US 2010O239583A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0239583 A1 MURTHY et al. (43) Pub. Date: Sep. 23, 2010

(54) ANTIBODIES AGAINST FLAGELLIN AND C07K I6/46 (2006.01) USES THEREOF CI2N 5/16 (2006.01) C7H 2L/04 (2006.01) (75) Inventors: KannegantiMURTHY, Stoneham, CI2N 5/3 (2006.01) MA (US); Andrew L. SALZMAN, AOIN 63/02 (2006.01) Herzliya (IL) AOIP I/00 (2006.01) A6IP3L/04 (2006.01) Correspondence Address: A6IP3L/08 (2006.01) LAHIVE & COCKFIELD, LLP A6IP3L/06 (2006.01) FLOOR 30, SUITE 3000 A6IPI3/02 (2006.01) ONE POST OFFICE SQUARE A6IP 7/00 (2006.01) BOSTON, MA 02109 (US) GOIN 33/53 (2006.01) (73) Assignee: NOTEKPHARMACEUTICALS CORPORATION, Beverly, MA (52) U.S. Cl...... 424/136.1; 530/388.4:530/389.5; (US) 530/387.9; 530/387.3; 530/388.15:435/340: 530/3917; 424/150.1; 424/164.1; 424/178.1; (21) Appl. No.: 12/717,351 536/23.53; 435/320.1; 435/7.1 (22) Filed: Mar. 4, 2010 (57) ABSTRACT Related U.S. Application Data The present invention provides a novel class of monoclonal (60) Provisional application No. 61/209,189, filed on Mar. antibodies which have a high affinity, broad spectrum neu 4, 2009. tralizing reactivity to flagellin from various Gram-negative O O including, but not limited to, E. coli, Salmonella, Publication Classification Serratia, Proteus, Enterobacter Citrobacter, Campylobacter (51) Int. Cl. and Pseudomonas. The present invention further provides A6K 39/395 (2006.01) methods of treating infections and diseases using anti-flagel C07K 6/12 (2006.01) lin antibodies in humans, other animals and birds. Patent Application Publication Sep. 23, 2010 Sheet 1 of 4 US 2010/02395.83 A1

mAb 743 anti-flagellin monoclonal antibody reacts strongly with type A flagellins and Weakly with type B

1. PAKwt (type A) 2. PAK (flagellin mutant) 3. PAO wt (type B) - 4. PAO (flagellin mutant) M 4 3 2 1 M

Fig. 1 Patent Application Publication Sep. 23, 2010 Sheet 2 of 4 US 2010/02395.83 A1

mAb 743 antibody cross-reacts with flagellins from various gram-negative bacteria O.800 | O.7OO- -- -(- Non Pathogenic E. Coli O600- -- Citobacter S. 0.500- -- s 0.400- -A - Pseudomonas aeruginosa 12691 g - A - Salmonella typhimurium 0.300- -O- 0.2OO- -O- Enteropathogenic E. Coli 0.100 0000-- -T- 0.01 uglml 0.1 uglml 1 ugliml

Fig. 2 Patent Application Publication Sep. 23, 2010 Sheet 3 of 4 US 2010/02395.83 A1

g.314 ?|N\/IV/OSOV/V/O,CIXIV/SN|?715)ST>ee Patent Application Publication Sep. 23, 2010 Sheet 4 of 4 US 2010/0239583 A1

mAb 743 inhibits flagellin activity in NO production assay

80 : FLAGELLIN FLAGELLIN+ 2 FLAGELLIN + Non mAb 743 Specific Mab

4060 2 y % % 2 O 2 32 % % 3 FLA (0.5 ng/ml) FLA (0.5 ng/ml) FLA (2.5 ng/ml) FLA (2.5 ng/ml) + 1 UAb + 5UAb + 1 ul Ab + 5u Ab F LA ( 9 | m Fig. 4 US 2010/02395.83 A1 Sep. 23, 2010

ANTIBODIES AGAINST FLAGELLIN AND including, but not limited to, gram-negative bacteria, Such as USES THEREOF E. coli, Salmonella, Serratia, Proteus, Enterobacter; Citro bacter; Campylobacter and Pseudomonas. Accordingly, the RELATED APPLICATION antibodies of the present invention can be used to treat, pre 0001. This application claims the benefit of U.S. provi vent and diagnose a variety of bacterial diseases associated sional application No. 61/209,189, filed Mar. 4, 2009, which with flagellin, including both infectious and non-infectious is incorporated by reference herein in its entirety. diseases in humans, other animals and birds. 0008 Antibodies of the invention generally are character GOVERNMENT FUNDING ized as having one or more of the following properties: (i) neutralization (i.e., inhibition) of bacterial flagellin, (includ 0002 This invention was made with government support ing flagellin bound to bacteria or “free', circulating flagellin under National Institutes of Health Grant it's R01 GM57407, in the systemic circulation); (ii) cross-reactivity with flagellin R43AI48249, R29GM54773 and R01 GM60699. The United from a broad spectrum of bacteria; (iii) inhibition of bacterial States government has certain rights in the invention. invasion into Susceptible epithelial cells; (iv) binding to flagellin with an affinity of at least 10'M', (v) reduction or BACKGROUND OF THE INVENTION prevention of flagellin-induced tissue injury; (vi) reduction or 0003. The human intestine is colonized by a large and prevention of flagellin-stimulated neutrophil infiltration; (vii) diverse population of commensal bacteria and, on occasion, is reduction or prevention of colonic mucosal congestion, ero exposed to potentially . One particular sion and/or hemorrhagic ulcerations associated with IBD; subset of intestinal bacteria have flagella, which are whip-like and (viii) reduction or prevention of cytokine production, organelles that attach to a rotatory motor embedded in the including MDA, IL-1 B, TNFC, MIP-1, MIP-2, IL-6 and IL-8, bacterial cell wall. Flagella provide bacteria with motility and and pro-inflammatory free radical synthesizing enzymes, enable these microbes to reach, adhere and eventually invade such as the inducible nitric-oxide synthases; (ix) ability to or colonize a particular niche in their host. An individual opSonize bacteria; and (X) ability to promote macrophage flagellum is composed of approximately 20,000 subunits of ingestion of bacteria. U.S. patent application Ser. No. 12/231, the monomeric protein flagellin. Due to physical constraints 777, U.S. patent application Ser. No. 12/231,797, PCT Appli by its function, flagellin has a relatively conserved structure cation No.: PCT/US2008/075387, and PCT Application No. among widely diverse bacterial species (Steiner, T. S. Infect PCT/US2008/075383, teach anti-flagellin antibodies and all Immun. 2007 February: 75(2):545-52). are incorporated herein by reference in their entireties. 0004 Flagellin is highly antigenic and is a major immu 0009 From a structural standpoint, particular representa noglobulin target in a variety of infectious events (Sitaraman tive antibodies of the invention include a heavy chain variable et al., Am J Physiol Gastrointest Liver Physiol. 2005 Febru region comprising an amino acid sequence which is at least ary; 288(2):G403-6). As such, it is a potent and direct activa 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identi tor of the innate immune system. From the perspective of the cal to the heavy chain variable region amino acid sequence set host, flagellin is a microbial-associated molecular pattern forth in SEQ ID NO:1. Other particular antibodies of the (MAMP) i.e., a microbial-associated determinant that can be present invention include a light chain variable region com perceived by the innate immune system, typically by pattern prising an amino acid sequence which is at least 80% (e.g., recognition receptors. Flagellin therefore serves as a danger 85%, 90%. 95%, 96%, 97%, 98% or 99%) identical to the signal across a wide variety of eukaryotes and is a potent light chain variable region amino acid sequence set forth in inducer of inflammatory effector responses in the mammalian SEQ ID NO:2. The antibodies may include a heavy chain gut (Neish, A. S., Am J Physiol Gastrointest Liver Physiol. only, a light chain only, or they may also include both of the 2007 February; 292(2):G462-6). aforementioned heavy chain and light chain variable regions. 0005 Specifically, upon detection of miniscule levels of 0010 Specifically, in one embodiment, the invention pro the monomeric protein flagellin, the mammalian germline vides an isolated monoclonal antibody that binds to flagellin encoded cell surface receptor Toll-like receptor 5 (TRL5) can comprising a heavy chain variable region comprising an directly promote a mucosal inflammatory response and trig amino acid sequence set forth in SEQID NO:1 (or an amino ger a massive induction of host gene expression designed to acid sequence at least 80% identical thereto). In another arm and protect the host against the invading microbe. The embodiment, the invention provides an isolated monoclonal resulting inflammatory cascade triggered by flagellin can be antibody that binds to flagellin comprising a light chain vari profound, causing clinical manifestations and tissue damage able region comprising an amino acid sequence set forth in (Gewirtz, A. T., Am J Physiol Gastrointest Liver Physiol. SEQID NO:2 (or an amino acid sequence at least 80% iden 2007 March; 292(3):G706-10). tical thereto). Inafurther embodiment, the invention provides 0006 Current treatments to offset the deleterious effects an isolated monoclonal antibody that binds to flagellin com of an inflammatory cascade stimulated by the flagellin prising a heavy and light chain variable region comprising the include anti-inflammatory drugs, immune system Suppres amino acid sequences set forth in SEQ ID NOs: 1 and 2. sors and other over-the-counter (OTC) drugs. These thera respectively (or amino acid sequences at least 80% identical pies, however, have clear drawbacks in that they are associ thereto). In yet another embodiment, the invention provides, ated with undesirable side effects and are merely palliative in an isolated monoclonal antibody that binds to flagellin and nature. Accordingly, improved agents and therapeutic treat comprises a heavy chain variable region comprising SEQID ments would be beneficial. NO:1, a light chain variable region comprising SEQID NO:2. or a combination thereof. SUMMARY OF THE INVENTION 0011. The variable heavy and light chain regions of the 0007. The present invention provides antibodies that bind antibodies typically include one or more complementarity to flagellin and neutralize a broad spectrum of bacteria determining regions (CDRS). These include one or more US 2010/02395.83 A1 Sep. 23, 2010

CDR1, CDR2, and CDR3 regions. Accordingly, other par 00.15 Antibodies of the present invention include all ticular antibodies of the present invention include one or more known immunoglobulin forms and other protein scaffolds with antibody-like properties. For example, the antibody can CDR sequences selected from a heavy chain variable region be a murine antibody, a human antibody, a humanized anti CDR1 comprising SEQ ID NO:5; a heavy chain variable body, a chimericantibody or a protein scaffold with antibody region CDR2 comprising SEQID NO:7; a heavy chain vari like properties, such as fibronectin or Ankyrin repeats. The able region CDR3 comprising SEQ ID NO:9; a light chain antibody also can have any of the following isotypes: IgG1. variable region CDR1 comprising SEQ ID NO:11; a light IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgASec, Ig|D and IgE. chain variable region CDR2 comprising SEQ ID NO:13; a Antibodies of the invention also include antibody fragments, such as an Fab, Fab'2, ScFv, SMIP affibody, avimer, nano light chain variable region CDR3 comprising SEQID NO:15: body or a domain antibody. and combinations thereof. 0016. Antibodies of the invention can be administered 0012. The antibodies may also comprise one or more alone or in combination with other therapeutic agents. For CDRs which are at least 80% (e.g., 85%, 90%. 95%, 96%, example, the antibodies can be administered in combination with (i.e., together with or linked to) cytotoxins, antibacterial 97%, 98% or 99%) identical to any of the aforementioned agents, including antibiotics and/or other therapeutic anti CDRs, or combinations of CDRs. For example, in one bodies. In one embodiment, the antibody is linked to a second embodiment, the isolated monoclonal antibody, or antigen antibody (i.e., thereby forming a bispecific antibody) or other binding portion thereof, comprises a heavy chain variable binding agent that binds to a different target (e.g., an Fc region that comprises CDR1, CDR2, and CDR3 sequences, receptor on an immune cell) or a different epitope on flagellin. and a light chain variable region that comprises CDR1. 0017. In yet another aspect, the present invention provides CDR2, and CDR3 sequences, wherein the heavy chain vari isolated nucleic acids encoding the aforementioned antibod able region CDR3 sequence comprises SEQ ID NO:9 (or ies of the invention. In particular embodiments, the nucleic acid encodes a heavy chain variable region comprising a conservative modifications thereof) and the light chain vari nucleotide sequence which is at least 80% (e.g., 85%, 90%, able region CDR3 sequence comprises SEQ ID NOS:15 (or 95%, 96%, 97%, 98% or 99%) identical to, or which hybrid conservative modifications thereof). Inafurther embodiment, izes under high stringency conditions to, SEQID NO:3: or a the antibody comprises the aforementioned CDR3 sequences light chain variable region comprising a nucleotide sequence and, optionally, further comprises a heavy chain variable which is at least 80% (e.g., 85%, 90%, 95%,96%.97%.98% region CDR2 sequence comprising SEQID NO:7 (or conser or 99%) identical to, or which hybridizes under high strin vative modifications thereof), and a light chain variable gency conditions to, SEQID NO:4; or a combination of these region CDR2 sequence comprising SEQID NO:13 (or con heavy and light variable regions. servative modifications thereof). In yet a further embodiment, 0018. The present invention also provides hybridomas that the antibody comprises the aforementioned CDR3 and CDR2 express and/or produce the aforementioned antibodies. sequences and, optionally, further comprises a heavy chain 0019. Further provided by the invention are kits compris variable region CDR1 sequence comprising SEQID NO:5 (or ing one or more of the aforementioned antibodies (or immu conservative modifications thereof), and a light chain variable noconjugates or bispecific antibodies), optionally, with region CDR1 sequence comprising SEQID NO:11 (or con instructions for use in treating or diagnosing bacterial dis servative modifications thereof). eases associated with flagellin in humans, other animals and birds. 0013 Also provided by the present invention are antibod 0020. The present invention also provides methods of pro ies that bind to the same or overlapping epitopes bound by any ducing the particular antibodies of the invention by providing of the aforementioned antibodies. In a particular embodi an isolated cell which expresses the antibody of interest and ment, these antibodies cross-react with a variety of gram isolating the antibody from the cell, thereby producing the negative bacteria, including Proteus Vulgaris, non-patho antibody. genic E. Coli, , Serratia marcenscens, 0021. As noted above, antibodies of the present invention , , Helicobacter can be used in a broad variety of diagnostic and therapeutic pylori, Pseudomonas aeruginosa, Salmonella typhimurium, applications, or used in the manufacture of one or more medi Salmonella muenchen, Proteus mirabilis and Enteropatho caments for diagnostic or therapeutic applications. These genic E. Coli. In another particular embodiment, the antibod applications include treatment and prevention of both infec ies bind to an epitope on flagellin of Salmonella muenchen tious and non-infectious bacterial diseases associated with which includes all or a portion of (e.g., is located between) flagellin. Particular non-infectious diseases include, but are amino acids 41-52 (SEQID NO:23). not limited to, inflammatory bowel diseases (IBDs), such as 0014. In another aspect, the invention pertains to antibod Crohn's Disease and colitis. Other particular diseases include ies that compete for binding to flagellin with the anti-flagellin gram negative bacterial infections (e.g., enterobacterial infec antibodies described herein. In a particular embodiment, the tions) sepsis and septic shock, in particular. Still other par antibody competes for binding to flagellin with an antibody ticular diseases include Anthrax, Bacterial Meningitis, Botu lism, , Cat Scratch Disease, , Diphtheria, comprising heavy and/or light chain variable regions com Epidemic , Impetigo, Legionellosis, Leprosy, Lep prising the amino acid sequences set forth in SEQID NOs: 1 tospirosis, Listeriosis, Lyme Disease, , MRSA and 2, respectively, or amino acid sequences at least 80% infection, Nocardiosis, Pertussis, , Pneumococcal identical thereto. Other antibodies of the invention bind to an , Psittacosis, , Rocky Mountain Spotted epitope on flagellin recognized by the antibodies described Fever (RMSF), , Scarlet Fever, , herein. In another particular embodiment, the antibody binds Syphilis, Tetanus, Trachoma, Tuberculosis, , to an epitope on flagellin recognized by an antibody compris , Urinary Tract Infections and Necrotizing ing heavy and/or light chain variable regions comprising the enterocolitis. amino acid sequences set forth in SEQ ID NOS:1 and 2. 0022. Other features and advantages of the invention will respectively, or amino acid sequences at least 80% identical be apparent from the following detailed description, and from thereto. the claims. US 2010/02395.83 A1 Sep. 23, 2010

BRIEF DESCRIPTION OF THE DRAWINGS 0023 FIG. 1 shows the results of a Western Blot demon - Continued strating that mAb 743 reacts strongly with Pseudomonas DGTTSAVSGVAITDTGSTGAGTAAGTTTFTEANDTVAKIDISTAKGAOSA aeruginosa type A flagellin and weakly with Pseudomonas aeruginosa type B flagellin. WLVIDEAIKOIDAORADLGAVONRFDNTINNLKNIGENVSAARGRIEDTD 0024 FIG. 2 is a graph showing the specific, wide-spread, FAAETANLTKNOWLOOAGTAILAQANOLPOSVLSLLR reactivity of mAb 743 to a variety of gram-negative bacteria (Spangenberg, C. et al., (1996). FEBS Lett. 396, in a live bacterial ELISA assay. 213 - 217) 0025 FIG.3 shows the epitope binding region of mAb 743 on Salmonella muenchen flagellin. (GI: 1655807) 0026 FIG. 4 is a graph showing that anti-flagellin mAb (SEQ ID NO: 21) 743 inhibits flagellin activity in an NO production assay. MAQVINTNSLSLITONNLNKNOSALSSSIERLSSGLRINSAKDDAAGOAI DETAILED DESCRIPTION OF THE INVENTION ANRFTSNIKGLTOAARNANDGISVAQTTEGALSEINNNLORIRELTVOAT 0027. In order that the present invention may be more TGTNSDSDLDSIODEIKSRLDEIDRVSGOTOFNGVNVLAKDGSMKIQVGA readily understood, certain terms are first defined. Additional INDGETITIDLKKIDSDTLGLNGFNWNGKGTITNKAATWSDLTSAGAKLNT definitions are set forth throughout the detailed description. TTGLYDLKTENTLLTTDAAFDKLGNGDKWTWGGWDYTYNAKSGDFTTTKS I. Definitions TAGTGVDAAAOAADSASKRDALAATLHADVGKSVNGSYTTKDGTVSFETD 0028. As used herein, the term “flagellin' carries its art recognized meaning as referring to a monomeric Subunit of SAGNITIGGSQAYWDDAGNLTTNNAGSAAKADMKALLKAASEGSDGASLT bacterial flagellum. The term “flagellin' includes the mono meric protein flagellin bound to bacteria, free circulating FNGTEYTIAKATPATTTPVAPLIPGGITYOATVSKDVVLSETKAAAATSS flagellin, and flagellin subunits of an individual flagellum or ITFNSGWLSKTIGFTAGESSDAAKSYWDDKGGITNWADYTWSYSWNKDNG flagella. The amino acid sequences of flagellins from differ ent bacterial strains are known in the art and are widely SWTWAGYASATDTNKDYAPAIGTAWNWNSAGKITTETTSAGSATTNPLAA conserved, as discussed by Steiner, T. S. (Infect Immun. 2007 February: 75(2):545-52), the teachings of which are incorpo LDDAISSIDKFRSSLGAIONRLDSAVTNLNNTTTNLSEAOSRIODADYAT rated by reference herein. Preferred antibodies of the inven tion cross react with flagellins of multiple bacterial species, EWSNMSKAQIIOOAGNSVLAKANOWPOOVLSLLQG including, but not limited to, Proteus Vulgaris, non-patho Serratia marces cens (GI: 514988) genic E. Coli, Citrobacter freundii, Serratia marcescens, (SEQ ID NO: 22) Pseudomonas aeruginosa, Salmonella typhimurium, Proteus MAQVINTNSLSLMAONNLNKSQSSLGTAIERLSSGLRINSAKDDAAGOAI mirabilis, and Enteropathogenic E. Coli. Representative flagellin sequences, include, but are not limited to, the SDRFTANIKGLTOASRNANDGISLAQTTEGALNEVNDNLONIRRLTWOAQ sequences set forth below. NGSNSTSDLKSIODEITORMSEINRISEOTDFNGVKVLSSDOKLTIOVGA

Proteus mirabilis (GI: 1169696) NDGETIDIDLOGLTGFDVTENGTKIGSAIADKAMVKDDTGTDVAFDLGES (SEQ ID NO: 19) MAQVINTNYLSLWTONNLNKSOGTLGSAIERLSSGLRINSAKDDAAGOAI FOTGGALEKATLVSGKTKDGKEGYYIOTTDAATGAKTYATAKIDDKGVVT

