non-limiting examples of affinity tags include chitin binding protein (CBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, and glutathione-S- transferase (GST). Visual reporters typically result in a visual signal, such as a color change in the cell, or fluorescence or luminescence of the cell. For instance, the reporter LacZ, which encodes β-galactosidase, will turn a cell blue in the presence of a suitable substrate, such as X-gal. Other non-limiting examples of visual reporters include a fluorescent protein, luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase, and variants thereof. Selectable-marker reporters typically confer a selectable trait to the cell, such as drug resistance (e.g. antibiotic resistance).

[0049] In various embodiments, the tag is a fluorescent protein visual reporter. Non limiting examples of fluorescent protein visual reporters include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. ECFP, Cerulean, CyPet, AmCyanl , Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl , AsRed2, eqFP611 , mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.

[0050] In various embodiments, a protein may be fused to the tag through a peptide linker. The sequence of the linker peptide is chosen based on known structural and conformational contributions of peptide segments to allow for proper folding and prevent possible steric hindrance of the protein to be tagged and the tag polypeptide. Linker peptides are commonly used and known in the art, and may be from about 3 to about 40 amino acids in length.

[0051] In some embodiments, more than one tag is employed. For instance, an endogenous protein may be tagged with at least one, two, three, four, five, six, seven, eight, or nine tags. More than one tag may be expressed as a single polypeptide fused to an endogenous protein of interest. More than one tag fused to an endogenous protein may be expressed as a single polypeptide which is cleaved into the individual tag polypeptides after translation.

[0052] In some embodiments, the flagellin derivative comprises a flexible linker. In a further embodiment, the flexible linker comprises SEQ ID NO: 16. In yet a further embodiment, the flexible linker comprises SEQ ID NO:17.

[0053] In some embodiments, the flagellin derivative comprises or consists of any of the polypeptides listed in Table 1. In various embodiments, the flagellin derivative comprises or consists of polypeptides encoded by any one of SEQ ID NOs: 18-27. In other embodiments, the flagellin derivatives may be at least 30-99% identical to SEQ ID NOs: 18-27. For example, the flagellin derivatives may be at least 30%, or at least 35,%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or-99% identical to SEQ ID NOs: 18-27

Table 1: Illustrative Sequences

[0054] In some embodiments, the flagellin derivatives have improved functional and pharmacological properties which, for example, allow for improved medical effects. In some embodiments, the flagellin derivatives have improved NF-kB activation and radioprotection relative to CBLB502. In some embodiments, the flagellin derivatives have improved pharmacokinetics leading to a proportionally stronger pharmacodynamic response (as detected by, for example, cytokine assays).

[0055] In some embodiments, the flagellin derivatives have improved pharmacological properties, including reduced antigenicity and immunogenicity, which, for example, allows for use in wide variety of disease states and patient types. A reduced antigenicity and immunogenicity expands the medical applications for which the flagellin derivatives of the invention can be used including, for example, medical applications requiring recurrent administration. In some embodiments, the decreased antigenicity translates to improved resistance against the neutralizing action of preexisting human antibodies (e.g. anti-flagellin) as well as those induced in response to CBLB502 injection. In further embodiments, the flagellin derivatives have longer retention times in vivo. A longer retention time may allow the flagellin derivative to be effective with fewer doses or with doses spaced further apart. In some embodiments, these improved pharmacological properties are relative to SEQ ID NOs: 1 or 2.

Uses of Flagellin derivatives

[0056] In some embodiments, the flagellin derivatives may stimulate Toll-like receptor activity (e.g. TLR1 , and/or TLR2, and/or TLR3, and/or TLR4, and/or TLR5, and/or TLR6, and/or TLR7, and/or TLR8, and/or TLR9, and/or TLR10, and/or TLR11 , and/or TLR12, and/or TLR13). The TLR family is composed of at least ten members and is essential for innate immune defense against pathogens. The innate immune system recognizes conserved pathogen-associated molecular patterns (PAMPs). TLR may recognize a conserved structure that is particular to bacterial flagellin which may be composed of a large group of residues that are somewhat permissive to variation in amino acid content. Smith ef a/., Nat. Immunol. 4:1247-53 (2003) have identified 13 conserved amino acids in flagellin that are part of the conserved structure recognized by TLR5.

[0057] In some embodiments, the flagellin derivative activates TLR5 signaling. In some embodiments, the flagellin derivative activates TLR5 at the same levels, or levels similar to, CBLB502. Activation of TLR5 induces expression of the nuclear factor NF-κΒ, which in turn activates numerous inflammatory-related cytokines. In further embodiments, the flagellin derivatives induce expression of proinflammatory cytokines. In further embodiments, the flagellin derivatives induce expression of anti-inflammatory molecules. In another embodiment, the flagellin derivatives induce expression of anti-apoptotic molecules. In yet a further embodiment, the flagellin derivatives induce expression of anti-bacterial molecules. The targets of NF-κΒ, include, but are not limited to, IL- β, TNF-α, IL-6, IL-8, IL-18, G-CSF, TNFSF13B, keratinocyte chemoattractant (KC), BLIMP1 /PRDM1 , CCL5, CCL15, CCL17, CCL19, CCL20, CCL22, CCL23, CXCL1 ,CCL28, CXCL11 , CXCL10, CXCL3, CXCL1 , GRO- beta, GRO-gamma, CXCL1 , ICOS, IFNG, IL-1A, IL-1 B, IL1 RN, IL-2, IL-9, IL-10, IL-11 , IL-12, IL-12B, IL-12A, IL- 13, IL-15, IL-17, IL-23A, IL-27, EBI3, IFNB1 , CXCL5, KC, liGpl , CXCL5, CXCL6, LTA, LTB, CCL2, CXCL9, MCP-1/JE, CCL3, CCL4, CXCL3, CCL20, CXCL10, CXCL5, CCL5, CCL1 , TNFbeta, TNFSF10, TFF3, TNFSF15, CD86, complement component 8a, CCL27, defensin- 3, MIG, MIP-2, and/or NOD2/CARD15.

[0058] In some embodiments, activating TLR5 signaling may regulate CD4⁺ T-cell immune function by increasing the generation of regulatory T-cells (T_regs), decreasing LPS-induced ERK1/2 activation, and/or activating Natural Killer (NK) T-cells.

Diseases and Methods of Treatment/Prevention

[0059] In various embodiments, the flagellin derivatives (and/or additional agents) and methods described herein are applicable to variety of disease states. In one aspect, the invention provides a method of stimulating TLR5 signaling comprising administering a flagellin derivative of the invention to a subject in need thereof. Activating TLR5 signaling may have broad therapeutic applications, including, but not limited to treating cancer, protecting from radiation-induced or reperfusion-induced damage, acting as adjuvant in vaccines, or protecting cells from cytotoxic compounds.

[0060] In some embodiments, the flagellin derivatives of the invention, or fragments thereof may be provided as adjuvants to viral vaccines. In one embodiment, the flagellin derivatives or fragments thereof may be administered in conjunction with an influenza vaccine or antigen to elicit a greater host immune response to the influenza antigens. In yet a further embodiment, the flagellin derivatives of the invention, or fragments thereof may be provided as adjuvants to vaccines against parasites. In one embodiment, the flagellin derivatives or fragments thereof may be administered in conjunction with a Plasmodium vaccine or antigen to elicit a greater host immune response to the Plasmodium antigen.

[0061] In some embodiments, the flagellin derivatives of the invention may be administered to protect cells from toxic conditions. In some embodiments, the flagellin derivatives may prevent liver cells from Fas-mediated injury. The flagellin derivatives of the invention may cause a decrease in liver enzymes in the peripheral blood and caspase activation.

Cancers

[0062] In various embodiments, the present invention pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As used herein, "cancer" or "tumor" refers to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g. virus infected cells). A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, are able to out- compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0063] The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis.

[0064] The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor.

[0065] The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.

[0066] The cancer may have an origin from any tissue. The cancer may originate from, for example, melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal. The invaded tissue may express a TLR, while the cancer may or may not express a TLR.

[0067] Also provided herein is a method of reducing cancer recurrence, comprising administering to a mammal in need thereof a flagellin derivative of the invention. The cancer may be or may have been present in a tissue that either does or does not express TLR, such as TLR5. The method may also prevent cancer recurrence. The cancer may be an oncological disease. The cancer may be a dormant tumor, which may result from the metastasis of a cancer. The dormant tumor may also be left over from surgical removal of a tumor. The cancer recurrence may be tumor regrowth, a lung metastasis, or a liver metastasis.

[0068] Representative cancers and/or tumors of the present invention may or may not express TLR5, and may include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasts leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0069] The flagellin derivatives (and/or additional agents) and methods described herein are applicable to metastatic diseases, including cancers and/or tumors. "Metastasis" refers to the spread of cancer from a primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life -threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

[0070] Metastases may be detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.

[0071] In some embodiments, the invention relates to a method of treating a mammal suffering from a constitutively active NF-κΒ cancer comprising administering to the mammal a composition comprising a therapeutically effective amount of an agent that induces NF-κΒ activity, including the flagellin derivatives (and/or additional agents) described herein. The agent that induces NF-κΒ activity may be administered in combination with a cancer treatment.

[0072] In some embodiments, the present invention includes methods for treatment of side effects from cancer treatment comprising administering the flagellin derivative (and/or additional agents) described herein. In some embodiments, the side effects from cancer treatment include alopecia, myelosuppression, renal toxicity, weight lossy pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post-chemotherapy cognitive impairment.

[0073] In some embodiments, the present invention relates to a method of treating a mammal suffering from damage to normal tissue attributable to treatment of cancer, including but not limited to a constitutively active NF- KB cancer, comprising administering to the mammal a composition comprising a therapeutically effective amount of the flagellin derivative (and/or additional agents) described herein.

Ageing and Stress

[0074] In some embodiments, the present invention includes methods for modulation of cell aging comprising administering the flagellin derivative (and/or additional agents) described herein.

[0075] In some embodiments, the present invention includes methods for treatment of stress comprising administering the flagellin derivative (and/or additional agents) described herein. This invention also relates to a method of treating a subject suffering from damage to normal tissue attributable to stress, comprising administering to the mammal a composition comprising a therapeutically effective amount of a flagellin derivative (and/or additional agents). The stress may be attributable to any source including, but not limited to, radiation, wounding, poisoning, infection, and temperature shock.

[0076] In some embodiments, the flagellin derivative (and/or additional agents) may be administered at any point prior to exposure to the stress including, but not limited to, about 48 hr, about 46 hr, about 44 hr, about 42 hr, about 40 hr, about 38 hr, about 36 hr, about 34 hr, about 32 hr, about 30 hr, about 28 hr, about 26 hr, about 24 hr, about 22 hr, about 20 hr, about 18 hr, about 16 hr, about 14 hr, about 12 hr, about 10 hr, about 8 hr, about 6 hr, about 4 hr, about 3 hr, about 2 hr, or about 1 hour prior to exposure. In some embodiments, the flagellin derivative may be administered at any point after exposure to the stress including, but not limited to, about 1 hr, about 2 hr, about 3 hr, about 4 hr, about 6 hr, about 8 hr, about 10 hr, about 12 hr, about 14 hr, about 16 hr, about 18 hr, about 20 hr, about 22 hr, about 24 hr, about 26 hr, about 28 hr, about 30 hr, about 32 hr, about 34 hr, about 36 hr, about 38 hr, about 40 hr, about 42 hr, about 44 hr, about 46 hr, or about 48 hours after exposure.

Mitigation and Prevention of Radiation Damage

[0077] In still other embodiments, the present invention relates to treatment of radiation related diseases or damage. In specific embodiments, the present invention relates to mitigation of or prevention and/or protection from radiation related diseases.

[0078] In one embodiment, the present invention relates to the protection of cells from the effects of exposure to radiation. In some embodiments, the present invention pertains to a method of protecting a subject from radiation comprising administering a flagellin derivative (and/or additional agents) described herein. In some embodiments, the radiation is ionizing radiation. In some embodiments, the ionizing radiation is sufficient to cause gastrointestinal syndrome or hematopoietic syndrome. In some embodiments, the flagellin derivative (and/or additional agents) described herein is administered in combination with a radioprotectant e.g. an antioxidant (e.g. amifostine and vitamin E), a cytokine (e.g. a stem cell factor), etc. In some embodiments, the flagellin derivative (and/or additional agents) described herein is administered prior to, together with, or after radiation. In some embodiments, the flagellin derivative (and/or additional agents) described herein is administered in combination with a growth factor (e.g. keratinocyte growth factor), a steroid (e.g. 5- androstenediol), ammonium trichloro(dioxoethylene-0,0')tellurate, thyroid protecting agents (e.g. Potassium iodide (Kl)), anti-nausea agents, anti-diarrhea agents, analgesics, anxiolytics, sedatives, cytokine therapy, antibiotics, antifungal agents, and/or antiviral agents.

[0079] In some embodiments, the present invention pertains to a method of treating and/or mitigating apoptosis-mediated tissue damage in a subject, comprising administering to a subject in need thereof a composition comprising a flagellin derivative (and/or additional agents) described herein. In some embodiments the apoptosis is attributable to cellular stress. In some embodiments, the flagellin derivative (and/or additional agents) described herein is administered prior to, together with, or after the tissue damage. In some embodiments, the cellular stress is radiation. In some embodiments, the flagellin derivative (and/or additional agents) is administered in combination with a radioprotectant (e.g. an antioxidant (e.g. amifostine and vitamin E), a cytokine (e.g. a stem cell factor), etc.

[0080] Injury and death of normal cells from ionizing radiation is a combination of a direct radiation-induced damage to the exposed cells and an active genetically programmed cell reaction to radiation-induced stress resulting in a suicidal death or apoptosis. Apoptosis plays a key role in massive cell loss occurring in several radiosensitive organs (e.g., hematopoietic and immune systems, epithelium of digestive tract, etc.), the failure of which determines general radiosensitivity of the organism. In some embodiments, administration of the flagellin derivatives of the invention to a subject in need thereof suppresses apoptosis in cells. In some embodiments, the flagellin derivatives of the invention are administered to a subject undergoing cancer radiotherapy treatment to protect healthy cells from the damaging effects of the radiation treatment.

[0081] Exposure to ionizing radiation (IR) may be short- or long-term, and/or it may be applied as a single or multiple doses and/or it may be applied to the whole body or locally. The present invention, in some embodiments, pertains to nuclear accidents or military attacks, which may involve exposure to a single high dose of whole body irradiation (sometimes followed by a long-term poisoning with radioactive isotopes). The same is true (with strict control of the applied dose), for example, for pretreatment of patients for bone marrow transplantation when it is necessary to prepare hematopoietic organs for donor's bone marrow by "cleaning" them from the host blood precursors. Cancer treatment may involve multiple doses of local irradiation that greatly exceeds lethal dose if it were applied as a total body irradiation. Poisoning or treatment with radioactive isotopes results in a long-term local exposure to radiation of targeted organs (e.g., thyroid gland in the case of inhalation of ¹²⁵l). Further, there are many physical forms of ionizing radiation differing significantly in the severity of biological effects. [0082] At the molecular and cellular level, radiation particles are able to produce breakage and cross-linking in the DNA, proteins, cell membranes and other macromolecular structures. Ionizing radiation also induces the secondary damage to the cellular components by giving rise to the free radicals and reactive oxygen species (ROS). Multiple repair systems counteract this damage, such as, several DNA repair pathways that restore the integrity and fidelity of the DNA, and antioxidant chemicals and enzymes that scavenge the free radicals and ROS and reduce the oxidized proteins and lipids. Cellular checkpoint systems detect the DNA defects and delay cell cycle progression until damage is repaired or decision to commit cell to growth arrest or programmed cell death (apoptosis) is reached

[0083] Radiation can cause damage to mammalian organism ranging from mild mutagenic and carcinogenic effects of low doses to almost instant killing by high doses. Overall radiosensitivity of the organism is determined by pathological alterations developed in several sensitive tissues that include hematopoietic system, reproductive system and different epithelia with high rate of cell turnover.

[0084] Acute pathological outcome of gamma irradiation leading to death is different for different doses and may be determined by the failure of certain organs that define the threshold of organism's sensitivity to each particular dose. Thus, lethality at lower doses occurs from, for example, bone marrow aplasia, while moderate doses kill faster by inducing, for example, a gastrointestinal (Gl) syndrome. Very high doses of radiation can cause almost instant death by eliciting neuronal degeneration.

[0085] Organisms that survive a period of acute toxicity of radiation can suffer from long-term remote consequences that include radiation-induced carcinogenesis and fibrosis developing in exposed organs (e.g., kidney, liver or lungs) in the months and years after irradiation.

[0086] Cellular DNA is a major target of IR that causes a variety of types of DNA damage (genotoxic stress) by direct and indirect (e.g. free radical-based) mechanisms. All organisms maintain DNA repair system capable of effective recovery of radiation-damaged DNA; errors in DNA repair process may lead to mutations.

[0087] In some embodiments, the radiation exposure experienced by the subject is a consequence of cancer radiotherapy treatment. Tumors are generally more sensitive to gamma radiation and can be treated with multiple local doses that cause relatively low damage to normal tissue. Nevertheless, in some instances, damage of normal tissues is a limiting factor in application of gamma radiation for cancer treatment. The use of gamma- irradiation during cancer therapy by conventional, three-dimensional conformal or even more focused BeamCath delivery has also dose-limiting toxicities caused by cumulative effect of irradiation and inducing the damage of the stem cells of rapidly renewing normal tissues, such as bone marrow and gastrointestinal (Gl) tract. Administration of the flagellin derivatives of the invention may protect the patient's healthy cells from radiation damage without affecting the radiosensitivity of the tumor cells.

[0088] In some embodiments, the subject has been exposed to lethal doses of radiation. At high doses, radiation-induced lethality is associated with so-called hematopoietic and gastrointestinal radiation syndromes. Hematopoietic syndrome is characterized by loss of hematopoietic cells and their progenitors making it impossible to regenerate blood and lymphoid system. Death usually occurs as a consequence of infection (result of immunosuppression), hemorrhage and/or anemia. Gl syndrome is caused by massive cell death in the intestinal epithelium, predominantly in the small intestine, followed by disintegration of intestinal wall and death from bacteremia and sepsis. Hematopoietic syndrome usually prevails at the lower doses of radiation and leads to the more delayed death than Gl syndrome.

[0089] In the past, radioprotectants were typically antioxidants-both synthetic and natural. More recently, cytokines and growth factors have been added to the list of radioprotectants; the mechanism of their radioprotection is considered to be a result of facilitating the effects on regeneration of sensitive tissues. There is no clear functional distinction between both groups of radioprotectants, however, since some cytokines induce the expression of the cellular antioxidant proteins, such as manganese superoxide dismutase (MnSOD) and metallothionein.

[0090] The measure of protection for a particular agent may be expressed by dose modification factor (DMF or DRF). DMF is determined by irradiating the radio protector treated subject and untreated control subjects with a range of radiation doses and then comparing the survival or some other endpoints. DMF is commonly calculated for 30-day survival (LD50/30 drug-treated divided by LD50/30 vehicle-treated) and quantifies the protection of the hematopoietic system. In order to estimate gastrointestinal system protection, LD50 and DMF are calculated for 6- or 7-day survival.

[0091] The flagellin derivatives described herein possess strong pro-survival activity at the cellular level and on the organism as a whole. In response to super-lethal doses of radiation, the flagellin derivatives described herein may inhibit both gastrointestinal and hematopoietic syndromes, which are major causes of death from acute radiation exposure. As a result of these properties, the flagellin derivatives described herein may be used to treat the effects of natural radiation events and nuclear accidents. For example, the present invention provides for the treatment or prevention of acute radiation syndrome (ARS). Moreover, the flagellin derivatives described herein can be used in combination with other radioprotectants, thereby, dramatically increasing the scale of protection from ionizing radiation.

[0092] As opposed to conventional radioprotective agents (e.g., scavengers of free radicals), anti-apoptotic agents may not reduce primary radiation-mediated damage but may act against secondary events involving active cell reaction on primary damage, therefore complementing the existing lines of defense. Pifithrin-alpha, a pharmacological inhibitor of p53 (a key mediator of radiation response in mammalian cells), is an example of this new class of radioprotectants. However, the activity of p53 inhibitors is limited to protection of the hematopoietic system and has no protective effect in digestive tract (gastrointestinal syndrome), therefore reducing therapeutic value of these compounds.