ANRFTSNVNGLTOASRNANDGISIAOTTEGALNEINNNLORIRELTVOAK KGADVTDWKDPLATLDKALAOVDGLRSSLGAVONRFDSVISNLNSTVNNL NGTNSNSDITSIONEVKNVLDEINRISEOTOFNGVKVLSGEKSEMVIOVG ASQSRIODADYATEWSNMSRAHILOOAGTSVLAQANOSTONWLSLLR TNDNETIKFNLDKWDNDTLGWASDKLFDTKTEKKGWTAAGAGVTDAKKIN (Akatsuka, H. et al., (1995). Gene 163, 157-158

AAATLDMMWSLWKEFNLDGKPWTDKFIWTKGGKDYWATKSDFELDATGTK Salmonella muenchen (GI: 1333832) (SEQ ID NO: 23) LGLKASATTEFKVDAGKDWKTLNVKDDALATLDKAINTIDESRSKLGAIO MAQVINTNSLSLLTONNLNKSQSALGTAIERLSSGLRINSAKDDAAGOAI

NRFESTINNLNNTVNNLSASRSRILDADYATEVSNMSRGOILOOAGTSVL ANRFTANIKGLTOASRNANDGISIAQTTEGALNEINNNLORVRELAVOSA AQANOVPOTWLSLLR (Betas, et al. (1994). Gene 148, 33 - 41.) NGTNSOSDLDSIOAEITORLNEIDRVSGOTOFNGVKVLAODNTLTIOVGA Pseudomonas aeruginosa (GI: 3386643) NDGETIDIDLKEISSKTLGLDKLNVODAYTPKETAVTVDKTTYKNGTDTI (SEQ ID NO: 2O) MALTWNTNIASLNTQRNLNNSSASLNTSLORLSTGSRINSAKDDAAGLOI TAOSNTDIOTAIGGGATGVTGADIKFKDGOYYLDWKGGASAGVYKATYDE

ANRLTSQVNGLNVATKNANDGISLAOTAEGALOOSTNILORMRDLSLOSA TTKKVNIDTTDKTPLATAEATAIRGTATITHNOIAEWTKEGWDTTTWAAQ NGSNSDSERTALNGEVKOLOKELDRISNTTTFGGRKLLDGSFGVASFOVG LAAAGVTGADKDNTSLVKLSFEDKNGKVIDGGYAVKMGDDFYAATYDEKO SAANEIISWGIDEMSAESLNGTYFKADGGGAWTAATASGTWDIAIGITGG VOLLLNNHYTDGAGVLOTGAVKFGGANGKSEVVTATVGKTYLASDLDKHN SAVNVKVDMKGNETAEOAAAKIAAAVNDANVGIGAFTDGAOISYWSKASA US 2010/02395.83 A1 Sep. 23, 2010

catarrhalis, Hemophilus influenzae, , - Continued , Pseudomonas aeruginosa, Proteus FRTGGELKEWNTDKTENPLOKIDAALAOVDTLRSDLGAVONRFNSAITNL mirabilis, Enterobacter cloacae, Serratia marcescens Heli cobacter pylori, Salmonella enteritidis, and Salmonella GNTVNNLSSARSRIEDSDYATEVSNMSRAOILOOAGTSVLAOANOVPONV typhi. LSILR 0033. As used herein, “a bacterial infectious disease' is a (Wei, L.N. et al., (1985) . J. Mol. Biol. 186, disease or infection caused by bacteria. 791-8O3) 0034. As used herein, “a gram negative bacterial infec Salmonella typhimurium (GI: 153979) tion' is a disease or infection caused by gram negative bac (SEQ ID NO: 24) teria. MAQVINTNSLSLLTONNLNKSQSALGTAIERLSSGLRINSAKDDAAGOAI 0035. As used herein, “an enterobacterial infection' is an ANRFTANIKGLTOASRNANDGISIAQTTEGALNEINNNLORVRELAVOSA infection caused by . 0036. As used herein, “Enterobacteriaceae and “entero NSTNSOSDLDSIOAEITORLNEIDRVNGOTOFSGVKVLAODNTLTIOVGA bacteria' refer to a large family of bacteria, including many of NDGETIDIDLKOINSOTLGLDTLNVOOKYKVSDTAATWTGYADTTIALDN the more familiarpathogens, such as Salmonella and Escheri chia coli. Genetic Studies place them among the Proteobac STFKASATGLGGTDEKIDGDLKFDDTTGKYYAKWTWTGGTGKDGYYEWSW teria, and they are given their own order (Enterobacteriales). Members of the Enterobacteriaceae are rod-shaped, and are DKTNGEWTLAAWTPATWTTATALSGKMYSANPDSDIAKAALTAAGWTGTA typically 1-5 um in length. Like other , they SVVKMSYTDNNGKTIDGGLAWKVGDDYYSATODKDGSISIDTTKYTADNG have Gram-negative stains, and they are facultative anaer obes, fermenting Sugars to produce lactic acid and various TSKTALNKLGGADGKTEVVTIDGKTYNASKAAGHDFKAEPELAEOAAKTT other end products. They also reduce nitrate to nitrite. Unlike ENPLOKIDAALAQVDTLRSDLGAWONRFNSAITNLGNTVNNLSSARSRIE most similar bacteria, enterobacteria generally lack cyto chrome C oxidase, although there are exceptions (e.g., Plesi DSDYATEWSNMSRAQILOOAGTSVLAQANOWPONVLSLLR Omonas). Most have many flagella used to move about, but a (Joys, T. M. (1985). J. Biol. Chem. 260, 15758 few genera are non-motile. They are non-spore forming, and 15761. ) except for strains they are catalase-posi 0029. As used herein, the term “bacteria' or “bacterium tive. Many members of this family area normal part of the gut refers to unicellular prokaryotic microorganisms, i.e., organ flora found in the intestines of humans and other animals, isms without a cell nucleus or any other membrane-bound while others are found in water or soil, or are parasites on a organelles. Bacteria are typically a few micrometres in length variety of different animals and plants. and individual bacteria have a wide-range of shapes, ranging 0037 Examples of Enterobacteriaceae include, but are not from spheres to rods to spirals. Although the vast majority of limited to, Alishewanella, Alterococcus, Aquamonas, Arani bacteria are rendered harmless or beneficial by the protective cola, Arsenophonus, Azotivirga, Blochmannia, Brenneria, effects of the immune system, a few pathogenic bacteria Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, cause infectious diseases. Dickeya, Edwardsiella, Enterobacter, Erwinia (e.g. Erwinia 0030. As used herein, “gram-negative bacteria' or “gram amylovora), Escherichia (e.g. Escherichia coli), Ewingella, negative bacterium” refer to bacteria having characteristic Grimontella, Hafinia, Klebsiella (e.g. Klebsiella pneumo staining properties under the microscope, where they either niae), Kluyvera, Leclercia, Leminorella, Moellerella, Mor do not stain or are decolorized by alcohol during Gram’s ganella, Obesumbacterium, Pantoea, , Can method of staining. didatus Phlomobacter, Photorhabdus (e.g. Photorhabdus 0031 Gram-negative bacteria generally have the follow luminescens), Plesiomonas (e.g. ), ing characteristics: (1) their cell wall only contains a few Pragia Proteus (e.g. Proteus vulgaris), Providencia, Rah layers of peptidoglycan (which is present in much higher nella, Raoultella, Salmonella, Samsonia, Serratia (e.g. Ser levels in Gram-positive bacteria); (2) the cells are surrounded ratia marcescens), Shigella, Sodalis, Tatumella, Trabulsiella, by an outer membrane containing lipopolysaccharide (which Wigglesworthia, Xenorhabdus, Yersinia (e.g. ), consists of Lipid A, core polysaccharide, and O-polysaccha and Yokenella. ride) outside the peptidoglycan layer; (3) porins exist in the 0038 Examples of enterobacterial infections include, but outer membrane, which act like pores for particular mol are not limited to, Anthrax (by the bacterium Bacillus anthra ecules; (4) there is a space between the layers of peptidogly cis), Bacterial Meningitis (caused by a variety of bacteria, can and the secondary cell membrane called the periplasmic including, but not limited to, Neisseria meningitides, Strep space; (5) the S-layer is directly attached to the outer mem tococcus pneumoniae, Listeria monocytogenes, Pseudomo brane, rather than the peptidoglycan; (6) if present, flagella nas aeruginosa, Staphylococcus aureus, Streptococcus aga have four Supporting rings instead of two; (7) no teichoic lactiae and influenzae), Botulism (caused by acids or lipoteichoic acids are present; (8) lipoproteins are bacterium Clostridium botulinum), Brucellosis (caused by attached to the polysaccharide backbone, whereas in Gram bacteria of the genus Brucella), (caused positive bacteria no lipoproteins are present; and (9) most do by bacteria of the genus Campylobacter), Cat Scratch Disease not sporulate. (caused by hemselae and Bartonella clarridgeiae), 0032 Examples of gram-negative bacteria include, but are Cholera (caused by the bacterium ), Diphthe not limited to, Escherichia coli, Enterobacteriaceae, ria (caused by Corynebacterium diphtheriae), Epidemic Moraxella, Helicobacter, Burkholderia cepacia, Stenotroph Typhus (causative organism is Rickettsia prowazeki), Impe Omonas, Bdello vibrio, acetic acid bacteria, cyanobacteria, tigo (caused by several bacteria, including, Staphylococcus spirochaetes, green Sulfur and green non-sulfur bacteria, aureus and Streptococcus pyogenes), Legionellosis (caused , Neisseria meningitides, Moraxella by bacteria belonging to the genus Legionella), Leprosy US 2010/02395.83 A1 Sep. 23, 2010

(Hansen's Disease) (caused by the bacterium Mycobacterium Crohn's disease: (1) Ileocolitis (the most common form, leprae), Leptospirosis (caused by Spirochaetes of the genus which affects the ileum and the colon); (2) Ileitis (which Leptospira), Listeriosis (caused by the bacterium Listeria affects the ileum); (3) Gastroduodenal Crohn's Disease monocytogenes), Lyme Disease (caused by Spirochete bacte (which causes inflammation in the stomach and the duode ria from the genus Borrelia), Melioidosis (caused by the num); (4) Jejunoileitis (which causes spotty patches of bacterium Burkholderia pseudomallei), MRSA infection inflammation in the top half of the small intestine (i.e., the (caused by Staphylococcus aureus), Nocardiosis (bacterium jejunum); and (5) Crohn's (Granulomatous) Colitis (which of the genus Nocardia, most commonly Nocardia asteroides affects only affects the large intestine). or Nocardia brasiliensis), Pertussis () 0042. As used herein, “Ulcerative Colitis is a form of IBD (caused by the bacterium ), Plague that affects the colon (the large intestine) alone and inflam (caused by the enterobacteria Yersinia pestis), Pneumococcal mation is confined to the mucosa (the inner lining) of the pneumonia (caused by a variety ofbacteria, including, but not intestine. It can be difficult to diagnose because its symptoms limited to, Streptococcus pneumoniae, Staphylococcus are similar to other intestinal disorders and Crohn's Disease. aureus, , Klebsiella pneumoniae, 0043. The term “Toll-like receptor (TLR) as used herein, Escherichia coli, Pseudomonas aeruginosa, Moraxella refers to an important family of innate immune receptors that catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneu recognize pathogen-associated molecular patterns, i.e., evo moniae, and Legionella pneumophila), Psittacosis (caused by lutionarily conserved structures that are required for micro a bacterium called Chlamydophila psittaci), Q fever (caused bial fitness and are not present in the host. by infection with ), Rocky Mountain Spot 0044) The term “Toll-like receptor 5 (TLR5) as used ted Fever (RMSF) (by ), Salmonellosis herein, refers to the Toll-like receptor which recognizes and (caused by bacteria of the genus Salmonella), Scarlet Fever, binds bacterial flagellin from both gram-positive and gram Shigellosis (caused by bacteria of the genus Shigella), Syphi negative and activates host inflammatory responses. TLR5 is lis (caused by Treponema pallidum), Tetanus (Clostridium specifically expressed in monocytes, immature dendritic cells tetani), Trachoma, Tuberculosis (caused by mycobacteria, and epithelial cells. mainly Mycobacterium tuberculosis), Tularemia (by the bac 0045. The term “neutralizes and “inhibits” are used inter terium ), Typhoid Fever (caused by the changeably herein, and refer to any statistically significant bacterium Salmonella typhi), and Urinary Tract Infections decrease in the biological activity (e.g., motility) of flagellin, (caused by bacteria Such as Escherichia coli, Staphylococcus including full blocking of the activity. For example, “neutral Saprophyticus, Proteus mirabilis, Klebsiella pneumoniae, izes” or “inhibits' can refer to a decrease of about 10%, 15%, Enterobacter spp., Pseudomonas and Enterococcus). 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 0039. As used herein, “gram-positive bacteria' or “gram 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% in positive bacterium” refer to bacteria that retain the stain or flagellin activity. that are resistant to decolourisation by alcohol during Gram’s 0046. In particular embodiments of the invention, neutral method of staining. Gram-positive bacteria generally have the ization or inhibition of flagellin activity results in one or more following characteristics: (1) a very thick cell wall (pepti of the following effects: it prevents bacterial invasion into doglycan); (2) if a flagellum is present, it contains two rings Susceptible epithelial cells, reduces the symptoms of an for Support as opposed to four in Gram-negative bacteria enterobacterial infection or IBD in a subject, reduces the because Gram-positive bacteria have only one membrane extent and severity of flagellin-induced tissue injury, reduces layer; and (3) teichoic acids and lipoteichoic acids are flagellin-stimulated neutrophil infiltration, decreases colonic present, which serve to act as chelating agents, and also for mucosal congestion, erosion and hemorrhagic ulcerations certain types of adherence. Examples of gram-positive bac associated with IBD, inhibits or decrease the production of teria genera include, but are not limited to, Bacillus, Listeria, mediators (e.g., MDA, IL-1 B, TNFC, MIP-1, MIP-2 and Staphylococcus, Streptococcus, Enterococcus, and IL-8); and/or counteracts a reduction in body weight associ Clostridium. ated with IBD. 0040. As used herein, “Inflammatory Bowel Disease 0047. The term “antibody” or “immunoglobulin, as used (IBD) refers to a group of chronic intestinal diseases char interchangeably herein, includes whole antibodies and any acterized by inflammation of the bowel, i.e., the large or small antigen binding fragment (i.e., “antigen-binding portion') or intestine. The most common types of IBD are Ulcerative single chains thereof. An “antibody’ comprises at least two Colitis and Crohn's Disease. The symptoms of IBD include heavy (H) chains and two light (L) chains inter-connected by abdominal pain, diarrhea, bloody diarrhea, severe urgency to disulfide bonds. Each heavy chain is comprised of a heavy have a bowel movement, fever, loss of appetite, weight loss, chain variable region (abbreviated herein as V) and a heavy anemia. IBD can also cause intestinal complications includ chain constant region. The heavy chain constant region is ing profuse bleeding from the ulcers, perforation of the comprised of three domains, CH1, CH2 and CH3. Each light bowel, strictures and obstructions, fistulae, perianal disease, chain is comprised of a light chain variable region (abbrevi toxic megacolon and cancer. The disease can be limited to the ated herein as V) and a light chain constant region. The light intestine or affect the skin, joints, spine, liver, eyes, and other chain constant region is comprised of one domain, CL. The Organs. V and V regions can be further Subdivided into regions of 0041. As used herein, “Crohn's Disease' is a form of IBD hyperVariability, termed complementarity determining that causes severe irritation in the gastrointestinal tract. It regions (CDR), interspersed with regions that are more con usually affects the lower small intestine (i.e., the ileum) or the served, termed framework regions (FR). Each V, and V, is colon, but can affect other parts of the digestive system composed of three CDRs and four FRs, arranged from amino including the Small intestine, mouth, esophagus, and stom terminus to carboxy-terminus in the following order: FR1, ach. The inflammation in Crohn's Disease involves the entire CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions thickness of the bowel wall. There are five different types of of the heavy and light chains contain a binding domain that US 2010/02395.83 A1 Sep. 23, 2010

interacts with an antigen. The constant regions of the anti 0050. The term “recombinant antibody,” refers to antibod bodies may mediate the binding of the immunoglobulin to ies that are prepared, expressed, created or isolated by recom host tissues or factors, including various cells of the immune binant means, such as (a) antibodies isolated from an animal system (e.g., effector cells) and the first component (Cld) of (e.g., a mouse) that is transgenic or transchromosomal for the classical complement system. immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated 0048. The term “antigen-binding portion of an antibody from a host cell transformed to express the antibody, e.g., (or simply “antibody portion'), as used herein, refers to one or from a transfectoma, (c) antibodies isolated from a recombi more fragments of an antibody that retain the ability to spe nant, combinatorial antibody library (e.g., containing human cifically bind to an antigen (e.g., flagellin). It has been shown antibody sequences) using phage display, and (d) antibodies that the antigen-binding function of an antibody can be per prepared, expressed, created or isolated by any other means formed by fragments of a full-length antibody. Examples of that involve splicing of immunoglobulin gene sequences binding fragments encompassed within the term “antigen (e.g., human immunoglobulin genes) to other DNA binding portion of an antibody include (i) a Fab fragment, a sequences. Such recombinant antibodies may have variable monovalent fragment consisting of the V, V, CL and CH1 and constant regions derived from human germline immuno domains; (ii) a F(ab')2 fragment, a bivalent fragment compris globulin sequences. In certain embodiments, however, Such ing two Fab fragments linked by a disulfide bridge at the hinge recombinant human antibodies can be subjected to in vitro region; (iii) a Fd fragment consisting of the V, and CH1 mutagenesis and thus the amino acid sequences of the V and domains; (iv) a Fv fragment consisting of the V, and V. V, regions of the recombinant antibodies are sequences that, domains of a single arm of an antibody, (v) a dAb including while derived from and related to human germline V, and V. VHand VL domains; (vi) a dAb fragment (Ward et al. (1989) sequences, may not naturally exist within the human antibody Nature 341, 544-546), which consists of a V. domain; (vii) a germline repertoire in vivo. dAb which consists of a VH or a VL domain; and (viii) an 0051. The term "chimeric immunoglobulin' or antibody isolated complementarity determining region (CDR) or (ix) a refers to an immunoglobulin or antibody whose variable combination of two or more isolated CDRs which may regions derive from a first species and whose constant regions optionally be joined by a synthetic linker. Furthermore, derive from a second species. Chimeric immunoglobulins or although the two domains of the FV fragment, V, and V, are antibodies can be constructed, for example by genetic engi coded for by separate genes, they can be joined, using recom neering, from immunoglobulin gene segments belonging to binant methods, by a synthetic linker that enables them to be different species. made as a single protein chain in which the V, and V regions 0.052 The term “human antibody, as used herein, is pair to form monovalent molecules (known as single chain FV intended to include antibodies having variable regions in (scFV); see e.g., Bird et al. (1988) Science 242, 423-426; and which both the framework and CDR regions are derived from Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879 human germline immunoglobulin sequences as described, for 5883). Such single chain antibodies are also intended to be example, by Kabat et al. (See Kabat, et al. (1991) Sequences encompassed within the term “antigen-binding portion of an of proteins of Immunological Interest, Fifth Edition, U.S. antibody. These antibody fragments are obtained using con Department of Health and Human Services, NIH Publication ventional techniques known to those with skill in the art, and No. 91-3242). Furthermore, if the antibody contains a con the fragments are screened for utility in the same manner as stant region, the constant region also is derived from human are intact antibodies. Antigen-binding portions can be pro germline immunoglobulin sequences. The human antibodies duced by recombinant DNA techniques, or by enzymatic or may include amino acid residues not encoded by human chemical cleavage of intact immunoglobulins. germline immunoglobulin sequences (e.g., mutations intro 0049. The term “monoclonal antibody” as used herein duced by random or site-specific mutagenesis in vitro or by refers to an antibody obtained from a population of Substan somatic mutation in vivo). However, the term “human anti tially homogeneous antibodies, i.e., the individual antibodies body', as used herein, is not intended to include antibodies in comprising the population are identical except for possible which CDR sequences derived from the germline of another naturally occurring mutations that may be present in minor mammalian species, such as a mouse, have been grafted onto amounts. Monoclonal antibodies are highly specific, being human framework sequences. directed against a single antigenic site. Furthermore, in con 0053. The human antibody can have at least one or more trast to conventional (polyclonal) antibody preparations amino acids replaced with an amino acid residue, e.g., an which typically include different antibodies directed against activity enhancing amino acid residue which is not encoded different determinants (epitopes), each monoclonal antibody by the human germline immunoglobulin sequence. Typically, is directed against a single determinant on the antigen. Mono the human antibody can have up to twenty positions replaced clonal antibodies can be prepared using any art recognized with amino acid residues which are not part of the human technique and those described herein Such as, for example, a germline immunoglobulin sequence. In a particular embodi hybridoma method, as described by Kohler et al. (1975) ment, these replacements are within the CDR regions as Nature, 256:495, a transgenic animal, as described by, for described in detail below. example, (see e.g., Lonberg, et al. (1994) Nature 368(6474): 0054 The term “humanized immunoglobulin' or 856-859), recombinant DNA methods (see, e.g., U.S. Pat. No. “humanized antibody” refers to an immunoglobulin or anti 4,816.567), or using phage antibody libraries using the tech body that includes at least one humanized immunoglobulin or niques described in, for example, Clackson et al., Nature, antibody chain (i.e., at least one humanized light or heavy 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 chain). The term “humanized immunoglobulin chain” or 597 (1991). Monoclonal antibodies include chimeric anti "humanized antibody chain' (i.e., a “humanized immunoglo bodies, human antibodies and humanized antibodies and may bulin light chain” or “humanized immunoglobulin heavy occur naturally or be recombinantly produced. chain”) refers to an immunoglobulin orantibody chain (i.e., a US 2010/02395.83 A1 Sep. 23, 2010