[0093] The flagellin derivatives described herein may be used as a radioprotective agent to extend the range of tolerable radiation doses by increasing radioresistance of humans beyond the levels achievable by currently available measures (shielding and application of existing bioprotective agents) and drastically increase the chances of crew survival in case of nuclear accidents or large-scale solar particle events, for example.

[0094] The flagellin derivatives described herein are also useful for treating irreplaceable cell loss caused by low-dose irradiation, for example, in the central nervous system and reproductive organs. The flagellin derivatives described herein may also be used during cancer chemotherapy to treat the side effects associated with chemotherapy, including alopecia, myelosuppression, renal toxicity, weight loss_T pain, nausea, vomiting, diarrhea, constipation, anemia, malnutrition, hair loss, numbness, changes in tastes, loss of appetite, thinned or brittle hair, mouth sores, memory loss, hemorrhage, cardiotoxicity, hepatotoxicity, ototoxicity, and post- chemotherapy cognitive impairment.

[0095] In one embodiment, a mammal is treated for exposure to radiation, comprising administering to the mammal a composition comprising a therapeutically effective amount of a flagellin related composition. The flagellin derivative may be administered in combination with one or more radioprotectants. The one or more radioprotectants may be any agent that treats the effects of radiation exposure including, but not limited to, antioxidants, free radical scavengers and cytokines.

[0096] The flagellin derivatives described herein may inhibit radiation-induced programmed cell death in response to damage in DNA and other cellular structures. In some embodiments, the flagellin derivatives described herein may not deal with damage at the cellular and may not prevent mutations. Free radicals and reactive oxygen species (ROS) are the major cause of mutations and other intracellular damage. Antioxidants and free radical scavengers are effective at preventing damage by free radicals. The combination of a flagellin derivative and an antioxidant or free radical scavenger may result in less extensive injury, higher survival, and improved health for mammals exposed to radiation. Antioxidants and free radical scavengers that may be used in the practice of the invention include, but are not limited to, thiols, such as cysteine, cysteamine, glutathione and bilirubin; amifostine (WR-2721); vitamin A; vitamin C; vitamin E; and flavonoids such as Indian holy basil (Ocimum sanctum), orientin and vicenin.

[0097] The flagellin derivatives described herein may also be administered in combination with a number of cytokines and growth factors that confer radioprotection by replenishing and/or protecting the radiosensitive stem cell populations. Radioprotection with minimal side effects may be achieved by the use of stem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1 fragment IL-1 b-rd. Protection may be achieved through induction of proliferation of stem cells (all mentioned cytokines), and prevention of their apoptosis (SCF). The treatment allows accumulation of leukocytes and their precursors prior to irradiation thus enabling quicker reconstitution of the immune system after irradiation. SCF efficiently rescues lethally irradiated mice with DMF in range 1.3-1.35 and is also effective against gastrointestinal syndrome. Flt-3 ligand also provides strong protection in mice and rabbits.

[0098] Several factors, while not cytokines by nature, stimulate the proliferation of the immunocytes and may be used in combination with the flagellin derivatives described herein. For example, 5-AED (5- androstenediol) is a steroid that stimulates the expression of cytokines and increases resistance to bacterial and viral infections. Synthetic compounds, such as ammonium tri-chloro(dioxoethylene-0,0'-) tellurate (AS-101), may also be used to induce secretion of numerous cytokines and for combination with the flagellin derivatives described herein.

[0099] Growth factors and cytokines may also be used to provide protection against the gastrointestinal syndrome. Keratinocyte growth factor (KGF) promotes proliferation and differentiation in the intestinal mucosa, and increases the post-irradiation cell survival in the intestinal crypts. Hematopoietic cytokine and radioprotectant SCF may also increase intestinal stem cell survival and associated short-term organism survival.

[00100] The flagellin derivatives described herein may offer protection against both gastrointestinal (Gl) and hematopoietic syndromes. Such compositions may be used in combination with one or more inhibitors of Gl syndrome (including, but are not limited to, cytokines such as SCF and KGF).

[00101] The flagellin related composition may be administered at any point prior to exposure to radiation including, but not limited to, about 48 hours, about 46 hours, about 44 hours, about 42 hours, about 40 hours, about 38 hours, about 36 hours, about 34 hours, about 32 hours, about 30 hours, about 28 hours, about 26 hours, about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour prior to exposure. The flagellin derivative may be administered at any point after exposure to radiation including, but not limited to, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours after exposure to radiation.

[00102] In various embodiments, the present methods and compositions provide treatment or prevention of radiation-related disorders, such as ARS. In various embodiments, the treatments described herein reduce morbidity or mortality of an exposed population of human patients or accelerates recovery from symptoms of ARS. ARS often presents as a sequence of phased symptoms, which may vary with individual radiation sensitivity, type of radiation, and the radiation dose absorbed. Generally, without wishing to be bound by theory, the extent of symptoms will heighten and the duration of each phase will shorten with increasing radiation dose. ARS can be divided into three phases: prodromal phase (a.k.a. N-V-D stage), latent period and manifest illness. In various embodiments, the flagellin derivative (and/or additional agents), as described herein, may be administered to a human patient in any one of these three stages (i.e. the flagellin derivative (and/or additional agents) may be administered to a human patient in the prodromal phase, the flagellin derivative (and/or additional agents) may be administered to a human patient in latent period, or the flagellin derivative (and/or additional agents) may be administered to a human patient in manifest illness stage). [00103] In the prodromal phase there is often a relatively rapid onset of nausea, vomiting, and malaise. Use of antiemetics, (e.g. oral prophylactic antiemetics) such as granisetron (KYTRIL), ondansetron (ZOFRAN), and 5- HT3 blockers with or without dexamethasone, may be indicated in situations where high-dose radiological exposure has occurred, is likely, or is unavoidable. Accordingly, in various embodiments, the flagellin derivative (and/or additional agents) may be administered to a human patient in receiving an anti-emetic agent or the flagellin derivative (and/or additional agents) may be administered to a human patient in combination with an anti-emetic agent. For example, the flagellin derivative (and/or additional agents) may also be added to the following antiemetic regimens: Ondansetron: initially 0.15 mg/kg IV; a continuous IV dose option consists of 8 mg followed by 1 mg/h for the next 24 hours. Oral dose is 8 mg every 8 hours as needed or Granisetron (oral dosage form): dose is usually 1 mg initially, then repeated 12 hours after the first dose. Alternatively, 2 mg may be taken as one dose. IV dose is based on body weight; typically 10 g/kg (4.5 g/lb) of body weight.

[00104] In the latent period, a human patient may be relatively symptom free. The length of this phase varies with the dose. The latent phase is longest preceding the bone-marrow depression of the hematopoietic syndrome and may vary between about 2 and 6 weeks. The latent period is somewhat shorter prior to the gastrointestinal syndrome, lasting from a few days to a week. It is shortest of all preceding the neurovascular syndrome, lasting only a matter of hours. These times are variable and may be modified by the presence of other disease or injury. Manifest illness presents with the clinical symptoms associated with the major organ system injured (marrow, intestinal, neurovascular).

[00105] In some embodiments, the present invention relates to the mitigation of, or protection of cells from, the effects of exposure to radiation. In some embodiments, the present invention pertains to a method of mitigating and/or protecting a human patient from radiation comprising administering the flagellin derivative (and/or additional agents). In some embodiments, the radiation is ionizing radiation. In some embodiments, the ionizing radiation is sufficient to cause gastrointestinal syndrome or hematopoietic syndrome.

[00106] In some embodiments, the ARS comprises one of more of gastrointestinal syndrome; hematopoietic syndrome; neurovascular syndrome; apoptosis-mediated tissue damage, wherein the apoptosis is optionally attributable to cellular stress; and ionizing radiation induced apoptosis tissue damage.

[00107] Hematopoietic syndrome (a.k.a. bone marrow syndrome) is characterized by loss of hematopoietic cells and their progenitors making it impossible to regenerate blood and lymphoid system. This syndrome is often marked by a drop in the number of blood cells, i.e., aplastic anemia. This may result in infections (e.g. opportunistic infections) due to a low amount of white blood cells, bleeding due to a lack of platelets, and anemia due to few red blood cells in the circulation. These changes can be detected by blood tests after receiving a whole-body acute dose. Conventional trauma and burns resulting from a bomb blast are complicated by the poor wound healing caused by hematopoietic syndrome, increasing mortality. Death may occur as a consequence of infection (result of immunosuppression), hemorrhage and/or anemia. Hematopoietic syndrome usually prevails at the lower doses of radiation and leads to the more delayed death than Gl syndrome.

[00108] Gastrointestinal syndrome is caused by massive cell death in the intestinal epithelium, predominantly in the small intestine, followed by disintegration of intestinal wall and death from bacteriemia and sepsis. Symptoms of this form of radiation injury include nausea, vomiting, loss of appetite, loss of absorptive capacity, hemorrhage in denuded areas, and abdominal pain. Illustrative systemic effects of gastrointestinal syndrome include malnutrition, dehydration, renal failure, anemia, sepsis, etc. Without treatment (including, for example, bone marrow transplant), death is common (e.g. via infection from intestinal bacteria). In some embodiments, the flagellin derivative (and/or additional agents), may be used in combination with bone marrow transplant. In some embodiments, the flagellin derivative (and/or additional agents), may be used in combination with one or more inhibitors of Gl syndrome and/or any of the additional agents described herein.

[00109] Neurovascular syndrome presents with neurological symptoms such as dizziness, headache, or decreased level of consciousness, occurring within minutes to a few hours, and with an absence of vomiting. Additional symptoms include extreme nervousness and confusion; severe nausea, vomiting, and watery diarrhea; loss of consciousness; and burning sensations of the skin. Neurovascular syndrome is commonly fatal.