light or heavy chain, respectively) having a variable region 0061. As used herein, the term “switch sequence” refers to that includes a variable framework region substantially from those DNA sequences responsible for switch recombination. a human immunoglobulin or antibody and complementarity A 'switch donor sequence, typically a L Switch region, will determining regions (CDRS) (e.g., at least one CDR, prefer be 5' (i.e., upstream) of the construct region to be deleted ably two CDRs, more preferably three CDRs) substantially during the switch recombination. The “switch acceptor from a non-human immunoglobulin or antibody, and further region will be between the construct region to be deleted and includes constant regions (e.g., at least one constant region or the replacement constant region (e.g., Y, e, etc.). As there is no portion thereof, in the case of a light chain, and preferably specific site where recombination always occurs, the final three constant regions in the case of a heavy chain). The term gene sequence will typically not be predictable from the "humanized variable region' (e.g., “humanized light chain COnStruct. variable region' or “humanized heavy chain variable region') 0062 An “antigen' is an entity (e.g., a proteinaceous refers to a variable region that includes a variable framework entity or peptide) to which an antibody or antigen-binding region Substantially from a human immunoglobulin or anti portion thereof binds. In various embodiments of the present body and complementarity determining regions (CDRS) Sub invention, an antigen is flagellin. stantially from a non-human immunoglobulin or antibody. 0063. The term “epitope' or “antigenic determinant 0055. A “bispecific' or “bifunctional antibody” is an arti refers to a site on an antigen to which an immunoglobulin or ficial hybrid antibody having two different heavy/light chain antibody specifically binds. Epitopes can be formed both pairs and two different binding sites. Bispecific antibodies from contiguous amino acids or noncontiguous amino acids can be produced by a variety of methods including fusion of juxtaposed by tertiary folding of a protein. Epitopes formed hybridomas or linking of Fab' fragments. See, e.g., Songsiv from contiguous amino acids are typically retained on expo ilai & Lachmann, (1990) Clin. Exp. Immunol. 79, 315-321; Sure to denaturing solvents, whereas epitopes formed by ter Kostelny et al. (1992).J. Immunol. 148, 1547-1553. tiary folding are typically lost on treatment with denaturing Solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 0056. As used herein, a "heterologous antibody' is 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial defined in relation to the transgenic non-human organism or conformation. Methods for determining what epitopes are plant producing Such an antibody. bound by a given antibody (i.e., epitope mapping) are well 0057. An "isolated antibody, as used herein, is intended known in the art and include, for example, immunoblotting to refer to an antibody which is substantially free of other and immunoprecipitation assays, wherein overlapping or antibodies having different antigenic specificities (e.g., an contiguous peptides from flagellin are tested for reactivity isolated antibody that specifically binds to flagellin is sub with the given anti-flagellin antibody. Methods of determin stantially free of antibodies that specifically bind antigens ing spatial conformation of epitopes are also well known in other than flagellin). In addition, an isolated antibody is typi the art and include, for example, X-ray crystallography and cally substantially free of other cellular material and/or 2-dimensional nuclear magnetic resonance. See, e.g., Epitope chemicals. In one embodiment of the invention, a combina Mapping Protocols in Methods in Molecular Biology, Vol. 66, tion of "isolated monoclonal antibodies having different G. E. Morris, Ed. (1996). flagellin binding specificities are combined in a well defined 0064. Accordingly, also encompassed by the present composition. invention are antibodies that bind to an epitope on flagellin 0058 As used herein, “isotype” refers to the antibody which comprises all or a portion of an epitope recognized by class (e.g., IgM or IgG1) that is encoded by heavy chain the particular antibodies described herein (e.g., the same oran constant region genes. In one embodiment, an antibody or overlapping region or a region between or spanning the antigen binding portion thereof is of an isotype selected from region). In a particular embodiment, the antibodies bind to an an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an epitope which comprises all or a portion of amino acids 1-60 IgA2, an IgASec, an Ig), or an IgE antibody isotype. In some of flagellin from Salmonella (Genbank Accession No. embodiments, a monoclonal antibody of the invention is of GI: 1333832) (SEQ ID NO:23) or Pseudomonas (Genbank the IgG1 isotype. In other embodiments, a monoclonal anti Accession No. GI:3386643) (SEQ ID NO:20), or any other body of the invention is of the IgG2 isotype. homologous flagellin sequences from various bacterial spe 0059. As used herein, “isotype switching refers to the cies, such as an epitope that is between or includes all or a phenomenon by which the class, or isotype, of an antibody portion of amino acids 1-30 or 1-40 or 1-50 or 1-55 or 20-40 changes from one Ig class to one of the other Ig classes. or 30-40 or 30-50 or 35-45 or 37-43 or 31-47 or 41-52 or 0060. As used herein, “nonswitched isotype” refers to the 45-55 or 40-55 or 40-60 of flagellin from Salmonella (Gen isotypic class of heavy chain that is produced when no isotype bank Accession No. GI: 1333832) (SEQ ID NO:23) or Switching has taken place; the CH gene encoding the non Pseudomonas (Genbank Accession No. GI:3386643) (SEQ switched isotype is typically the first CH gene immediately ID NO:20). In a particularembodiment, the antibodies bind to downstream from the functionally rearranged VDJ gene. Iso an epitope which comprises all or a portion of (i.e., is located type Switching has been classified as classical or non-classi between, spans, or overlaps) with amino acids 41-53 of Sal cal isotype Switching. Classical isotype Switching occurs by monella muenchen flagellin (SEQ ID NO:23). recombination events which involve at least one switch 0065. In another embodiment, the invention provides anti sequence regions in a gene encoding an antibody. Non-clas bodies that compete for binding to flagellin (e.g., Salmonella sical isotype Switching may occur by, for example, homolo muenchen flagellin (SEQ ID NO:23)) with the antibodies gous recombination between human O, and human X (Ö-as described herein. Competing antibodies and antibodies that Sociated deletion). Alternative non-classical Switching recognize the same or an overlapping epitope can be identi mechanisms, such as intertransgene and/or interchromo fied using routine techniques such as an immunoassay, for Somal recombination, among others, may occur and effectu example, by showing the ability of one antibody to block the ate isotype Switching. binding of another antibody to a target antigen, i.e., a com US 2010/02395.83 A1 Sep. 23, 2010 petitive binding assay. Competitive binding is determined in M, 10 M or 10' M or even lower when determined by an assay in which the immunoglobulin under test inhibits surface plasmon resonance (SPR) technology in a BIACORE specific binding of a reference antibody to an antigen, Such as 3000 instrument using recombinant flagellin as the analyte flagellin. Numerous types of competitive binding assays are and the antibody as the ligand, and binds to the predetermined known, for example: Solid phase direct or indirect radioim antigen with an affinity that is at least two-fold greater than its munoassay (RIA), Solid phase direct or indirect enzyme affinity for binding to a non-specific antigen (e.g., BSA, immunoassay (EIA), Sandwich competition assay (see Stahli casein) other than the predetermined antigen or a closely et al., (1983) Methods in Enzymology 9:242); solid phase related antigen. direct biotin-avidin EIA (see Kirkland et al., (1986).J. Immu (0068. The term “K,” as used herein, is intended to refer mol. 137:3614); solid phase direct labeled assay, solid phase to the off rate constant for the dissociation of an antibody direct labeled sandwich assay (see Harlow and Lane, (1988) from the antibody/antigen complex. Antibodies: A Laboratory Manual, Cold Spring Harbor 0069. The term “EC50, as used herein, refers to the con Press); solid phase direct label RIA using I-125 label (see centration of an antibody or an antigen-binding portion Morel et al., (1988) Mol. Immunol. 25(1):7); solid phase thereof, which induces a response, either in an in vitro oran in direct biotin-avidin EIA (Cheung et al., (1990) Virology 176: vivo assay, which is 50% of the maximal response, i.e., half 546); and direct labeled RIA. (Moldenhauer et al., (1990) way between the maximal response and the baseline. Scand. J. Immunol. 32:77). Typically, such an assay involves 0070. As used herein, glycosylation pattern' is defined as the use of purified antigen (e.g., flagellin) bound to a solid the pattern of carbohydrate units that are covalently attached surface or cells bearing either of these, an unlabeled test to a protein, more specifically to an immunoglobulin protein. immunoglobulin and a labeled reference immunoglobulin. 0071. The term “naturally-occurring” as used herein as Competitive inhibition is measured by determining the applied to an object refers to the fact that an object can be amount of label bound to the solid surface or cells in the found in nature. For example, a polypeptide or polynucleotide presence of the test immunoglobulin. Usually the test immu sequence that is present in an organism (including viruses) noglobulin is present in excess. Usually, when a competing that can be isolated from a source in nature and which has not antibody is present in excess, it will inhibit specific binding of been intentionally modified by man in the laboratory is natu a reference antibody to a common antigen by at least 50-55%. rally-occurring. 55-60%, 60-65%, 65-70% 70-75% or more. 0072 The term “rearranged as used herein refers to a 0066. As used herein, the terms “specific binding.” “spe configuration of a heavy chain or light chain immunoglobulin cifically binds.” “selective binding,” and “selectively binds.” locus wherein a V segment is positioned immediately adja mean that an antibody or antigen-binding portion thereof, cent to a D-J or J segment in a conformation encoding essen exhibits appreciable affinity for aparticular antigenorepitope tially a complete V, or V, domain, respectively. A rearranged and, generally, does not exhibit significant cross-reactivity immunoglobulin gene locus can be identified by comparison with other antigens and epitopes. Appreciable' or preferred to germline DNA; a rearranged locus will have at least one binding includes binding with an affinity of at least 10, 107. recombined heptamer/nonamer homology element. 10, 10 M', or 10' M'. Affinities greater than 107M', 0073. The term “unrearranged’ or “germline configura preferably greater than 10 M are more preferred. Values tion” as used herein in reference to a V segment refers to the intermediate of those set forth herein are also intended to be configuration wherein the V segment is not recombined so as within the scope of the present invention and a preferred to be immediately adjacent to a D or J segment. binding affinity can be indicated as a range of affinities, for 0074 The term “nucleic acid molecule” as used herein, is example, 10° to 10'M', preferably 107 to 10'M', more intended to include DNA molecules and RNA molecules. A preferably 10 to 10'M'. Anantibody that “does not exhibit nucleic acid molecule may be single-stranded or double significant cross-reactivity” is one that will not appreciably stranded, but preferably is double-stranded DNA. bind to an undesirable entity (e.g., an undesirable proteina 0075. The term "isolated nucleic acid molecule,” as used ceous entity). Specific or selective binding can be determined herein in reference to nucleic acids encoding antibodies (e.g., according to any art-recognized means for determining Such V, V, CDR3) that bind to flagellin, is intended to refer to a binding, including, for example, according to Scatchard nucleic acid molecule in which the nucleotide sequences analysis and/or competitive binding assays. encoding the antibody are free of other nucleotide sequences 0067. The term “K, as used herein, is intended to refer to encoding antibodies that bind antigens other than flagellin, the dissociation equilibrium constant of aparticular antibody which other sequences may naturally flank the nucleic acid in antigen interaction or the affinity of an antibody for an anti human genomic DNA. gen. In one embodiment, the antibody or antigen binding 0076 Alternatively, antibodies can comprise an amino portion thereof according to the present invention binds an acid sequence which is encoded by a nucleotide sequence antigen (e.g., flagellin) with an affinity (K) of 50 nM or which hybridizes, e.g., hybridizes under Stringent conditions better (i.e., or less) (e.g., 40 nM or 30 nM or 20 nM or 10 nM to a nucleotide sequence disclosed herein. As used herein, the or less), as measured using a surface plasmon resonance assay term “hybridizes under stringent conditions” is intended to or a cell binding assay. In a particular embodiment, an anti describe conditions for hybridization and washing under body or antigen binding portion thereof according to the which nucleotide sequences at least 60% homologous to each present invention binds flagellin with an affinity (K) of 8 nM other typically remain hybridized to each other. Preferably, or better (e.g., 7 nM, 6 nM, 5 nM, 4 nM, 2nM, 1.5 nM, 1.4 the conditions are Such that sequences at least about 65%, nM, 1.3 nM, 1 nM or less), as measured by a surface plasmon more preferably at least about 70%, and even more preferably resonance assay or a cell binding assay. In other embodi at least about 75% or more homologous to each other typi ments, an antibody or antigen binding portion thereof binds cally remain hybridized to each other. Such stringent condi an antigen (e.g., flagellin) with an affinity (K) of approxi tions are known to those of ordinary skill in the art and can be mately less than 107M, such as approximately less than 10 found in Current Protocols in Molecular Biology, John Wiley US 2010/02395.83 A1 Sep. 23, 2010

& Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting I0082 Similarly, the consensus sequence for the CDRs can example of stringent hybridization conditions are hybridiza be derived by optimal alignment of the CDR amino acid tion in 6x sodium chloride/sodium citrate (SSC) at about 45° sequences of flagellin antibodies of the present invention. C., followed by one or more washes in 0.2xSSC, 0.1% SDS at I0083. For nucleic acids, the term “substantial homology’ 50-650 C. indicates that two nucleic acids, or designated sequences 0077. The term “modifying,” or “modification,” as used thereof, when optimally aligned and compared, are identical, herein, is intended to refer to changing one or more amino with appropriate nucleotide insertions or deletions, in at least acids in the antibodies. The change can be produced by add about 80% of the nucleotides, usually at least about 90% to ing, Substituting or deleting an amino acid at one or more 95%, and more preferably at least about 98% to 99.5% of the positions. The change can be produced using known tech nucleotides. Alternatively, Substantial homology exists when niques. Such as PCR mutagenesis. For example, in some the segments will hybridize under selective hybridization embodiments, an antibody identified using the methods of the conditions, to the complement of the Strand. invention can be modified, to thereby modify the binding I0084. The percent identity between two sequences is a affinity of the antibody to flagellin. function of the number of identical positions shared by the 0078. The present invention also encompasses “conserva sequences (i.e., 96 homology it of identical positions/total # tive amino acid Substitutions in the sequences of the anti of positionsX100), taking into account the number of gaps, bodies of the invention, i.e., nucleotide and amino acid and the length of each gap, which need to be introduced for sequence modifications which do not abrogate the binding of optimal alignment of the two sequences. The comparison of the antibody encoded by the nucleotide sequence or contain sequences and determination of percent identity between two ing the amino acid sequence, to the antigen, i.e., flagellin. sequences can be accomplished using a mathematical algo Conservative amino acid substitutions include the substitu rithm, as described in the non-limiting examples below. tion of an amino acid in one class by anamino acid of the same I0085. The percent identity between two nucleotide class, where a class is defined by common physicochemical sequences can be determined using the GAP program in the amino acid side chain properties and high Substitution fre GCG software, using a NWSgapdna. CMP matrix and a gap quencies in homologous proteins found in nature, as deter weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, mined, for example, by a standard Dayhoff frequency 4, 5, or 6. The percent identity between two nucleotide or exchange matrix or BLOSUM matrix. Six general classes of amino acid sequences can also be determined using the algo amino acid side chains have been categorized and include: rithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (ASn, which has been incorporated into the ALIGN program (ver Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, sion 2.0), using a PAM120 weight residue table, a gap length Val, Met); and Class VI (Phe, Tyr, Trp). For example, substi penalty of 12 and a gap penalty of 4. In addition, the percent tution of an Asp for another class III residue such as ASn, Gln, identity between two amino acid sequences can be deter or Glu, is a conservative Substitution. Thus, a predicted non mined using the Needleman and Wunsch (J. Mol. Biol. (48): essential amino acid residue in an anti-flagellin antibody of 444-453 (1970)) algorithm which has been incorporated into the present invention is preferably replaced with another the GAP program in the GCG Software package, using either amino acid residue from the same class. Methods of identi a Blossum 62 matrix or a PAM250 matrix, and a gap weight fying nucleotide and amino acid conservative Substitutions of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, which do not eliminate antigenbinding are well-known in the or 6. art (see, e.g., Brummell et al., Biochem. 32:1180-1187 I0086. The nucleic acid and protein sequences of the (1993); Kobayashietal. Protein Eng. 12(10):879-884 (1999); present invention can further be used as a “query sequence' to and Burks et al. Proc. Natl. Acad. Sci. USA 94:412–417 perform a search against public databases to, for example, (1997)). identify related sequences. Such searches can be performed 0079. The term “non-conservative amino acid substitu using the NBLAST and XBLAST programs (version 2.0) of tion” refers to the substitution of an amino acid in one class Altschul, et al. (1990).J. Mol. Biol. 215:403-10. BLAST with an amino acid from another class; for example, Substi nucleotide searches can be performed with the NBLAST tution of an Ala, a class II residue, with a class III residue Such program, score=100, wordlength=12 to obtain nucleotide as Asp, ASn, Glu, or Gln. sequences homologous to the nucleic acid molecules of the 0080. Alternatively, in another embodiment, mutations invention. BLAST protein searches can be performed with (conservative or non-conservative) can be introduced ran the XBLAST program, score=50, wordlength=3 to obtain domly along all or part of an anti-flagellin antibody coding amino acid sequences homologous to the protein molecules sequence. Such as by Saturation mutagenesis, and the result of the invention. To obtaingapped alignments for comparison ing modified anti-flagellin antibodies can be screened for purposes, Gapped BLAST can be utilized as described in binding activity. Altschulet al., (1997) Nucleic Acids Res. 25(17):3389-3402. 0081. A “consensus sequence' is a sequence formed from When utilizing BLAST and Gapped BLAST programs, the the most frequently occurring amino acids (or nucleotides) in default parameters of the respective programs (e.g., XBLAST a family of related sequences (See e.g., Winnaker, From and NBLAST) can be used. Genes to Clones (Verlagsgesellschaft, Weinheim, Germany I0087. The nucleic acids may be present in whole cells, in 1987). In a family of proteins, each position in the consensus a cell lysate, or in a partially purified or Substantially pure sequence is occupied by the amino acid occurring most fre form. A nucleic acid is "isolated” or “rendered substantially quently at that position in the family. If two amino acids occur pure' when purified away from other cellular components or equally frequently, either can be included in the consensus other contaminants, e.g., other cellular nucleic acids or pro sequence. A "consensus framework of an immunoglobulin teins, by standard techniques, including alkaline/SDS treat refers to a framework region in the consensus immunoglobu ment, CsCl banding, column chromatography, agarose gel lin sequence. electrophoresis and others well known in the art. See, F. US 2010/02395.83 A1 Sep. 23, 2010

Ausubel, et al., ed. Current Protocols in Molecular Biology, (0091. The term “recombinant host cell’ (or simply “host Greene Publishing and Wiley Interscience, New York (1987). cell”), as used herein, is intended to refer to a cell into which 0088. The nucleic acid compositions of the present inven a recombinant expression vector has been introduced. It tion, while often in a native sequence (except for modified should be understood that such terms are intended to refer not restriction sites and the like), from either cDNA, genomic or only to the particular Subject cell but to the progeny of such a mixtures thereof may be mutated, in accordance with stan cell. Because certain modifications may occur in Succeeding dard techniques to provide gene sequences. For coding generations due to either mutation or environmental influ sequences, these mutations, may affect amino acid sequence ences, such progeny may not, in fact, be identical to the parent as desired. In particular, DNA sequences Substantially cell, but are still included within the scope of the term “host homologous to or derived from native V. D. J., constant, cell as used herein. Switches and other Such sequences described herein are con 0092. The terms “treat,” “treating,” and “treatment,” as templated (where "derived indicates that a sequence is iden used herein, refer to therapeutic or preventative measures tical or modified from another sequence). described herein. The methods of “treatment' employ admin I0089. The term “operably linked” refers to a nucleic acid istration to a Subject, an antibody of the present invention, for sequence placed into a functional relationship with another example, a Subject having an infection or disease associated nucleic acid sequence. For example, DNA for a presequence with flagellin or predisposed to having Such an infection or or secretory leader is operably linked to DNA for a polypep disease, in order to prevent, cure, delay, reduce the severity of tide if it is expressed as a preprotein that participates in the or ameliorate one or more symptoms of the infection or dis secretion of the polypeptide; a promoter or enhancer is oper ease in order to prolong the survival of a subject beyond that ably linked to a coding sequence if it affects the transcription expected in the absence of Such treatment. of the sequence; or a ribosome binding site is operably linked (0093. The terms “effective amount” and “therapeutically to a coding sequence if it is positioned so as to facilitate effective amount’ as used herein, refers to that amount of an translation. Generally, “operably linked means that the DNA antibody thereof that binds flagellin, which is sufficient to sequences being linked are contiguous, and, in the case of a effect treatment, prognosis or diagnosis of an infection or secretory leader, contiguous and in reading phase. However, disease associated with flagellin, as described herein, when enhancers do not have to be contiguous. Linking is accom administered to a subject. A therapeutically effective amount plished by ligation at convenient restriction sites. If such sites will vary depending upon the Subject and the infection or do not exist, the synthetic oligonucleotide adaptors or linkers disease condition being treated, the weight and age of the are used in accordance with conventional practice. A nucleic subject, the severity of the infection or disease condition, the acid is “operably linked' when it is placed into a functional manner of administration and the like, which can readily be relationship with another nucleic acid sequence. For instance, determined by one of ordinary skill in the art. The dosages for a promoter or enhancer is operably linked to a coding administration can range from, for example, about 1 ng to sequence if it affects the transcription of the sequence. With about 10,000 mg, about 5ng to about 9,500 mg, about 10 ng respect to transcription regulatory sequences, operably linked to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng means that the DNA sequences being linked are contiguous to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng and, where necessary to join two protein coding regions, to about 6,500 mg, about 100ng to about 6,000 mg, about 200 contiguous and in reading frame. For Switch sequences, oper ng to about 5,500 mg, about 300 ng to about 5,000 mg, about ably linked indicates that the sequences are capable of effect 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg. ing Switch recombination. about 1 lug to about 3,500 mg, about 5ug to about 3,000 mg. 0090. The term “vector, as used herein, is intended to about 10 ug to about 2,600 mg, about 20 ug to about 2.575 mg. refer to a nucleic acid molecule capable of transporting about 30 ug to about 2.550 mg, about 40 ug to about 2,500 mg. another nucleic acid to which it has been linked. One type of about 50 ug to about 2.475 mg, about 100 ug to about 2,450 vector is a “plasmid, which refers to a circular double mg, about 200 ug to about 2,425 mg, about 300 ug to about stranded DNA loop into which additional DNA segments 2,000, about 400 ug to about 1,175 mg, about 500 ug to about may be ligated. Another type of vector is a viral vector, 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to wherein additional DNA segments may be ligated into the about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 viral genome. Certain vectors are capable of autonomous mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about replication in a host cell into which they are introduced (e.g., 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg. bacterial vectors having a bacterial origin of replication and about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg. episomal mammalian vectors). Other vectors (e.g., non-epi about 4.5 mg to about 900 mg, about 5 mg to about 875 mg. Somal mammalian vectors) can be integrated into the genome about 10 mg to about 850 mg, about 20 mg to about 825 mg. of a host cell upon introduction into the host cell, and thereby about 30 mg to about 800 mg, about 40 mg to about 775 mg. are replicated along with the host genome. Moreover, certain about 50 mg to about 750 mg, about 100 mg to about 725 mg. vectors are capable of directing the expression of genes to about 200 mg to about 700 mg, about 300 mg to about 675 which they are operatively linked. Such vectors are referred to mg, about 400 mg to about 650 mg, about 500 mg. or about herein as “recombinant expression vectors' (or simply, 525 mg to about 625 mg. of an antibody of the present inven “expression vectors'). In general, expression vectors of util tion. Dosage regimens may be adjusted to provide the opti ity in recombinant DNA techniques are often in the form of mum therapeutic response. An effective amount is also one in plasmids. The terms, “plasmid' and “vector” may be used which any toxic or detrimental effects (i.e., side effects) of an interchangeably. However, the invention is intended to antibody are minimized and/or outweighed by the beneficial include Such other forms of expression vectors, such as viral effects. vectors (e.g., replication defective retroviruses, adenoviruses 0094. The term “patient' includes human and other mam and adeno-associated viruses), which serve equivalent func malian Subjects that receive either prophylactic ortherapeutic tions. treatment. US 2010/02395.83 A1 Sep. 23, 2010

0095. As used herein, the term “subject' includes any flagellin from Salmonella (Genbank Accession No. human or non-human animal. For example, the methods and GI: 1333832) (SEQ ID NO:23) or Pseudomonas (Genbank compositions of the present invention can be used to treat a Accession No. GI:3386643) (SEQ ID NO:20). Subject having a bacterial disease. In a particular embodi 0103 Alternatively, lymphocytes may be immunized in ment, the Subject is a human. The term “non-human animal' vitro. Lymphocytes can then be fused with myeloma cells includes all vertebrates, e.g., mammals and non-mammals, using a suitable fusing agent, such as polyethylene glycol, to Such as non-human primates, sheep, rabbits, dogs, cows, form a hybridoma cell (Goding, Monoclonal Antibodies: chickens, amphibians, reptiles, etc. Principles and Practice, pp. 59-103 (Academic Press, 1986)). 0096. The term “sample” refers to tissue, body fluid, or a Culture medium in which hybridoma cells are growing is cell from a patient or a subject. Normally, the tissue or cell assayed for production of monoclonal antibodies directed will be removed from the patient, but in vivo diagnosis is also against the antigen. After hybridoma cells are identified that contemplated. Other patient samples, include blood, urine, produce antibodies of the desired specificity, affinity, and/or tear drops, serum, cerebrospinal fluid, feces, sputum, cell activity, the clones may be subcloned by limiting dilution extracts, lymph, gynecological fluid, ocular fluid, and fluid procedures and grown by Standard methods (Goding, Mono collected by peritoneal rinsing, etc. clonal Antibodies: Principles and Practice, pp. 59-103 (Aca 0097. The term “therapeutic agent” refers to any agent demic Press, 1986)). Suitable culture media for this purpose which acts in conjunction with or synergistically with the include, for example, D-MEM or RPMI-1640 medium. In antibody to treat or prevent an infection-associated infection addition, the hybridoma cells may be grown in vivo as ascites or disease. Therapeutic agents include, but are not limited to, tumors in an animal. The monoclonal antibodies secreted by chemotherapeutic agents, cytotoxic agents, anti-inflamma the Subclones can be separated from the culture medium, tory agents, e.g., a steroidal or nonsteroidal inflammatory ascites fluid, or serum by conventional immunoglobulin puri agent, or a cytotoxin antimetabolites (e.g., methotrexate, fication procedures such as, for example, protein 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil A-Sepharose, hydroxylapatite chromatography, gel electro decarbazine), alkylating agents (e.g., mechlorethamine, phoresis, dialysis, or affinity chromatography. thioepa chlorambucil, melphalan, carmustine (BSNU) and 0104. In another embodiment, antibodies (and binding lomustine (CCNU), cyclothosphamide, busulfan, dibromo fragments thereof) that bind flagellin can be isolated from mannitol, Streptozotocin, mitomycin C, and cis-dichlorodi antibody phage libraries generated using the techniques amine platinum (II) (DDP) cisplatin), anthracyclines (e.g., described in, for example, McCafferty et al., Nature, 348: daunorubicin (formerly daunomycin) and doxorubicin), anti 552-554 (1990). Clackson et al., Nature, 352:624-628 (1991), biotics (e.g., dactinomycin (formerly actinomycin), bleomy Marks et al., J. Mol. Biol., 222:581-597 (1991) and Hoet etal cin, mithramycin, and anthramycin (AMC)), and anti-mitotic (2005) Nature Biotechnology 23, 344-348; U.S. Pat. Nos. agents (e.g., Vincristine and vinblastine). 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. 0098. The term “cytotoxin' or “cytotoxic agent” includes Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. any agent that is detrimental to (e.g., kills) cells. Examples Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. include taxol, cytochalasin B, gramicidin D, ethidium bro Pat. Nos. 5,885,793; 6,521,404: 6,544,731; 6,555,313; 6,582, mide, emetime, mitomycin, etoposide, tenoposide, Vincris 915 and 6.593,081 to Griffiths et al. Additionally, production tine, vinblastine, colchicin, doxorubicin, daunorubicin, dihy of high affinity (nM range) human antibodies by chain shuf droxy anthracin dione, mitoxantrone, mithramycin, fling (Marks et al., Bio/Technology, 10:779-783 (1992)), as actinomycin D, 1-dehydrotestosterone, glucocorticoids, well as combinatorial infection and in vivo recombination as procaine, tetracaine, lidocaine, propranolol, and puromycin a strategy for constructing very large phage libraries (Water and analogs or homologs thereof. house et al., Nuc. Acids. Res., 21:2265-2266 (1993)), may 0099 Various aspects of the invention are described in also be used. further detail in the following subsections. 0105. In a particular embodiment, the antibodies of the invention are fully human antibodies. Such antibodies can be II. Methods for Producing Anti-Flagellin Antibodies produced using a variety of known methods, for example, the 0100 (i) Monoclonal Antibodies phage display technique described by Hoet et al., Supra. This 0101 Monoclonal antibodies of the invention can be pro technique involves the generation of a human Fab library duced using a variety of known techniques, such as those having a unique combination of immunoglobulin sequences described in the examples, as well as the standard Somatic cell isolated from human donors and having synthetic diversity in hybridization technique described by Kohler and Milstein the heavy-chain CDRs is generated. The library is then (1975) Nature 256: 495, viral or oncogenic transformation of screened for Fabs that bind to flagellin. B lymphocytes or phage display technique using libraries of 010.6 Additionally, fully human antibodies directed human antibody genes. In particular embodiments, the anti against flagellin can be generated using transgenic or tran bodies are fully human monoclonal antibodies. Schromosomic mice carrying parts of the human immune 0102) Accordingly, in one embodiment, a hybridoma system rather than the mouse system (see e.g., Lonberg, et al. method is used for producing an antibody that binds flagellin. (1994) Nature 368(6474): 856-859; Lonberg, N. etal. (1994), In this method, a mouse or other appropriate host animal can supra; reviewed in Lonberg, N. (1994) Handbook of Experi be immunized with flagellin protein (or a fragment of flagel mental Pharmacology 1 13:49-101; Lonberg, N. and Huszar, lin) in order to elicitlymphocytes that produce or are capable D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. of producing antibodies that will specifically bind to this and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546. antigen. Suitable flagellin protein can be obtained using a See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; variety of methods, purified from a source, produced recom 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; binantly or chemically synthesized. In a particular embodi 5,874.299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. ment of the present invention, antibodies are raised against No. 5,545,807 to Surani et al., PCT Publication Nos. WO US 2010/02395.83 A1 Sep. 23, 2010

92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO producing structurally and functionally related antibodies. 98/24884 and WO 99/45962, all to Lonberg and Kay; and For example, antibodies interact with target antigens pre PCT Publication No. WO 01/14424 to Korman et al.). dominantly through amino acid residues that are located in 0107. Other techniques for generating fully human anti the six heavy and light chain complementarity determining bodies of the invention include the use of a mouse that carries regions (CDRS). For this reason, the amino acid sequences human immunoglobulin sequences on transgenes and tran within CDRs are more diverse between individual antibodies Schomosomes, such as a mouse that carries a human heavy than sequences outside of CDRs. Because CDR sequences chain transgene and a human light chain transchromosome are responsible for most antibody-antigen interactions, it is (see e.g., PCT Publication WO 02/43478 to Ishida et al.). possible to express recombinant antibodies that mimic the 0108 Still further, alternative transgenic animal systems properties of specific naturally occurring antibodies by con expressing human immunoglobulin genes are available in the structing expression vectors that include CDR sequences art and can be used to raise anti-flagellin antibodies of the from the specific naturally occurring antibody grafted onto invention. For example, an alternative transgenic system framework sequences from a different antibody with different referred to as the Xenomouse (Abgenix, Inc.) can be used; properties (see, e.g., Riechmann, L. et al., 1998, Nature 332: such mice are described in, for example, U.S. Pat. Nos. 5,939, 323-327; Jones, P. et al., 1986, Nature 321:522-525; Tamura 598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to et al., J. Immunol., 2000 Feb. 1; 164(3):1432-41; and Queen, Kucherlapati et al. C. et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029 0109 Moreover, alternative transchromosomic animal 10033). Such framework sequences can be obtained from systems expressing human immunoglobulin genes are avail public DNA databases that include germline antibody gene able in the art and can be used to raise anti-flagellin antibodies Sequences. of the invention. For example, mice carrying both a human 0113. Thus, in one embodiment, one or more structural heavy chain transchromosome and a human light chain tran features of the particular anti-flagellin antibodies of the Schromosome can be used; as described in Tomizuka et al. invention are used to create structurally related anti-flagellin (2000) Proc. Natl. Acad. Sci. USA 97.722-727. Furthermore, antibodies that retain the functional properties of the parent cows carrying human heavy and light chain transchromo antibodies of the invention, such as binding to the same somes have been described in the art (Kuroiwa et al. (2002) epitope or overlapping epitopes bound by the anti-flagellin Nature Biotechnology 20:889-894) and can be used to raise antibodies exemplified herein, as well as competing for anti antibodies of the present invention. gen-binding with the anti-flagellin antibodies exemplified 0110. In yet another embodiment, antibodies of the herein. present invention can be prepared using a transgenic plant 0114. In another embodiment, one or more structural fea and/or cultured plant cells (such as, for example, tobacco, tures of the particular antibodies of the invention are used to maize and duckweed) that produce such antibodies. For create structurally related anti-flagellin antibodies that retain example, transgenic tobacco leaves expressing antibodies or functional properties of the parent antibodies of the invention, antigen binding portions thereof can be used to produce Such Such as (i) neutralizing flagellin; (ii) inhibiting the activity of antibodies by, for example, using an inducible promoter (see, flagellin; (iii) cross-reacting with a broad spectrum of gram e.g., Cramer et al., Curr: Top. Microbol. Immunol. 240:95118 negative bacteria; (iv) inhibiting bacterial invasion into Sus (1999)). Also, transgenic maize can be used to express Such ceptible epithelial cells; (v) binding to flagellin with an affin antibodies and antigen binding portions thereof (see, e.g., ity of at least 10 M'; (vi) reducing the symptoms of an Hood et al., Adv. Exp. Med. Biol. 464:127 147 (1999)). Anti enterobacterial infection or IBD in a subject; (vii) reducing bodies can also be produced in large amounts from transgenic the extent and severity of flagellin-induced tissue injury; (viii) plant seeds including antibody portions, such as single chain reducing flagellin-stimulated neutrophil infiltration; (ix) antibodies (scFv's), for example, using tobacco seeds and decreasing colonic mucosal congestion, erosion and hemor potato tubers (see, e.g., Conrad et al., Plant Mol. Biol. 38:101 rhagic ulcerations associated with IBD: (x) inhibiting or 109 (1998)). Methods of producing antibodies or antigen decreasing the production of mediators (e.g., MDA, IL-1B. binding portions in plants can also be found in, e.g., Fischeret TNFC, MIP-1, MIP-2 and IL-8); and (xi) counteracting a al., Biotechnol. Appl. Biochem. 30:99 108 (1999), Ma et al., reduction in body weight associated with IBD. Trends Biotechnol. 13:5227 (1995); Ma et al., Plant Physiol. 0115 Methods known in the art for creating such struc 109:341 6 (1995); Whitelam et al., Biochem. Soc. Trans. tural and functional related antibodies include, for example, 22:940944 (1994) and U.S. Pat. Nos. 6,040,498 and 6,815, Marks et al. (Biotechnology (NY). 1992 July; 10(7):779-83) 184. (monoclonal antibodies diversification by shuffling light 0111. The binding specificity of the antibodies of the chain variable regions, then heavy chain variable regions with present invention can be identified using any technique fixed CDR3 sequence changes), Jespers et al., (Biotechnology including those disclosed here, can be determined by immu (NY). 1994 September; 12(9):899-903) (selection of human noprecipitation or by an in vitro binding assay, Such as radio antibodies from phage display repertoires to a single epitope immunoassay (RIA) or enzyme-linked immunoabsorbent of an antigen), Sharon et al., (Proc Natl AcadSci USA. 1986 assay (ELISA). The binding affinity of a monoclonal anti April; 83(8):2628–31) (site-directed mutagenesis of an invari body or portion thereof can be determined by the Scatchard ant amino acid residue at the variable-diversity segments analysis of Munson et al., Anal. Biochem., 107:220 (1980). junction of an antibody); Casson et al., (JImmunol. 1995 Dec. Art recognized techniques can also be used to alter or opti 15; 155(12):5647-54) (evolution of loss and change of speci mize particular binding specificities and/or affinities (see, for ficity resulting from random mutagenesis of an antibody example, Carter PJ, Nature Reviews Immunology 6:343-357 heavy chain variable region). (2006)). 0116. In one embodiment, one or more CDR regions of 0112 In certain embodiments, partial antibody sequences antibodies of the invention can be combined recombinantly derived from antibodies of the invention may be used for with known human framework regions and CDRs to create US 2010/02395.83 A1 Sep. 23, 2010 additional, recombinantly-engineered, anti-flagellin antibod improved internalization capability and/or increased comple ies of the invention. The heavy and light chain variable frame ment-mediated cell killing and antibody-dependent cellular work regions can be derived from the same or different anti cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176: 1191 body sequences. 1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 0117. It is well known in the art that antibody heavy and (1992). Homodimeric antibodies with enhanced anti-tumor light chain CDR3 domains play a particularly important role activity may also be prepared using heterobifunctional cross in the binding specificity/affinity of an antibody for an anti linkers as described in Wolffetal. Cancer Research 53:2560 gen. See, for example, Brummel et al. (Biochemistry. 1993 2565 (1993). Alternatively, an antibody can be engineered Feb. 2: 32(4): 1180-7), which showed that binding activity is which has dual Fc regions and may thereby have enhanced retained in a wide range of CDR3 mutants for each of the four complement lysis and ADCC capabilities. See Stevenson et residues that directly hydrogen bond to the antigen. Only al. Anti-Cancer Drug Design 3:219-230 (1989). Gly' could not be replaced without significant loss of affin 0.122 Also encompassed by the present invention are ity (see also, Hall et al., J. Immunol., 149:1605-1612 (1992); bispecific antibodies and immunoconjugates, as discussed Polymenis et al., J. Immunol., 152:5318-5329 (1994); Jahn et below. al., Immunobiol., 193:400-419 (1995); Klimka et al., Brit. J. (0123 (ii) Bispecific Antibodies Cancer, 83:252-260 (2000); Beiboer et al., J. Mol. Biol, 296: 0.124. The present invention also provides bispecific anti 833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA, bodies which include at least one anti-flagellin antibody of 95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., 116: the invention linked to one or more antibodies which bind to 2161-2162 (1994); Ditzel et al., J. Immunol., 157:739-749 a second target (such as an immune cell (e.g., an Fc receptor (1996)). Accordingly, in certain embodiments, antibodies can on an immune cell) or a different epitope on flagellin). Bispe be prepared to include the heavy and/or light chain CDR3s of cific antibodies can be prepared as full length antibodies or the antibodies of the present invention (e.g., SEQID NOs: 9 antibody fragments (e.g. F(ab') bispecific antibodies). In a and 15). The antibodies can further include the heavy and/or particular embodiment, the invention provides mAb 743 light chain CDR2s of the antibodies of the present invention linked to one more antibodies which binds to a different (e.g., SEQ ID NOS:7 and 13). The antibodies can further epitope on flagellin. include the heavy and/or light chain CDR1s of the antibodies 0.125 Methods for making bispecific antibodies are well of the present invention (e.g., SEQID NOS:5 and 11). known in the art. For example, production of full length 0118. The CDR1, 2, and/or 3 regions of the engineered bispecific antibodies can be based on the coexpression of two antibodies described above can comprise the exact amino immunoglobulin heavy chain-light chain pairs, where the two acid sequences as those disclosed herein (e.g., CDRS of chains have different specificities (see, e.g., Millstein et al., monoclonal antibody 743 (“m Ab 743), set forth in SEQID Nature, 305:537-539 (1983)). Further details of generating NOs: 5, 7, 9, 11, 13 and 15. However, the ordinarily skilled bispecific antibodies can be found, for example, in Suresh et artisan will appreciate that some deviation from the exact al., Methods in Enzymology, 121:210 (1986) and in Brennan CDR sequences may be possible while still retaining the et al., Science, 229: 81 (1985), which describes a chemical ability of the antibody to bind flagellin effectively (e.g., con linkage process for making bispecific antibodies. Various servative amino acid substitutions). Accordingly, in another techniques for making and isolating bispecific antibody frag embodiment, the engineered antibody may be composed of ments directly from recombinant cell culture have also been one or more CDRs that are, for example, 90%. 95%, 98%, described. For example, bispecific antibodies have been pro 99% or 99.5% identical to one or more CDRS of mAb 743. duced using leucine Zippers (see, e.g., Kostelny et al., J. 0119. In another embodiment, one or more residues of a Immunol., 148(5):1547-1553 (1992)). Another strategy for CDR may be altered to modify binding to achieve a more making bispecific antibody fragments by the use of single favored on-rate of binding. Using this strategy, an antibody chain FV (SFV) dimers has also been reported (see, e.g., Gru having ultra high binding affinity of for example, 10'M' or ber et al., J. Immunol., 152:5368 (1994)). more, can be achieved. Affinity maturation techniques, well 0.126 (iii) Immunoconjugates known in the art and those described herein, can be used to I0127. In another aspect, the present invention provides alter the CDR region(s) followed by screening of the resultant immunoconjugates that bind to flagellin and target therapeu binding molecules for the desired change in binding. Accord tic agents (e.g., a toxin) to particular classes of bacteria. ingly, as CDR(S) are altered, changes in binding affinity as Immunoconjugates can be formed by conjugating (e.g., well as immunogenicity can be monitored and scored Such chemically linking or recombinantly expressing) antibodies that an antibody optimized for the best combined binding and of the invention to Suitable therapeutic agents. In a particular low immunogenicity are achieved. embodiment, the invention provides mAb 743 linked to a 0120 In addition to, or instead of, modifications within the therapeutic agent. Suitable agents include, for example, a CDRs, modifications can also be made within one or more of cytotoxic agent, a toxin (e.g. an enzymatically active toxin of the framework regions, FR1, FR2, FR3 and FR4, of the heavy bacterial, fungal, plant or animal origin, or fragments and/or the light chain variable regions of an antibody, so long thereof), and/or a radioactive isotope (i.e., a radioconjugate). as these modifications do not eliminate the binding affinity of Enzymatically active toxins and fragments thereof which can the antibody. be used include diphtheria A chain, nonbinding active frag 0121. In another embodiment, it may be desirable to ments of diphtheria toxin, exotoxin Achain (from Pseudomo modify the antibody of the invention with respect to effector nas aeruginosa), ricin A chain, abrin A chain, modeccin A function, so as to enhance the effectiveness of the antibody in chain, alpha-sarcin, Aleurites fordii proteins, dianthin pro treating an infection in a Subject, for example. For example teins, Phytolaca americana proteins (PAPI, PAPII, and PAP cysteine residue(s) may be introduced in the Fc region, S), momordica charantia inhibitor, curcin, crotin, Sapaonaria thereby allowing interchain disulfide bond formation in this officinalis inhibitor, gelonin, mitogellin, restrictocin, pheno region. The homodimeric antibody thus generated may have mycin, enomycin and the tricothecenes. A variety of radio US 2010/02395.83 A1 Sep. 23, 2010