[00110] In some embodiments, the present invention provides a method for reducing the risk of death following exposure to irradiation comprising administering an effective amount of the flagellin derivative (and/or additional agents) In some embodiments, the radiation is potentially lethal, and, optionally, occurs as the result of a radiation disaster. In various embodiments, the flagellin derivative (and/or additional agents) is administered within about 25 hours following radiation exposure. In some embodiments, the present invention provides a method for reducing the risk of death following exposure to potentially lethal irradiation occurring as the result of a radiation disaster, comprising administering the flagellin derivative (and/or additional agents) within about 25 hours following radiation exposure.

[00111] In various embodiments, the flagellin derivative (and/or additional agents) is administered to a patient who has been exposed to a high dose of radiation, namely a whole body dose. In various embodiments, the high dose of radiation may not be uniform. In various embodiments, the ARS is a result of a high dose of radiation. In various embodiments, the high dose of radiation is about 2.0 Gy, or about 2.5 Gy, or about 3.0 Gy, or about 3.5 Gy, or about 4.0 Gy, or about 4.5 Gy, or about 5 Gy, or about 10 Gy, or about 15 Gy, or about 20 Gy, or about 25 Gy, or about 30 Gy. In various embodiments, the high dose of radiation is about 5 to about 30 Gy, or about 10 to 25 Gy, or about 15 to 20 Gy. In some embodiments, the high dose of radiation is assessed by one or more of physical dosimetry and/or biological dosimetry (e.g. multiparameter dose assessments), cytogenics (e.g. chromosomal analysis for, for example, blood samples (including, by way of non-limiting example, dicentric analysis).

[00112] In various embodiments, whole-body radiation doses can be divided into sublethal (<2 Gy), potentially lethal (2-10 Gy), and supralethal (>10 Gy).

Reperfusion Injuries

[00113] In some embodiments, the present invention pertains to a method of treating the effects of reperfusion on a subject's tissue comprising administering the flagellin derivative (and/or additional agents) described herein. The flagellin derivatives (and/or additional agents) described herein may be administered in combination with an antioxidant, such as, for example, amifostine and vitamin E.

[00114] Reperfusion may be caused by an injury, which may be ischemia or hypoxia. The ischemia may result from a condition such as, for example, tachycardia, infarction, hypotension, embolism, thromboemoblism (blood clot), sickle cell disease, localized pressure to extremities to the body, and tumors. The hypoxia may be selected from hypoxemic hypoxia (carbon monoxide poisoning; sleep apnea, chronic obstructive pulmonary disease, respiratory arrest; shunts), anemic hypoxia (O2 content low), hypoxemic hypoxia, and histotoxic hypoxia. The localized pressure may be due to a tourniquet.

[00115] The flagellin derivatives (and/or additional agents) described herein may be administered prior to, together with, or after the influx of oxygen. The tissue may be for example, the Gl tract, lung, kidney, liver, cardiovascular system, blood vessel endothelium, central nervous system, peripheral nervous system, muscle, bone, and hair follicle.

[00116] Reperfusion may damage a body component when blood supply returns to the body component after the injury. The effects of reperfusion may be more damaging to the body component than the injury itself. There are several mechanism and mediators of reperfusion including, for example, oxygen free radicals, intracellular calcium overload, and endothelial dysfunction. Excessive quantities of reactive oxygen species, when reintroduced into a previously injured body component, undergo a sequential reduction leading to the formation of oxygen free radicals. Potent oxidant radicals, such as superoxide anion, hydroxyl radical, and peroxynitrite may be produced within the first few minutes of reflow to the body component and may play a crucial role in the development of reperfusion injury. Oxygen free radicals also can be generated from sources other than reduction of molecular oxygen. These sources include enzymes, such as, for example, xanthine oxidase, cytochrome oxidase, and cyclooxygenase, and the oxidation of catecholamines.

[00117] Reperfusion is also a potent stimulus for neutrophil activation and accumulation, which in turn serve as potent stimuli for reactive oxygen species production. Specifically, the main products of the neutrophil respiratory burst are strong oxidizing agents including hydrogen peroxide, free oxygen radicals and hypochlorite. Neutrophils are the most abundant type of phagocyte, normally representing 50 to 60% of the total circulating leukocytes, and are usually the first cells to arrive at the site of injured body component. Oxygen-derived free radicals produce damage by reacting with polyunsaturated fatty acids, resulting in the formation of lipid peroxides and hydroperoxides that damage the body component and impair the function of membrane-bound enzyme systems. Free radicals stimulate the endothelial release of platelet activating factor and chemokines such as neutrophil activator factor, chemokine (C-X-C motif) ligand 1 , and chemokine (C-X-C motif) ligand 1 which attracts more neutrophils and amplifies the production of oxidant radicals and the degree of reperfusion injury. Reactive oxygen species also quench nitric oxide, exaggerating endothelial injury and tissue cell dysfunction. In addition to an increased production, there is also a relative deficiency in endogenous oxidant scavenging enzymes, which further exaggerates free radical-mediated cardiac dysfunction. [00118] Reperfusion may further result in marked endothelial cell dysfunction. Endothelial dysfunction facilitates the expression of a prothrombotic phenotype characterized by platelet and neutrophil activation, important mediators of reperfusion. Once neutrophils make contact with the dysfunctional endothelium, they are activated, and in a series of well-defined steps (rolling, firm adherence, and transmigration) they migrate into areas of tissue injury through endothelial cell junctions as part of the innate immune response.

[00119] Changes in intracellular calcium homeostasis play an important role in the development of reperfusion. Reperfusion may be associated with an increase in intracellular calcium; this effect may be related to increased sarcolemmal calcium entry through L-type calcium channels or may be secondary to alterations in sarcoplasmic reticulum calcium cycling. In addition to intracellular calcium overload, alterations in myofilament sensitivity to calcium have been implicated in reperfusion. Activation of calcium-dependent proteases (calpain I) with resultant myofibril proteolysis has been suggested to underscore reperfusion injury, as has proteolysis of troponin.

[00120] Reperfusion of tissue cells subjected to an injury has an altered cellular metabolism, which in turn may contribute to delayed functional recovery. For example, an injury may induce anaerobic metabolism in the cell with a net production of lactate. Lactate release persists during reperfusion, suggesting a delayed recovery of normal aerobic metabolism. Likewise, the activity of mitochondrial pyruvate dehydrogenase (PDH) may be inhibited up to 40% after an injury and may remain depressed for up to 30 minutes after reperfusion.

[00121] Each of these events during reperfusion can lead to stress to the tissue cells and programmed cell death (apoptosis) and necrosis of the tissue cells. Apoptosis normally functions to "clean" tissues from wounded and genetically damaged cells, while cytokines serve to mobilize the defense system of the organism against the pathogen. However, under conditions of severe injury both stress response mechanisms can by themselves act as causes of death.

[00122] In various embodiments, the effects of reperfusion may be caused by an injury to the body. The injury may be due to ischemia, hypoxia, an infarction, or an embolism. Treatment of the injury may lead to reperfusion and further damage to the body component.

[00123] Ischemia may be an absolute or relative shortage of blood supply to a body component. Relative shortage may be a mismatch, however small, of blood supplied (oxygen delivery) to a body component versus blood required to a body component for the adequate oxygenation. Ischemia may also be an inadequate flow of blood to a part of the body due to a constriction or blockage of blood vessels supplying it and may affect any body component in the body. Insufficient blood supply causes body components to become hypoxic, or, if no oxygen is supplied at all, anoxic. This may cause necrosis. The mechanisms of ischemia may vary greatly. For example, ischemia to any body component may be due to tachycardia (abnormally rapid beating of the heart), atherosclerosis (lipid-laden plaque obstructing the lumen of arteries), hypotension (low blood pressure in septic shock, heart failure), thromboembolisms (blood clots), outside compression of blood vessels (tumor), embolisms (foreign bodies in the circulation, e.g., amniotic fluid embolism), sickle cell disease (abnormally shaped hemoglobin), infarctions, induced g-forces which restrict the blood flow and force the blood to extremities of the body, localized extreme cold due to frostbite, ice, improper cold compression therapy, and any other force that restricts blood flow to the extremities such as a tourniquet. Force to restrict blood flow to extremities may be required due to severe lacerations, incisions, puncture such as a knifing, crushing injuries due to blunt force trauma, and ballistic trauma due to gunshot or shrapnel wounds. Ischemia may be a feature of heart diseases, ischemic colitis, transient ischemia attacks, cerebrovascular accidents, acute renal injury, ruptured arteriovenous malformations, and peripheral artery occlusive disease.

[00124] Hypoxia may be a deprivation of adequate supply of oxygen. Hypoxia may be pathological condition in which the body as a whole (generalized hypoxia) or region of the body (tissue hypoxia) is deprived of adequate oxygen supply. A variation in levels of arterial oxygen may be due to a mismatch between supply and demand of oxygen by body components. A complete deprivation of oxygen supply is anoxia. Hypoxia may be hypoxemic hypoxia, anemic hypoxia, hypoxemic hypoxia, histotoxic hypoxia, histotoxic hypoxia, and ischemic hypoxia.

[00125] Hypoxemic hypoxia may be an inadequate supply of oxygen to the body as a whole caused by low partial pressure of oxygen in arterial blood. Hypoxemic hypoxia may be due to low partial pressure of atmospheric oxygen such as at high altitudes, replacement of oxygen in breathing mix of a modified atmosphere such as a sewer, replacement of oxygen intentionally as in recreational use of nitrous oxide, a decrease in oxygen saturation of the blood due to sleep apnea, or hypopnea, inadequate pulmonary ventilation such as chronic obstructive pulmonary disease or respiratory arrest, anatomical or mechanical shunts in the pulmonary circulation or a right to left shunt in the heart and lung. Shunts may cause collapsed alveoli that are still perfused or a block in ventilation to an area of the lung. Shunts may present blood meant for the pulmonary system to not be ventilated and prevent gas exchange because the blood vessels empty into the left ventricle and the bronchial circulation, which supplies the bronchi with oxygen.

[00126] Anemia hypoxia may be the total oxygen content is reduced but the arterial oxygen pressure is normal. Hypoxemic hypoxia may be when blood fails to deliver oxygen to target body components. Hypoxemic hypoxia may be caused by carbon monoxide poisoning which inhibits the ability of hemoglobin to release the oxygen bound to it, or methaemoglobinaemia, an abnormal hemoglobin that accumulates in the blood. Histotoxic hypoxia may be due to being unable to effectively use oxygen due to disabled oxidative phosphorylation enzymes.