nuclides are available for the production of radioconjugated one or a combination of antibodies of the invention thereof, of anti-flagellin antibodies. Examples include 'Bi, ''I, In, the present invention, formulated together with a pharmaceu OY and Re. tically acceptable carrier. In one embodiment, the composi 0128 Immunoconjugates of the invention can be made tions include a combination of multiple (e.g., two or more) using a variety of bifunctional protein coupling agents such as isolated antibodies of the invention, which bind different N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), epitopes on flagellin. iminothiolane (IT), bifunctional derivatives of imidoesters 0.132. As used herein, “pharmaceutically acceptable car (such as dimethyl adipimidate HCL), active esters (such as rier includes any and all solvents, dispersion media, coat disuccinimidyl Suberate), aldehydes (such as glutarelde ings, antibacterial and antifungal agents, isotonic and absorp hyde), bis-azido compounds (such as bis(p-azidobenzoyl) tion delaying agents, and the like that are physiologically hexanediamine), bis-diazonium derivatives (such as bis-(p- compatible. Preferably, the carrier is suitable for intravenous, diazoniumbenzoyl)-ethylenediamine), diisocyanates (such intramuscular, Subcutaneous, parenteral, spinal or epidermal as tolyene 2,6-diisocyanate), and bis-active fluorine com administration (e.g., by injection or infusion). Depending on pounds (such as 1,5-difluoro-2,4-dinitrobenzene). For the route of administration, the active compound, i.e., anti example, a ricin immunotoxin can be prepared as described in body, bispecific and multispecific molecule, may be coated in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled a material to protect the compound from the action of acids 1-isothiocyanatobenzyl-3-methyldiethylene triaminepen and other natural conditions that may inactivate the com taacetic acid (MX-DTPA) is an exemplary chelating agent for pound. conjugation of radionucleotide to the antibody (see, e.g., 0.133 A“pharmaceutically acceptable salt” refers to a salt WO94/11026). that retains the desired biological activity of the parent com pound and does not impart any undesired toxicological III. Methods for Screening Anti-Flagellin Antibodies effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 0129. Subsequent to producing antibodies that bind to 66:1-19). Examples of such salts include acid addition salts flagellin, the antibodies can be screened and selected for and base addition salts. Acid addition salts include those various properties, such as (i) their effect on bacterial invasion derived from nontoxic inorganic acids, Such as hydrochloric, into susceptible epithelial cells, (ii) inhibition of flagellin nitric, phosphoric, Sulfuric, hydrobromic, hydroiodic, phos stimulated NO or IL-8 production from epithelial cells, (iii) phorous and the like, as well as from nontoxic organic acids bacterial opsonophagocytosis, (iv) macrophage ingestion of Such as aliphatic mono- and dicarboxylic acids, phenyl-Sub bacteria, (v) superoxide production, (vi) ability to neutralize stituted alkanoic acids, hydroxy alkanoic acids, aromatic flagellin, (vii) ability to inhibit the activity of flagellin, (viii) acids, aliphatic and aromatic sulfonic acids and the like. Base cross-reactivity with a broad spectrum of gram-negative bac addition salts include those derived from alkaline earth met teria, (ix) ability to inhibit bacterial invasion into susceptible als, such as Sodium, potassium, magnesium, calcium and the epithelial cells, (x) ability to bind to flagellin with an affinity like, as well as from nontoxic organic amines, such as N,N'- of at least 10 M', (xi) capability of reducing the symptoms dibenzylethylenediamine, N-methylglucamine, chlorop of an enterobacterial infection or IBD in a subject, (xii) capa rocaine, choline, diethanolamine, ethylenediamine, procaine bility of reducing the extent and severity of flagellin-induced and the like. tissue injury, (xiii) capability of reducing flagellin-stimulated 0.134 Pharmaceutical compositions of the invention can neutrophil infiltration; (xiv) capability of decreasing colonic be administered alone or in combination therapy, i.e., com mucosal congestion, erosion and hemorrhagic ulcerations bined with other agents. For example, the combination associated with IBD: (XV) capability of inhibiting or decreas therapy can include a composition of the present invention ing the production of mediators (e.g., MDA, IL-1B, TNFC. with at least one or more additional therapeutic agents. Such MIP-1, MIP-2 and IL-8); and (xvi) capability of counteract as chemotherapeutic agents. The pharmaceutical composi ing a reduction in body weight associated with IBD, using a tions of the invention can also be administered in conjunction variety of assays that are well known in the art. Assays for with radiation therapy. screening for Such properties include the assays exemplified 0.135 Pharmaceutical compositions of the invention can and described herein, as well as those well known in the art, administered by a variety of methods known in the art. As will Such as binding to immobilized recombinant or bacterial be appreciated by the skilled artisan, the route and/or mode of flagellin on ELISA, binding to recombinant or bacterial administration will vary depending upon the desired results. extracts on SDS-PAGE, affinity binding determinations to The active compounds can be prepared with carriers that will purified antigens by BIACoreanalysis. protect the compound against rapid release, such as a con 0130 Antibodies or antigen binding portions thereofthat trolled release formulation, including implants, transdermal bind to the same or overlapping epitopes as one or more patches, and microencapsulated delivery systems. Biode antibodies of the present invention can also be identified gradable, biocompatible polymers can be used, such as eth using standard techniques known in the art and described ylene vinyl acetate, polyanhydrides, polyglycolic acid, col herein. For example, in order to screen for antibodies which lagen, polyorthoesters, and polylactic acid. Many methods bind to the same oran overlapping epitope on flagellin bound for the preparation of such formulations are patented or gen by an antibody of interest, a cross-blocking assay, Such as that erally known to those skilled in the art. See, e.g., Sustained described in Antibodies, A Laboratory Manual, Cold Spring and Controlled Release Drug Delivery Systems, J. R. Robin Harbor Laboratory, Ed Harlow and David Lane (1988), can son, ed., Marcel Dekker, Inc., New York, 1978. be performed. 0.136 To administer a compound of the invention by cer tain routes of administration, it may be necessary to coat the IV. Pharmaceutical Compositions compound with, or co-administer the compound with, a mate 0131. In another aspect, the present invention provides rial to prevent its inactivation. For example, the compound compositions, e.g., a pharmaceutical composition, containing may be administered to a Subject in an appropriate carrier, for US 2010/02395.83 A1 Sep. 23, 2010 example, liposomes, or a diluent. Pharmaceutically accept on (a) the unique characteristics of the active compound and able diluents include Saline and aqueous buffer Solutions. the particular therapeutic effect to be achieved, and (b) the Liposomes include water-in-oil-in-water CGF emulsions as limitations inherent in the art of compounding Such an active well as conventional liposomes (Strejan et al. (1984) J. Neu compound for the treatment of sensitivity in individuals. roimmunol. 7:27). 0142. Examples of pharmaceutically-acceptable antioxi 0.137 Pharmaceutically acceptable carriers include sterile dants include: (1) water Soluble antioxidants, such as ascorbic aqueous Solutions or dispersions and sterile powders for the acid, cysteine hydrochloride, Sodium bisulfate, Sodium met extemporaneous preparation of sterile injectable solutions or abisulfite, sodium sulfite and the like; (2) oil-soluble antioxi dispersion. The use of such media and agents for pharmaceu dants, such as ascorbyl palmitate, butylated hydroxyanisole tically active Substances is known in the art. Except insofar as (BHA), butylated hydroxytoluene (BHT), lecithin, propyl any conventional media or agent is incompatible with the gallate, alpha-tocopherol, and the like; and (3) metal chelat active compound, use thereof in the pharmaceutical compo ing agents, such as citric acid, ethylenediamine tetraacetic sitions of the invention is contemplated. Supplementary acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the active compounds can also be incorporated into the compo like. sitions. 0143. Therapeutic compositions of the present invention 0.138. Therapeutic compositions typically must be sterile include those Suitable for oral, nasal, topical (including buc and stable under the conditions of manufacture and storage. cal and Sublingual), rectal, vaginal and/or parenteral admin The composition can beformulated as a solution, microemul istration. The formulations may conveniently be presented in Sion, liposome, or other ordered structure Suitable to high unit dosage form and may be prepared by any methods known drug concentration. The carrier can be a solvent or dispersion in the art of pharmacy. The amount of active ingredient which medium containing, for example, water, ethanol, polyol (for can be combined with a carrier material to produce a single example, glycerol, propylene glycol, and liquid polyethylene dosage form will vary depending upon the Subject being glycol, and the like), and suitable mixtures thereof. The treated, and the particular mode of administration. The proper fluidity can be maintained, for example, by the use of amount of active ingredient which can be combined with a a coating Such as lecithin, by the maintenance of the required carrier material to produce a single dosage form will gener particle size in the case of dispersion and by the use of Sur ally be that amount of the composition which produces a factants. In many cases, it will be preferable to include iso therapeutic effect. Generally, out of one hundred percent, this tonic agents, for example, Sugars, polyalcohols such as man amount will range from about 0.001 percent to about ninety nitol, sorbitol, or sodium chloride in the composition. percent of active ingredient, preferably from about 0.005 Prolonged absorption of the injectable compositions can be percent to about 70 percent, most preferably from about 0.01 brought about by including in the composition an agent that percent to about 30 percent. delays absorption, for example, monostearate salts and gela 0144. The phrases “parenteral administration' and tin “administered parenterally as used herein means modes of 0139 Sterile injectable solutions can be prepared by administration other than enteral and topical administration, incorporating the active compound in the required amount in usually by injection, and includes, without limitation, intra an appropriate solvent with one or a combination of ingredi venous, intramuscular, intraarterial, intrathecal, intracapsu ents enumerated above, as required, followed by sterilization lar, intraorbital, intracardiac, intradermal, intraperitoneal, microfiltration. Generally, dispersions are prepared by incor transtracheal. Subcutaneous, Subcuticular, intraarticular, Sub porating the active compound into a sterile vehicle that con capsular, Subarachnoid, intraspinal, epidural and intrasternal tains a basic dispersion medium and the required other ingre injection and infusion. dients from those enumerated above. In the case of sterile 0145 Examples of Suitable aqueous and nonaqueous car powders for the preparation of sterile injectable solutions, the riers which may be employed in the pharmaceutical compo preferred methods of preparation are vacuum drying and sitions of the invention include water, ethanol, polyols (Such freeze-drying (lyophilization) that yield a powder of the as glycerol, propylene glycol, polyethylene glycol, and the active ingredient plus any additional desired ingredient from like), and Suitable mixtures thereof, vegetable oils, such as a previously sterile-filtered solution thereof. olive oil, and injectable organic esters, such as ethyl oleate. 0140 Dosage regimens are adjusted to provide the opti Proper fluidity can be maintained, for example, by the use of mum desired response (e.g., a therapeutic response). For coating materials, such as lecithin, by the maintenance of the example, a single bolus may be administered, several divided required particle size in the case of dispersions, and by the use doses may be administered over time or the dose may be of Surfactants. proportionally reduced or increased as indicated by the exi 0146 These compositions may also contain adjuvants gencies of the therapeutic situation. For example, the human Such as preservatives, wetting agents, emulsifying agents and antibodies of the invention may be administered once or twice dispersing agents. Prevention of presence of microorganisms weekly by Subcutaneous injection or once or twice monthly may be ensured both by Sterilization procedures, Supra, and by Subcutaneous injection. by the inclusion of various antibacterial and antifungal 0141. It is especially advantageous to formulate parenteral agents, for example, paraben, chlorobutanol, phenol Sorbic compositions in dosage unit form for ease of administration acid, and the like. It may also be desirable to include isotonic and uniformity of dosage. Dosage unit form as used herein agents, such as Sugars, sodium chloride, and the like into the refers to physically discrete units Suited as unitary dosages for compositions. In addition, prolonged absorption of the inject the Subjects to be treated; each unit contains a predetermined able pharmaceutical form may be brought about by the inclu quantity of active compound calculated to produce the sion of agents which delay absorption Such as aluminum desired therapeutic effect in association with the required monostearate and gelatin. pharmaceutical carrier. The specification for the dosage unit 0147 When the antibodies of the present invention are forms of the invention are dictated by and directly dependent administered as pharmaceuticals, to humans and animals, US 2010/02395.83 A1 Sep. 23, 2010 they can be given alone or as a pharmaceutical composition which discloses an osmotic drug delivery system. Many other containing, for example, 0.001 to 90% (more preferably, Such implants, delivery systems, and modules are known to 0.005 to 70%, such as 0.01 to 30%) of active ingredient in those skilled in the art. combination with a pharmaceutically acceptable carrier. 0151. In certain embodiments, antibodies of the invention 0148 Regardless of the route of administration selected, can be formulated to ensure proper distribution in vivo. For antibodies of the present invention and/or the pharmaceutical example, the blood-brain barrier (BBB) excludes many compositions thereof, are formulated into pharmaceutically highly hydrophilic compounds. To ensure that the therapeutic acceptable dosage forms by conventional methods known to compounds of the invention cross the BBB (if desired), they those of skill in the art. can beformulated, for example, in liposomes. For methods of 0149 Actual dosage levels of the active ingredients in the manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4.522,811; pharmaceutical compositions of the present invention may be 5,374,548; and 5,399.331. The liposomes may comprise one varied so as to obtainanamount of the active ingredient which or more moieties which are selectively transported into spe is effective to achieve the desired therapeutic response for a cific cells or organs, thus enhance targeted drug delivery (see, particular patient, composition, and mode of administration, e.g., V.V. Ranade (1989).J. Clin. Pharmacol. 29.685). Exem without being toxic to the patient. The selected dosage level plary targeting moieties include folate or biotin (see, e.g., will depend upon a variety of pharmacokinetic factors includ U.S. Pat. No. 5,416,016 to Low et al.); mannosides ing the activity of the particular compositions of the present (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. invention employed, or the ester, salt or amide thereof, the 153:1038): antibodies (P. G. Bloeman et al. (1995) FEBS Lett. route of administration, the time of administration, the rate of 357:140; M. Owais et al. (1995) Antimicrob. Agents excretion of the particular compound being employed, the Chemother: 39:180); surfactant protein A receptor (Briscoe et duration of the treatment, other drugs, compounds and/or al. (1995) Am. J. Physiol. 1233:134), different species of materials used in combination with the particular composi which may comprise the formulations of the inventions, as tions employed, the age, sex, weight, condition, general well as components of the invented molecules: p120 health and prior medical history of the patient being treated, (Schreier et al. (1994).J. Biol. Chem. 269:9090); see also K. and like factors well known in the medical arts. A physician or Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J.J. Veterinarian having ordinary skill in the art can readily deter Killion; I. J. Fidler (1994) Immunomethods 4:273. mine and prescribe the effective amount of the pharmaceuti cal composition required. For example, the physician or vet V. Methods of Using Anti-Flagellin Antibodies erinarian could start doses of the compounds of the invention 0152 The present invention provides methods of using employed in the pharmaceutical composition at levels lower antibodies that bind to and neutralize bacterial flagellin in a than that required in order to achieve the desired therapeutic variety of therapeutic and diagnostic applications. effect and gradually increase the dosage until the desired 0153 Suitable diseases that can be treated and/or diag effect is achieved. In general, a suitable daily dose of a com nosed using the antibodies provided herein include, for position of the invention will be that amount of the compound example, IBD, Ulcerative Colitis and Crohn's Disease, as which is the lowest dose effective to produce a therapeutic well as infectious diseases, including, but not limited to, gram effect. Such an effective dose will generally depend upon the negative bacterial infections (e.g., enterobacterial infections), factors described above. It is preferred that administration be sepsis, septic shock, Anthrax (by the bacterium Bacillus intravenous, intramuscular, intraperitoneal, or subcutaneous, anthracis), Bacterial Meningitis (caused by a variety of bac preferably administered proximal to the site of the target. If teria, including, but not limited to, Neisseria meningitides, desired, the effective daily dose of a therapeutic composition Streptococcus pneumoniae, Listeria monocytogenes, may be administered as two, three, four, five, six or more Pseudomonas aeruginosa, Staphylococcus aureus, Strepto Sub-doses administered separately at appropriate intervals coccus agalactiae and Haemophilus influenzae). Botulism throughout the day, optionally, in unit dosage forms. While it (caused by bacterium Clostridium botulinum), Brucellosis is possible for a compound of the present invention to be (caused by bacteria of the genus Brucella), Campylobacte administered alone, it is preferable to administer the com riosis (caused by bacteria of the genus Campylobacter), Cat pound as a pharmaceutical formulation (composition). Scratch Disease (caused by and Bar 0150. Therapeutic compositions can be administered with tonella clarridgeiae), Cholera (caused by the bacterium medical devices known in the art. For example, in a preferred Vibrio cholerae), Diphtheria (caused by Corynebacterium embodiment, a therapeutic composition of the invention can diphtheriae), (causative organism is Rick be administered with a needleless hypodermic injection ettsia prowazeki), Impetigo (caused by several bacteria, device, such as the devices disclosed in U.S. Pat. Nos. 5,399, including, Staphylococcus aureus and Streptococcus pyo 163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, genes), Legionellosis (caused by bacteria belonging to the or 4.596,556. Examples of well-known implants and modules genus Legionella), Leprosy (Hansen's Disease) (caused by useful in the present invention include: U.S. Pat. No. 4.487, the bacterium Mycobacterium leprae), Leptospirosis (caused 603, which discloses an implantable micro-infusion pump for by Spirochaetes of the genus Leptospira), Listeriosis (caused dispensing medication at a controlled rate; U.S. Pat. No. by the bacterium Listeria monocytogenes), Lyme Disease 4.486,194, which discloses a therapeutic device for adminis (caused by Spirochete bacteria from the genus Borrelia), tering medications through the skin; U.S. Pat. No. 4.447.233, Melioidosis (caused by the bacterium Burkholderia which discloses a medication infusion pump for delivering pseudomalilei), MRSA infection (caused by Staphylococcus medication at a precise infusion rate; U.S. Pat. No. 4.447.224, aureus), Nocardiosis (bacterium of the genus Nocardia, most which discloses a variable flow implantable infusion appara commonly Nocardia asteroides or Nocardia brasiliensis), tus for continuous drug delivery; U.S. Pat. No. 4,439,196, Pertussis (Whooping Cough) (caused by the bacterium Bor which discloses an osmotic drug delivery system having detella pertussis), Plague (caused by the enterobacteria Yers multi-chamber compartments; and U.S. Pat. No. 4.475,196, inia pestis), Pneumococcal pneumonia (caused by a variety of US 2010/02395.83 A1 Sep. 23, 2010 bacteria, including, but not limited to, Streptococcus pneu moniae, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas (SEQ ID NO: 25) : aeruginosa, , Chlamydophila pneumo aaggaaaagat catggcacaagt cattaatacaaac agcctgtc.gctgtt niae, Mycoplasma pneumoniae, and Legionella pneumo gacccagaataac citgaacaaatcc.cagtc.cgctctgggcaccgctatog phila), Psittacosis (caused by a bacterium called Chlamydo phila psittaci), Q fever (caused by infection with Coxiella agcgtctgtc.ttc.cggtctgcgt at Caacagcgcgaaagacgatgcggca burnetii), Rocky Mountain Spotted Fever (RMSF) (by Rick ggit caggcgattgct aaccgttt Caccg.cgaa catcaaaggtotgactica ettsia rickettsii), Salmonellosis (caused by bacteria of the genus Salmonella), Scarlet Fever, Shigellosis (caused by bac ggct tcc.cgtaacgctaacgacggitat ct c cattgcgcagaccactgaag teria of the genus Shigella), Syphilis (caused by Treponema pallidum), Tetanus (Clostridium tetani), Trachoma, Tubercu gcgc.gctgaacgaaatcaacaacaac ctgcagcgtgtgcgtgaactggcg losis (caused by mycobacteria, mainly Mycobacterium tuber gttcagtctgctaacgg tactaactic ccagtctgaccttgactictat coa culosis), Tularemia (by the bacterium Francisella tularensis), Typhoid Fever (caused by the bacterium Salmonella typhi), ggctgaaatcacc cagogtctgaacgaaatcgaccgtgt at Coggtcaga and Urinary Tract Infections (caused by bacteria such as Escherichia coli, Staphylococcus saprophyticus, Proteus ct cagttcaacggcgtgaaagtic ctggcgcagga caacaccCtgaccatc mirabilis, Klebsiella pneumoniae, Enterobacter spp., Caggttggtgccaacgac Pseudomonas and Enterococcus). 0160 The antigen was prepared by expression of a cDNA 0154 Antibodies of the present invention are particularly clone obtained by PCR amplification of DNA from S. useful for treating enterobacterial infections, and can be muenchen using a sense primer designated 1S (5'-CGC selected for broad reactivity with multiple entobacterial GGATCCCAATGGCACAAGTCATTAATACAAACA) strains, such Alishewanella, Alterococcus, Aquamonas, Ara (SEQ ID NO:17) and an antisense primer designated 468A nicola, Arsenophonus, Azotivirga, Blochmannia, Brenneria, (5'-TCCG Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter; CTCGAGTTAAATAGTTTCACCGTCGTTGGCACC) Dickeya, Edwardsiella, Enterobacter, Erwinia, Escherichia, (SEQ ID NO:18). Underlined nucleotides represent adaptor sequences added to the ends of primers to maintain proper Ewingella, Grimontella, Hafnia, Klebsiella, Kluyvera, reading frame and facilitate cloning (BamHI recognition sites Leclercia, Leminorella, Moellerella, Morganella, Obesum on sense primers and XhoI sites on antisense primers). The bacterium, Pantoea, Pectobacterium, Candidatus Phlomo template DNA for PCR was plasmid CL402, a clone of bacter, Photorhabdus, Plesiomonas, Pragia Proteus, Provi pBR322 containing a 3.8-kb EcoRI fragment of S. muenchen dencia, Rahnella, Raoultella, Salmonella, Samsonia, chromosomal DNA that harbors the 1.5-kb flagellin gene. Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, Wiggles PCR-generated flagellin DNA were digested with BamHI worthia, Xenorhabdus, Yersinia and Yokenella. plus XhoI, gel purified, and subcloned into the BamHI/XhoI O155 The antibodies can be administered alone or with sites at the 3' end of the His tag in expression vectorpFT 30C other therapeutic agents, which act in conjunction with or (Novagen, San Diego, Calif.). The correct reading frame and integrity of subcloned DNA was verified by DNA sequence synergistically with the antibodies, to treat diseases. Such analysis. The recombinant plasmids were then introduced therapeutic agents include, for example, toxins, chemothera into Escherichia coli BL21 (DE3) (Novagen) by transforma peutic agents, Small molecules and radiation tion and selected in the presence of kanamycin (50 ug/ml). 0156 Also within the scope of the present invention are kits comprising antibodies (or immunoconjugates or bispe Expression and Purification of Recombinant Antigen cific antibodies) of the invention which optionally include 0.161. A single colony of E. coli containing the recombi instructions for use in treating a disease associated with nant plasmid was grown at 37°C. in Luria broth containing 50 ug/ml kanamycin to an A600 of 0.5 and then induced for 3 h flagellin. The kits may include a label indicating the intended with 0.5 mM isopropyl-1-thio-b-D-galactopyranoside. Fol use of the contents of the kit. The term label includes any lowing induction, bacteria were harvested and washed with writing, marketing materials or recorded material Supplied on phosphate-buffered saline (PBS, pH 7.2). Cell-free lysates or with the kit, or which otherwise accompanies the kit. were prepared in 6 M guanidine chloride containing 5 mM 0157. Other embodiments of the present invention are imidazole and 0.1% Nonidet P-40 (binding buffer). After described in the following Examples. removing the insoluble material by centrifugation, the lysate 0158. The present invention is further illustrated by the was applied to a nickel-nitrilotriacetic acid-agarose (Qiagen, following examples which should not be construed as further Valencia, Calif.) column, washed extensively with binding limiting. The contents of Sequence Listing, figures and all buffer, and then eluted with binding buffer containing 200 mM imidazole. The purified proteins were extensively dia references, patents and published patent applications cited lyzed against PBS, and protein concentrations were deter throughout this application are expressly incorporated herein mined by the Bradford method. The final proteins were ana by reference. lyzed by 10% SDS-PAGE and visualized with Coomassie Blue staining to assess protein purity, integrity, and concen EXAMPLES tration. Example 1 Immunization (0162 Female BALB/c mice of 12 week old were immu Generation of Anti-Flagellin Antibodies nized with 50 ug of fusion protein in complete adjuvant. On Antigen Construction day 14, 28 and 42 mice were boosted with 50 ug of protein in incomplete adjuvant. Three days after final boost, spleen cells 0159. The gene fragment corresponding to amino acids will be prepared for fusion with SP2/O myeloma cells 1-156 of the flagellin gene of Salmonella muenchen was used (ATCC). Antibody titer in serum was measured by ELISA as an antigen (Genbank Accession No. GI: 47233) and using recombinant S. muenchen flagellin as antigen. US 2010/02395.83 A1 Sep. 23, 2010