[00127] Infarction is a type of pathological condition that can cause ischemia. Infarction may be a macroscopic area of necrotic tissue caused the loss of an adequate blood supply due to an occlusion. The infarction may be a white infarction composed of platelets and causes necrosis in organ tissues such as heart, spleen, and kidneys. The infarction may be a red infarction composed of red blood cells and fibrin strands in organ tissues of the lung. Disease associated with infarction may include myocardial infarction, pulmonary embolism, cerebrovascular accident (stroke), acute renal failure, peripheral artery occlusive disease (example being gangrene), antiphospholipid syndrome, sepsis, giant cell arthritis, hernia, and volvulus. [00128] Embolism is a type of pathological condition that can cause ischemia. Embolism may be an object that migrates from one part of the body and causes an occlusion or blockage of a blood vessel in another part of the body. An embolism may be thromboembolism, fat embolism, air embolism, septic embolism, tissue embolism, foreign body embolism, amniotic fluid embolism. Thromboembolism may be a blood clot that is completely or partially detached from the site of thrombosis. Fat embolism may be endogenous fat tissues that escape into the blood circulation. The fracture of bones is one example of a leakage of fat tissue into the ruptured vessels and arteries. Air embolism may be a rupture of alveoli and inhaled air that leaks into the blood vessels. The puncture of the subclavian vein or intravenous therapy are examples of leakage of air into the blood vessels. A gas embolism may be gasses such as nitrogen and helium because insoluble and forming small bubbles in the blood.

Pharmaceutically Acceptable Salts and Excipients

[00129] The flagellin derivatives (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

[00130] Pharmaceutically acceptable salts include, by way of non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, a-hydroxybutyrate, butyne-1 ,4- dicarboxylate, hexyne-1 ,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenyl propionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1 -sulfonate, naphthalene-2-sulfonate, naphthalene-1 ,5-sulfonate, xylenesulfonate, and tartarate salts.

[00131] The term "pharmaceutically acceptable salt" also refers to a salt of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group, and a base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert- butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

[00132] In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

[00133] Further, any flagellin derivatives (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.

[00134] Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

Formulations, Administration, Dosing, and Treatment Regimens

[00135] The present invention includes the described flagellin derivatives (and/or additional agents) in various formulations. Any flagellin related composition(and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

[00136] Where necessary, the flagellin derivatives (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection. [00137] The formulations comprising the flagellin derivatives (and/or additional agents) of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, eic, followed by tableting using conventional methods known in the art)

[00138] In one embodiment, any flagellin derivative (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

[00139] Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection. The mode of administration can be left to the discretion of the practitioner, and depends in-part upon the site of the medical condition. In most instances, administration results in the release of any agent described herein into the bloodstream.

[00140] Any flagellin derivative (and/or additional agents) described herein can be administered orally. Such flagellin derivative (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, eic, and can be used to administer.

[00141] In specific embodiments, it may be desirable to administer locally to the area in need of treatment.

[00142] In one embodiment, any flagellin derivative (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for oral administration to humans. Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can comprise one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving any flagellin derivative (and/or additional agents) described herein are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, eic, and mixtures thereof.

[00143] Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

[00144] The dosage of any flagellin derivative (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any agent described herein, can be administered prior to (e.g., about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks before), concurrently with, or subsequent to (e.g., about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks after) the administration of an additional therapeutic agent, to a subject in need thereof. In various embodiments any agent described herein is administered about 1 minute apart, about 10 minutes apart, about 30 minutes apart, less than about 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, no more than about 24 hours apart or no more than about 48 hours apart.

[00145] The amount of any flagellin derivative (and/or additional agents) described herein that is admixed with the carrier materials to produce a single dosage can vary depending upon the subject being treated and the particular mode of administration. In vitro or in vivo assays can be employed to help identify optimal dosage ranges. [00146] In general, the doses that are useful are known to those in the art. For example, doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (December 27, 2011), the contents of which are incorporated by reference in its entirety.

[00147] The dosage of any flagellin derivative (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

[00148] Generally, when orally administered to a mammal, the dosage of any flagellin derivative (and/or additional agents) described herein may be about 0.001 mg/kg/day to about 100 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, or about 0.1 mg/kg/day to about 10 mg/kg/day. When orally administered to a human, the dosage of any agent described herein is normally about 0.001 mg to about 1000 mg per day, about 1 mg to about 600 mg per day, or about 5 mg to about 30 mg per day.

[00149] For administration of any flagellin derivative (and/or additional agents) described herein by parenteral injection, the dosage is normally about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Injections may be given up to four times daily. Generally, when orally or parenterally administered, the dosage of any agent described herein is normally about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day. A dosage of up to about 3000 mg per day can be administered.

[00150] In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat ef a/., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).

[00151] Any flagellin derivative (and/or additional agents) described herein can be administered by controlled- release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled- or sustained-release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the agents described herein. The invention thus provides single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled- or sustained-release.

[00152] Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

[00153] In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy ef a/., 1985, Science 228:190; During ef a/., 1989, Ann. Neurol. 25:351 ; Howard et al., 1989, J. Neurosurg. 71 :105).

[00154] In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used.

[00155] Administration of any flagellin derivative (and/or additional agents) described herein can, independently, be about one to about four times daily or about one to about four times per month or about one to about six times per year or about once every two, three, four or five years. Administration can be for the duration of about one day or about one month, about two months, about three months, about six months, about one year, about two years, about three years, and may even be for the life of the subject. Chronic, long-term administration will be indicated in many cases. The dosage may be administered as a single dose or divided into multiple doses. In general, the desired dosage should be administered at set intervals for a prolonged period, usually at least over several weeks or months, although longer periods of administration of several months or years or more may be needed.

[00156] The dosage regimen utilizing any flagellin derivative (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any flagellin derivative (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any flagellin derivative (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.

Combination Therapies and Conjugation [00157] In some embodiments, the invention provides for flagellin derivatives and methods that further comprise administering an additional agent to a subject. In some embodiments, the invention pertains to coadministration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or coadministered.

[00158] In some embodiments, any flagellin derivative described herein acts synergistically when coadministered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy. In various embodiments, any agent referenced herein may be used in combination with any of the flagellin derivatives described herein.

[00159] In some embodiments, the present invention pertains to chemotherapeutic agents as additional agents.

[00160] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrol ino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paditaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paditaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-a, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy.

[00161] In some embodiments, the flagellin derivative (and/or additional agents) described herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non- classical amino acids.

[00162] In still other embodiments, the flagellin derivative (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in exemplary embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to a composition described herein.

[00163] The flagellin derivative (and/or additional agents) described herein may thus be modified post- translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

[00164] Exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1- methyl nitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (0,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.

[00165] Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxols, irinotecan, 5- fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine and vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin conjugated monoclonal antibodies, tumor antigen specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN®, or any combination thereof. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the therapeutic agents (e.g. antibodies) to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[00166] Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67, Yttrium-88, Yttrium-90, lodine-125, lodine-131 , Samarium-153, Lutetium-177, Rhenium- 186 or Rhenium-188, and alpha-emitters such as Astatine-211 , Lead-212, Bismuth-212, Bismuth-213 or Actinium-225. [00167] Exemplary detectable moieties further include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, lodine-125, Carbon-14, Sulfur-35, Tritium and Phosphorus-32.

Viral Vectors Encoding Therapeutic Agents and Cells Expressing Same

[00168] In various embodiments, the flagellin derivatives of the present invention are expressed by viral vectors and transformed cells. For example, the viral vectors and transformed human cells described herein may express the present compositions. In an embodiment, the viral vector or human cells expressing the therapeutic agent are capable of expressing the agent proximal to a tumor. The cells can be modified in vivo, or alternatively cells modified ex vivo can be administered to a patient by a variety of methods, such as by injection.

[00169] In one embodiment, the cell is a tumor cell. For ex vivo transformation, such tumor cells can be irradiated to eliminate the ability of the cell to replicate, as known in the art, while maintaining the transient expression of the therapeutic agent after administration. For in vivo transformation, non-integrative expression vectors may be preferred.

[00170] In certain embodiments, the tumor cell is autologous or endogenous. In the former instance, the tumor cell is taken from a patient, transfected or transduced with a construct encoding the therapeutic agent and re-introduced to the patient, for example after irradiation. In the latter instance, the tumor cell is transformed in vivo by local administration of an appropriate construct as described herein.

[00171] In an alternative embodiment, the modified tumor cell is allogeneic. The allogeneic tumor cell thus can be maintained in a cell line. In this instance, the tumor cell can be selected from the cell line, irradiated, and introduced to the patent.

[00172] Modified human cells capable of producing the flagellin derivatives can be made by transfecting or transducing the cells with an expression vector encoding the therapeutic agent. Expression vectors for the expression of the flagellin derivative, or a combination of therapeutic agents can be made by methods well known in the art.

[00173] In various embodiments, the flagellin derivatives can be administered to a patient in the form of one or more nucleic acid construct.

[00174] In one embodiment, the construct comprises a retroviral vector. Retroviral vectors are capable of permanently integrating DNA encoding flagellin derivatives into the cell genome. Thus, in the case of ex vivo manipulation of autologous or allogeneic cells, stable cell lines that constitutively produce the flagellin derivatives (and/or additional agents) can be prepared. In an embodiment, the cells are irradiated prior to administration to a patient. The irradiated cells produce the flagellin derivatives for a limited period of time.

[00175] In one embodiment, the expression construct comprises an SFV vector, which demonstrates high levels of transient expression in mammalian cells. The SFV vector is described, for example, in Lundstrom, Expert Opin. Biol. Ther. 3:771-777 (2003), incorporated herein by reference in its entirety. Thus, in the case of in vivo manipulation of endogenous cells in a patient, transient expression of high levels of the flagellin derivatives (and/or additional agents) can be accomplished.

[00176] Systems capable of expressing recombinant protein in vivo are known in the art. By way of example, the system can use the 2A mediated antibody expression system disclosed in Fang et al., Nature Biotech. 23(5): 584-590 (2005) and U.S. Patent Publication No. 2005/0003506, the disclosures of which are expressly incorporated by reference herein in their entirety. Other systems known in the art are contemplated, and can also be adapted to produce the flagellin derivatives (and/or additional agents) in vivo as described herein.

[00177] In various embodiments, administration of the flagellin derivative (and/or additional agents) expressing cells disclosed herein or the agents of the invention disclosed herein can be combined with administration of cytokines that stimulate antigen-presenting cells such as granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interferon, efc, or cellular vaccines capable of expressing such cytokines. In some embodiments, the flagellin derivative (and/or additional agents) expressing cells are further modified to express such cytokines. Additional proteins and/or cytokines known to enhance T cell proliferation and secretion, such as IL-1 , IL-2, B7, anti-CD3 and anti-CD28 can be employed simultaneously or sequentially with the flagellin derivatives (and/or additional agents) of the invention to augment the immune response, and/or stimulate co-stimulatory pathways and/or induce activation/proliferation of effector T cells.

Vectors and Methods of Transformation

[00178] Expression vectors encoding the flagellin derivatives may be viral or non-viral. Viral vectors are preferred for use in vivo. Expression vectors of the invention comprise a nucleic acid encoding the flagellin derivatives (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.

[00179] Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid.

[00180] An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell. [00181] In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[00182] In an embodiment, the present invention contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. When in the proximity of a tumor cell, a cell transformed with an expression vector for the flagellin derivatives comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Exemplary inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.

[00183] Expression control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).

[00184] As used herein, "operable linkage" refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream"). Expression control regions can also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

[00185] Expression systems functional in human cells are well known in the art, and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a B7-H4 ligand coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

[00186] Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.

[00187] There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also preferred for in vivo transduction. In some situations it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu ei a/., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner ei a/., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

[00188] Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby ei a/., Nucleic Acids Res. 26:391- 406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk ei a/., TIG 15:326-332, 1999; Kootstra ei a/., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, ei a/., Cell, 29:227-234, 1982), R (Matsuzaki, ei a/., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth ei a/., J. Mol. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family (Plasterk ei a/., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra ei a/., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). . Viral Vectors

[00189] In one aspect, the invention provides expression vectors for the expression of the flagellin derivatives that are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21 : 1 17, 122, 2003.

[00190] Exemplary viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are preferred, such as a viruses and adenoviruses, with a viruses being especially preferred. Exemplary types of a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV), with SFV being especially preferred. For in vitro uses, viral vectors that integrate into the host genome are preferred, such as retroviruses, AAV, and Antiviruses.

[00191] In an embodiment, the viral vector provides for transient high level expression in a transduced human cell.

[00192] In one embodiment, the viral vector does not provide for integration of the flagellin derivative (and/or additional agents) encoding nucleic acid into the genome of a transduced human cell.

[00193] In another embodiment, the viral vector provides for integration of the flagellin derivatives (and/or additional agents) encoding nucleic acid into the genome of a transduced human cell.

[00194] In one embodiment, the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.

[00195] In another embodiment, the invention provides methods of transducing a human cell ex vivo, comprising contacting a human cell ex vivo with the viral vector of the invention. In one embodiment, the human cell is a tumor cell. In one embodiment, the human cell is allogeneic. In one embodiment, the tumor cell is derived from the patient. In one embodiment, the human cell is a non-tumor cell, such as, e.g., an antigen presenting cell (APC), or a T cell.

[00196] Virus particle coats may be modified to alter specificity and improve cell/tissue targeting, as is well known in the art. Viral vectors may also be delivered in other vehicles, for example, liposomes. Liposomes may also have targeting moieties attached to their surface to improve cell/tissue targeting.

[00197] In some embodiments, the present invention provides human cells expressing the therapeutic agent of the invention. In various embodiments, the human cells express the agent proximal to a tumor cell of, for example, a patient.

Diagnostic and Predictive Methods

[00198] In some aspects, the invention provides a method for identifying a subject who may respond to treatment with a TLR5 agonist. In some embodiments, the present invention provides a method of determining if a patient's tumor expresses TLR5. [00199] TLR5 expression may be a predictive marker for determining the grade and/or progression of a patient's tumor or dysplasia. In some embodiments, the flagellin derivative (and/or additional agents) described herein are useful in determining a tumor grade and/or stage of a particular cancer.

[00200] Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade may vary with each type of cancer and are known in the art.

[00201] Histologic grade, also called differentiation, refers to how much the tumor cells resemble normal cells of the same tissue type. Nuclear grade refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing.

[00202] Based on the microscopic appearance of cancer cells, pathologists commonly describe tumor grade by four degrees of severity: Grades 1 , 2, 3, and 4. The cells of Grade 1 tumors resemble normal cells, and tend to grow and multiply slowly. Grade 1 tumors are generally considered the least aggressive in behavior. Conversely, the cells of Grade 3 or Grade 4 tumors do not look like normal cells of the same type. Grade 3 and 4 tumors tend to grow rapidly and spread faster than tumors with a lower grade. The American Joint Committee on Cancer recommends the following guidelines for grading tumors: GX-grade cannot be assessed (Undetermined grade); G1 -well-differentiated (Low grade); G2-moderately differentiated (Intermediate grade); G3-poorly differentiated (High grade); and G4-u nd iffe re nt iated (High grade).

[00203] Grading systems are different for each type of cancer. For example, pathologists use the Gleason system to describe the degree of differentiation of prostate cancer cells. The Gleason system uses scores ranging from Grade 2 to Grade 10. Lower Gleason scores describe well-differentiated, less aggressive tumors. Higher scores describe poorly differentiated, more aggressive tumors. Other grading systems include, for example, the Bloom-Richardson system for breast cancer and the Fuhrman system for kidney cancer.

[00204] Cancer survival rates or survival statistics may refer to the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. For example the overall five-year survival rate for bladder cancer is 80 percent, i.e. 80 of every 100 of people diagnosed with bladder cancer were living five years after diagnosis and 20 out of every 100 died within five years of a bladder cancer diagnosis. Other types of survival rates may be used, for example: disease-free survival rate (number of people with cancer who achieve remission) and progression-free survival rate, (number of people who still have cancer, but their disease is not progressing).

[00205] In some embodiments, the flagellin derivative (and/or additional agents) described herein are useful in establishing a tumor grade for the purposes of diagnosis or prognosis of a particular cancer, including prognosing the survival rate, disease-free survival rate and/or progression-free survival rate prior to, during and/or after administration of a flagellin derivative (and/or additional agents) disclosed herein and/or prior to, during and/or after administration of an anti-cancer agent or therapy. [00206] In some embodiments, the flagellin derivative (and/or additional agents) described herein are used as part of a method of scoring tumor grades to assist in the selection and/or predict the outcome of treatment. For example, the flagellin derivatives (and/or additional agents) described herein may be used to diagnose or identify the cancer from a patient as stage I (e.g. not locally advanced) predicting the need for less aggressive treatment. Alternatively, the therapeutic agent described herein may be used to diagnose or identify the cancer from a patient as stage II or III, (e.g. the cancer may be locally advanced) predicting the need for more aggressive treatment. Similarly, the flagellin derivatives (and/or additional agents) described herein may be used to diagnose or identify the cancer from a patient as stage IV, or is metastatic, predicting the need for very aggressive treatment.

[00207] In some embodiments, the cancer is non-resectable. A non-resectable cancer is a malignancy which cannot be surgically removed, due either to the number of metastatic foci, or because it is in a surgical danger zone. In some embodiments, the therapeutic agent described herein is used as part of a method of treating tumors to assist in selecting the nature and/or timing/administration of treatment including, for example, administering anti-cancer agents which reduce tumor volume, prior to chemotherapeutic and/or radiation treatment, and/or increase or decrease the dose of chemotherapy or radiation administered to a patient.

[00208] In some embodiments, the cancer is multidrug resistant. For example, the patient may have undergone one or more cycles of chemotherapy, without substantial response. Alternatively or in addition, the tumor has one or more markers of multidrug resistance. Thus, as used herein, the term multidrug resistant means a cancer exhibiting non-responsiveness to at least one cycle of combination chemotherapy, or alternatively, has scored (diagnostically) as resistant to at least two of (including comparable agent to) docetaxel, paclitaxel, doxorubicin, epirubicin, carboplatin, cisplatin, vinblastine, vincristine, oxaliplatin, carmustine, fluorouracil, gemcitabine, cyclophosphamide, ifosfamide, topotecan, erlotinib, etoposide, and mitomycin. In some embodiments, the therapeutic agents described herein are useful in establishing whether the tumor is responsive to one or more chemotherapeutics, radiation therapy and/or other anti-cancer therapy.

[00209] In other embodiments, the cancer is a recurrence following conventional chemotherapy of an initial cancer. Often, recurrent cancer has developed drug resistance, and thus is particularly difficult to treat and often comes with a poor prognosis for survival.

[00210] In some embodiments, the flagellin derivative (and/or additional agents) described herein are used as part of a method of tumor evaluation which takes the place of a performance status. Performance status can be quantified using any system and methods for scoring a patient's performance status which are known in the art. The measure is often used to determine whether a patient can receive chemotherapy, dose adjustment, and/or to determine intensity of palliative care. There are various scoring systems, including the Karnofsky score and the Zubrod score. Parallel scoring systems include the Global Assessment of Functioning (GAF) score, which has been incorporated as the fifth axis of the Diagnostic and Statistical Manual (DSM) of psychiatry. [00211] Higher performance status (e.g., at least about 80%, or at least about 70% using the Karnofsky scoring system) may indicate treatment to prevent progression of the disease state, and enhance the patient's ability to accept chemotherapy and/or radiation treatment. For example, when the therapeutic agent described herein indicates higher performance status, the patient is ambulatory and capable of self care. In other embodiments, when the therapeutic agent described herein indicates a low performance status (e.g., less than about 50%, less than about 30%, or less than about 20% using the Karnofsky scoring system), the patient is largely confined to bed or chair and is disabled even for self-care.

[00212] The Karnofsky score runs from 100 to 0, where 100 is "perfect" health and 0 is death. The score may be employed at intervals of 10, where: about 100% is normal, no complaints, no signs of disease; about 90% is capable of normal activity, few symptoms or signs of disease, about 80% is normal activity with some difficulty, some symptoms or signs; about 70% is caring for self, not capable of normal activity or work; about 60% is requiring some help, can take care of most personal requirements; about 50% requires help often, requires frequent medical care; about 40% is disabled, requires special care and help; about 30% is severely disabled, hospital admission indicated but no risk of death; about 20% is very ill, urgently requiring admission, requires supportive measures or treatment; and about 10% is moribund, rapidly progressive fatal disease processes.