Preparation of Monoclonal Antibodies by 35 cycles 94° C. for 30 seconds; 60° C. for 30 secs: 72° C. 0163 Anti-flagellin monoclonal antibodies were pro for 30 seconds; followed by 7 min at 72° C. Each PCR duced using previously described methods (Harlow and reaction used one adaptor-specific primer and one immuno Lane, Antibodies, CSH laboratories). Spleens from immu globulin constant region-specific primer (proprietary). The nized mice were broken apart with Sterile forceps and passed PCR products (approximate size of 500 bp) were extracted through a sterile stainless-steel strainer by pressing the spleen from a 2% agarose gel and purified using Qiaquick gel extrac tissue with the glass plunger. Splenocytes were collected, tion kit (Qiagen cat #28704). washed once in serum-free DMEM medium and then fused with SP2/O myeloma cells using sterile PEG (polyethylene Cloning glycol) solution. After fusion cells were seeded in 96-well microtiter plates and selected against HAT medium. Hybri (0170 Purified PCR products of the correct size for the doma culture supernatants were screened by ELISA using 96 heavy and light chain variable regions were ligated into Invit well plates coated with recombinant flagellin and amplified rogen's TOPO TA cloning vectors and transformed into the positive ones and tested for Ig-subclass (Southern Bio TOP10 cells according to manufacturer's instructions. The technology Associates). The candidate hybridoma lines pro clones were screened by PCR (using the previously described ducing IgG Subclass were selected and cloned by limited reaction conditions) to find those with the correct-sized insert dilution. (approximately 650 bp). Light chain clones containing the correct-sized insert, were grown in Luria broth containing Purification of Monoclonal Antibodies competent cells (Invitrogen OneShot Chemically Competent 0164. The murine monoclonal antibody to flagellin was E. Coli.), purified using a MiniPrep kit (Invitrogen cat it purified using standard immunology techniques. In brief, K2100-11) and cycle sequenced (Dartmouth MicroSequenc hybridoma cells were grown in roller bottles for 14 days in ing Facility, Lebanon, N.H.). Heavy chain clones which con BD medium and tissue culture supernatants wee collected. tained the correct-sized insert, were similarly grown and puri Antibodies were further purified by passing over a 10 ml fied and cycle sequenced. protein G-Sepharose affinity column, washed extensively with PBS, and eluted with 0.1 M glycine (pH 2.5). After Sequences dialysis of the eluate against PBS, antibodies titers were tested by ELISA and tested the purity by SDS-polyacryla 0171 The sequences were aligned using Sequencher soft mide gels. ware (Gene Codes Corporation). The results are as follows: Example 2 mAb 743 light chain consensus nucleotide sequence: Hybridoma Sequencing (SEQ ID NO : 4) ATTAGCCAGGAACAAAAATTCAAAGACAAAATGGATTTTCAGGTGCAGAT (0165 mRNA Preparation (0166 mRNA was extracted from 3x10 thawed hybri TTTCAGCTTCCTGCTAATCAGTTTCTCAGTCATAATGTCCAGAGGACAAA doma cells expressing murine mAb 743 using Norgen’s Cyto TTGTTCTCACCCAGTCTCCAGCAATCATGTCTGTATCTCCGGGGGAGAAA plasmic & Nuclear RNA Purification Kit using the standard procedure. Cells were thawed at room temperature and cen GT CACCTTGACCTGCAATGCCAGCTCAAGTGTAAGTTCCAGCTACTTATA trifuged briefly in a microcentrifuge for 10 minutes. Approxi mately 0.2 ml of lysis buffer containing 2 ul beta-mercapto CTGGTATCGGCAGAAACCAGGATCTTCCCCCAAC CTCTGGATTTATAGCA ethanol was added to the pellet and the mixture transferred to CATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCT a 1.5 mileppendorf tube. The tube was vortexed for 15 secs and centrifuged for 3 minutes at room temperature and trans GGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGTTGC ferred to a clean 1.5 ml tube. Binding solution (0.2 ml con CTCTTATTTCTGCCATCAGTGGAGTAGTTACCCGCTCACGTTCGGTGCTG taining beta-mercaptoethanol) was added to Supernatant, mixed by vortexing for 10 secs, followed by addition of 160 GGACCAAGCTGGAGCTGAAG ul of 100% ethanol and vortexed for 10 secs. The extracted mAb 743 light chain amino acid sequence: RNA was purified on a spin column according to kit direc (SEQ ID NO: 2) tions. ISOEOKFKDKMDFOVOIFSFLLISFSVIMSRGOIVLTOSPAIMSVSPGEK 0167. The light and heavy chains were prepared using distinct mRNA samples in separate reactions. The contami VTLTCNASSSWSSSYLYWYROKPGSSPNLWIYSTSNLASGVPARFSGSGS nating light chain variable region pseudogene mRNA pro GTSYSLTISSMEAEDVASYFCHQWSSYPLTFGAGTKLELK duced by the myeloma cells was digested RNase H (propri etary). mAb 743 light chain CDR1 amino acid sequence: (SEQ ID NO: 11) 0168 Following digestion 5' RACE was performed using NASSSWSSSYLY an Ambion FirstChoice RLM-RACE kit according to manu facturer's instruction. mAb 743 light chain CDR2 amino acid sequence: (SEQ ID NO: 13) RT-PCR STSNLAS mAb 743 light chain CDR3 amino acid sequence: 0169 Reverse transcription was performed using random (SEQ ID NO: 15) decamer primers provided in the Ambion kit. The product HOWSSYPLT cDNA was amplified using outer and inner PCR reactions. Thermocycler was programmed 3 minutes at 94°C. followed US 2010/02395.83 A1 Sep. 23, 2010

0176 Segments of monoclonal antibody heavy and light - Continued chain variable region amino acid sequences can then be com mAb 743 heavy chain consensus nucleotide sequence: pared with corresponding segments of human variable region (SEQ ID NO : 3) sequences in order to identify potential heavy and light chain GCGGCCGCGAATTCGCCCTTCGCGGATCCGAACACTGCGTTTGCTGGCTT human sequences for possible inclusion in the humanized TGATGAAAATAAGGTCACTGTTCTCACTATAGTTACTGAGCACACAGACC Sequences. 0177. A series of at least ten of each humanized heavy and TCACCATGGGATGGAGCTCTATCATCCTCTTCTTGTTAGCAACAGCTACA light chain variable regions can then be designed entirely GTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAG from segments of human variable region sequences. Alterna tive variants will differ in the inclusion of residues which GCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACTCCTTCA might be critical to restoration of the original monoclonal CCAGCGACTGGATGAACTGGGTGAAACAGAGGCCTGGACAGGGCCTTGAG antibody binding efficiency with the objective that the num ber of alterations in the frameworks needed to restore binding TGGATTGGCATAATTCATCCTTCCGACAGTGAAACTAAAATAAATCAATA efficiency will be kept to the minimum and generation of T-cell epitopes avoided. Potential T cell epitopes as deter TTTCAAGGACAAGGCCACATTGACTATAGACAAATCCTCCAGCACAGCCT mined by in silico methods can be considered in the selection ACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGT of alternative variants. 0.178 B. Construction of Humanized Heavy and Light GTTAGATGGGTGGGCTACCACGCTCAGGTCTGGGGCCAAGGCACCACTCT Chains CACCGTC 0179 Humanized variable regions can be constructed by PCR-ligation of long synthetic oligonucleotides. The initial mAb 743 heavy chain amino acid sequence: (SEQ ID NO: 1) heavy and light chain variable region genes can be used as AAANSPFADPNTAFAGFDENKWTWLTIWTEHTDLTMGWSSIILFLLATAT templates for construction of additional sequences by mutagenesis using overlapping PCR with mutagenic oligo GVHSQWOLOOPGAELVRPGASWKLSCKASGYSFTSDWMNWWKQRPGOGLE nucleotide primers. Restriction enzyme sites can then be engineered upstream and downstream of each of the variable WIGIIHPSDSETKINOYFKDKATLTIDKSSSTAYMOLSSPTSEDSAVYYC heavy and light chains for cloning into the appropriate expres WRWVGYHAQVWGQGTTL TV sion vector (e.g., an Antitope expression vector). The entire mAb 743 heavy chain CDR1 amino acid sequence: DNA sequence can be confirmed to be correct for each modi (SEO ID NO; 5) fied variable region cassette. GYSFTSDWMN 0180 C. Construction of Expression Plasmids Encoding mAb 743 heavy chain CDR2 amino acid sequence: Humanized Antibodies with Human Constant Regions (SEO ID NO: 7) 0181 At least ten humanized variable regions can be IIHPSDSETKINOYFKD transferred into mammalian expression vectors as follows. First, the DNA sequences for each variable region can be mAb 743 heavy chain CDR3 amino acid sequence: inserted into mammalian expression vectors between an (SEO ID NO: 9) upstream cytomegalovirus immediate/early promoter/en WVGYHAOV hancer (CMV-ie) plus the immunoglobulin signal sequence and a downstream the immunoglobulin constant region. The Example 3 heavy chain vector includes a genomic human IgG constant Generation of Humanized Antibodies region of choice (IgG1, or IgG4) and the dhfr gene for selec tion in mammalian cells. The light chain vector includes the 0172 Antibodies of the invention can also be humanized genomic human K constant region. using a variety of known techniques known in the art, such as 0182 DNA samples can then be prepared for transfection those taught in U.S. Pat. No. 5.225,539 to Winter, and U.S. into mammalian cells. The humanized antibody heavy and Pat. Nos. 5,530,101: 5,585,089: 5,693,762 and 6,180,370 to light chain-encoding plasmids can be co-transfected into Queen et al., the substance of which is incorporated herein by mammalian cells by electroporation. reference. Additionally, the antibodies of the invention can be humanized using composite human antibody technologies, as 0183 D. Generation of Humanized Antibody-Producing described below. Cell Lines and Selection of Lead Humanized Antibody 0173 A. Design of Humanized Heavy and Light Chains 0.184 Individual heavy and light chain plasmids can be 0.174. The sequences of mAb 743 (or other anti-flagellin paired in order to produce a final series of antibodies com antibodies) heavy and light chain variable regions can be bining variant humanized variable region sequences. These analyzed to identify CDRS, unusual amino acids and residues combinations may also include chimeric heavy and light critical to binding as follows. chains in order to determine the effects of individual modified 0175 First, protein models of the murine antibody vari humanized chains on binding efficiency. able regions can be generated using existing antibody struc 0185. Heavy and light chain plasmid DNA pairs can then tures as templates. Structural information from the protein be transfected into mammalian cells by electroporation, model can then be used to identify and compare residues stable cell lines can be selected and tested for antibody pro critical for antibody conformation and binding with structur duction. Cell lines producing humanized antibodies compris ally equivalent residues from existing antibody structures and ing combinations of heavy and light chains can then be sequence databases. These amino acids can then be candi expanded and antibody samples (typically 100 ug) purified. dates for inclusion in one or more variants of the final human 0186 Antibodies can then be tested in a binding assay in ized sequences. order to determine the humanized best antibody lead. The US 2010/02395.83 A1 Sep. 23, 2010 20 entire coding sequence of the lead humanized antibody can nella typhimurium, Proteus mirabilis, Enteropathogenic E. then be subcloned into an appropriate mammalian expression Coli) in a live bacterial ELISA assay. mab 743 bound cross Vector. reactively to a broad variety of different bacteria with highest binding observed towards Proteus Vulgaris, Salmonella typh Example 4 imurium and Serratia marcenscens (FIG. 2). Generation of Human Antibodies 0187. The present invention also contemplates the genera Example 7 tion of fully human antibodies. Such antibodies can be gen erated using a variety of techniques known in the art, such as Epitope Mapping Studies those described above, including, but, not limited to the tech niques described by Hoet etal (2005) Nature Biotechnology 0191 The epitope of Salmonella muenchen flagellin 23, 344-348, Lonberg, et al. (1994) Nature 368(6474): 856 bound by mab 743 was determined by immunoblotting and 859; Lonberg, N. etal. (1994), supra; reviewed in Lonberg, N. immunoprecipitation assays. Specifically, fine epitope map (1994) Handbook of Experimental Pharmacology 1 13:49 ping was performed by assessing the binding of antibodies to 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. various peptides corresponding to various regions of flagellin 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. protein. Recombinant proteins containing amino acids corre Acad. Sci. 764:536-546. See further, U.S. Pat. Nos. 5,545, sponding to 1-40, 1-156, 41-156, 78-156 and 53-505 were 806; 5,569,825;5,625,126;5,633,425; 5,789,650; 5,877,397; expressed in E. coli and purified to homogeneity using Ni 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lon affinity columns. Protein amounts equivalent to 1 Lug were berg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT separated on an 8-1.6% Tris-glycine gradient gel (Novex, San Publication Nos. WO 92/03918, WO 93/12227, WO Diego, Calif.). The proteins were then transferred to nitrocel 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, lulose (Novex) using the Novex Xcell Mini-Gel system. all to Lonberg and Kay; and PCT Publication No. WO Membranes were then blocked with 1% bovine serum albu 01/14424 to Korman et al., the substance of which is incor min (BSA) in PBS-T (0.05% tween 20) for 1 hour on an porated herein by reference. orbital shaker. The blocked membranes were then incubated with INO-743 (0.1 g/ml) in 1% BSA/PBS-T for 1 hour, Example 5 washed with PBS-T to remove unboundantibodies, and incu Binding Studies bated with Rabbit anti-mouse IgG-HRP 1:5000 in 1% BSA/ PBS-T for 1 hour. The blots were washed as above and ECL 0188 The binding of mAb 743 to Pseudomonas aerugi nosa type A flagellin and type B flagellin was assessed via chemiluminescent reagent added to the membrane for 1 SDS-PAGE and Western Blotting. Bacterial cells grown over minute. The blots were then exposed to X-ray film. night in Luria Broth were harvested by centrifugation and 0.192 As shown in FIG. 3, mab 743 mapped to amino then stored at -800 C. After thawing, the pellets were sus acids 41-53 of Salmonella muenchen (SEQID NO:23). pended in phosphate buffered saline (PBS) and sonicated to break chromosomal DNA. Bacterial lysates were boiled in Example 8 equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), and 20 In Vitro Nitric Oxide Production Assay ug protein loaded per lane on an 8-1.6% Tris-glycine gradient gel (Novex, San Diego, Calif. The proteins were then trans 0193 An in vitro nitric oxide (NO) production assay was ferred to nitrocellulose (Novex) using the Novex Xcell Mini used to assess the functional characteristics of murine mAb Gel system. Membranes were then blocked with 1% bovine 743. A non-specific monoclonal antibody and flagellin alone serum albumin (BSA) in PBS-T (0.05% tween 20) for 1 hour on an orbital shaker. The blocked membranes were then incu were used as controls. DLD-1 cells (ATCC) were grown at bated with INO-743 (0.1 ug/ml) in 1% BSA/PBS-T for 1 37° C. in 5% CO2 in Dulbecco's modified Eagle's medium hour, washed with PBS-T to remove unboundantibodies, and (DMEM, Invitrogen) and were supplemented with 10% FBS, incubated with Rabbit anti-mouse IgG-HRP 1:5000 in 1% 4 mM glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter BSA/PBS-T for 1 hour. The blots were washed as above and glucose, 1 mM Sodium pyruvate, and antibiotics. Cells, ECL chemiluminescent reagent added to the membrane for 1 between passages 5 and 15, were seeded at a density of 50,000 minute. The blots were then exposed to X-ray film. cells/cm2 in 96-well plates and allowed to grow 72-96 hours 0189 As shown in FIG. 1, m.Ab 743 reacts strongly with to confluence before use. Growth medium was changed the Pseudomonas aeruginosa type A flagellin and binds weakly day before use. Cells were washed once with DMEM without with Pseudomonas aeruginosa type B flagellin. FBS (but containing antibiotics) before the addition of flagel lin proteins. Flagellinto be tested were added to 100 ul growth Example 6 medium containing 0.5% FBS and 100 U/ml IFN-Y in each well. To neutralize NO production activity, the recombinant Anti-Flagellin Monoclonal Antibody 743 Cross-Re flagellin was incubated with antibodies for 1 hour at room acts with a Broad Spectrum of Gram-Negative Bac temperature and then added to DLD-1 cells in 96 well plate. teria After 20 hours of incubation at 370 C, the culture medium was 0190. Dose-response binding relative to mAb 743 was removed and tested for NO2-/NO3-concentration by Greiss assessed towards a panel of different gram-negative bacteria assay (Salzman et al. Am J Physiol. 268, 361-73 (1995)). (i.e., Proteus Vulgaris, non-pathogenic E. Coli, Citrobacter; (0194 As depicted in FIG.4, mAb 743 inhibited flagellin Serratia marcenscens, Pseudomonas aeruginosa, Salmo activity in the NO production assay. US 2010/02395.83 A1 Sep. 23, 2010 21