[00213] The Zubrod scoring system for performance status includes: 0, fully active, able to carry on all pre- disease performance without restriction; 1 , restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work; 2, ambulatory and capable of all self-care but unable to carry out any work activities, up and about more than about 50% of waking hours; 3, capable of only limited self-care, confined to bed or chair more than about 50% of waking hours; 4, completely disabled, cannot carry on any self-care, totally confined to bed or chair; 5, dead.

[00214] In some embodiments, histological samples of tumors are graded using the therapeutic agent described herein according to Elston & Ellis, Histopathology, 1991 , 19:403-10, which is hereby incorporated by reference in its entirety. In some embodiments, the therapeutic agent described herein is useful in establishing a tumor grade for the purposes of diagnosis or prognosis of a particular cancer.

[00215] In some embodiments, the flagellin derivatives (and/or additional agents) described herein are useful for evaluating a subject and/or a specimen from a subject (e.g. a cancer patient). In some embodiments, evaluation is one or more of diagnosis, prognosis, and/or response to treatment.

[00216] Diagnosis refers to the process of attempting to determine or identify a possible disease or disorder, such as, for example, cancer. Prognosis refers to the predicting of a likely outcome of a disease or disorder, such as, for example, cancer. A complete prognosis often includes the expected duration, the function, and a description of the course of the disease, such as progressive decline, intermittent crisis, or sudden, unpredictable crisis. Response to treatment is a prediction of a patient's medical outcome when receiving a treatment. Responses to treatment can be, by way of non-limiting example, pathological complete response, survival, and probability of recurrence. [00217] In various embodiments, the diagnostic and predictive methods described herein comprise evaluating a presence, absence, or level of a protein. In another embodiment, the methods described herein comprise evaluating a presence, absence, or level of expression of a nucleic acid. The compositions described herein may be used for these measurements. For example, in some embodiments, the methods described herein comprise contacting a specimen of the tumor or cells cultured from the tumor with a therapeutic agent as described herein.

[00218] In some embodiments, the present invention includes the measurement of a tumor specimen, including biopsy or surgical specimen samples. In some embodiments, the biopsy is a human biopsy. In various embodiments, the biopsy is any one of a frozen tumor tissue specimen, cultured cells, circulating tumor cells, and a formalin-fixed paraffin-embedded tumor tissue specimen. In some embodiments, the tumor specimen may be a biopsy sample, such as a frozen tumor tissue (cryosection) specimen. As is known in the art, a cryosection may employ a cryostat, which comprises a microtome inside a freezer. The surgical specimen is placed on a metal tissue disc which is then secured in a chuck and frozen rapidly to about -20°C to about -30°C. The specimen is embedded in a gel like medium consisting of, for example, poly ethylene glycol and polyvinyl alcohol. The frozen tissue is cut frozen with the microtome portion of the cryostat, and the section is optionally picked up on a glass slide and stained. In some embodiments, the tumor specimen may be a biopsy sample, such as cultured cells. These cells may be processed using the usual cell culture techniques that are known in the art. These cells may be circulating tumor cells. In some embodiments, the tumor specimen may be a biopsy sample, such as a formalin-fixed paraffin-embedded (FFPE) tumor tissue specimen. As is known in the art, a biopsy specimen may be placed in a container with formalin (a mixture of water and formaldehyde) or some other fluid to preserve it. The tissue sample may be placed into a mold with hot paraffin wax. The wax cools to form a solid block that protects the tissue. This paraffin wax block with the embedded tissue is placed on a microtome, which cuts very thin slices of the tissue. In certain embodiments, the tumor specimen contains less than about 100 mg of tissue, or in certain embodiments, contains about 50 mg of tissue or less. The tumor specimen (or biopsy) may contain from about 20 mg to about 50 mgs of tissue, such as about 35 mg of tissue. The tissue may be obtained, for example, as one or more (e.g., 1 , 2, 3, 4, or 5) needle biopsies (e.g., using a 14-gauge needle or other suitable size). In some embodiments, the biopsy is a fine-needle aspiration in which a long, thin needle is inserted into a suspicious area and a syringe is used to draw out fluid and cells for analysis. In some embodiments, the biopsy is a core needle biopsy in which a large needle with a cutting tip is used during core needle biopsy to draw a column of tissue out of a suspicious area. In some embodiments, the biopsy is a vacuum-assisted biopsy in which a suction device increases the amount of fluid and cells that is extracted through the needle. In some embodiments, the biopsy is an image-guided biopsy in which a needle biopsy is combined with an imaging procedure, such as, for example, X ray, computerized tomography (CT), magnetic resonance imaging (MRI) or ultrasound. In other embodiments, the sample may be obtained via a device such as the MAMMOTOME® biopsy system, which is a laser guided, vacuum-assisted biopsy system for breast biopsy.

[00219] In some embodiments, the diagnostic and predictive methods and/or evaluation may direct treatment (including treatment with the therapeutic agents described herein). In one embodiment, the evaluation may direct the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also called adjuvant care, is treatment that is given in addition to the primary, main or initial treatment. By way of non-limiting example, adjuvant therapy may be an additional treatment usually given after surgery where all detectable disease has been removed, but where there remains a statistical risk of relapse due to occult disease. In some embodiments, the therapeutic agents described herein are used as an adjuvant therapy in the treatment of a cancer. In some embodiments, the therapeutic agents described herein are used as the sole adjuvant therapy in the treatment of a cancer. In some embodiments, the therapeutic agents described herein are withheld as an adjuvant therapy in the treatment of a cancer. For example, if a patient is unlikely to respond to a therapeutic agent described herein or will have a minimal response, treatment may not be administered in the interest of quality of life and to avoid unnecessary toxicity from ineffective chemotherapies. In such cases, palliative care may be used.

[00220] In some embodiments the therapeutic agents described herein are administered as a neoadjuvant therapy prior to resection. In certain embodiments, neoadjuvant therapy refers to therapy to shrink and/or downgrade the tumor prior to any surgery. In some embodiments, neoadjuvant therapy means chemotherapy administered to cancer patients prior to surgery. In some embodiments, neoadjuvant therapy means a therapeutic agent described herein is administered to cancer patients prior to surgery. Types of cancers for which neoadjuvant chemotherapy is commonly considered include, for example, breast, colorectal, ovarian, cervical, bladder, and lung. In some embodiments, the therapeutic agents described herein are used as a neoadjuvant therapy in the treatment of a cancer. In some embodiments, the use is prior to resection. In some embodiments, the therapeutic agents described herein are withheld as a neoadjuvant therapy in the treatment of a cancer. For example, if a patient is unlikely to respond to a therapeutic agent described herein or will have a minimal response, treatment may not be administered in the interest of quality of life and to avoid unnecessary toxicity from ineffective chemotherapies. In such cases, palliative care may be used.

Subjects and/or Animals

[00221] In some embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In some embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g. GFP). In some embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.

[00222] In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient.

[00223] In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

[00224] In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.

Ms

[00225] The invention provides kits that can simplify the administration of any agent described herein. An exemplary kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.

Definitions

[00226] The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.

[00227] As used herein, "a," "an," or "the" can mean one or more than one.

[00228] Further, the term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language "about 50" covers the range of 45 to 55.

[00229] As used herein, an "extremophile" refers to an organism that thrives in physically or geochemically extreme conditions that are detrimental to most life on Earth. A "thermophile" is a type of extremophile and refers to an organism that can thrive at temperatures between about 45 to about 122°C. A "hyperthermophile" refers to an extreme thermophile that requires a very high temperature (e.g., about 80°C to about 105°C) for growth.

[00230] An "effective amount," when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

[00231] As used herein, something is "decreased" if a read-out of activity and/or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in some embodiments, activity is decreased and some downstream read-outs will decrease but others can increase.

[00232] Conversely, activity is "increased" if a read-out of activity and/or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

[00233] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "include," and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms "can" and "may" and their variants are intended to be non- limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[00234] Although the open-ended term "comprising," as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of or "consisting essentially of."

[00235] As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[00236] The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents (e.g. flagellin derivatives (and/or additional agents) described herein) for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A "pharmacologically effective amount," "pharmacologically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. For example, administration of therapeutic agents to a patient suffering from cancer provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the disease, e.g., a decrease in tumor burden, a decrease in circulating tumor cells, an increase in progression free survival. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[00237] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[00238] In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

[00239] In certain embodiments, a pharmacologically effective amount that will treat cancer will modulate the symptoms typically by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In exemplary embodiments, such modulations will result in, for example, statistically significant and quantifiable changes in the numbers of cancerous cells.

[00240] This invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1 : Engineering of flaqellin derivatives with improved efficacy and reduced antigenicity

[00241] Various flagellin constructs were engineered from flagellin derived from Lactobacillus ruminis (Lru), Thermotoga petrophila (Tpe), or Carboxydothermus hydrogenoformans (Chy). The first set of constructs included the ND0, ND1 , CD0, and CD1 domains {i.e., Lru283, Tpe270, or Chy275; see Figure 1 A). To further minimize the size of the flagellin derivatives, a second set of constructs were developed which lacked the NDO and CDO domains and included only the ND1 and CD1 domains (i.e., Tpe159 or Chy162; see Figure 1 B). The amino acid sequences of these constructs are provided in Table 1. The flagellin constructs were expressed in E. coli, purified, and characterized. Figure 2 shows purified preparations of the flagellin constructs after metal affinity chromatography using either the Pierce HisPur Co++ column or the Sigma His-select plate.

[00242] A mammalian cell-based reporter assay was used to determine the ability of the flagellin constructs to induce NF-κΒ. Specifically, the activities were tested in HEK293 cells that have been engineered to induce activation of a NF-κΒ regulated reporter gene (lacZ) via binding to the TLR5 receptor (i.e., HEK293-hTLR5:: NF- κΒ-lacZ reporter cells). Further, to test the antigenicity of the constructs, the assays were carried out in the presence of increasing amounts various anti-CBLB502 or anti-flagellin antibodies.