Example 9 Additional In Vitro Functional Assays SUMMARY OF SEQUENCE LISTING 0.195 A number of in-vitro functional assays can be per formed to assess the functional characteristics of the anti- SEQ ID NO: SEQUENCE flagellin antibody of the present invention (e.g., murine mAb 1 NO-743 VHaa 743). Such assays can include, but are not limited to, assays 2 NO-743 WLaa designed to assess the effect of anti-flagellin antibodies on (1) : N: y bacterial invasion into susceptible epithelial cells, (2) inhibi- 5 NO-743 VHaa CDR1 tion of flagellin-stimulated NO or IL-8 production from epi- 6 NO-743 VHnt CDR1 thelial cells, (3) bacterial opsonophagocytosis, (3) macroph- 7 NO-743 VHaa CDR2 age ingestion of bacteria, (4) bacterial "killing” and (5) 8 NO-743 VHnt CDR2 9 NO-743 VHaa CDR3 Superoxide production. 10 NO-743 VHnt CDR3 11 NO-743 WLala CDR1 12 NO-743 VLint CDR1 EQUIVALENTS 13 NO-743 WLala CDR2 14 NO-743 VLint CDR2 0196. Those skilled in the art will recognize, or be able to 15 NO-743 WLaa CDR3 ascertain using no more than routine experimentation, many 16 NO-743 VLint CDR3 equivalents of the specific embodiments of the invention 17 Sense Primer Designated 1s described herein. Such equivalents are intended to be encom- 18 AntisenseDesignated Primer 468A passed by the following claims. Any combination of the 19 Proteus mirabilis embodiments disclosed in the dependent claims are contem- (GI: 1169696) plated to be within the scope of the invention. 2O Pseudomonas aeruginosa (GI: 3386643) 21 Escherichia coi (GI: 1655807) INCORPORATION BY REFERENCE 22 Serraia marcescens (GI: 514988) 0197) The contents of all references, patents and published 2423 Salmonella muenchentyphimurium aa (GI: 153979)1333832) patent applications cited throughout this application, as well 25 Salmonella muenchen nt (GI: 47233) as the figures and Sequence Listing, are expressly incorpo rated herein by reference.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 2O

<21 Os SEQ ID NO 1 &211s LENGTH: 169 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs SEQUENCE: 1 Ala Ala Ala Asn. Ser Pro Phe Ala Asp Pro Asn. Thir Ala Phe Ala Gly 1. 5 1O 15 Phe Asp Glu Asn Llys Val Thr Val Lieu. Thir Ile Val Thr Glu. His Thr 2O 25 3 O Asp Lieu. Thir Met Gly Trp Ser Ser Ile Ile Leu Phe Leu Lleu Ala Thr 35 4 O 45 Ala Thr Gly Val His Ser Glin Val Glin Lieu. Glin Gln Pro Gly Ala Glu SO 55 60

Lieu Val Arg Pro Gly Ala Ser Val Llys Lieu. Ser Cys Lys Ala Ser Gly 65 70 7s 8O

Tyr Ser Phe Thr Ser Asp Trp Met Asn Trp Val Lys Glin Arg Pro Gly 85 90 95 Gln Gly Lieu. Glu Trp Ile Gly Ile Ile His Pro Ser Asp Ser Glu Thr 1OO 105 110 US 2010/02395.83 A1 Sep. 23, 2010 22

- Continued Lys Ile Asin Glin Tyr Phe Lys Asp Lys Ala Thr Lieu. Thir Ile Asp Llys 115 12 O 125 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp 13 O 135 14 O Ser Ala Val Tyr Tyr Cys Val Arg Trp Val Gly Tyr His Ala Glin Val 145 150 155 160 Trp Gly Glin Gly Thr Thr Lieu. Thr Val 1.65

<210s, SEQ ID NO 2 &211s LENGTH: 14 O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 2 Ile Ser Glin Glu Gln Llys Phe Lys Asp Llys Met Asp Phe Glin Val Glin 1. 5 1O 15 Ile Phe Ser Phe Leu Lleu. Ile Ser Phe Ser Val Ile Met Ser Arg Gly 2O 25 3O Glin Ile Val Lieu. Thr Glin Ser Pro Ala Ile Met Ser Val Ser Pro Gly 35 4 O 45 Glu Lys Val Thr Lieu. Thr Cys Asn Ala Ser Ser Ser Val Ser Ser Ser SO 55 6 O Tyr Lieu. Tyr Trp Tyr Arg Gln Llys Pro Gly Ser Ser Pro Asn Lieu. Trp 65 70 7s 8O Ile Tyr Ser Thr Ser Asn Lieu Ala Ser Gly Val Pro Ala Arg Phe Ser 85 90 95 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Lieu. Thir Ile Ser Ser Met Glu 1OO 105 11 O Ala Glu Asp Val Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro 115 12 O 125 Lieu. Thir Phe Gly Ala Gly Thr Lys Lieu. Glu Lieu Lys 13 O 135 14 O

<210s, SEQ ID NO 3 &211s LENGTH: 507 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 3 gcggcc.gcga attcgc.cctt cqcggat.ccg aac actg.cgt ttgctggctt tatgaaaat 6 O aagg to actg ttct cact at agttactgag cacacagacic ticaccatggg atggagctict 12 O atcatcct ct tcttgttago aac agctaca ggtgtcc act c cc aggt cca actgcagoag 18O Cctggggctg agctggtgag gcctgagct t cagtgaagc tigt cctgcaa ggcttctggc 24 O tact cott ca C cagogactg gatgaactgg gtgaaacaga ggcctggaca gggccttgag 3OO tggattggca taattcatcc titc.cgacagt gaalactaaaa taaat caata tittcaaggac 360 aaggccacat tdactataga caaatcct co agcacagcct acatgcaact cagcago cc.g 42O a catctgagg actictgcggit ctatt actgt gttagatggg togctacca cqcticaggit c 48O US 2010/02395.83 A1 Sep. 23, 2010 23

- Continued tggggccaag gcaccact ct caccgt.c O7

<210s, SEQ ID NO 4 &211s LENGTH: 42O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 4 attago cagg aacaaaaatt caaagacaaa atggattitt.c aggtgcagat titt cagott c 6 O ctgctaatca gtttct cagt cataatgtcc agaggacaaa ttgttct cac ccagt ct coa 12 O gcaatcatgt Citgitat ct co gggggaga aa gtcaccttga Cctgcaatgc cagct caagt 18O gtaagttcca gct acttata citggitat cqg cagaaac cag gatct tcc.cc caacctctgg 24 O atttatagca catcca acct ggcttctgga gtc. cctgctic gct tcagtgg cagtgggtct 3OO gggacct citt act citct cac aat cagoagc atggaggctgaagatgttgc ct cittatttic 360 tgc.cat cagt ggagtagtta ccc.gct cacg titcggtgctg ggaccaa.gct ggagctgaag 42O

<210s, SEQ ID NO 5 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 5 Gly Tyr Ser Phe Thr Ser Asp Trp Met Asn 1. 5 1O

<210s, SEQ ID NO 6 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 6 Ile Ile His Pro Ser Asp Ser Glu Thir Lys Ile Asn Glin Tyr Phe Lys 1. 5 1O 15 Asp

<210s, SEQ ID NO 7 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OO > SEQUENCE: 7 Trp Val Gly Tyr His Ala Glin Val 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 12 212. TYPE: PRT US 2010/02395.83 A1 Sep. 23, 2010 24

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 8 Asn Ala Ser Ser Ser Val Ser Ser Ser Tyr Lieu. Tyr 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 9

Ser Thir Ser Asn Lieu. Ala Ser 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 10 His Glin Trp Ser Ser Tyr Pro Leu. Thr 1. 5

<210s, SEQ ID NO 11 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 11 cgcggat.ccc aatggcacaa gttcattaata caaaca 36

<210s, SEQ ID NO 12 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 12 tcc.gct coag ttaaatagitt to accotcgt tdgcacc 37

<210s, SEQ ID NO 13 &211s LENGTH: 365 212. TYPE: PRT <213> ORGANISM: Proteus mirabilis

<4 OOs, SEQUENCE: 13 Met Ala Glin Val Ile Asn Thr Asn Tyr Lieu. Ser Lieu Val Thr Glin Asn 1. 5 1O 15 Asn Lieu. Asn Llys Ser Glin Gly. Thir Lieu. Gly Ser Ala Ile Glu Arg Lieu US 2010/02395.83 A1 Sep. 23, 2010 25

- Continued

2O 25 3O Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Glin 35 4 O 45 Ala Ile Ala Asn Arg Phe Thir Ser Asn Val Asn Gly Lieu. Thr Glin Ala SO 55 6 O Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Glin Thir Thr Glu Gly 65 70 7s 8O Ala Lieu. Asn. Glu Ile Asn. Asn. Asn Lieu. Glin Arg Ile Arg Glu Lieu. Thr 85 90 95 Val Glin Ala Lys Asn Gly Thr Asn. Ser Asn. Ser Asp Ile Thir Ser Ile 1OO 105 11 O Glin Asn. Glu Val Lys Asn Val Lieu. Asp Glu Ile Asn Arg Ile Ser Glu 115 12 O 125 Glin Thr Glin Phe Asn Gly Val Llys Val Lieu. Ser Gly Glu Lys Ser Glu 13 O 135 14 O Met Val Ile Glin Val Gly Thr Asn Asp Asn Glu Thir Ile Llys Phe Asn 145 150 155 160 Lieu. Asp Llys Val Asp Asn Asp Thir Lieu. Gly Val Ala Ser Asp Llys Lieu. 1.65 17O 17s Phe Asp Thir Lys Thr Glu Lys Lys Gly Val Thr Ala Ala Gly Ala Gly 18O 185 19 O Val Thr Asp Ala Lys Lys Ile Asn Ala Ala Ala Thr Lieu. Asp Met Met 195 2OO 2O5 Val Ser Lieu Val Lys Glu Phe Asn Lieu. Asp Gly Llys Pro Val Thir Asp 21 O 215 22O Llys Phe Ile Val Thir Lys Gly Gly Lys Asp Tyr Val Ala Thir Lys Ser 225 23 O 235 24 O Asp Phe Glu Lieu. Asp Ala Thr Gly. Thir Lys Lieu. Gly Lieu Lys Ala Ser 245 250 255 Ala Thir Thr Glu Phe Llys Val Asp Ala Gly Lys Asp Wall Lys Thir Lieu 26 O 265 27 O Asn. Wall Lys Asp Asp Ala Lieu Ala Thir Lieu. Asp Lys Ala Ile Asn Thr 27s 28O 285 Ile Asp Glu Ser Arg Ser Llys Lieu. Gly Ala Ile Glin Asn Arg Phe Glu 29 O 295 3 OO

Ser Thir Ile Asn. Asn Lieu. Asn. Asn. Thir Wall Asn. Asn Lieu. Ser Ala Ser 3. OS 310 315 32O Arg Ser Arg Ile Lieu. Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met 3.25 330 335 Ser Arg Gly Glin Ile Lieu. Glin Glin Ala Gly. Thir Ser Val Lieu Ala Glin 34 O 345 35. O Ala Asn Glin Val Pro Glin Thr Val Lieu. Ser Lieu. Lieu. Arg 355 360 365

<210s, SEQ ID NO 14 &211s LENGTH: 387 212. TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa <4 OOs, SEQUENCE: 14 Met Ala Lieu. Thr Val Asn. Thir Asn. Ile Ala Ser Lieu. Asn Thr Glin Arg 1. 5 1O 15 US 2010/02395.83 A1 Sep. 23, 2010 26

- Continued Asn Lieu. Asn. Asn. Ser Ser Ala Ser Lieu. Asn. Thir Ser Lieu. Glin Arg Lieu 2O 25 3O Ser Thr Gly Ser Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Lieu. 35 4 O 45 Glin Ile Ala Asn Arg Lieu. Thir Ser Glin Val Asn Gly Lieu. Asn. Wall Ala SO 55 6 O Thir Lys Asn Ala Asn Asp Gly Ile Ser Lieu Ala Glin Thr Ala Glu Gly 65 70 7s 8O Ala Lieu. Glin Glin Ser Thr Asn. Ile Lieu. Glin Arg Met Arg Asp Lieu. Ser 85 90 95 Lieu. Glin Ser Ala Asn Gly Ser Asn. Ser Asp Ser Glu Arg Thir Ala Lieu. 1OO 105 11 O Asn Gly Glu Val Lys Glin Lieu Gln Lys Glu Lieu. Asp Arg Ile Ser Asn 115 12 O 125 Thir Thir Thr Phe Gly Gly Arg Llys Lieu. Leu Asp Gly Ser Phe Gly Val 13 O 135 14 O Ala Ser Phe Glin Val Gly Ser Ala Ala Asn. Glu Ile Ile Ser Val Gly 145 150 155 160 Ile Asp Glu Met Ser Ala Glu Ser Lieu. Asn Gly Thr Tyr Phe Lys Ala 1.65 17O 17s Asp Gly Gly Gly Ala Val Thr Ala Ala Thr Ala Ser Gly Thr Val Asp 18O 185 19 O Ile Ala Ile Gly Ile Thr Gly Gly Ser Ala Val Asn Val Llys Val Asp 195 2OO 2O5 Met Lys Gly Asn. Glu Thir Ala Glu Glin Ala Ala Ala Lys Ile Ala Ala 21 O 215 22O Ala Val Asn Asp Ala Asn Val Gly Ile Gly Ala Phe Thr Asp Gly Ala 225 23 O 235 24 O Glin Ile Ser Tyr Val Ser Lys Ala Ser Ala Asp Gly Thr Thr Ser Ala 245 250 255 Val Ser Gly Val Ala Ile Thr Asp Thr Gly Ser Thr Gly Ala Gly Thr 26 O 265 27 O Ala Ala Gly. Thir Thr Thr Phe Thr Glu Ala Asn Asp Thr Val Ala Lys 27s 28O 285 Ile Asp Ile Ser Thr Ala Lys Gly Ala Glin Ser Ala Val Lieu Val Ile 29 O 295 3 OO Asp Glu Ala Ile Lys Glin Ile Asp Ala Glin Arg Ala Asp Lieu. Gly Ala 3. OS 310 315 32O Val Glin Asn Arg Phe Asp Asn. Thir Ile Asin Asn Lieu Lys Asn. Ile Gly 3.25 330 335 Glu Asn Val Ser Ala Ala Arg Gly Arg Ile Glu Asp Thr Asp Phe Ala 34 O 345 35. O Ala Glu Thir Ala Asn Lieu. Thir Lys Asn Glin Val Lieu. Glin Glin Ala Gly 355 360 365

Thir Ala Ile Lieu. Ala Glin Ala Asn. Glin Lieu. Pro Glin Ser Wall Lieu. Ser 37 O 375 38O

Lieu. Lieu. Arg 385

<210s, SEQ ID NO 15 &211s LENGTH: 585 212. TYPE: PRT US 2010/02395.83 A1 Sep. 23, 2010 27

- Continued

<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 15

Met Ala Glin Wall Ile Asn. Thir Asn. Ser Lieu. Ser Lieu. Ile Thr Glin Asn 1. 5 1O 15 Asn Lieu. Asn Lys Asn Glin Ser Ala Lieu. Ser Ser Ser Ile Glu Arg Lieu 2O 25 3O Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Glin 35 4 O 45 Ala Ile Ala Asn Arg Phe Thir Ser Asn. Ile Lys Gly Lieu. Thr Glin Ala SO 55 6 O Ala Arg Asn Ala Asn Asp Gly Ile Ser Val Ala Glin Thir Thr Glu Gly 65 70 7s 8O Ala Lieu. Ser Glu Ile Asn. Asn. Asn Lieu. Glin Arg Ile Arg Glu Lieu. Thr 85 90 95 Val Glin Ala Thir Thr Gly Thr Asn. Ser Asp Ser Asp Lieu. Asp Ser Ile 1OO 105 11 O Glin Asp G Ile Llys Ser Arg Lieu. Asp Glu Ile Asp Arg Val Ser Gly 12 O 125 Glin Thr Glin Phe Asn Gly Val Asn Val Lieu Ala Lys Asp Gly Ser Met 13 O 135 14 O Lys Ile Glin Val Gly Ala Asn Asp Gly Glu Thir Ile Thir Ile Asp Lieu. 145 150 155 160 Llys Lys Ile Asp Ser Asp Thr Lieu. Gly Lieu. Asn Gly Phe Asin Val Asn 1.65 17O 17s Gly Lys Gly. Thir Ile Thr Asn Lys Ala Ala Thr Val Ser Asp Lieu. Thir 18O 185 19 O Ser Ala Gly Ala Lys Lieu. Asn. Thir Thir Thr Gly Lieu. Tyr Asp Lieu Lys 195 2OO 2O5 Thr Glu Asn. Thir Lieu. Lieu. Thir Thr Asp Ala Ala Phe Asp Llys Lieu. Gly 21 O 215 22O Asn Gly Asp Llys Val Thr Val Gly Gly Val Asp Tyr Thr Tyr Asn Ala 225 23 O 235 24 O Lys Ser Gly Asp Phe Thir Thr Thr Lys Ser Thr Ala Gly Thr Gly Val 245 250 255 Asp Ala Ala Ala Glin Ala Ala Asp Ser Ala Ser Lys Arg Asp Ala Lieu 26 O 265 27 O Ala Ala Thir Lieu. His Ala Asp Val Gly Lys Ser Val Asn Gly Ser Tyr 27s 28O 285 Thir Thr Lys Asp Gly Thr Val Ser Phe Glu Thr Asp Ser Ala Gly Asn 29 O 295 3 OO Ile Thir Ile Gly Gly Ser Glin Ala Tyr Val Asp Asp Ala Gly Asn Lieu. 3. OS 310 315 32O Thir Thr Asn. Asn Ala Gly Ser Ala Ala Lys Ala Asp Met Lys Ala Lieu. 3.25 330 335 Lieu Lys Ala Ala Ser Glu Gly Ser Asp Gly Ala Ser Lieu. Thir Phe Asn 34 O 345 35. O Gly Thr Glu Tyr Thr Ile Ala Lys Ala Thr Pro Ala Thr Thr Thr Pro 355 360 365 Val Ala Pro Leu. Ile Pro Gly Gly Ile Thr Tyr Glin Ala Thr Val Ser 37 O 375 38O US 2010/02395.83 A1 Sep. 23, 2010 28