[00243] Figures 3A-3D show that Lru283, Tpe270, Chy275, Tpe159w and Chy162w all activated the NF-KB regulated lacZ reporter gene in vitro. Surprisingly, the truncated constructs Tpe159w and Chy162w were just as active as the longer constructs Lru283, Tpe270, or Chy275. This is in direct contrast to previous studies by, for example, Eaves-Pyles 2003, Murthy 2004, and DiDonato 2005, which indicate that deletions removing more than approximately 55 amino acids from either the N-terminus or C-terminus of flagellin would render the protein inactive. In fact, the activities of Tpe270, Chy275, Tpe159w and Chy162w were comparable and even higher than CBLB502. The truncated flagellin construct Chy162w derived from Carboxydothermus hydrogenoformans appeared to be the most active flagellin derivative.

[00244] The antigenicity of the flagellin constructs was also tested. Specifically, it was discovered that none of the flagellin constructs were neutralized by any of the anti-CBLB502 or anti-flagellin antibodies (either mouse mAb or human pAb; either pre-existing or CBLB502-induced). In contrast, the anti-CBLB502 or anti-flagellin antibodies completely neutralized the in vitro activity of CBLB502. See Figures 3A-3D. The antigenicity of the flagellin constructs were further tested using CBLB502-boosted sera or pre-immune normal human sera. As indicated in Figures 4A-4E and 5A-5E, whereas the activity of CBLB502 was neutralized by both CBLB502- boosted sera or pre-immune normal human sera, the Tpe270, Chy275, Tpe159w and Chy162w variants remained active in the presence of both types of sera.

[00245] Based on these observations, additional truncated variants of Chy162w were constructed (i.e., ChyU137, ChyN108, ChyZ94, Fir161 B, and Fir161 MNB; see Figures 6A-6D). The amino acid sequences of the variants are provided in Table 1. The flagellin constructs were expressed in E. coli, purified, and characterized. Figure 7 shows purified preparations of the Chy162w variants after metal affinity chromatography using the Sigma His-select iLAP-5 column. As seen, ChyU137, ChyN108, ChyZ94 were barely if at all expressed or purified. Due to their smaller sizes, these variants may have been lost or gone into other fractions besides the 2M urea fraction. [00246] Purified fractions of the Chy162w variants were also tested for their ability to activate a NF-KB regulated lacZ reporter using an in vitro assay as previously described. As seen in Figures 8A-8F, only the ChyU137 variant showed in vitro activity.

[00247] Further protein purifications were carried out to obtain additional fractions (e.g., 2M urea, soluble, and pellet fractions) of Tpe270, Chy275, Tpe159w, Chy162w, ChyU137, ChyN108, ChyZ94, R 61 B, and Fir161 MNB. See Figure 9. In addition, the activity of these additional fractions were also tested, particularly the activities of the soluble fractions were compared to the 2M urea fractions. As seen in Figures 10A-10D, the ChyU137 and ChyN108 fractions appeared to have some activity after proper purifications. These variants may be further stabilized to provide moderate activity and low molecular weight TLR5 ligands. Further, even the smallest ChyZ94 variant exhibited some activity in vitro.

EQUIVALENTS

[00248] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[00249] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

[00250] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[00251] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00252] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:

1. A composition comprising an engineered flagellin derivative from a thermophilic microorganism comprising a NDO domain, a ND1 domain, a CD1 domain and a CDO domain or fragments thereof.

2. The composition of claim 1 , wherein the microorganism is a hyperthermophile.

3. The composition of claim 1 or 2, wherein the microorganism is selected from Firmicutes, Thermotogae, or Aquificae.

4. The composition of claim 3, wherein the microorganism is Thermotoga petrophila.

5. The compositionof claim 3, wherein the microorganism is Carboxydothermus hydrogenoformans.

6. A composition comprising flagellin derivative from a microorganism well-tolerated by human comprising a tag, a NDO domain, a ND1 domain, a linker, a CD1 domain and a CDO domain or fragments thereof.

7. The composition of claim 6, wherein the microorganism is from a human microflora.

8. The composition of claim 7, wherein the microorganism is from Lactobacillus.

9. The composition of claim 8, wherein the microorganism is Lactobacillus ruminis.

10. The composition of any one of claims 1-9, wherein the flagellin derivative comprises a deletion of one or more residues in one or more domains.

11. The composition of claim 10, wherein the flagellin derivative comprises a deletion of one or more residues in a N-terminal domain.

12. The composition of claim 11 , wherein the flagellin derivative comprises a deletion of one or more residues in the NDO domain.

13. The composition of claim 12, wherein the flagellin derivative comprises a deletion of the entire NDO domain.

14. The composition of any of claims 1-13, wherein the flagellin derivative comprises a deletion of one or more residues in a C-terminal domain.

15. The composition of claim 14, wherein the flagellin derivative comprises a deletion of one or more residues in the CDO domain.

16. The composition of claim 15, wherein the flagellin derivative comprises a deletion of the entire CDO domain.

17. The composition of claim 10, wherein the flagellin derivative consists essentially of a NDO, ND1 , CDO, and CD1 domain.

18. The composition of claim 10, wherein the flagellin derivative consists essentially of a ND1 and CD1 domain.

19. The composition of any one of the above claims, wherein the flagellin derivative comprises a sequence selected from SEQ ID NOs: 18-27.

20. The composition of claim 19, wherein the flagellin derivative comprises a sequence that is 30-99% identical to SEQ ID NOs: 18-27.

21. The composition of any of the above claims, wherein the flagellin derivative retains the ability to activate TLR5 signaling.

22. The composition of any of the above claims, wherein the flagellin derivative activates TLR5 signaling at a level the same as, or similar to, that of a SEQ ID NO: 2.

23. The composition of any of the above claims, wherein the flagellin derivative comprises mutations that decrease the antigenicity and immunogenicity of the construct compared with SEQ ID NO: 2.

24. The composition of any of the above claims, wherein the flagellin derivative demonstrates improved pharmacokinetics compared with SEQ ID NO: 2.

25. The composition of any of the above claims, wherein the flagellin derivative demonstrates increased retention in the host.

26. The composition of any of the above claims, wherein the flagellin derivative comprises mutations in epitopes recognized by B cells.

27. The composition of any of the above claims, wherein the flagellin derivative comprises mutations in epitopes recognized by T cells.

28. The composition of any of the above claims, wherein the flagellin derivative further comprises a tag.

29. The composition of claim 28, wherein the flagellin derivative comprises a N-terminal tag.

30. The composition of claim 28, wherein the flagellin derivative comprises a C-terminal tag.

31. The composition of any of the above claims, wherein the flagellin derivative comprises a flexible linker.

32. The composition of claim 31 , wherein the flexible linker comprises SEQ ID NO: 16.

33. The composition of claim 31 , wherein the flexible linker comprises SEQ ID NO:17.

34. The composition of any of the above claims, wherein the flagellin derivative induces expression of one or more of cytokines.

35. The composition of claim 34, wherein the flagellin derivative induces expression of one or more of the cytokines selected from IL-6, IL-12, IL-8, keratinocyte chemoattractant (KC), IL-10, G-CSF, MCP-1 , TNF-a, MIG, and MIP-2.

36. A pharmaceutical composition comprising the flagellin derivative of any of the above claims and a pharmaceutically accepted carrier.

37. A method of stimulating TLR5 signaling comprising administering any one of a flagellin derivative derived from SEQ ID NOs: 18-27 to a subject in need thereof.

38. The method of claim 37, wherein the subject suffers from cancer.

39. The method of claim 38, wherein the cancer comprises a tumor which expresses TLR5 or a tumor which does not expresses TLR5.

40. The method of claim 38, wherein the cancer is selected from breast cancer, lung cancer, colon cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkett's lymphoma, acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, promyelocytic leukemia, astrocytoma, neuroblastoma, glioma, schwannomas, fibrosarcoma, rhabdomyoscarcoma, osteosarcoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, and cancers of the gastrointestinal tract or the abdominopelvic cavity.

41. The method of claim 37, wherein the subject suffers from radiation-induced damage.

42. The method of claim 41 , wherein the subject has been subjected to a lethal dose of radiation.

43. The method of claim 41 , wherein the subject is undergoing radiation treatment.

44. The method of claim 41 , wherein the flagellin derivative is administered prior to exposure to radiation.

45. The method of claim 41 , wherein the flagellin derivative is administered during exposure to radiation.

46. The method of claim 41 , wherein the flagellin derivative is administered after exposure to radiation.

47. The method of claim 37, wherein the subject suffers from reperfusion injury.

48. The method of claim 47, wherein the reperfusion injury is caused by ischemia or hypoxia.

49. The method of claim 47, wherein the flagellin derivative is administered prior to the influx of oxygen.

50. The method of claim 47, wherein the flagellin derivative is administered during the influx of oxygen.

51. The method of claim 47, wherein the flagellin derivative is administered after the influx of oxygen.

52. The method of claims 37-51 , wherein the flagellin derivative is administered in conjunction with other therapeutics and/or treatments.

53. The method of claim 52, wherein the flagellin derivative is administered in conjunction with chemotherapy.

54. The method of claim 52, wherein the flagellin derivative is administered with radiation treatment.

55. The method of claim 52, wherein the flagellin derivative is administered in conjunction with an antioxidant.

56. The method of claim 52, wherein the flagellin derivative is administered prior to administration of other therapeutics and/or treatments.

57. The method of claim 52, wherein the flagellin derivative is administered at the same time as other therapeutics and/or treatments.

58. The method of claim 52, wherein the flagellin derivative is administered after administration of other therapeutics and/or treatments.

59. A method of treating cancer comprising administering a flagellin derivative selected from SEQ ID NOs: 18-27 to a subject in need thereof.

60. A method of treating radiation-induced damage comprising administering a flagellin derivative selected from SEQ ID NOs: 18-27 to a subject in need thereof.

61. A method of treating reperfusion injury comprising administering a flagellin derivative selected from SEQ ID NOs: 18-27 to a subject in need thereof.

62. A method of treating or preventing of acute radiation syndrome (ARS) comprising administering a flagellin derivative selected from SEQ ID NOs: 18-27 to a subject in need thereof.