- Continued Lys Asp Val Val Lieu. Ser Glu Thir Lys Ala Ala Ala Ala Thir Ser Ser 385 390 395 4 OO Ile Thr Phe Asin Ser Gly Val Lieu Ser Lys Thr Ile Gly Phe Thr Ala 4 OS 41O 415 Gly Glu Ser Ser Asp Ala Ala Lys Ser Tyr Val Asp Asp Llys Gly Gly 42O 425 43 O Ile Thr Asn Val Ala Asp Tyr Thr Val Ser Tyr Ser Val Asn Lys Asp 435 44 O 445 Asn Gly Ser Val Thr Val Ala Gly Tyr Ala Ser Ala Thr Asp Thr Asn 450 45.5 460 Lys Asp Tyr Ala Pro Ala Ile Gly Thr Ala Val Asn Val Asn. Ser Ala 465 470 47s 48O Gly Lys Ile Thr Thr Glu Thir Thr Ser Ala Gly Ser Ala Thr Thr Asn 485 490 495 Pro Lieu Ala Ala Lieu. Asp Asp Ala Ile Ser Ser Ile Asp Llys Phe Arg SOO 505 51O Ser Ser Lieu. Gly Ala Ile Glin Asn Arg Lieu. Asp Ser Ala Val Thr Asn 515 52O 525 Lieu. Asn. Asn. Thir Thir Thr Asn Lieu. Ser Glu Ala Glin Ser Arg Ile Glin 53 O 535 54 O Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Glin Ile 5.45 550 555 560 Ile Glin Glin Ala Gly Asn. Ser Val Lieu Ala Lys Ala Asn Glin Val Pro 565 st O sts Glin Glin Val Lieu. Ser Lieu. Lieu. Glin Gly 58O 585

<210s, SEQ ID NO 16 &211s LENGTH: 348 212. TYPE: PRT <213> ORGANISM: Serratia marces cens

<4 OOs, SEQUENCE: 16

Met Ala Glin Wall Ile Asn. Thir Asn. Ser Lieu. Ser Lieu Met Ala Glin Asn 1. 5 1O 15 Asn Lieu. Asn Llys Ser Glin Ser Ser Lieu. Gly Thr Ala Ile Glu Arg Lieu 2O 25 3O Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Glin 35 4 O 45 Ala Ile Ser Asp Arg Phe Thr Ala Asn. Ile Lys Gly Lieu. Thr Glin Ala SO 55 6 O Ser Arg Asn Ala Asn Asp Gly Ile Ser Lieu Ala Glin Thir Thr Glu Gly 65 70 7s 8O Ala Lieu. Asn. Glu Val Asn Asp Asn Lieu. Glin Asn. Ile Arg Arg Lieu. Thr 85 90 95 Val Glin Ala Glin Asn Gly Ser Asn. Ser Thir Ser Asp Lieu Lys Ser Ile 1OO 105 11 O Glin Asp Glu Ile Thr Glin Arg Met Ser Glu Ile Asn Arg Ile Ser Glu 115 12 O 125 Glin Thr Asp Phe Asn Gly Val Llys Val Lieu. Ser Ser Asp Glin Llys Lieu. 13 O 135 14 O Thir Ile Glin Val Gly Ala Asn Asp Gly Glu Thir Ile Asp Ile Asp Lieu. 145 150 155 160 US 2010/02395.83 A1 Sep. 23, 2010 29

- Continued

Gln Gly Lieu. Thr Gly Phe Asp Val Thr Glu Asn Gly Thr Lys Ile Gly 1.65 17O 17s Ser Ala Ile Ala Asp Lys Ala Met Val Lys Asp Asp Thr Gly Thr Asp 18O 185 19 O Val Ala Phe Asp Lieu. Gly Glu Ser Phe Glin Thr Gly Gly Ala Lieu. Glu 195 2OO 2O5 Lys Ala Thr Lieu Val Ser G Llys Thir Lys Asp Gly Lys Glu Gly Tyr 21 O 2 : 22O Tyr Ile Glin Thr Thr Asp Ala Ala Thr Gly Ala Lys Thr Tyr Ala Thr 225 23 O 235 24 O Ala Lys Ile Asp Asp Llys Gly Val Val Thir Lys Gly Ala Asp Val Thr 245 250 255 Asp Wall Lys Asp Pro Lieu Ala Thr Lieu. Asp Lys Ala Lieu Ala Glin Val 26 O 265 27 O Asp Gly Lieu. Arg Ser Ser Lieu. Gly Ala Val Glin Asn Arg Phe Asp Ser 27s 28O 285

Wall Ile Ser Asn Lieu. Asn. Ser Thr Wall Asn. Asn Lieu. Ser Ala Ser Glin 29 O 295 3 OO Ser Arg Ile Glin Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser 3. OS 310 315 32O Arg Ala His Ile Lieu. Glin Glin Ala Gly. Thir Ser Val Lieu Ala Glin Ala 3.25 330 335 Asn Glin Ser Thr Glin Asn Val Lieu. Ser Lieu. Lieu. Arg 34 O 345

<210s, SEQ ID NO 17 &211s LENGTH: 505 212. TYPE: PRT <213s ORGANISM: Salmonella mulenchen

<4 OOs, SEQUENCE: 17

Met Ala Glin Wall Ile Asn. Thir Asn. Ser Lieu. Ser Lieu. Lieu. Thr Glin Asn 1. 5 1O 15 Asn Lieu. Asn Llys Ser Glin Ser Ala Lieu. Gly Thr Ala Ile Glu Arg Lieu 2O 25 3O Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Glin 35 4 O 45 Ala Ile Ala Asn Arg Phe Thr Ala Asn. Ile Lys Gly Lieu. Thr Glin Ala SO 55 6 O Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Glin Thir Thr Glu Gly 65 70 7s 8O Ala Lieu. Asn. Glu Ile Asn. Asn. Asn Lieu. Glin Arg Val Arg Glu Lieu Ala 85 90 95 Val Glin Ser Ala Asn Gly Thr Asn. Ser Glin Ser Asp Lieu. Asp Ser Ile 1OO 105 11 O Glin Ala Glu Ile Thr Glin Arg Lieu. Asn. Glu Ile Asp Arg Val Ser Gly 115 12 O 125 Glin Thr Glin Phe Asn Gly Val Llys Val Lieu Ala Glin Asp Asn. Thir Lieu. 13 O 135 14 O Thir Ile Glin Val Gly Ala Asn Asp Gly Glu Thir Ile Asp Ile Asp Lieu. 145 150 155 160 Lys Glu Ile Ser Ser Llys Thr Lieu. Gly Lieu. Asp Llys Lieu. Asn Val Glin US 2010/02395.83 A1 Sep. 23, 2010 30

- Continued

1.65 17O 17s Asp Ala Tyr Thr Pro Lys Glu Thir Ala Val Thr Val Asp Llys Thr Thr 18O 185 19 O Tyr Lys Asn Gly Thr Asp Thr Ile Thr Ala Glin Ser Asn Thr Asp Ile 195 2OO 2O5 Glin Thr Ala Ile Gly Gly Gly Ala Thr Gly Val Thr Gly Ala Asp Ile 21 O 215 22O Llys Phe Lys Asp Gly Glin Tyr Tyr Lieu. Asp Wall Lys Gly Gly Ala Ser 225 23 O 235 24 O Ala Gly Val Tyr Lys Ala Thr Tyr Asp Glu Thir Thr Llys Llys Val Asn 245 250 255 Ile Asp Thir Thr Asp Llys Thr Pro Lieu Ala Thr Ala Glu Ala Thr Ala 26 O 265 27 O Ile Arg Gly Thr Ala Thir Ile Thr His Asn Glin Ile Ala Glu Val Thr 27s 28O 285 Lys Glu Gly Val Asp Thir Thir Thr Val Ala Ala Glin Lieu Ala Ala Ala 29 O 295 3 OO Gly Val Thr Gly Ala Asp Lys Asp Asn. Thir Ser Lieu Val Lys Lieu. Ser 3. OS 310 315 32O Phe Glu Asp Lys Asn Gly Llys Val Ile Asp Gly Gly Tyr Ala Val Lys 3.25 330 335 Met Gly Asp Asp Phe Tyr Ala Ala Thr Tyr Asp Glu Lys Glin Val Glin 34 O 345 35. O Lieu. Lieu. Lieu. Asn. Asn His Tyr Thr Asp Gly Ala Gly Val Lieu. Glin Thr 355 360 365 Gly Ala Val Llys Phe Gly Gly Ala Asn Gly Lys Ser Glu Val Val Thr 37 O 375 38O Ala Thr Val Gly Llys Thr Tyr Lieu Ala Ser Asp Lieu. Asp Llys His Asn 385 390 395 4 OO Phe Arg Thr Gly Gly Glu Lieu Lys Glu Val Asn. Thir Asp Llys Thr Glu 4 OS 41O 415 Asn Pro Lieu. Glin Lys Ile Asp Ala Ala Lieu Ala Glin Val Asp Thir Lieu 42O 425 43 O Arg Ser Asp Lieu. Gly Ala Val Glin Asn Arg Phe Asn. Ser Ala Ile Thr 435 44 O 445 Asn Lieu. Gly Asn Thr Val Asn. Asn Lieu. Ser Ser Ala Arg Ser Arg Ile 450 45.5 460 Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala Glin 465 470 47s 48O Ile Lieu. Glin Glin Ala Gly. Thir Ser Val Lieu Ala Glin Ala Asn. Glin Val 485 490 495 Pro Glin Asn Val Lieu. Ser Lieu. Lieu. Arg SOO 505

<210s, SEQ ID NO 18 &211s LENGTH: 490 212. TYPE: PRT <213> ORGANISM: Salmonella typhimurium <4 OOs, SEQUENCE: 18

Met Ala Glin Wall Ile Asn. Thir Asn. Ser Lieu. Ser Lieu. Lieu. Thr Glin Asn 1. 5 1O 15 US 2010/02395.83 A1 Sep. 23, 2010 31

- Continued Asn Lieu. Asn Llys Ser Glin Ser Ala Lieu. Gly Thr Ala Ile Glu Arg Lieu 2O 25 3O Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala Gly Glin 35 4 O 45 Ala Ile Ala Asn Arg Phe Thr Ala Asn. Ile Lys Gly Lieu. Thr Glin Ala SO 55 6 O Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Glin Thir Thr Glu Gly 65 70 7s 8O Ala Lieu. Asn. Glu Ile Asn. Asn. Asn Lieu. Glin Arg Val Arg Glu Lieu Ala 85 90 95 Val Glin Ser Ala Asn. Ser Thr Asn. Ser Glin Ser Asp Lieu. Asp Ser Ile 1OO 105 11 O Glin Ala Glu Ile Thr Glin Arg Lieu. Asn. Glu Ile Asp Arg Val Asn Gly 115 12 O 125 Glin Thr Glin Phe Ser Gly Val Llys Val Lieu Ala Glin Asp Asn. Thir Lieu. 13 O 135 14 O Thir Ile Glin Val Gly Ala Asn Asp Gly Glu Thir Ile Asp Ile Asp Lieu. 145 150 155 160 Lys Glin Ile Asn. Ser Glin Thr Lieu. Gly Lieu. Asp Thir Lieu. Asn Val Glin 1.65 17O 17s Gln Lys Tyr Llys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala 18O 185 19 O Asp Thir Thir Ile Ala Lieu. Asp Asn. Ser Thr Phe Lys Ala Ser Ala Thr 195 2OO 2O5 Gly Lieu. Gly Gly Thr Asp Glu Lys Ile Asp Gly Asp Lieu Lys Phe Asp 21 O 215 22O Asp Thir Thr Gly Lys Tyr Tyr Ala Lys Val Thr Val Thr Gly Gly Thr 225 23 O 235 24 O Gly Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp Llys Thr Asn Gly Glu 245 250 255

Wall. Thir Lieu Ala Ala Wall Thr Pro Ala Thir Wall. Thir Thir Ala Thir Ala 26 O 265 27 O Lieu. Ser Gly Lys Met Tyr Ser Ala Asn Pro Asp Ser Asp Ile Ala Lys 27s 28O 285 Ala Ala Lieu. Thir Ala Ala Gly Val Thr Gly Thr Ala Ser Val Val Lys 29 O 295 3 OO Met Ser Tyr Thr Asp Asn. Asn Gly Llys Thir Ile Asp Gly Gly Lieu Ala 3. OS 310 315 32O Val Llys Val Gly Asp Asp Tyr Tyr Ser Ala Thr Glin Asp Lys Asp Gly 3.25 330 335 Ser Ile Ser Ile Asp Thir Thr Lys Tyr Thr Ala Asp Asn Gly. Thir Ser 34 O 345 35. O Llys Thr Ala Lieu. Asn Llys Lieu. Gly Gly Ala Asp Gly Llys Thr Glu Val 355 360 365 Val Thir Ile Asp Gly Llys Thr Tyr Asn Ala Ser Lys Ala Ala Gly His 37 O 375 38O Asp Phe Lys Ala Glu Pro Glu Lieu Ala Glu Glin Ala Ala Lys Thir Thr 385 390 395 4 OO Glu Asn Pro Lieu. Glin Lys Ile Asp Ala Ala Lieu Ala Glin Val Asp Thr 4 OS 41O 415 Lieu. Arg Ser Asp Lieu. Gly Ala Val Glin Asn Arg Phe Asn. Ser Ala Ile US 2010/02395.83 A1 Sep. 23, 2010

- Continued

42O 425 43 O Thir Asn Lieu. Gly Asn Thr Val Asn. Asn Lieu. Ser Ser Ala Arg Ser Arg 435 44 O 445 Ile Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala 450 45.5 460 Glin Ile Lieu. Glin Glin Ala Gly. Thir Ser Val Lieu Ala Glin Ala Asn Glin 465 470 47s 48O Val Pro Glin Asn Val Lieu. Ser Lieu. Lieu. Arg 485 490

<210s, SEQ ID NO 19 &211s LENGTH: 468 &212s. TYPE: DNA <213s ORGANISM: Salmonella mulenchen

<4 OOs, SEQUENCE: 19 aaggaaaaga t catggcaca agt cattaat acaaacagoc titcgctgtt gacccagaat 6 O alacctgaaca aatcc.cagtic cqctctgggc accgctatcg agcgtctgtc. ttc.cggtctg 12 O cgitat caa.ca gcgcgaaaga catgcggca ggt caggcga ttgct aaccg titt Caccgc.g 18O alacatcaaag gtctgactica ggctt cocgt aacgctaacg acggitat ct c cattgcgcag 24 O accactgaag gcgc.gctgaa caaatcaac aacaacctgc agcgtgtgcg tdaactggcg 3OO gttcagtctg ctaacggtac taact cocag totgaccttg actictat coa ggctgaaatc 360 acccagcgt.c talacgaaat caccgtgta t ccggtcaga Ctcagttcaa C9gcgtgaaa 42O gtcCtggcgc aggaca acac cct gaccatc Caggttggtg C caacgac 468

<210s, SEQ ID NO 2 O &211s LENGTH: 23 212. TYPE: PRT <213s ORGANISM: Salmonella mulenchen

<4 OOs, SEQUENCE: 2O Arg Lieu. Ser Ser Gly Lieu. Arg Ile Asn. Ser Ala Lys Asp Asp Ala Ala 1. 5 15 Gly Glin Ala Ile Ala Asn Arg

1. An isolated monoclonal antibody that binds to flagellin, the heavy chain variable region CDR1 comprises SEQID wherein the antibody comprises aheavy chain variable region NO:5; comprising an amino acid sequence set forth in SEQ ID the heavy chain variable region CDR2 comprises SEQID NO:1. NO:6; 2. An isolated monoclonal antibody that binds to flagellin, the heavy chain variable region CDR3 comprises SEQID wherein the antibody comprises a light chain variable region NO:7; comprising an amino acid sequence set forth in SEQ ID the light chain variable region CDR1 comprises SEQ ID NO:2. NO:8: 3. An isolated monoclonal antibody that binds to flagellin, the light chain variable region CDR2 comprises SEQ ID wherein the antibody comprises a heavy and light chain vari NO:9; and able region comprising the amino acid sequences set forth in the light chain variable region CDR3 comprises SEQ ID SEQ ID NOS:1 and 2, respectively. NO:10. 4. (canceled) 9. An isolated antibody that binds to an epitope on flagellin 5. (canceled) recognized by the antibody of claim 3 or 8. 6. (canceled) 10. An isolated antibody that competes for binding to 7. (canceled) flagellin with the antibody of claim 3 or 8. 8. An isolated monoclonal antibody that binds to flagellin 11. An isolated antibody that specifically binds to an and comprises heavy and light chain variable region CDR1. epitope located between amino acids 41-53 of flagellin from CDR2, and CDR3 sequences, wherein: Salmonella (SEQ ID NO:17). US 2010/02395.83 A1 Sep. 23, 2010

12. The antibody of any one of claims 3, 8, or 11, wherein 26. An expression vector comprising the nucleic acid mol the antibody is selected from the group consisting of a human ecule of claim 23. antibody, a humanized antibody, a chimeric antibody, and a 27. A kit comprising one or more isolated monoclonal murine antibody. antibodies of any of claims 3 or 8-11, an immunoconjugate of 13. The antibody of any one of claims 3, 8, or 11, wherein claim 16, or the bispecific molecule of claim 18, and option the antibody is selected from the group consisting of a Fab, ally comprising instructions for use in treating or diagnosing Fab'2, Schv, SMIP affibody, avimer, nanobody, and a domain a disease or infection associated with flagellin. antibody. 28. An isolated cell expressing the antibody of claim3 or 8. 14. The antibody of any one of claims 3, 8 or 11, wherein 29. A method of producing the antibody of claim 3 or 8. the antibody isotype is selected from the group consisting of 30. A method of treating a disease or infection associated an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an with flagellin comprising administering to the Subject athera IgA2, an IgASec, an Ig|D, and an IgE antibody. peutically effective amount of an isolated antibody of claim 3 15. A hybridoma which produces the antibody of claim3 or or 8. 8. 31. A method of treating a gram negative bacterial infection 16. An immunoconjugate comprising the antibody of any in a subject, comprising administering to the Subject a thera one of claims 3 or 8-11, linked to a therapeutic agent. peutically effective amount of an isolated antibody of claim 3 17. (canceled) or 8. 18. A bispecific molecule comprising the antibody of any 32. A method of neutralizing enterobacteria comprising one of claims 3 or 8-11, linked to a molecule having a binding contacting the enterobacteria with an isolated antibody of any specificity which is different from said antibody. of claim 3 or 8. 19. A composition comprising an antibody of any of claims 33. The method of claim 31, wherein the gram negative 3 or 8-11, an immunoconjugate of claim 16, or the bispecific bacterial infection is an enterobacterial infection selected molecule of claim 18, and a pharmaceutically effective car from the group consisting of Anthrax, Bacterial Meningitis, 1. Botulism, Brucellosis, Cat Scratch Disease, Cholera, Diph 20. The composition of claim 19, further comprising a theria, Epidemic Typhus, Impetigo, Legionellosis, Leprosy, therapeutic agent selected from the group consisting of a Leptospirosis, Listeriosis, Lyme Disease, Melioidosis, second antibody and an antibiotic. MRSA infection, Nocardiosis, Pertussis, Plague, Pneumo 21. (canceled) coccal pneumonia, Psittacosis, Q fever, Rocky Mountain 22. (canceled) Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, 23. An isolated nucleic acid molecule encoding a variable Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tula region of an antibody that binds to flagellin, wherein the remia, Typhoid Fever, sepsis, septic shock and Urinary Tract nucleic acid comprises the nucleotide sequence set forth in Infections. SEQID NOs:3 or 4. 34. (canceled) 24. (canceled) 25. (canceled)